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University of Cambridge
1.
Erard, Nicolas Pascal Jean.
Optimization of molecular tools for high-throughput genetic screening.
Degree: PhD, 2018, University of Cambridge
URL: https://www.repository.cam.ac.uk/handle/1810/271895 
► Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is…
(more)
▼ Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is broadly agreed on, the efficacy of currently available tools limits the strength of conclusions drawn from these experiments.
This thesis describes a method to optimize molecular tools for high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, we built a library of potent shRNAs to reliably and efficiently knock-down any gene in the human and mouse genomes. We then went on to apply a similar approach to identify sgRNAs with increased activity. We complemented this with conservation and repair prediction to increase the likelihood of generating functional knock-outs. With these tools in hand, we constructed, sequence-verified and validated arrayed shRNA and sgRNA libraries targeting any protein coding gene in the human genome. These resources allow large-scale screens to be performed in a multiplexed or arrayed format in a variety of biological contexts.
I have also applied these tools to identify therapeutic targets to circumvent cancer resistance to treatment in two different contexts. To overcome the shortfalls of single target therapy, I have developed multiplexed multidimensional shRNA screening strategy, where two genes are knocked down simultaneously in each cell. This strategy allows the identification of gene pairs that could be targeted in tandem to maximize therapeutic benefits. As a proof of concept, I have used it with a subset of druggable genes in melanoma cell lines. Moreover, we have applied our genome wide shRNA libraries to a different resistance context, stroma-mediated resistance to gemcitabine in PDAC. In this project, we performed screens in a PDAC-CAF coculture setting to try and identify cancer vulnerabilities specifically in the presence of stroma.
Overall, the tools developed in this thesis allow for the efficient knockdown or knockout of any gene, both in an individual or combinatorial setting. Apart from providing a resource that will be useful for many fields, we have performed several proof-of-concept studies where we have applied our tools to identify potential cancer drug targets.
Subjects/Keywords: Cancer; shRNA; CRISPR
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APA (6th Edition):
Erard, N. P. J. (2018). Optimization of molecular tools for high-throughput genetic screening. (Doctoral Dissertation). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/271895
Chicago Manual of Style (16th Edition):
Erard, Nicolas Pascal Jean. “Optimization of molecular tools for high-throughput genetic screening.” 2018. Doctoral Dissertation, University of Cambridge. Accessed April 16, 2021.
https://www.repository.cam.ac.uk/handle/1810/271895.
MLA Handbook (7th Edition):
Erard, Nicolas Pascal Jean. “Optimization of molecular tools for high-throughput genetic screening.” 2018. Web. 16 Apr 2021.
Vancouver:
Erard NPJ. Optimization of molecular tools for high-throughput genetic screening. [Internet] [Doctoral dissertation]. University of Cambridge; 2018. [cited 2021 Apr 16].
Available from: https://www.repository.cam.ac.uk/handle/1810/271895.
Council of Science Editors:
Erard NPJ. Optimization of molecular tools for high-throughput genetic screening. [Doctoral Dissertation]. University of Cambridge; 2018. Available from: https://www.repository.cam.ac.uk/handle/1810/271895
2.
김, 은영.
Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells.
Degree: 2011, Ajou University
URL: http://repository.ajou.ac.kr/handle/201003/4405
;
http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011837
► 일반적으로 Cellular senescence란 시간이 지나면 자연적으로 세포 증식이 억제되고 세포내 항상성 유지가 어렵게 되는 것을 말한다. 이러한 cellular senescence는 tumour cell에서도 일어나는데 tumour cell에서의 senescence는…
(more)
▼ 일반적으로 Cellular senescence란 시간이 지나면 자연적으로 세포 증식이 억제되고 세포내 항상성 유지가 어렵게 되는 것을 말한다. 이러한 cellular senescence는 tumour cell에서도 일어나는데 tumour cell에서의 senescence는 그 결과가 tumour-suppression 으로서도 작용하지만 tumour-promotion 으로서도 작용되어질 수 있다고 알려져 있다. 본 연구에서 사용한 유전자인 TIS21은 TPA에 의해 유도되어지는 21번째 sequences로서 anti-proliferative family에 속하는 단백질이다. 이 유전자는 mouse에서는 TIS21(TPA inducible sequences 21), human에서는 BTG2(B-cell translocation gene-2), rat에서는 PC3(pheocromocytoma clone-3)로 존재하는 동등 유전자이다. TIS21는 세포내의 전사 보조 인자, 신경세포 분화인자 및 성장억제인자로서 thymocyte 의 stage-specific expansion 을 매개하는 중요인자이고 조혈모세포 증식억제인자로 보고된 바 있다. 이 외에도TIS21은 암 억제, 세포사멸(Apoptosis), cell cycle 조절인자, DNA 수복 조절인자, transcriptional co-regulator, 배아세포의 분화 기작 조절인자로서의 기능을 가지고 있다. 본 연구에서 주목한 TIS21의 기능은 TIS21이 cell cycle 조절인자들을 조절함으로서 세포의 노화에 관여하고 있다는 점이다. 정상 세포에서 TIS21과 동등유전자인 BTG2가 세포노화를 유도한다는 사실에 기반을 두어 암세포에서의 TIS21의 역할을 규명하고자 본 연구를 진행하게 되었다. 암세포의 세포노화환경을 만들고자 사용한 Doxorubicin은 대표적인 anti-cancer drug로서 DNA 염기서열에 직접적으로 삽입되어 α-helix구조의 변형을 초래하여 DNA damage를 일으킨다고 알려져 있다. Doxorubicin은 농도에 따라 세포노화를 일으키거나 세포사멸을 일으킬 수 있다. 저농도의 Doxorubicin은 세포노화를 유도할 수 있지만 Doxorubicin에 의해 유도되는 노화기전은 아직 정확히 규명되지 않았다. 본 연구에서 간암세포주인 Huh7 에 저농도의 doxorubicin을 이용하여 유도되어진 세포노화현상에 anti-proliferative gene 인 TIS21이 미치는 효과를 확인하였다. Huh7 세포에서 TIS21단독으로는 세포노화 과정에 관여하지만 흥미롭게도 Doxorubicin을 이용해 노화를 유도한 Huh7 간암세포에서는 오히려 Doxorubicin에 의해 유도되어지는 노화를 억제하는 결과를 보여주었다. 이를 몇 가지 대표적인 노화 표지들을 이용하여 실험하였다. 먼저 TIS21을 단독으로 발현시켰을 경우 control에 비해 SA-β-galactosidase 활성도가 미약하게 증가하였고, proliferation이 억제되었으며 , actin filament가 가늘어지고 끊어지는 현상이 관찰되었다. 이와는 반대로 doxorubicin으로 노화를 유도한 간암세포에 TIS21을 과발현 시킨 경우 SA-β-galactosidase 활성도가 떨어졌고, doxorubicin에 의해 유도되어지는 actin filament의 remodeling이 감소하였으며, doxorubicin에 의한 세포 내 활성산소 (ROS)의 발생이 억제되었다. 이러한 결과들을 토대로, TIS21단독으로는 간암세포의 노화에 주된 역할을 하기엔 역부족이지만 간암세포노화과정에 기여하고, anti-cancer drug인 doxorubicin에 의해 유도되어지는 간암세포노화에서 TIS21은 doxorubicin에 의해 유도되어지는 간암세포의 노화를 억제한다고 생각되어진다. 또한 이를 short-hairpin RNA를 이용하여 TIS21의 발현을 Knockdown 시켜 이를 확인하는 단계를 진행 중이다. 이러한 이유로 TIS21 발현에 의한 Huh7 간암세포의 노화 조절을 규명하고자 하였다.
국문요약 ⅰ
차례 ⅲ
그림 차례 Ⅴ
Ⅰ. 서론 1
A. 세포노화 1
B. TIS21 2
Ⅱ. 재료 및 방법(혹은 연구대상 및 방법) 5
A. 세포 주 및 세포배양 5
B. 세포 수 집계 (Cell number counting) 5
C. TIS21 adenovirus (Ad) 5
D. Senescence associated-β-galactosidase (SA-β-gal) assay 5
E. 면역형광세포염색 (Immunocytochemistry) 6
F. Western blot analysis 6
G. 세포 내 활성산소 (ROS)의 양 측정 7
H. Doxorubicin-induced senescence 7
I. RNA분리와 RT-PCR 7
J. Lentivirus를 이용한 shTIS21 제작 8
Ⅲ. 결과 9
Part 1. TIS21 induces senescence of Huh7 cells and reversal of senescence phenotype by shTIS21 in Huh7 cells with TIS21
A. TIS21에 의한 Huh7 세포에서의 세포증식 억제 9
B. TIS21에 의한 Huh7 세포에서의 SA-β-galactosidase 활성도 증가 9
C. Huh7 세포내 actin filament 분포에서의 TIS21의역할 9
D. Lentivirus를 이용한 shTIS21 제작 10
E. shTIS21 에 의한 세포증식 복구 10
F. shTIS21 에 의한 F-actin의 형태와 위치 복구 10
G. TIS21에 의한 Huh7 세포노화 유도 가설도 11
Part 2. Effect of TIS21 on the senescence of Huh7 cells by treatment with doxorubicin
A. Huh7세포에서 Doxorubicin을 이용한 노화 유도 19
B. Doxorubicin-induced senescence Huh7 세포에서의 TIS21의역할 19
C. TIS21을 과 발현시킨 doxorubicin-induced senescence Huh7…
Advisors/Committee Members: 대학원 의생명과학과, 200524160, 김, 은영.
Subjects/Keywords: TIS21; doxorubicin; 세포노화유도; Huh7; shRNA
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
김, . (2011). Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells. (Thesis). Ajou University. Retrieved from http://repository.ajou.ac.kr/handle/201003/4405 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011837
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
김, 은영. “Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells.” 2011. Thesis, Ajou University. Accessed April 16, 2021.
http://repository.ajou.ac.kr/handle/201003/4405 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011837.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
김, 은영. “Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells.” 2011. Web. 16 Apr 2021.
Vancouver:
김 . Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells. [Internet] [Thesis]. Ajou University; 2011. [cited 2021 Apr 16].
Available from: http://repository.ajou.ac.kr/handle/201003/4405 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011837.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
김 . Regulation of Senescence Phenotypes by TIS21 gene in Huh7 Human Liver Cancer Cells. [Thesis]. Ajou University; 2011. Available from: http://repository.ajou.ac.kr/handle/201003/4405 ; http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000011837
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Cambridge
3.
Erard, Nicolas Pascal Jean.
Optimization of molecular tools for high-throughput genetic screening.
Degree: PhD, 2018, University of Cambridge
URL: https://doi.org/10.17863/CAM.18903
;
https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744534
► Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is…
(more)
▼ Forward genetic screening allows for the identification of any genes important for a particular biological process or phenotype. While the power of this approach is broadly agreed on, the efficacy of currently available tools limits the strength of conclusions drawn from these experiments. This thesis describes a method to optimize molecular tools for high-throughput screening, both for shRNA and sgRNA based reagents. Using large shRNA efficacy datasets, we first designed an algorithm predicting the potency of shRNAs based on sequence determinants. Combined with a novel shRNA backbone that further improves the processing of synthetic shRNAs, we built a library of potent shRNAs to reliably and efficiently knock-down any gene in the human and mouse genomes. We then went on to apply a similar approach to identify sgRNAs with increased activity. We complemented this with conservation and repair prediction to increase the likelihood of generating functional knock-outs. With these tools in hand, we constructed, sequence-verified and validated arrayed shRNA and sgRNA libraries targeting any protein coding gene in the human genome. These resources allow large-scale screens to be performed in a multiplexed or arrayed format in a variety of biological contexts. I have also applied these tools to identify therapeutic targets to circumvent cancer resistance to treatment in two different contexts. To overcome the shortfalls of single target therapy, I have developed multiplexed multidimensional shRNA screening strategy, where two genes are knocked down simultaneously in each cell. This strategy allows the identification of gene pairs that could be targeted in tandem to maximize therapeutic benefits. As a proof of concept, I have used it with a subset of druggable genes in melanoma cell lines. Moreover, we have applied our genome wide shRNA libraries to a different resistance context, stroma-mediated resistance to gemcitabine in PDAC. In this project, we performed screens in a PDAC-CAF coculture setting to try and identify cancer vulnerabilities specifically in the presence of stroma. Overall, the tools developed in this thesis allow for the efficient knockdown or knockout of any gene, both in an individual or combinatorial setting. Apart from providing a resource that will be useful for many fields, we have performed several proof-of-concept studies where we have applied our tools to identify potential cancer drug targets.
Subjects/Keywords: 616.99; Cancer; shRNA; CRISPR
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Erard, N. P. J. (2018). Optimization of molecular tools for high-throughput genetic screening. (Doctoral Dissertation). University of Cambridge. Retrieved from https://doi.org/10.17863/CAM.18903 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744534
Chicago Manual of Style (16th Edition):
Erard, Nicolas Pascal Jean. “Optimization of molecular tools for high-throughput genetic screening.” 2018. Doctoral Dissertation, University of Cambridge. Accessed April 16, 2021.
https://doi.org/10.17863/CAM.18903 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744534.
MLA Handbook (7th Edition):
Erard, Nicolas Pascal Jean. “Optimization of molecular tools for high-throughput genetic screening.” 2018. Web. 16 Apr 2021.
Vancouver:
Erard NPJ. Optimization of molecular tools for high-throughput genetic screening. [Internet] [Doctoral dissertation]. University of Cambridge; 2018. [cited 2021 Apr 16].
Available from: https://doi.org/10.17863/CAM.18903 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744534.
Council of Science Editors:
Erard NPJ. Optimization of molecular tools for high-throughput genetic screening. [Doctoral Dissertation]. University of Cambridge; 2018. Available from: https://doi.org/10.17863/CAM.18903 ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.744534
University of Texas – Austin
4.
-8375-338X.
Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses.
Degree: PhD, Cell and Molecular Biology, 2016, University of Texas – Austin
URL: http://hdl.handle.net/2152/68600
► Some eukaryotic viruses express small RNAs called microRNAs (miRNAs) to regulate host and viral gene expression. Due to their small genomic footprint, ability to regulate…
(more)
▼ Some eukaryotic viruses express small RNAs called microRNAs (miRNAs) to regulate host and viral gene expression. Due to their small genomic footprint, ability to regulate numerous genes, and lack of immunogenicity, miRNAs are an apt gene regulatory mechanism for viruses. Nevertheless, few viral miRNAs have been studied in vivo and the biological functions of most remain unknown. All known viral miRNAs are generated via host machinery. Most viral miRNAs mature through the canonical miRNA biogenesis pathway, whereas a few viruses generate miRNAs using noncanonical miRNA mechanisms. The noncanonical biogenesis mechanisms and the variety of miRNA alleles associated with some viruses provide powerful tools for probing mammalian small RNA biology.
In this dissertation, I analyze the biogenesis and function of miRNAs encoded by diverse tumor viruses. In chapter 2, I utilize the Simian virus 40 (SV40) primary miRNA (pri-miRNA) as a system to provide new mechanistic insights relevant to canonical pri-miRNA processing. This work reveals that multiple pri-miRNA structures coordinate processing by the Microprocessor complex. In chapter 3, I characterize the biogenesis of noncanonical miRNAs encoded by bovine leukemia virus (BLV), a deltaretrovirus. This work demonstrates that the BLV pre-miRNAs are directly transcribed by RNA polymerase III from proviral genomes, circumventing the requirement of mRNA cleavage for miRNA production. In chapter 4, using BLV and Adenoviral miRNAs, I demonstrate that dual-specificity phosphatase 11 (DUSP11) promotes accumulation and activity of small RNAs derived from diverse 5´-triphosphorylated precursors in the RNA-induced silencing complex (RISC). In chapter 5, I develop a novel method to express short hairpin RNAs (shRNAs) using the architecture of the BLV miRNA genes, thereby decreasing the template space required for
shRNA expression. This work applies to gene silencing strategies where smaller gene cassettes are desirable. In chapter 6, I report that the miRNAs encoded by murine polyomavirus (MuPyV) are not required for viral persistence. Instead, the MuPyV miRNAs promote viral shedding during the acute phase of infection in vivo. This work provides a fundamental understanding of the functional role of polyomavirus-encoded miRNAs.
Combined, this work advances the fields of experimental gene silencing, small RNA biology, and virus-host interactions.
Advisors/Committee Members: Sullivan, Christopher S. (advisor), Dudley, Jaquelin (committee member), Upton, Jason (committee member), Ehrlich, Lauren (committee member), Russell, Rick (committee member), Ulug, Emin (committee member).
Subjects/Keywords: miRNA; Polyomavirus; BLV; shRNA; RNAi
Record Details
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
-8375-338X. (2016). Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses. (Doctoral Dissertation). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/68600
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Chicago Manual of Style (16th Edition):
-8375-338X. “Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses.” 2016. Doctoral Dissertation, University of Texas – Austin. Accessed April 16, 2021.
http://hdl.handle.net/2152/68600.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
MLA Handbook (7th Edition):
-8375-338X. “Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses.” 2016. Web. 16 Apr 2021.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Vancouver:
-8375-338X. Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses. [Internet] [Doctoral dissertation]. University of Texas – Austin; 2016. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/2152/68600.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Council of Science Editors:
-8375-338X. Characterization of the biogenesis and function of miRNAs encoded by diverse tumor viruses. [Doctoral Dissertation]. University of Texas – Austin; 2016. Available from: http://hdl.handle.net/2152/68600
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
5.
Jivrajani Mehul.
Targeted Delivery of shRNA to Cancer Cell;.
Degree: 2015, Nirma University
URL: http://shodhganga.inflibnet.ac.in/handle/10603/44918
Subjects/Keywords: Cancer; Cell; shRNA
Record Details
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Share »
Record Details
Similar Records
Cite
« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mehul, J. (2015). Targeted Delivery of shRNA to Cancer Cell;. (Thesis). Nirma University. Retrieved from http://shodhganga.inflibnet.ac.in/handle/10603/44918
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Mehul, Jivrajani. “Targeted Delivery of shRNA to Cancer Cell;.” 2015. Thesis, Nirma University. Accessed April 16, 2021.
http://shodhganga.inflibnet.ac.in/handle/10603/44918.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Mehul, Jivrajani. “Targeted Delivery of shRNA to Cancer Cell;.” 2015. Web. 16 Apr 2021.
Vancouver:
Mehul J. Targeted Delivery of shRNA to Cancer Cell;. [Internet] [Thesis]. Nirma University; 2015. [cited 2021 Apr 16].
Available from: http://shodhganga.inflibnet.ac.in/handle/10603/44918.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Mehul J. Targeted Delivery of shRNA to Cancer Cell;. [Thesis]. Nirma University; 2015. Available from: http://shodhganga.inflibnet.ac.in/handle/10603/44918
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Tasmania
6.
Casey, NP.
The Therapeutic potential of
lentivector-delivered RNAi.
Degree: 2010, University of Tasmania
URL: https://eprints.utas.edu.au/10783/1/01_Casey_front.pdf
;
https://eprints.utas.edu.au/10783/2/01_Casey_whole.pdf
► Many forms of leukaemia are caused by chromosomal translocations, which result in specific and characteristic genomic sequences. Where these sequences are unique to the leukaemic…
(more)
▼ Many forms of leukaemia are caused by chromosomal translocations, which
result in specific and characteristic genomic sequences. Where these sequences are
unique to the leukaemic cells, they represent good candidates for targeting by sequencespecific
techniques, such as RNA-Interference (RNAi). RNAi is a mechanism inherent
in eukaryotic cells which silences target mRNAs based on homology to a dsRNA
template. This template may be introduced artificially by a number of methods, and so
this mechanism can be manipulated to regulate the expression of target genes. One of
the most efficient methods of introducing RNAi templates is by expression of shorthairpin
RNA (shRNA) cassettes from DNA plasmids or vectors.
Lentiviral vectors are based on viruses that integrate into the DNA of the host
cell, and are a highly efficient class of vectors for transducing and providing stable
transgene expression in a range of cell types. They are particularly effective at
tansducing haematopoietic cells, which have proven difficult to transduce by other
methods. By fine-tuning the methods of vector production and transduction, a range of
human leukaemic cells lines were able to be transduced with unprecedented efficiency
in the present study. Lentiviral transduction was combined with rapid puromycin
selection to generate a pure population of transduced cells with minimal expansion of
the cell population.
The strategy of expressing shRNAs from retroviral and lentiviral vectors
combined with puromycin selection was used to target three well-characterised fusion
genes; Bcr-Abl, PML/RARα and RUNX1/ETO, in three human leukaemic cell lines. In
two of these, the shRNA was able to efficiently and effectively down-regulate the target
mRNA, and inhibit the proliferation of the transduced leukaemic cells. In the third case,
RUNX1/ETO, no effective shRNA design could be identified.
Finally, concerns over the safety of integration targeting by current gene therapy
vectors motivated an investigation of the activity of a novel integrase enzyme from the
Ty3 retrotransposon found in yeast. In yeast cells, the integration-mediating enzyme of
this retrotransposon has very specific targeting characteristcs, which, if retained in
human cells, would provide a very safe gene therapy vector. It was found that this
enzyme is indeed active in human cells, and therefore has potential in the context of
human gene therapy.
Subjects/Keywords: lentivirus; vector; RNAi; shRNA; chrosomal-translocation; leukaemia
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Casey, N. (2010). The Therapeutic potential of
lentivector-delivered RNAi. (Thesis). University of Tasmania. Retrieved from https://eprints.utas.edu.au/10783/1/01_Casey_front.pdf ; https://eprints.utas.edu.au/10783/2/01_Casey_whole.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Casey, NP. “The Therapeutic potential of
lentivector-delivered RNAi.” 2010. Thesis, University of Tasmania. Accessed April 16, 2021.
https://eprints.utas.edu.au/10783/1/01_Casey_front.pdf ; https://eprints.utas.edu.au/10783/2/01_Casey_whole.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Casey, NP. “The Therapeutic potential of
lentivector-delivered RNAi.” 2010. Web. 16 Apr 2021.
Vancouver:
Casey N. The Therapeutic potential of
lentivector-delivered RNAi. [Internet] [Thesis]. University of Tasmania; 2010. [cited 2021 Apr 16].
Available from: https://eprints.utas.edu.au/10783/1/01_Casey_front.pdf ; https://eprints.utas.edu.au/10783/2/01_Casey_whole.pdf.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Casey N. The Therapeutic potential of
lentivector-delivered RNAi. [Thesis]. University of Tasmania; 2010. Available from: https://eprints.utas.edu.au/10783/1/01_Casey_front.pdf ; https://eprints.utas.edu.au/10783/2/01_Casey_whole.pdf
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Manchester
7.
Blaser, Julian.
The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia.
Degree: 2012, University of Manchester
URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:182916
► Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide…
(more)
▼ Phosphoinositides (PIs) are pivotal lipid molecules
with both scaffolding and signalling functions regulating key
aspects of cellular physiology. For example, phosphatidylinositol
(3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase
(PI3K), is an essential mediator of the PI3K/AKT signalling
pathway, which is crucial for cell proliferation, survival and
apoptosis. Constitutive activation of this signalling cascade has
been identified in acute myeloid leukaemia (AML), the most common
haematopoietic malignancy in adults, and experimental deletion of
the PI3K antagonists PTEN and SHIP cause leukaemia in mice.
However, little is known regarding the role of other PI modulator
proteins in AML. Thus, in this thesis, a lentivirally delivered
small hairpin RNA (
shRNA) library targeting 103 genes (345 pLKO
knockdown constructs) with presumed or established roles in PI
metabolism was utilised to screen for genes required for AML blast
cell viability/proliferation and differentiation.First, knockdown
constructs were tested for their impact on proliferation/viability
in seven human AML cell lines by measuring fold change in
fluorescence of the cell viability dye alamarBlue relative to
controls (cells transduced with a non-targeting control hairpin)
over three days. This identified 13 candidate genes selected with
the criterion that two or more knockdown constructs per gene reduce
cell viability/proliferation relative to control by ≥50 % across
all cell lines. From these candidate genes, PIP4K2A, INPP5B and
IMPAD1 were selected for downstream validation experiments, which
reproduced the observation from the primary screen. For INPP5B and
IMPAD1, knockdown constructs also reduced clonogenic potential of
primary human AML samples but only showed a modest effect on normal
CD34+ haematopoietic stem or progenitor cells (HSPCs) in a
methylcellulose based assay. This could be recapitulated in a
murine setting where knockdown constructs targeting both genes
reduced clonogenic potential of murine MLL- AF9 AML cells with
little effect on normal KIT+ HSPCs. In line with this, Inpp5b
knockout KIT+ BM cells either failed to immortalise or weakly
immortalised, following forced expression of the powerful MLL-AF9
oncogene.A further screen was performed to identify regulators of
THP-1 blast cell differentiation, by seeding knockdown construct
transduced cells into methylcellulose based semisolid media. After
ten days of incubation the degree of macrophage differentiation was
evaluated by light microscopy and an arbitrary differentiation
score was given. With the criterion that ≥2 knockdown constructs
per gene received the highest differentiation score, reflecting
terminal macrophage differentiation of all seeded cells, SBF2 was
identified as the top-scoring hit. Validation experiments have
confirmed macrophage differentiation based on cytospin preparations
of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have
shown that knockdown constructs targeting PIP4K2A and SBF2 delayed
or abrogated in vivo leukaemogenesis.Thus this work has…
Advisors/Committee Members: SOMERVAILLE, TIMOTHY T, Somervaille, Tim, Divecha, Nullin.
Subjects/Keywords: RNAi; shRNA; acute myeloid leukaemia; phosphoinositides
Record Details
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Blaser, J. (2012). The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:182916
Chicago Manual of Style (16th Edition):
Blaser, Julian. “The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia.” 2012. Doctoral Dissertation, University of Manchester. Accessed April 16, 2021.
http://www.manchester.ac.uk/escholar/uk-ac-man-scw:182916.
MLA Handbook (7th Edition):
Blaser, Julian. “The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia.” 2012. Web. 16 Apr 2021.
Vancouver:
Blaser J. The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia. [Internet] [Doctoral dissertation]. University of Manchester; 2012. [cited 2021 Apr 16].
Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:182916.
Council of Science Editors:
Blaser J. The utilisation of shRNA screens to investigate the role
of phosphoinositide modulator genes in actue myeloid
leukaemia. [Doctoral Dissertation]. University of Manchester; 2012. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:182916
Queens University
8.
Lian, Eric.
Characterization of isoform specific RET knockdown in cancer cell lines
.
Degree: Pathology and Molecular Medicine, 2013, Queens University
URL: http://hdl.handle.net/1974/8237
► The REarranged in Transfection (RET) tyrosine kinase is an important signalling protein for the development of neural crest-derived tissues such as the enteric and sympathetic…
(more)
▼ The REarranged in Transfection (RET) tyrosine kinase is an important signalling protein for the development of neural crest-derived tissues such as the enteric and sympathetic nervous systems. RET is constitutively activated in multiple human tumour types, such as thyroid carcinomas and some non-small cell lung cancers. RET has 3 distinct isoforms, RET9, RET43 and RET51, which are named after the lengths of their unique C-terminal tails. Here, we investigate the role of RET in the TT thyroid carcinoma cell line, where it is a driver of tumourigenesis, and in the MiaPaCa-2 pancreatic carcinoma cell line, where RET is not driving tumour initiation, but may nonetheless have a profound effect on tumour progression. We generated lentiviral constructs for shRNAs that target either RET9 or RET51 specifically, or a common region shared by all RET isoforms. TT and MiaPaCa-2 cells were transduced using these lentiviral particles to create stable cell lines containing knockdowns of total RET, RET9, or RET51. Using a variety of morphological and biochemical assays, we found that RET expression is critical for TT cell survival, and that both RET9 and RET51 play significant roles in driving cell proliferation in TT cells. Conversely, RET is not critical for MiaPaCa-2 cell survival, and RET knockdown had no effect on MiaPaCa-2 proliferation. MiaPaCa-2 cells instead underwent dramatic morphological changes, from their normal spindle-like mesenchymal appearance to an increasingly flattened and epithelioid character, in response to RET9, RET51 or total-RET knockdown. The observed morphological changes were coupled with significantly reduced invasiveness through matrigel towards a source of chemoattractant, suggesting a critical role for RET in mediating cell invasiveness. These results suggest that RET may not only drive tumourigenesis, but can also enhance disease progression when expressed in other tumour types. We predict that RET may play critical roles in perineural invasion in pancreatic cancers, a process where cells invade along peripheral nerve fibers by following an increasing concentration of chemoattractants secreted by nerve and glial cells. Thus, RET may be a valuable target to slow, or stop this process, which would have significant clinical implications in a wide variety of cancers.
Subjects/Keywords: Migration
;
RET
;
Cancer
;
Isoforms
;
shRNA
;
Invasion
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Record Details
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Lian, E. (2013). Characterization of isoform specific RET knockdown in cancer cell lines
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/8237
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Lian, Eric. “Characterization of isoform specific RET knockdown in cancer cell lines
.” 2013. Thesis, Queens University. Accessed April 16, 2021.
http://hdl.handle.net/1974/8237.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Lian, Eric. “Characterization of isoform specific RET knockdown in cancer cell lines
.” 2013. Web. 16 Apr 2021.
Vancouver:
Lian E. Characterization of isoform specific RET knockdown in cancer cell lines
. [Internet] [Thesis]. Queens University; 2013. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1974/8237.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Lian E. Characterization of isoform specific RET knockdown in cancer cell lines
. [Thesis]. Queens University; 2013. Available from: http://hdl.handle.net/1974/8237
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of New South Wales
9.
Ledger, Scott.
Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection.
Degree: Clinical School - St Vincent's Hospital, 2016, University of New South Wales
URL: http://handle.unsw.edu.au/1959.4/56230
;
https://unsworks.unsw.edu.au/fapi/datastream/unsworks:40347/SOURCE02?view=true
► HIV currently infects 35 million people worldwide and antiretroviral treatments are expensive, lifelong, and fail to provide a cure. Gene therapy provides an alternate approach…
(more)
▼ HIV currently infects 35 million people worldwide and antiretroviral treatments are expensive, lifelong, and fail to provide a cure. Gene therapy provides an alternate approach to the treatment of HIV. I investigated the effects of two therapeutic genes both singly and in tandem: the fusion inhibiting membrane anchored peptide maC46 (C46), and a short-hairpin RNA to CCR5 (sh5) which downregulates expression of the CCR5 receptor. These genes were carried on lentiviral vectors which were transduced into cell lines and PBMC. The vectors were named Control, C46, sh5 and Dual. The therapeutic genes conferred selective advantages in the presence of the R5tropic HIV-1BaL, with the Dual construct displaying the strongest selective advantage as Dual gene-containing cells expanded significantly faster than either single-gene vector. Both the sh5 and Dual vectors conferred a survival advantage for the cultures, with significant resistance against cell death, as well as strong inhibition of viral replication. The therapeutic genes also conferred protection against pseudotyped HIV in single round infection assays, with the Dual construct performing the best and with the greatest consistency. While most protection conferred in Molt4/CCR5 was paralleled in PBMC, the sh5 construct yielded differing results in PBMC. The most likely explanation is that among the cells that comprise PBMC, some of them may express other receptors which can be utilised by the HIV strains used. Cells containing the therapeutic genes were also challenged to assess their potential to trap HIV on their surface. Results indicated the C46 (and Dual)-marked cells mediated viral trapping of HIV-1NL4-3. However, HIV-1BaL virion trapping was observed on sh5 and Dual marked cells, yet C46-marked cells behaved differently. Microscopy indicated potential viral trapping on C46-marked cells, while flow cytometric analysis showed that this virus was more likely within the cell membrane. This indicated that the processes of viral entry may differ (by more than just co-receptor use) between HIV-1NL4-3 and HIV-1BaL.The work conducted here points to there being complex interactions between virus and host cells around the points of viral attachment and entry. However, even with these undefined processes taking place, the combined gene therapy consistently provided the best protection on every metric assessed.
Advisors/Committee Members: Symonds, Geoff, UNSW, Murray, John, UNSW.
Subjects/Keywords: shRNA; HIV; Gene therapy; CCR5; C46; lentivirus
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Ledger, S. (2016). Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection. (Doctoral Dissertation). University of New South Wales. Retrieved from http://handle.unsw.edu.au/1959.4/56230 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:40347/SOURCE02?view=true
Chicago Manual of Style (16th Edition):
Ledger, Scott. “Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection.” 2016. Doctoral Dissertation, University of New South Wales. Accessed April 16, 2021.
http://handle.unsw.edu.au/1959.4/56230 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:40347/SOURCE02?view=true.
MLA Handbook (7th Edition):
Ledger, Scott. “Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection.” 2016. Web. 16 Apr 2021.
Vancouver:
Ledger S. Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection. [Internet] [Doctoral dissertation]. University of New South Wales; 2016. [cited 2021 Apr 16].
Available from: http://handle.unsw.edu.au/1959.4/56230 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:40347/SOURCE02?view=true.
Council of Science Editors:
Ledger S. Use of Short-Hairpin RNA to CCR5, and maC46 Entry Inhibitor Gene-Therapies against HIV infection. [Doctoral Dissertation]. University of New South Wales; 2016. Available from: http://handle.unsw.edu.au/1959.4/56230 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:40347/SOURCE02?view=true
University of Manchester
10.
Blaser, Julian.
The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia.
Degree: PhD, 2013, University of Manchester
URL: https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html
;
https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764253
► Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide…
(more)
▼ Phosphoinositides (PIs) are pivotal lipid molecules with both scaffolding and signalling functions regulating key aspects of cellular physiology. For example, phosphatidylinositol (3,4,5)-trisphosphate, generated by phosphoinositide 3-kinase (PI3K), is an essential mediator of the PI3K/AKT signalling pathway, which is crucial for cell proliferation, survival and apoptosis. Constitutive activation of this signalling cascade has been identified in acute myeloid leukaemia (AML), the most common haematopoietic malignancy in adults, and experimental deletion of the PI3K antagonists PTEN and SHIP cause leukaemia in mice. However, little is known regarding the role of other PI modulator proteins in AML. Thus, in this thesis, a lentivirally delivered small hairpin RNA (shRNA) library targeting 103 genes (345 pLKO knockdown constructs) with presumed or established roles in PI metabolism was utilised to screen for genes required for AML blast cell viability/proliferation and differentiation. First, knockdown constructs were tested for their impact on proliferation/viability in seven human AML cell lines by measuring fold change in fluorescence of the cell viability dye alamarBlue relative to controls (cells transduced with a non-targeting control hairpin) over three days. This identified 13 candidate genes selected with the criterion that two or more knockdown constructs per gene reduce cell viability/proliferation relative to control by greater than or equal to50 % across all cell lines. From these candidate genes, PIP4K2A, INPP5B and IMPAD1 were selected for downstream validation experiments, which reproduced the observation from the primary screen. For INPP5B and IMPAD1, knockdown constructs also reduced clonogenic potential of primary human AML samples but only showed a modest effect on normal CD34+ haematopoietic stem or progenitor cells (HSPCs) in a methylcellulose based assay. This could be recapitulated in a murine setting where knockdown constructs targeting both genes reduced clonogenic potential of murine MLL- AF9 AML cells with little effect on normal KIT+ HSPCs. In line with this, Inpp5b knockout KIT+ BM cells either failed to immortalise or weakly immortalised, following forced expression of the powerful MLL-AF9 oncogene. A further screen was performed to identify regulators of THP-1 blast cell differentiation, by seeding knockdown construct transduced cells into methylcellulose based semisolid media. After ten days of incubation the degree of macrophage differentiation was evaluated by light microscopy and an arbitrary differentiation score was given. With the criterion that greater than or equal to2 knockdown constructs per gene received the highest differentiation score, reflecting terminal macrophage differentiation of all seeded cells, SBF2 was identified as the top-scoring hit. Validation experiments have confirmed macrophage differentiation based on cytospin preparations of SBF2 knockdown THP-1 cells. Moreover, xenograft assays have shown that knockdown constructs targeting PIP4K2A and SBF2 delayed or…
Subjects/Keywords: 616.99; phosphoinositides; acute myeloid leukaemia; RNAi; shRNA
Record Details
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Record Details
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« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Blaser, J. (2013). The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764253
Chicago Manual of Style (16th Edition):
Blaser, Julian. “The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia.” 2013. Doctoral Dissertation, University of Manchester. Accessed April 16, 2021.
https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764253.
MLA Handbook (7th Edition):
Blaser, Julian. “The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia.” 2013. Web. 16 Apr 2021.
Vancouver:
Blaser J. The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia. [Internet] [Doctoral dissertation]. University of Manchester; 2013. [cited 2021 Apr 16].
Available from: https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764253.
Council of Science Editors:
Blaser J. The utilisation of shRNA screens to investigate the role of phosphoinositide modulator genes in actue myeloid leukaemia. [Doctoral Dissertation]. University of Manchester; 2013. Available from: https://www.research.manchester.ac.uk/portal/en/theses/the-utilisation-of-shrna-screens-to-investigate-the-role-of-phosphoinositide-modulator-genes-in-actue-myeloid-leukaemia(87b33955-d4e9-4398-bd1d-58e4c5142eb3).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.764253
11.
Helena Bacha Lopes.
Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia.
Degree: 2018, University of São Paulo
URL: http://www.teses.usp.br/teses/disponiveis/58/58136/tde-19032019-163700/
► As integrinas constituem uma família de receptores de membrana que tem como função primária a adesão de células a proteínas da matriz extracelular e alguns…
(more)
▼ As integrinas constituem uma família de receptores de membrana que tem como função primária a adesão de células a proteínas da matriz extracelular e alguns de seus membros estão envolvidos nos processos de diferenciação osteoblástica e formação óssea, eventos diretamente relacionados à osseointegração de implantes de titânio (Ti). Sabe-se que superfícies de Ti com nano e microtopografia podem favorecer a diferenciação osteoblástica e a mineralização da matriz extracelular. No entanto, os mecanismos celulares envolvidos nesses processos não são completamente entendidos. Neste contexto, os objetivos deste estudo foram: (1) caracterizar as superfícies de Ti com nano (Ti-Nano) e microtopografia (Ti-Micro), (2) investigar a participação da integrina V na diferenciação osteoblástica induzida pelo Ti-Nano e (3) investigar a participação da integrina β3 na diferenciação osteoblástica induzida por Ti-Nano e Ti-Micro. Para isso, discos de Ti-Nano e
Ti-Micro foram preparados por ataque ácido com H2SO4/H2O2 ou com HNO3/H2SO4 / HCl, respectivamente, e caracterizados quanto à topografia, rugosidade e composição química de superfície. Discos de Ti usinados foram usados com controle (Ti-Controle) em alguns experimentos. Células-tronco mesenquimais derivadas de medula óssea de ratos foram cultivadas sobre as três superfícies de Ti e foi avaliada a expressão gênica de componentes envolvidos na via de sinalização das integrinas por PCR array. Com base nos resultados do PCR array, as integrinas αV e β3 foram selecionadas e silenciadas por RNA de interferência (shRNA) ou CRISPR/Cas9, respectivamente, em células pré-osteoblásticas da linhagem MC3T3-E1 para investigarmos a participação dessas integrinas na diferenciação osteoblástica induzida por superfícies de Ti com diferentes topografias. Os resultados deste estudo mostraram que os tratamentos empregados foram eficientes para a produção de superfícies de Ti com
topografias nas escalas nano e micrométrica. Além disso, foi demonstrado que o maior potencial osteogênico do Ti-Nano se deve, ao menos em parte, à integrina αV, uma vez que seu silenciamento reduziu a diferenciação osteoblástica induzida pela nanotopografia. Por fim, também demonstramos que a via de sinalização ativada pela integrina β3 exerce um papel fundamental no potencial osteogênico do Ti-Nano, mas não do Ti-Micro. O silenciamento da integrina β3 reduziu a diferenciação osteoblástica, concomitantemente com a regulação negativa da expressão de vários componentes das vias de sinalização de Wnt e de BMP, apenas nas células crescidas sobre a nanotopografia. Em conjunto, nossos resultados revelam um novo mecanismo para explicar a maior diferenciação osteoblástica induzida pelo Ti-Nano, que envolve uma complexa rede regulatória ativada pela maior expressão das integrinas αV e β3, esta última gerando ativação da transdução de sinal das
vias de Wnt e de BMP
Integrins are a family of membrane receptors that primarily mediate cell adhesion to extracellular matrix proteins and…
Advisors/Committee Members: Márcio Mateus Beloti, Adalberto Luiz Rosa, Cristina Antoniali Silva, Tarcilia Aparecida da Silva.
Subjects/Keywords: CRISPR/Cas9; Integrina; Nanotopografia; Osteoblasto; ShRNA; Titânio; CRISPR/Cas9; Integrin; Nanotopography; Osteoblast; ShRNA; Titanium
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❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Lopes, H. B. (2018). Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia. (Doctoral Dissertation). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/58/58136/tde-19032019-163700/
Chicago Manual of Style (16th Edition):
Lopes, Helena Bacha. “Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia.” 2018. Doctoral Dissertation, University of São Paulo. Accessed April 16, 2021.
http://www.teses.usp.br/teses/disponiveis/58/58136/tde-19032019-163700/.
MLA Handbook (7th Edition):
Lopes, Helena Bacha. “Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia.” 2018. Web. 16 Apr 2021.
Vancouver:
Lopes HB. Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia. [Internet] [Doctoral dissertation]. University of São Paulo; 2018. [cited 2021 Apr 16].
Available from: http://www.teses.usp.br/teses/disponiveis/58/58136/tde-19032019-163700/.
Council of Science Editors:
Lopes HB. Participação de integrinas na diferenciação osteoblástica induzida por superfícies de titânio com nano e microtopografia. [Doctoral Dissertation]. University of São Paulo; 2018. Available from: http://www.teses.usp.br/teses/disponiveis/58/58136/tde-19032019-163700/
12.
Amanda Ikegami.
Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal.
Degree: 2019, University of São Paulo
URL: http://www.teses.usp.br/teses/disponiveis/85/85131/tde-10052019-155321/
► O carcinoma de células renais (CCR) é responsável por, aproximadamente, 1 a 3% das neoplasias malignas humanas e, dentre os tumores urológicos, é o mais…
(more)
▼ O carcinoma de células renais (CCR) é responsável por, aproximadamente, 1 a 3% das neoplasias malignas humanas e, dentre os tumores urológicos, é o mais agressivo. A heterogeneidade biológica, a resistência aos fármacos e os efeitos colaterais à quimioterapia são os maiores obstáculos ao tratamento eficaz do CCR. Várias moléculas vêm sendo correlacionadas com o fenótipo agressivo deste tumor, sendo uma delas o fator de transcrição NF-kB. Estudos demonstraram a ativação de NF-kB no CCR e muitos apontaram NF-kB1 (p50) como uma molécula importante na progressão tumoral e metástase. Neste trabalho, foi utilizada a técnica de silenciamento gênico de interferência por RNA - Short hairpin RNA (shRNA) - para diminuir a expressão de NF-kB1 em células murinas de carcinoma de células renais (Renca). Verificou-se que, in vitro, a diminuição da expressão do gene NF-kB1 reduz a capacidade proliferativa das células Renca e aumenta a taxa de apoptose
tardia/necrose e a quantidade de células na fase G2/M do ciclo celular. Além disso, as análises de Western blot revelaram aumento significativo da expressão proteica de Ciclina B1 e Bax. A regulação negativa da p50 também alterou a capacidade clonogênica das células que, conjugada à irradiação ionizante, levou à diminuição significativa da fração de sobrevivência das células Renca e diminuiu a viabilidade celular verificada através do ensaio de MTS. Os ensaios in vivo mostraram que as células com baixa expressão de NF-kB1 (Renca-shRNA- NF-kB1) apresentam tumorigenicidade significativamente menor que as células controle e as análises de imunohistoquímica mostraram aumento significativo das áres necróticas dos tumores Renca-shRNA-NF-kB1, além da diminuição da marcação de Ki-67, um marcador de proliferação celular. Os resultados indicam que a diminuição da expressão de NF-kB1 pode suprimir a tumorigênese do CCR, induzindo apoptose tardia/necrose, podendo ser um potencial alvo
terapêutico para este carcinoma.
Renal cell carcinoma (RCC) is responsible for approximately 1 to 3% of human malignancies and, among urological tumors, is the most aggressive. Biological heterogeneity, drug resistance, and side effects to chemotherapy are major obstacles to effective RCC treatment. Several molecules have been correlated with the aggressive phenotype of this tumor, one of them being the transcription factor NF-kB. Studies have demonstrated the activation of NF-kB in the RCC and many have pointed to NF-kB1 (p50) as an important molecule in tumor progression and metastasis. In this study, the gene silencing technique of RNA Short hairpin RNA (shRNA) interference was used to decrease the expression of NF-kB1 in murine cells of renal cell carcinoma (Renca). It has been found that, in vitro, the decrease in NF-kB1 gene expression reduces the proliferative capacity of Renca cells and increases the rate of late apoptosis/necrosis and the number of cells in G2/M phase
of the cell cycle. In addition, Western blotting analyzes revealed a significant increase in the protein expression of Cyclin B1 and…
Advisors/Committee Members: Maria Helena Bellini Marumo, Michele Longoni Calió, Rafael Vicente de Padua Ferreira.
Subjects/Keywords: carcinoma de células renais; NF-kB1; proliferação; shRNA; NF-kB1; proliferation; renal cell carcinoma; shRNA
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APA (6th Edition):
Ikegami, A. (2019). Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal. (Masters Thesis). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/85/85131/tde-10052019-155321/
Chicago Manual of Style (16th Edition):
Ikegami, Amanda. “Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal.” 2019. Masters Thesis, University of São Paulo. Accessed April 16, 2021.
http://www.teses.usp.br/teses/disponiveis/85/85131/tde-10052019-155321/.
MLA Handbook (7th Edition):
Ikegami, Amanda. “Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal.” 2019. Web. 16 Apr 2021.
Vancouver:
Ikegami A. Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal. [Internet] [Masters thesis]. University of São Paulo; 2019. [cited 2021 Apr 16].
Available from: http://www.teses.usp.br/teses/disponiveis/85/85131/tde-10052019-155321/.
Council of Science Editors:
Ikegami A. Avaliação in vitro e in vivo do impacto do silenciamento do gene NF-kB1 no ciclo celular, capacidade proliferativa e na radiossensibilidade do adenocarcinoma renal. [Masters Thesis]. University of São Paulo; 2019. Available from: http://www.teses.usp.br/teses/disponiveis/85/85131/tde-10052019-155321/
Johannes Gutenberg Universität Mainz
13.
Wrede, Christine.
Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie.
Degree: 2011, Johannes Gutenberg Universität Mainz
URL: http://ubm.opus.hbz-nrw.de/volltexte/2012/3253/
► Retrovirale Vektoren basierend auf dem murinen Leukämievirus (MLV) gehören zu den zurzeit am häufigsten verwendeten Vektoren in der Gentherapie. MLV besitzt einen natürlichen Tropismus für…
(more)
▼ Retrovirale Vektoren basierend auf dem murinen Leukämievirus (MLV) gehören zu den zurzeit am häufigsten verwendeten Vektoren in der Gentherapie. MLV besitzt einen natürlichen Tropismus für sich teilende Zellen und ist somit besonders für die Krebs-Gentherapie geeignet.rnIn der vorliegenden Arbeit wurde zuerst der direkte Transport von pri-miRNA durch deren Aufnahme in MLV-Partikel untersucht, aber keine positiven Effekte beobachtet. Dabei blieb unklar, ob keine Verpackung der pri-miRNA erfolgte, oder die pri-miRNA nach Transduktion der Zellen nicht funktionell war.rnReplizierende MLVs sind eine vielversprechende Alternative zu replikationsinkompetenten Vektoren. Sie können das Transgen im gewünschten Gewebe verteilen und durch Integration ins Genom stabil exprimieren. Es wurden verschiedene Ansätze zur Herstellung von onkolytisch wirkenden MLVs untersucht. Dabei wurde gezeigt, dass der Einsatz des viralen Proteins R (VPR) als toxisches Gen eine Anzucht VPR-kodierender Viren erschwert, da bereits die VPR-exprimierenden Zellen abgetötet werden. Das Ergebnis zeigt den Bedarf weiterer Optimierungen, z.B. durch geeignete Anzuchtzellen oder induzierbare Promotoren zur Transgenexpression.rnEs konnte gezeigt werden, dass Expressionskassetten mit antitumoralen sh/miRNAs als therapeutisches Effektormolekül gegen die Proteinkinase PLK1 und den Transkriptionsfaktor STAT3 erfolgreich durch replizierende MLVs in Zielzellen übertragen werden und die Herabregulation der Genprodukte zu einer deutlichen Wachstumshemmung der Tumorzellen führt. Dabei konnten Expressionskassetten bis zu einer Größe von 1,6kb stabil in die 3´-UTR von Env inseriert werden. Es konnte ein reduziertes Tumorwachstum von HT1080-Zellen in SCID-Mäusen nach intratumoraler Applikation von aMLV, welches für eine miRNA gegen PLK1 kodiert, erreicht werden ohne dass die Viren mutierten (Schaser et al., 2011). Durch eine intravenöse Verabreichung der Viren oder der Applikation von vorinfizierten Tumorzellen in SCID-Mäuse mutierten die miRNA-Expressionskassetten aus ungeklärten Gründen vollständig. Durch die Balance zwischen Virusverbreitung und induziertem Zelltod sind modifizierte MLVs eine perfekte Waffe gegen entartete Zellen.rnrn
Retroviral vectors based on the murine leukemia virus (MLV) are among the currently most used vectors in gene therapy. MLV has a naturally occurring tropism for dividing cells and therefore is especially suitable for tomour therapy.rnIn this thesis at first the direct transport of pre-miRNA via incorporation in the virus particle was investigated. Here no positive effects were observed, whereas it could not be determined whether there was no incorporation of pre-miRNA into the virus particles or wether the pre-miRNA was non-functional after transduction of the cells.rnReplication competent MLV are a promising alternative to replication incompetent vectors. They are able to efficiently distribute the transgene in the tissue and to stably express it via integration into the host genome. Here different approaches to generate oncolytic MLV…
Subjects/Keywords: murines Leukemievirus (MLV); shRNA; viraler Vektor; Gentherapie; murine leukaemia virus (MLV); shRNA; viral vector; gentherapy; Life sciences
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Wrede, C. (2011). Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie. (Doctoral Dissertation). Johannes Gutenberg Universität Mainz. Retrieved from http://ubm.opus.hbz-nrw.de/volltexte/2012/3253/
Chicago Manual of Style (16th Edition):
Wrede, Christine. “Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie.” 2011. Doctoral Dissertation, Johannes Gutenberg Universität Mainz. Accessed April 16, 2021.
http://ubm.opus.hbz-nrw.de/volltexte/2012/3253/.
MLA Handbook (7th Edition):
Wrede, Christine. “Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie.” 2011. Web. 16 Apr 2021.
Vancouver:
Wrede C. Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie. [Internet] [Doctoral dissertation]. Johannes Gutenberg Universität Mainz; 2011. [cited 2021 Apr 16].
Available from: http://ubm.opus.hbz-nrw.de/volltexte/2012/3253/.
Council of Science Editors:
Wrede C. Transfer von antitumoralen Effektoren mittels viraler Vektoren zur experimentellen Tumortherapie. [Doctoral Dissertation]. Johannes Gutenberg Universität Mainz; 2011. Available from: http://ubm.opus.hbz-nrw.de/volltexte/2012/3253/
14.
Valencia Garcia, Sara.
Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model.
Degree: Docteur es, Neurosciences, 2014, Université Claude Bernard – Lyon I
URL: http://www.theses.fr/2014LYO10324
► Les circuits neuronaux responsables du sommeil paradoxal (SP) et de l'atonie musculaire qui le caractéristique sont l'objet de nombreuses recherches expérimentales, notamment en raison de…
(more)
▼ Les circuits neuronaux responsables du sommeil paradoxal (SP) et de l'atonie musculaire qui le caractéristique sont l'objet de nombreuses recherches expérimentales, notamment en raison de l'existence de plusieurs pathologies invalidantes associées. Cette thèse de Neurobiologie s'inscrit plus spécifiquement dans la description anatomique et fonctionnelle du réseau neuronal responsable de l'atonie musculaire et son potentiel dysfonctionnement dans les troubles comportementaux en SP (RBD, REM sleep Behavior Disorder). Pour ce faire, nous avons combiné plusieurs techniques faisant appel à la neuroanatomie fonctionnelle, au traçage rétrograde de voies nerveuses, à l'hybridation in situ à la polysomnographie et à l'inactivation irréversible de populations neuronales ciblées moléculairement à l'aide de virus adéno-associés contenant des short hairpin RNAs (AAV-shRNA) chez le rat libre de ses mouvements. Nous avons ainsi montré que, contrairement à l'hypothèse généralement admise, le noyau sublatérodorsal pontique (SLD) n'est pas le générateur du SP. En effet, l'inactivation neurochimique de ses neurones glutamatergiques ou sa lésion totale diminuent les quantités de SP sans le supprimer, indiquant que le SLD n'est pas suffisant pour la genèse du SP. En revanche, ces expériences démontrent son implication directe dans la mise en place de l'atonie musculaire lors du SP. En effet, la déconnexion neurochimique des neurones glutamatergiques du SLD provoque pendant le SP l'apparition intermittente de tonus musculaire accompagné de comportements moteurs anormaux. En parallèle, nos travaux de thèse ont permis d'apporter des données expérimentales nouvelles sur la localisation, au sein de la formation réticulée bulbaire ventrale et non dans la moelle épinière, des interneurones GABA/glycine responsables de l'hyperpolarisation des motoneurones somatiques pendant le SP. En effet, ces neurones réticulaires sont exclusivement recrutés pendant le SP et envoient des projections monosynaptiques inhibitrices vers les motoneurones somatiques lombaires. De plus, leur déconnexion neurochimique ciblée déclenche des comportements moteurs anormaux sous-tendus par le maintien d'un tonus musculaire irrégulier pendant le SP. L'analyse actimétrique de ces comportements moteurs oniriques induits expérimentalement montre qu'ils sont très semblables à ceux observés après l'inactivation du SLD et à ceux décrits chez les patients RBD. Les données rapportées dans cette thèse permettent de mieux comprendre les mécanismes neurobiologiques générant le SP et ceux contribuant au contrôle moteur pendant le SP. Par la même occasion, nos travaux ont permis de valider deux modèles rongeurs du RBD humain, ouvrant ainsi des perspectives expérimentales pour l'élaboration de traitements ciblés de cette pathologie affectant le SP
A growing number of studies investigate the neuronal network responsible for paradoxical (PS) (or REM) sleep genesis and muscle atonia specific of this sleep state. The aim of this thesis was to characterize at the anatomical and functional…
Advisors/Committee Members: Fort, Patrice (thesis director), Luppi, Pierre-Hervé (thesis director).
Subjects/Keywords: Tronc cérébral; C-Fos; Glutamate; Glycine; AAV-shRNA; Motoneurones somatiques; Brainstem; C-Fos; Glutamate; Glycine; AAV-shRNA; Somatic motoneurons; 612.8
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Valencia Garcia, S. (2014). Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model. (Doctoral Dissertation). Université Claude Bernard – Lyon I. Retrieved from http://www.theses.fr/2014LYO10324
Chicago Manual of Style (16th Edition):
Valencia Garcia, Sara. “Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model.” 2014. Doctoral Dissertation, Université Claude Bernard – Lyon I. Accessed April 16, 2021.
http://www.theses.fr/2014LYO10324.
MLA Handbook (7th Edition):
Valencia Garcia, Sara. “Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model.” 2014. Web. 16 Apr 2021.
Vancouver:
Valencia Garcia S. Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model. [Internet] [Doctoral dissertation]. Université Claude Bernard – Lyon I; 2014. [cited 2021 Apr 16].
Available from: http://www.theses.fr/2014LYO10324.
Council of Science Editors:
Valencia Garcia S. Décryptage du réseau neuronal responsable de l’atonie musculaire pendant le sommeil paradoxal chez le rat : création d’un modèle rongeur du RBD (REM sleep Behavior Disorder) : Neuronal network of paradoxical sleep muscle atonia : a pre-requirement in the creation of a RBD (REM sleep Behavior Disorder) rodent model. [Doctoral Dissertation]. Université Claude Bernard – Lyon I; 2014. Available from: http://www.theses.fr/2014LYO10324
15.
Helary, Louise.
Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development.
Degree: Docteur es, Aspects moléculaires et cellulaires de la biologie, 2019, Limoges
URL: http://www.theses.fr/2019LIMO0053
► Même si les acteurs majeurs du développement musculaire ont été identifiés et les voies de transductions décrites, d’autres régulateurs restent encore à découvrir. Un crible…
(more)
▼ Même si les acteurs majeurs du développement musculaire ont été identifiés et les voies de transductions décrites, d’autres régulateurs restent encore à découvrir. Un crible ARNi pratiqué sur un modèle cellulaire couramment utilisé, la lignée myoblastique C2C12, a identifié 20 nouveaux gènes potentiellement impliqués dans la myogenèse in vitro. Au cours de ma thèse, deux de ces gènes ont été invalidés sur modèle souris en utilisant la technologie CRISPR/Cas9 pour valider in vivo leur implication. Pour l’un d’entre eux, seuls les animaux hétérozygotes ont pu être étudiés puisqu’une létalité précoce a été observée chez les homozygotes mutés. Aucune anomalie du développement musculaire n’a été mise en évidence. Une étude plus fine dans les premières phases du développement embryonnaire nous a permis de montrer le rôle indispensable de cette protéine précocement. L’étude du second gène – dont les analyses se poursuivent – semble confirmer in vivo le rôle de ce gène au cours de la myogenèse. Pour éviter la survenue de létalité embryonnaire et observer rapidement les effets de l’invalidation d’autres gènes, une technique de transgenèse somatique s’appuyant sur l’ARN interférence a été mis en place via l’injection de lentivirus contenant une cassette d’expression de shRNA directement dans le tibialis antérieur des souris. La validation de cette approche a été faite sur le gène de la myostatine, régulateur négatif du développement musculaire, et a montré une diminution de l’expression du gène associée à une augmentation de l’aire des fibres musculaires. La même approche appliquée à trois autres gènes renforce l’hypothèse de l’implication d’un des gènes dans le développement musculaire. Cette approche permet donc un crible rapide « in vivo » de gènes identifiés in vitro. Cependant, certaines améliorations doivent être apportées au protocole au regard des résultats obtenus.
Even if the major actors and transduction pathways of muscle development have been identified, there are still unknown regulatory factors. An in vitro RNAi screening performed on C2C12 myoblastic cells has permitted to identify 20 novel genes potentially implicated in myogenesis. During my thesis, two of these genes were invalidated on mouse model using CRISPR/Cas9 technology in order to confirm their implication in vivo. For the first gene, due to an early lethality occurring in homozygous mutated animals, only heterozygous animals were studied and there was no muscular development anomaly detected. A refined study of earlier stages of embryonic development permitted to show the essential role of the protein in these phases. The study of the second gene, still in progress, seems to confirm in vivo the implication of the gene on the myogenesis. In order to avoid embryonic lethality due to germline invalidation and to observe more rapidly the effects of gene invalidation in muscle, we developed a technique of somatic transgenesis based on RNA interference. Lentivirus containing a shRNA expression cassette was injected directly into the tibialis anterior…
Advisors/Committee Members: Véronique, Blanquet (thesis director), Duchesne-Collardot, Amandine (thesis director).
Subjects/Keywords: Développement musculaire; Knock-out; ShRNA; CRISPR/Cas9; DNAJC2; Muscle development; Knock-out; ShRNA; CRISPR/Cas9; DNAJC2; 599
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Helary, L. (2019). Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development. (Doctoral Dissertation). Limoges. Retrieved from http://www.theses.fr/2019LIMO0053
Chicago Manual of Style (16th Edition):
Helary, Louise. “Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development.” 2019. Doctoral Dissertation, Limoges. Accessed April 16, 2021.
http://www.theses.fr/2019LIMO0053.
MLA Handbook (7th Edition):
Helary, Louise. “Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development.” 2019. Web. 16 Apr 2021.
Vancouver:
Helary L. Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development. [Internet] [Doctoral dissertation]. Limoges; 2019. [cited 2021 Apr 16].
Available from: http://www.theses.fr/2019LIMO0053.
Council of Science Editors:
Helary L. Validation in vivo de l'implication de nouveaux gènes impliqués dans le développement musculaire des mammifères : In vivo validation of the implication of new genes in mammalian muscle development. [Doctoral Dissertation]. Limoges; 2019. Available from: http://www.theses.fr/2019LIMO0053
Loyola University Chicago
16.
Case, Alicia Marie.
Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex.
Degree: PhD, Neuroscience, 2011, Loyola University Chicago
URL: https://ecommons.luc.edu/luc_diss/192
► Recovery after central nervous system (CNS) injury has long been a challenge for clinical investigators. Blockade of the oligodendrocyte-associated inhibitor Nogo-A has shown great…
(more)
▼ Recovery after central nervous
system (CNS) injury has long been a challenge for clinical
investigators. Blockade of the oligodendrocyte-associated inhibitor
Nogo-A has shown great promise in promoting neuronal regeneration,
sprouting, and plasticity, as well as functional recovery in rodent
and primate models of CNS injury. The high expression of Nogo-A in
neurons of the postnatal CNS led us to look for potential roles of
this protein in this stage of development. We hypothesized that
postnatal, neuronal NogoA influences the density and morphology of
dendritic spines in the developing CNS, in part, by regulating the
maturation and stability of glutamatergic synaptic input. To
examine the roles of Nogo-A at the excitatory synapse of the
neocortex, we used RNAi directed against Nogo-A and delivered it to
the developing rat sensorimotor cortex via AAV2/8, a neurotropic
vector. This resulted in lowered density of dendritic spines, which
are known to house over 90% of excitatory connections onto
pyramidal neurons. This decrease was particularly evident with
thin- and mushroom-shaped spines in dendrites of the apical arbor.
A decrease in protrusions with moderately-wide head widths and very
long necks was also noted. We then used vesicular glutamate
transporter 1 (vGlut1) as a marker for potential excitatory
synapses. Knocking down Nogo-A in postnatal pyramidal neurons of
the sensorimotor cortex led to a decrease in the number of vGlut1
puncta identifying a potential presynaptic partner, in opposition
to the apical dendritic shaft. The decreased vGlut1 in the apical
arbor likely represents a loss of potential synapses that may have
a strong influence on direct current injected into the dendrite. We
further examined regions of transduced cortex via qRT-PCR for
message levels of molecules important for plasticity at the
excitatory synapse, including Neuroligin-1, NMDA receptor subunits
NR2A and NR2B, and PSD-95. We found that mRNA of Neuroligin-1 and
NR2B was substantially reduced, with no changes to NR2A or PSD-95
expression. These results suggest that neuronal Nogo-A may act to
maintain elements of the neocortical excitatory synapse during
development. This finding represents a novel role for Nogo-A in the
intact CNS.
Subjects/Keywords: AAV; dendritic spine; Development; Nogo-A; shRNA; synapse; Neuroscience and Neurobiology
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Case, A. M. (2011). Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex. (Doctoral Dissertation). Loyola University Chicago. Retrieved from https://ecommons.luc.edu/luc_diss/192
Chicago Manual of Style (16th Edition):
Case, Alicia Marie. “Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex.” 2011. Doctoral Dissertation, Loyola University Chicago. Accessed April 16, 2021.
https://ecommons.luc.edu/luc_diss/192.
MLA Handbook (7th Edition):
Case, Alicia Marie. “Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex.” 2011. Web. 16 Apr 2021.
Vancouver:
Case AM. Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex. [Internet] [Doctoral dissertation]. Loyola University Chicago; 2011. [cited 2021 Apr 16].
Available from: https://ecommons.luc.edu/luc_diss/192.
Council of Science Editors:
Case AM. Effects of Neuronal Nogo-A on Properties of Excitatory
Synapses of the Sensorimotor Cortex. [Doctoral Dissertation]. Loyola University Chicago; 2011. Available from: https://ecommons.luc.edu/luc_diss/192
Penn State University
17.
Kazi, Abid A.
ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.
Degree: 2011, Penn State University
URL: https://submit-etda.libraries.psu.edu/catalog/11847
► PRAS40 and DEPTOR are mTOR binding proteins that affect cell metabolism. Under catabolic conditions such as sepsis and glucocorticoid excess, there is an increase in…
(more)
▼ PRAS40 and DEPTOR are mTOR binding proteins that affect cell metabolism. Under catabolic conditions such as sepsis and glucocorticoid excess, there is an increase in total DEPTOR protein and a reduction in phosphorylation of PRAS40, suggesting that these proteins may modulate the mTOR-mediated protein synthetic response under normal and diseased conditions. The hypothesis of the present study was that knock down (KD) of PRAS40 or DEPTOR in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. PRAS40 and DEPTOR KD was achieved using lentiviral particles containing
shRNA to target the mouse PRAS40 and DEPTOR mRNA sequence, whereas control cells were transfected with a scrambled control
shRNA. KD reduced PRAS40 and DEPTOR mRNA and protein content by 90%. PRAS40 KD did not result in increased phosphorylation of mTOR substrates or increased protein synthesis, whereas, DEPTOR KD increased both phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and protein synthesis. The responsiveness of PRAS40 and DEPTOR KD myocytes to anabolic (IGF-I) and catabolic (AICAR) stimuli was unaltered. Both PRAS40 and DEPTOR KD myoblasts were larger in diameter and exhibited an increased mean cell volume compared to scramble control. PRAS40 KD cells had decreased phosphorylation (S807/S811) of pRb protein. In contrast, DEPTOR KD cells had an increased phosphorylation (S807/S811) of pRb protein which is critical for the G1-S phase transition, coincident with an increased percentage of cells in the S phase. Neither PRAS40 nor DEPTOR KD altered myoblast apoptosis as evidenced by the lack of change for cleaved caspase-3. Although DEPTOR KD myoblasts did not alter autophagy as determined by a lack of change in the ratio of LC3BII/LC3BI, PRAS40 KD myoblasts had a reduced ratio of LC3BII/LC3BI. While PRAS40 KD delayed myotube formation concurrent with delayed proliferation, DEPTOR KD had the opposite effect on myogenesis and proliferation. Finally, while in vivo DEPTOR KD (~50% reduction) by electroporation into the muscle of C57/BL6 mice did not alter weight or protein synthesis in the control muscle, it prevented atrophy produced by 3 days of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that PRAS40 and DEPTOR are important regulators of protein metabolism in myocytes and demonstrate that, while PRAS40 is required for normal myoblast growth and function, decreasing DEPTOR expression is sufficient to ameliorate the atrophic response produced by immobilization.
Advisors/Committee Members: Charles H Lang, Dissertation Advisor/Co-Advisor, Charles H Lang, Committee Chair/Co-Chair, Scot R Kimball, Committee Member, Lisa M Shantz, Committee Member, Timothy M Ritty, Committee Member.
Subjects/Keywords: protein synthesis; mTOR; knockdown; lentivirus; shRNA; sepsis; disuse atrophy
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APA (6th Edition):
Kazi, A. A. (2011). ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. (Thesis). Penn State University. Retrieved from https://submit-etda.libraries.psu.edu/catalog/11847
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Kazi, Abid A. “ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.” 2011. Thesis, Penn State University. Accessed April 16, 2021.
https://submit-etda.libraries.psu.edu/catalog/11847.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Kazi, Abid A. “ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
.” 2011. Web. 16 Apr 2021.
Vancouver:
Kazi AA. ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. [Internet] [Thesis]. Penn State University; 2011. [cited 2021 Apr 16].
Available from: https://submit-etda.libraries.psu.edu/catalog/11847.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Kazi AA. ROLE OF PRAS40 AND DEPTOR – TWO mTOR BINDING PROTEINS IN C2C12 MYOCYTES
. [Thesis]. Penn State University; 2011. Available from: https://submit-etda.libraries.psu.edu/catalog/11847
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
18.
M. Rava'.
FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER.
Degree: 2015, Università degli Studi di Milano
URL: http://hdl.handle.net/2434/254403
► The molecular mechanisms and pathways responsible for the progression of hepatocellular carcinoma (HCC) remain to be fully characterized. Among the genetic lesions associated with HCC…
(more)
▼ The molecular mechanisms and pathways responsible for the progression of hepatocellular carcinoma (HCC) remain to be fully characterized. Among the genetic lesions associated with HCC progression, Shukla et al. (2013) identified insertions of the L1 transposon proximal to the gene encoding the zinc-finger DNA-binding protein ST18 (suppression of tumorigenicity 18) and suggested that this actually functions as an oncogene in HCC. However, functional evidence for a cancer-promoting activity of ST18 and insight into its mode of action are missing. Here, I pursued the functional characterization of ST18 in a mouse model of HCC based on ex vivo transformation and subcutaneous transplantation of embryonic hepatoblasts. ST18 was undetectable in either normal liver or cultured hepatoblasts, but was induced in the subcutaneous tumors. ST18 was also expressed in either chronically or acutely inflamed mouse livers (as assessed in Mdr2-/- or LPS-treated mice) as well as in human Progressive Familial Intrahepatic Cholestasis 2 (PFIC2: a condition associated with chronic inflammation), suggesting its induction by inflammatory stimuli. The knockdown of ST18 delayed tumor formation or, if induced in already formed tumors, led to rapid hemorrhage, pervasive morphological changes in the tumor cells reminiscent of an epithelial-to-mesenchymal transition (EMT) and eventually tumor regression. RNA profiling revealed that ST18 silencing caused expression of EMT-associated genes, among others. Previous studies have linked inflammation to the induction of EMT in other epithelia: we hypothesize that the concomitant activation of ST18 constitutes a safeguard against EMT, inactivation of this control mechanism causing the dramatic phenotypic switch observed in our model. These data warrant further evaluation of the mode of action of ST18 and of its potential value as a therapeutic target in HCC.
Advisors/Committee Members: added supervisor: S. Campaner, supervisor: B. Amati.
Subjects/Keywords: ST18; HCC; shRNA; inflammation; EMT; RNAseq; Settore BIO/11 - Biologia Molecolare
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APA (6th Edition):
Rava', M. (2015). FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER. (Thesis). Università degli Studi di Milano. Retrieved from http://hdl.handle.net/2434/254403
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Rava', M.. “FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER.” 2015. Thesis, Università degli Studi di Milano. Accessed April 16, 2021.
http://hdl.handle.net/2434/254403.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Rava', M.. “FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER.” 2015. Web. 16 Apr 2021.
Vancouver:
Rava' M. FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER. [Internet] [Thesis]. Università degli Studi di Milano; 2015. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/2434/254403.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Rava' M. FUNCTIONAL DISSECTION OF ST18 IN LIVER CANCER. [Thesis]. Università degli Studi di Milano; 2015. Available from: http://hdl.handle.net/2434/254403
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
19.
U.A. Cammarata.
CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS.
Degree: 2016, Università degli Studi di Milano
URL: http://hdl.handle.net/2434/365771
► Acute myeloid leukemia (AML) is a malignant tumor characterized by the uncontrolled proliferation of immature hematopoietic cells (blasts) that colonize the bone marrow leading to…
(more)
▼ Acute myeloid leukemia (AML) is a malignant tumor characterized by the uncontrolled proliferation of immature hematopoietic cells (blasts) that colonize the bone marrow leading to a malfunctional hematopoiesis. Despite the efforts to search for new therapies and the identification of new targets, leukemias are still largely incurable. The main reason for treatment failure is the high frequency of relapses after remission induced by the first chemotherapy treatment. Recent studies suggest that the high rate of relapse is due to the presence of cells with limitless regenerative capacity, Leukemia Initiating Cells (LIC), that are very resistant to chemotherapy. Moreover, since the leukemic blasts are highly heterogeneous both from a biological and genetic point of view, AML evolves continuously under both environmental and external stimuli (eg, chemotherapy).
In order to study the clonal evolution of AML and to find new therapeutic targets that allow to eradicate leukemic blasts, this study has set the following objectives: i) monitor LICs growth in vivo through the transplantation of traceable leukemic cells; ii) investigate the clonal evolution of the disease; iii) identify genes critical to tumor growth in vivo that can become new therapeutic targets.
To address these issues, we have used two lentiviral libraries (Cellecta Inc.): i) a clonal tracking library composed by 30 million different barcodes; ii) an
shRNA library composed by ~1000 different
shRNA able to silence ~100 genes. The libraries were used to infect xenotransplants of patient-derived leukemias and murine leukemias obtained from mouse models. The infected blasts were transplanted in recipient mice and, following leukemia development in vivo, the DNA extracted from infected blasts has been used, through massive sequencing techniques, to investigate leukemia growth and evolution (clonal tracking library) and genes required for AML growth (
shRNA screening).
Our clonal tracking analysis highlighted a strong clonal selection troughout leukemia evolution with the disappearance even of clones that were present at very high frequency within the tumor population. Nonetheless, the clonal composition of all tumors appeared to be very similar in terms of number of clones, of their frequency distributions and even of
13
their identity. Taken all together, our data strongly support a model in which each LIC is endowed with an intrinsic and highly variable replicative potential, with the vast majority of LICs that are not immortal and do not possess an indefinite self-renewal ability As a consequence, each leukemia possesses a definite and small number of LICs able to propagate the disease long-term. These LICs tend to overgrow all other clones and to constitute a very large proportion of the tumor.
Concerning the
shRNA screening, we believe we set up an innovative in vivo approach that allowed us to identify putative therapeutic targets involved in the growth of primary tumors in their natural environment and that is mainly selective for targeting the CSC…
Advisors/Committee Members: supervisor: P. G. Pelicci, E. Colombo, C. Ronchini, PELICCI, PIER GIUSEPPE, PELICCI, PIER GIUSEPPE, COLOMBO, EMANUELA, RONCHINI, CHIARA.
Subjects/Keywords: Clonal tracking, shRNA screening, AML, Leukemia; Settore BIO/11 - Biologia Molecolare
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APA (6th Edition):
Cammarata, U. (2016). CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS. (Thesis). Università degli Studi di Milano. Retrieved from http://hdl.handle.net/2434/365771
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Cammarata, U.A.. “CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS.” 2016. Thesis, Università degli Studi di Milano. Accessed April 16, 2021.
http://hdl.handle.net/2434/365771.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Cammarata, U.A.. “CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS.” 2016. Web. 16 Apr 2021.
Vancouver:
Cammarata U. CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS. [Internet] [Thesis]. Università degli Studi di Milano; 2016. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/2434/365771.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Cammarata U. CLONAL TRACKING AND HIGH THROUGHPUT SHRNA SCREENING IN AMLS. [Thesis]. Università degli Studi di Milano; 2016. Available from: http://hdl.handle.net/2434/365771
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Cambridge
20.
Bertero, Alessandro.
Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells.
Degree: PhD, 2016, University of Cambridge
URL: https://www.repository.cam.ac.uk/handle/1810/274120
► Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has…
(more)
▼ Human pluripotent stem cells (hPSCs) are an invaluable model for cellular and developmental biology, and hold great potential for translational applications. While great progress has been made in elucidating the signalling pathways regulating pluripotency and differentiation, our mechanistic understanding of the downstream regulations is still incomplete. Moreover, studies aimed at clarifying these aspects are severely impeded by the lack of efficient methods to conditionally modulate gene expression in hPSCs and hPSC-derived cells. In this dissertation I provide new insights into the molecular mechanisms controlled by the Activin/Nodal-SMAD2/3 signalling pathway, whose activity dictates the balance between hPSC pluripotency and differentiation. First, I show that SMAD2/3 modulates the chromatin epigenetic landscape of hPSCs by cooperating with the pluripotency factor NANOG to recruit the DPY30-COMPASS complex and promote histone 3 lysine 4 trimethylation (H3K4me3). This regulation promotes expression of pluripotency genes, while poising developmental regulators for activation during differentiation. Secondly, I describe a novel efficient approach for inducible gene knockdown in hPSCs and hPSC-derived cells. By taking advantage of this technology, I demonstrate that DPY30 is required for early differentiation of hPSCs into certain mesoderm and endoderm derivatives. Finally, I report the first large-scale proteomic identification of SMAD2/3 interacting proteins in both undifferentiated and differentiating hPSCs. This analysis not only confirms that SMAD2/3 interacts with multiple epigenetic modifiers involved in hPSC fate choices, but also implicates SMAD2/3 in several functions other than transcriptional regulation. In particular, I describe how SMAD2/3 physically and functionally interacts with the METTL3-METTL14-WTAP complex to promote the formation of N6-methyladenosine (m6A). This epitranscriptional modification antagonizes the expression of selected mRNAs, including pluripotency factors whose transcription is promoted by SMAD2/3. Therefore, this provides a negative feedback that facilitates rapid exit from pluripotency upon inhibition of Activin/Nodal signalling. Overall, the work presented in this dissertation advances the stem cell field in two ways. First, it demonstrates that the Activin/Nodal-SMAD2/3 pathway finely orchestrates the balance between pluripotency and differentiation by shaping both the epigenome and the epitranscriptome of hPSCs. Secondly, it provides a novel powerful technology to facilitate further studies of the mechanisms that regulate cell fate decisions.
Subjects/Keywords: stem cells; smad; DPY30; TGF beta; m6A; epigenetics; epitranscriptome; shRNA; knockdown
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Bertero, A. (2016). Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells. (Doctoral Dissertation). University of Cambridge. Retrieved from https://www.repository.cam.ac.uk/handle/1810/274120
Chicago Manual of Style (16th Edition):
Bertero, Alessandro. “Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells.” 2016. Doctoral Dissertation, University of Cambridge. Accessed April 16, 2021.
https://www.repository.cam.ac.uk/handle/1810/274120.
MLA Handbook (7th Edition):
Bertero, Alessandro. “Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells.” 2016. Web. 16 Apr 2021.
Vancouver:
Bertero A. Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells. [Internet] [Doctoral dissertation]. University of Cambridge; 2016. [cited 2021 Apr 16].
Available from: https://www.repository.cam.ac.uk/handle/1810/274120.
Council of Science Editors:
Bertero A. Activin/Nodal Signalling Controls the Epigenome and Epitranscriptome of Human Pluripotent Stem Cells. [Doctoral Dissertation]. University of Cambridge; 2016. Available from: https://www.repository.cam.ac.uk/handle/1810/274120
University of California – San Francisco
21.
Blau, James Alan.
Pooled screening to reveal the primary effectors of miR-142.
Degree: Pharmaceutical Sciences and Pharmacogenomics, 2015, University of California – San Francisco
URL: http://www.escholarship.org/uc/item/59b4f61r
► Although microRNAs are key regulators of gene expression, few studies have thoroughly evaluated the upstream regulators of microRNA activity. We seek to understand the upstream…
(more)
▼ Although microRNAs are key regulators of gene expression, few studies have thoroughly evaluated the upstream regulators of microRNA activity. We seek to understand the upstream regulators of miR-142, a microRNA thought to be important for B cell lymphogenesis and lymphocyte proliferation. We performed sequential whole-genome RNAi and focused CRISPR screens in a human B cell line to identify genes affecting miR-142 activity on a reporter construct bearing several perfect binding sites. Top hits include known core microRNA pathway genes, but also other genes such as PSME4, FXR1, SKIV2L, and FAM208A. The observation of FXR1 as a top screen hit is intriguing given that it has been observed to post-transcriptionally promote the expression of several neuronal microRNAs, but has not been demonstrated for other microRNAs in general. The results gathered thus far demonstrate the power of using pooled high-throughput screening and genome-editing approaches to discover regulators of microRNA biogenesis and activity.
Subjects/Keywords: Molecular biology; CRISPR-Cas9; miR-142; pooled screen; RNAi; shRNA
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APA ·
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MLA ·
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Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Blau, J. A. (2015). Pooled screening to reveal the primary effectors of miR-142. (Thesis). University of California – San Francisco. Retrieved from http://www.escholarship.org/uc/item/59b4f61r
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Blau, James Alan. “Pooled screening to reveal the primary effectors of miR-142.” 2015. Thesis, University of California – San Francisco. Accessed April 16, 2021.
http://www.escholarship.org/uc/item/59b4f61r.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Blau, James Alan. “Pooled screening to reveal the primary effectors of miR-142.” 2015. Web. 16 Apr 2021.
Vancouver:
Blau JA. Pooled screening to reveal the primary effectors of miR-142. [Internet] [Thesis]. University of California – San Francisco; 2015. [cited 2021 Apr 16].
Available from: http://www.escholarship.org/uc/item/59b4f61r.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Blau JA. Pooled screening to reveal the primary effectors of miR-142. [Thesis]. University of California – San Francisco; 2015. Available from: http://www.escholarship.org/uc/item/59b4f61r
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Melbourne
22.
Pierce, Thomas Patrick.
Use of RNAi in the mouse: inducible and reversible inhibition of Stat3.
Degree: 2012, University of Melbourne
URL: http://hdl.handle.net/11343/37193
► The signal transducer and activator of transcription 3 (Stat3) is a latent transcription factor that is activated during tissue inflammation and in a wide range…
(more)
▼ The signal transducer and activator of transcription 3 (Stat3) is a latent transcription factor that is activated during tissue inflammation and in a wide range of human malignancies. Activation of Stat3 within the tumour microenvironment leads to the transcriptional induction of a network of Stat3 target genes. Among the transcriptional targets of Stat3 are genes that promote cell survival, cell cycle progression and factors that induce angiogenesis and suppress tumour surveillance by the adaptive immune system. Consequently, activation of Stat3 within the tumour microenvironment enables tumour cells to acquire several of the fundamental biological hallmarks of cancer. The Stat3 pathway therefore represents an attractive target for cancer therapeutics and inhibitors targeting several members of the pathway are currently under development. The work presented in this thesis aimed to develop pre-clinical mouse models in which the activity of Stat3 can be inhibited in a drug-like manner. A recently described method for RNA interference (RNAi)-based gene targeting was employed in order to achieve tetracycline (Tet)-inducible and reversible inhibition of Stat3 expression. To knockdown Stat3 expression, several short hairpin RNAs (shRNAs) were designed to specifically target the Stat3 mRNA. The efficacy of each of these shRNAs was assessed in two independent cell lines, using immunoblot analysis and Stat3 reporter gene assays to detect a reduction in Stat3 protein expression and function. The Stat3.1348 shRNA emerged as the most effective shRNA tested. Expression of Stat3.1348 in mouse embryonic stem cells resulted in cellular differentiation, providing additional evidence of effective Stat3 impairment by Stat3.1348. A mouse strain was generated in which a Tet-regulated Stat3.1348 construct was stably integrated into the genome at the collagen A1 locus.
Furthermore, with the aim of facilitating tissue-specific Stat3 knockdown, a transgenic mouse strain harbouring an intestine-specific Tet-transactivator was generated and the function of this tissue-specific Tet-transactivator was compared to two published Tet-transactivator strains with more ubiquitous expression patterns. Mice harbouring the Stat3.1348 allele were crossed with Tet-transactivator strain with the most rebust intestinal activity, R26-M2rtTA. No reduction in Stat3 protein expression or function was detected in the gastrointestinal tract after treatment of R26-M2rtTA/Stat3.1348 mice with doxycycline to induce shRNA expression. In a parallel study, the utility of a retrovirally-delivered Stat3.1348 construct targeting Stat3 was explored in the mouse 4T1 breast tumour model. This tumour cell line exhibits constitutive Stat3 activity and 4T1 tumour growth in the mouse mammary fat pad has been previously been shown to dependent on Stat3 expression. However, despite achieving effective reduction of Stat3 expression in 4T1 cells using retroviral Stat3.1348 expression constructs, tumour growth was not found to be altered…
Subjects/Keywords: RNAi; RNA interference; shRNA; Stat3; miR-21; mouse models; gene knockdown
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APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Pierce, T. P. (2012). Use of RNAi in the mouse: inducible and reversible inhibition of Stat3. (Doctoral Dissertation). University of Melbourne. Retrieved from http://hdl.handle.net/11343/37193
Chicago Manual of Style (16th Edition):
Pierce, Thomas Patrick. “Use of RNAi in the mouse: inducible and reversible inhibition of Stat3.” 2012. Doctoral Dissertation, University of Melbourne. Accessed April 16, 2021.
http://hdl.handle.net/11343/37193.
MLA Handbook (7th Edition):
Pierce, Thomas Patrick. “Use of RNAi in the mouse: inducible and reversible inhibition of Stat3.” 2012. Web. 16 Apr 2021.
Vancouver:
Pierce TP. Use of RNAi in the mouse: inducible and reversible inhibition of Stat3. [Internet] [Doctoral dissertation]. University of Melbourne; 2012. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/11343/37193.
Council of Science Editors:
Pierce TP. Use of RNAi in the mouse: inducible and reversible inhibition of Stat3. [Doctoral Dissertation]. University of Melbourne; 2012. Available from: http://hdl.handle.net/11343/37193
University of Edinburgh
23.
Brightwell, Sara.
Identifying novel regulators of reprogramming using RNA interference.
Degree: PhD, 2015, University of Edinburgh
URL: http://hdl.handle.net/1842/16156
► Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and…
(more)
▼ Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.
Subjects/Keywords: 572.8; reprogramming; iPSC; induced pluripotent stem cells; shRNA
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APA (6th Edition):
Brightwell, S. (2015). Identifying novel regulators of reprogramming using RNA interference. (Doctoral Dissertation). University of Edinburgh. Retrieved from http://hdl.handle.net/1842/16156
Chicago Manual of Style (16th Edition):
Brightwell, Sara. “Identifying novel regulators of reprogramming using RNA interference.” 2015. Doctoral Dissertation, University of Edinburgh. Accessed April 16, 2021.
http://hdl.handle.net/1842/16156.
MLA Handbook (7th Edition):
Brightwell, Sara. “Identifying novel regulators of reprogramming using RNA interference.” 2015. Web. 16 Apr 2021.
Vancouver:
Brightwell S. Identifying novel regulators of reprogramming using RNA interference. [Internet] [Doctoral dissertation]. University of Edinburgh; 2015. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1842/16156.
Council of Science Editors:
Brightwell S. Identifying novel regulators of reprogramming using RNA interference. [Doctoral Dissertation]. University of Edinburgh; 2015. Available from: http://hdl.handle.net/1842/16156
University of New South Wales
24.
Ramadas, Radhika.
Identification of modulators of chemotherapeutic resistance using a random shRNA library.
Degree: Biotechnology & Biomolecular Sciences, 2012, University of New South Wales
URL: http://handle.unsw.edu.au/1959.4/52276
;
https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10948/SOURCE01?view=true
► High-throughput gene silencing using RNA interference (RNAi) libraries is a rapidly expanding strategy to study the molecular pathways underlying human diseases. A majority of RNAi…
(more)
▼ High-throughput gene silencing using RNA interference (RNAi) libraries is a rapidly expanding strategy to study the molecular pathways underlying human diseases. A majority of RNAi libraries contain expression constructs designed to target specific genes or gene sub-classes. The use of random RNAi libraries offers a more wide-reaching alternative to identify novel therapeutic targets. However, most of the previously reported protocols for the generation of random RNAi libraries remain highly complex. In this thesis, the development of a simplified and efficient method for the construction of a random
shRNA-encoding library is reported. To prove the functionality of the random
shRNA-encoding library, it was used to investigate the molecular components and mechanisms used by cancer cells to acquire resistance to three anti-cancer drugs from two drug classes with distinct mechanisms of action. Using independent selection assays, cells transduced with the random
shRNA-encoding library were exposed to cisplatin, carboplatin or paclitaxel, and resistant cells isolated. A total of 159 unique
shRNA-encoding inserts were recovered, sequenced and used to perform homology-based searching of protein- and RNA-encoding databases. Several of the inserts were enriched in cells displaying resistance to the specific chemotherapeutic agents. The predicted target sequences included both previously reported targets implicated in cellular resistance mechanisms and novel targets, thus proving the feasibility of this approach in identifying new targets for therapeutic agents. The cell-based RNAi screen and subsequent bioinformatic analysis produced a list of novel gene targets for further investigation of the role each plays in chemotherapeutic drug resistance.
Advisors/Committee Members: Arndt, Greg, Children's Cancer Institute Australia for Medical Research, UNSW, Dawes, Ian, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW.
Subjects/Keywords: Chemotherapeutic drug resistance; RNA interference; Random shRNA-encoding library
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APA (6th Edition):
Ramadas, R. (2012). Identification of modulators of chemotherapeutic resistance using a random shRNA library. (Doctoral Dissertation). University of New South Wales. Retrieved from http://handle.unsw.edu.au/1959.4/52276 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10948/SOURCE01?view=true
Chicago Manual of Style (16th Edition):
Ramadas, Radhika. “Identification of modulators of chemotherapeutic resistance using a random shRNA library.” 2012. Doctoral Dissertation, University of New South Wales. Accessed April 16, 2021.
http://handle.unsw.edu.au/1959.4/52276 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10948/SOURCE01?view=true.
MLA Handbook (7th Edition):
Ramadas, Radhika. “Identification of modulators of chemotherapeutic resistance using a random shRNA library.” 2012. Web. 16 Apr 2021.
Vancouver:
Ramadas R. Identification of modulators of chemotherapeutic resistance using a random shRNA library. [Internet] [Doctoral dissertation]. University of New South Wales; 2012. [cited 2021 Apr 16].
Available from: http://handle.unsw.edu.au/1959.4/52276 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10948/SOURCE01?view=true.
Council of Science Editors:
Ramadas R. Identification of modulators of chemotherapeutic resistance using a random shRNA library. [Doctoral Dissertation]. University of New South Wales; 2012. Available from: http://handle.unsw.edu.au/1959.4/52276 ; https://unsworks.unsw.edu.au/fapi/datastream/unsworks:10948/SOURCE01?view=true
25.
Lahmar, Qods.
Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent.
Degree: Docteur es, Immunologie, 2013, Aix Marseille Université
URL: http://www.theses.fr/2013AIXM4029
► Chez les métazoaires, les cellules spécialisées se caractérisent par la sortie du cycle cellulaire alors que les cellules souches et progénitrices se caractérisent par un…
(more)
▼ Chez les métazoaires, les cellules spécialisées se caractérisent par la sortie du cycle cellulaire alors que les cellules souches et progénitrices se caractérisent par un intense potentiel d'auto-renouvellement, lequel est perdu durant la différenciation. L'auto-renouvellement est contrôlé par une combinaison de facteurs intrinsèques et extrinsèques qui déclenchent une prolifération cellulaire équilibrée. Dans ce contexte, nous avons montré que le knock-out des facteurs de transcription MafB et c-Maf dans les monocytes, résulte en une expansion prolongée des monocytes et macrophages matures en culture, sans aucun signe de perte du phénotype différencié ou de la fonction. Etant donné que les macrophages sont impliqués dans la majorité des maladies dégénératives, les maladies inflammatoires ainsi que la biologie du cancer, l'amplification de macrophages consisterait en un atout considérable pour les applications thérapeutiques. Dans cette optique, et comme les macrophages sont également connus pour promouvoir le développement tumoral, nous avons étudié le comportement des macrophages Maf-DKO dans le contexte tumoral. Initialement, nous avons montré que les macrophages Maf-DKO sont capables d'empêcher l'installation de la tumeur ainsi que de réduire une masse tumorale établie, et ce indépendamment du model tumoral étudié. Ceci consiste en une nouvelle approche thérapeutique contre le cancer. Nous nous sommes ensuite intéressés à fournir une « preuve de principe » quant à la prolifération des monocytes humains après inhibition de l'expression des gènes MafB et c-Maf humains et à étudier leur potentiel dans des applications thérapeutiques.
In metazoans, specialized cells are typically withdrawn from the cell cycle, whereas stem cells and progenitor cells have extensive self-renewal potential that is usually lost on differentiation. Self-renewal is controlled by a combination of cell-intrinsic and extrinsic signals that trigger balanced cellular proliferation. In this context, we previously reported that the knock-out of two monocytic transcription factors, MafB and c-Maf, enables extended expansion of mature monocytes and macrophages in culture without loss of differentiated phenotype and function. As macrophages are involved in degenerative diseases, inflammatory diseases and cancer biology, amplified macrophages may provide potential therapeutic applications. In this context and since macrophages are also known to enhance tumor development, we aim to investigate Maf-DKO macrophages behavior in a tumor context Initially, we have shown that regardless of tumor model (ID8 ovarian carcinoma or B16 melanoma), Maf-DKO macrophages have the ability to prevent tumor growth and reduce established tumor mass in tumor bearing mice. The potential provides a novel therapeutic approach for cancer cell therapies. Next we aimed to provide a proof of principle for the amplification of human monocytes by the inhibition of MafB/c-Maf genes and to investigate their potential in therapeutic applications. So far, we have shown that the…
Advisors/Committee Members: Sieweke, Michael H. (thesis director).
Subjects/Keywords: Macrophages; Cancer; Maf; Facteurs de transcription; Applications therapeutiques; ShRNA; Lentivirus; Talen; Macrophages; Cancer; Maf; Transcription factors; Therapeutic applications; ShRNA; Lentivirus; Talen; 571
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APA (6th Edition):
Lahmar, Q. (2013). Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent. (Doctoral Dissertation). Aix Marseille Université. Retrieved from http://www.theses.fr/2013AIXM4029
Chicago Manual of Style (16th Edition):
Lahmar, Qods. “Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent.” 2013. Doctoral Dissertation, Aix Marseille Université. Accessed April 16, 2021.
http://www.theses.fr/2013AIXM4029.
MLA Handbook (7th Edition):
Lahmar, Qods. “Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent.” 2013. Web. 16 Apr 2021.
Vancouver:
Lahmar Q. Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent. [Internet] [Doctoral dissertation]. Aix Marseille Université 2013. [cited 2021 Apr 16].
Available from: http://www.theses.fr/2013AIXM4029.
Council of Science Editors:
Lahmar Q. Analyse du potentiel des macrophages double-déficients en MafB et c-Maf en tant qu'agent de thérapie cellulaire : Analyse of the potential of MafB/c-Maf double deficient macrophages as cellular therapeutic agent. [Doctoral Dissertation]. Aix Marseille Université 2013. Available from: http://www.theses.fr/2013AIXM4029
26.
Luís Bruno da Cruz e Alves de Moraes.
Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico.
Degree: 2018, University of São Paulo
URL: http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07052019-110949/
► O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice…
(more)
▼ O adenocarcinoma ductal pancreático (PDAC, pancreatic ductal adenocarcinoma), o tipo mais prevalente de câncer do pâncreas, é uma neoplasia extremamente agressiva e com elevado índice de letalidade. Há uma necessidade premente de identificação de vulnerabilidades no PDAC que possam ser exploradas como alvos terapêuticos, e a utilização de modelos pré-clínicos que recapitulem a complexidade biológica e heterogeneidade clínica da doença é um aspecto central para a realização dessa tarefa. Os xenotransplantes de tecido tumoral derivado de pacientes (PDX, patient-derived tumor tissue xenografts), realizados em camundongos imunodeficientes, replicam com grande similaridade as principais características do tumor original e, assim, constituem uma ferramenta valiosa para o teste de drogas e estudos funcionais. Neste trabalho, 17 amostras cirúrgicas de PDAC humano foram implantadas subcutaneamente em camundongos nude atímicos. Sete tumores (41%) foram
enxertados com sucesso e têm sido mantidos em sucessivas gerações de animais receptores. O exame histológico de seis desses xenoenxertos identificou características morfológicas compatíveis com os padrões reconhecidos no PDAC humano, assim como uma consistente similaridade de seu status de diferenciação histológica em relação aos perfis verificados nos tumoresoriginais. O cultivo in vitro de células derivadas de um dos xenotumores resultou em uma nova linhagem de câncer de pâncreas, com morfologia e cinética de crescimento comparáveis às de outras linhagens celulares de câncer pancreático. O potencial tumorigênico dessa nova linhagem foi validado in vivo, com uma consistente formação de tumores após inoculação em camundongos nude. A fim de aproveitar esse recurso para a investigação de potenciais alvos terapêuticos no PDAC, um rastreamento de vulnerabilidades moleculares foi realizado por meio de silenciamento gênico em larga-escala com RNA de interferência (RNAi). Uma biblioteca
lentiviral de 4492 shRNAs (short hairpin RNAs), alvejando cerca de 350 genes envolvidos na regulação epigenética, foi empregada para a triagem de genes de suscetibilidade nas células derivadas de PDX, e em outras cinco linhagens tumorais pancreáticas (AsPC-1, BxPC-3, Capan-1, MIA PaCa-2 e PANC-1). Inicialmente, foi realizada uma série de experimentos preliminares, visando à amplificação e controle de qualidade da biblioteca de silenciamento, à produção de vetores lentivirais e à padronização das condições experimentais para a transdução e seleção das células-alvo. Apenas três das linhagens avaliadas (AsPC-1, MIA PaCa-2 e PANC-1) mostraram-se permissíveis à transdução pelos vetores lentivirais, e foram assim utilizadas no screening de alvos epigenéticos. A análise dos dados obtidos nesse ensaio está em curso e os resultados serão utilizados para a definição de potenciais alvos candidatos. Em conclusão, recursos valiosos para apoiar a pesquisa sobre o câncer de pâncreas foram
desenvolvidos. A coleção de PDXs estabelecida, bem como a linhagem celular recém-derivada, constituem uma fonte permanente e…
Advisors/Committee Members: Eduardo Moraes Reis, Maria Mitzi Brentani, Miriam Galvonas Jasiulionis Leon, Deborah Schechtman, Mari Cleide Sogayar.
Subjects/Keywords: Câncer de pâncreas; Epigenética; Linhagem celular tumoral; RNA de interferência; shRNA; Xenoenxerto; Cancer cell line; Epigenetics; Pancreatic cancer; RNA interference; shRNA; Xenograft
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APA (6th Edition):
Moraes, L. B. d. C. e. A. d. (2018). Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico. (Doctoral Dissertation). University of São Paulo. Retrieved from http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07052019-110949/
Chicago Manual of Style (16th Edition):
Moraes, Luís Bruno da Cruz e Alves de. “Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico.” 2018. Doctoral Dissertation, University of São Paulo. Accessed April 16, 2021.
http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07052019-110949/.
MLA Handbook (7th Edition):
Moraes, Luís Bruno da Cruz e Alves de. “Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico.” 2018. Web. 16 Apr 2021.
Vancouver:
Moraes LBdCeAd. Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico. [Internet] [Doctoral dissertation]. University of São Paulo; 2018. [cited 2021 Apr 16].
Available from: http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07052019-110949/.
Council of Science Editors:
Moraes LBdCeAd. Desenvolvimento de xenotransplantes de tumores pancreáticos humanos para varredura genética de alvos moleculares com potencial terapêutico. [Doctoral Dissertation]. University of São Paulo; 2018. Available from: http://www.teses.usp.br/teses/disponiveis/46/46131/tde-07052019-110949/
27.
Quick-Cleveland, Jen.
Primary microRNA processing in humans: biology, biotechnology and disease.
Degree: Biological Chemistry, 2017, UCLA
URL: http://www.escholarship.org/uc/item/4sh028f3
► MicroRNAs (miRNAs) are a class of non-coding RNAs that tune gene expression by negatively regulating at least 60% of protein-coding genes. In animals, they are…
(more)
▼ MicroRNAs (miRNAs) are a class of non-coding RNAs that tune gene expression by negatively regulating at least 60% of protein-coding genes. In animals, they are required for development and cell physiology. Due to their role in controlling gene expression, dysregulation of miRNA biogenesis can cause genetic disorders, immunity deficits, neurological problems, and cancers. miRNA genes are transcribed into primary miRNA (pri-miRNAs), which undergo a multi-step maturation process. The first step occurs in the nucleus, where the pri-miRNA is cleaved by the Microprocessor Complex (MC). The MC contains the ribonuclease Drosha and an RNA-binding hemoprotein DGCR8. The MC specifically recognizes the characteristic pri-miRNA hairpin to initiate miRNA maturation. This thesis centers on the nuclear step of miRNA biogenesis. My research has two main areas of focus; 1) dissecting the molecular and structural determinants that govern pri-miRNA processing, and 2) investigating the regulation of pri-miRNA processing in normal physiology and diseases.The MC identifies pri-miRNA substrates from a myriad of other RNAs. DGCR8 plays an important role in pri-miRNA recognition, but the mechanism was unknown. DGRC8 contains two double-stranded RNA-binding domains (dsRBDs), but these domains do not provide specificity. It has been shown that “junctions” between single-stranded and double-stranded regions in pri-miRNAs are important features that define MC substrates. However, the protein moiety that recognizes pri-miRNA junctions had not been identified. We discovered that DGCR8 contains an RNA-binding heme domain (Rhed) that directly and specifically binds pri-miRNA junctions. Further, we showed that the Rhed and its RNA-binding surface are required for efficient pri-miRNA processing.Previous studies of our lab showed that the pri-miRNA processing activity of DGCR8 specifically requires heme in its Fe(III) redox state. However, it is unknown how much Fe(III) heme is available in cells and how much is required to support pri-miRNA processing. During our investigation of the Rhed-pri-miRNA interface, we performed mutagenesis on DGCR8 that led to identification of single mutations with reduced heme affinity but nearly full processing activity in cells. This meant that the Fe(III) heme affinities of these mutants are sufficient for them to acquire heme and to process pri-miRNAs. In contrast, all heme-binding-deficient mutants of DGCR8 we previously characterized are inactive in cells and have lower affinities for Fe(III) heme. We discovered that the Fe(III) heme affinity threshold for activating DGCR8 is shifted by overall heme availability changes in cells. These results indicate the presence of an available ferric heme pool that distinctly determines pri-miRNA processing efficiency in cells. Our study suggests cellular redox state and currently unknown Fe(III) heme-specific transporter proteins may be important for regulating miRNA maturation. Secondary structure is a defining characteristic of pri-miRNA substrates. Substantial effort has been…
Subjects/Keywords: Molecular biology; Biochemistry; DGCR8; Fe(III) heme; microRNA; miRNA and cancer; shRNA
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APA ·
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Export
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APA (6th Edition):
Quick-Cleveland, J. (2017). Primary microRNA processing in humans: biology, biotechnology and disease. (Thesis). UCLA. Retrieved from http://www.escholarship.org/uc/item/4sh028f3
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Quick-Cleveland, Jen. “Primary microRNA processing in humans: biology, biotechnology and disease.” 2017. Thesis, UCLA. Accessed April 16, 2021.
http://www.escholarship.org/uc/item/4sh028f3.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Quick-Cleveland, Jen. “Primary microRNA processing in humans: biology, biotechnology and disease.” 2017. Web. 16 Apr 2021.
Vancouver:
Quick-Cleveland J. Primary microRNA processing in humans: biology, biotechnology and disease. [Internet] [Thesis]. UCLA; 2017. [cited 2021 Apr 16].
Available from: http://www.escholarship.org/uc/item/4sh028f3.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Quick-Cleveland J. Primary microRNA processing in humans: biology, biotechnology and disease. [Thesis]. UCLA; 2017. Available from: http://www.escholarship.org/uc/item/4sh028f3
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Universidade de Lisboa
28.
Brito, Francisco Maria de Aboim Borges Fialho de.
Transcriptome profiling of spinal muscular atrophy models using RNA-Seq.
Degree: 2014, Universidade de Lisboa
URL: https://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/15998
► Tese de mestrado, Bioinformática e Biologia Computacional, Universidade de Lisboa, Faculdade de Ciências, 2014
Spinal Muscular Atrophy (SMA) is a neurodegenerative disorder that represents the…
(more)
▼ Tese de mestrado, Bioinformática e Biologia Computacional, Universidade de Lisboa, Faculdade de Ciências, 2014
Spinal Muscular Atrophy (SMA) is a neurodegenerative disorder that represents the second most common cause of hereditary infant death. It is caused by the reduced expression of the ubiquitous protein SMN (Survival of Motor Neuron), which is known to have a central function in the assembly of ribonucleoprotein complexes involved in pre-mRNA splicing. More recently, this protein has been reported to be involved in trafficking of mRNA molecules along neuron axons. Although the SMA causing gene has been identified for over a decade, the exact mechanisms that lead to the specific death of motor neurons remain unclear. A long-standing hypothesis suggests that the disease emerges from motor-neuron specific changes in pre-mRNA splicing that affect key genes required for the survival of these cells. A possible approach to identify these genes is whole-transcriptome profiling. Nowadays, one of the most powerful tools for transcriptome profiling is next generation RNA sequencing (RNA-Seq), which can provide data with minimal biological variation between replicates, resulting in a precise comparison of different phenotypes. However this is not a bias-free technique and library preparation and sequencing problems can introduce several artifacts which need to be addressed. Here we present an RNA-Seq study of disease models based on D. melanogaster and H. sapiens iPSC cultures developed to help unravel the pathways related to SMN down-regulation and SMA by identifying changes in gene expression and transcript isoform expression. During the development of the analysis pipeline for Drosophila, several difficulties were encountered, emerging from the inherent complexity of the process of preparing tissue specific RNA samples requiring dissection and pooling of multiple larvae brains, and the presence of an shRNA expression vector. This resulted in intra-treatment variance that needed to be addressed and stabilized. Furthermore, we found that some widely used algorithms for discovery of novel transcript isoforms can perform poorly on Drosophila, requiring the selection of alternative approaches. Similarly, the human analysis pipeline showed a high amount of variance due to the limited number of individuals used to create iPSC libraries, introducing bias in the analysis. Finally, we showed via comparison of differentially expressed ortholog genes that changes caused by SMN down-regulation affect several conserved genes across species, making Drosophila a favourable approach for modelling SMA.
A Atrofia Muscular Espinhal (SMA) é uma doença neuro-degenerativa que representa a segunda causa mais comum de morte infantil hereditária. É causada pela expressão reduzida da proteína ubíqua SMN (Survival of Motor neuron), que tem uma função central na assemblagem de complexos ribonucleoproteicos envolvidos no splicing do pre-mRNA. Recentemente, esta proteína também foi reportada como estando envolvida no tráfego de moléculas de…
Advisors/Committee Members: Carvalho, Margarida Henriques da Gama, 1972-, Fonseca, Andreia J. Amaral.
Subjects/Keywords: RNA-Seq; Spinal muscular antrophy; shRNA; D. melanogaster; H. sapiens; Teses de mestrado - 2014
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APA ·
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Export
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APA (6th Edition):
Brito, F. M. d. A. B. F. d. (2014). Transcriptome profiling of spinal muscular atrophy models using RNA-Seq. (Thesis). Universidade de Lisboa. Retrieved from https://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/15998
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Chicago Manual of Style (16th Edition):
Brito, Francisco Maria de Aboim Borges Fialho de. “Transcriptome profiling of spinal muscular atrophy models using RNA-Seq.” 2014. Thesis, Universidade de Lisboa. Accessed April 16, 2021.
https://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/15998.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Brito, Francisco Maria de Aboim Borges Fialho de. “Transcriptome profiling of spinal muscular atrophy models using RNA-Seq.” 2014. Web. 16 Apr 2021.
Vancouver:
Brito FMdABFd. Transcriptome profiling of spinal muscular atrophy models using RNA-Seq. [Internet] [Thesis]. Universidade de Lisboa; 2014. [cited 2021 Apr 16].
Available from: https://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/15998.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Brito FMdABFd. Transcriptome profiling of spinal muscular atrophy models using RNA-Seq. [Thesis]. Universidade de Lisboa; 2014. Available from: https://www.rcaap.pt/detail.jsp?id=oai:repositorio.ul.pt:10451/15998
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Toronto
29.
Ma, Xiangyuan.
Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation.
Degree: 2014, University of Toronto
URL: http://hdl.handle.net/1807/67885
► N-glycosylation is a co-translational modification that covalently attaches oligosaccharide to and regulates proper folding, trafficking, and surface residency of secreted proteins. MGAT5 encodes a glycosyltransferase…
(more)
▼ N-glycosylation is a co-translational modification that covalently attaches oligosaccharide to and regulates proper folding, trafficking, and surface residency of secreted proteins. MGAT5 encodes a glycosyltransferase that synthesizes β1,6GlcNAc-branched N-glycans in medial Golgi. MGAT5 is frequently up-regulated in and associated with the progression of multiple types of human carcinomas. In order to identify Mgat5 interacting genes and signaling pathways, immortalized mouse embryonic fibroblast (MEF) cell lines that are Mgat5 wild-type and null were established on p53 null genetic background. Pooled lentiviral shRNA drop-out screens revealed that mTOR pathway was essential in the wild-type MEFs, whereas signaling dependency shifts to RAS-MEK-ERK pathway was observed in the null cells. Metabolite profiling of the MEFs and human cell lines with MGAT5 knock-down showed drastic changes in tricarboxylic acid cycle metabolites and amino acids upon MGAT5 disruption. Together, these approaches permitted a global survey of genetic and metabolic changes that occur when MGAT5 is disturbed.
M.Sc.
Advisors/Committee Members: Dennis, W James, Molecular and Medical Genetics.
Subjects/Keywords: Cell Signaling; Metabolism; Mgat5; N-glycosylation; Pooled Lentiviral shRNA Drop-out Screen; 0307
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APA ·
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MLA ·
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CSE |
Export
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APA (6th Edition):
Ma, X. (2014). Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/67885
Chicago Manual of Style (16th Edition):
Ma, Xiangyuan. “Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation.” 2014. Masters Thesis, University of Toronto. Accessed April 16, 2021.
http://hdl.handle.net/1807/67885.
MLA Handbook (7th Edition):
Ma, Xiangyuan. “Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation.” 2014. Web. 16 Apr 2021.
Vancouver:
Ma X. Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation. [Internet] [Masters thesis]. University of Toronto; 2014. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1807/67885.
Council of Science Editors:
Ma X. Identification of Genes and Pathways Linking Cancer Metabolism to Cell Surface Dynamics through Protein N-glycosylation. [Masters Thesis]. University of Toronto; 2014. Available from: http://hdl.handle.net/1807/67885
University of Toronto
30.
Ramakrishnan, Ashwin.
Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia.
Degree: 2014, University of Toronto
URL: http://hdl.handle.net/1807/68527
► Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by a wide variety of cytogenetic abnormalities. Treatment has been largely unchanged for the past…
(more)
▼ Acute myeloid leukemia (AML) is a highly heterogeneous disease characterized by a wide variety of cytogenetic abnormalities. Treatment has been largely unchanged for the past two decades and prognosis is still poor; thus, new therapeutics is needed for this disease. Here, we explore the possibility of targeting the Mitochondrial Processing Peptidase (MPP) as a potential therapeutic target for AML. MPP is a heterodimeric zinc metallopeptidase that cleaves presequences following the import of nuclear encoded matrix space precursors. We demonstrate that MPP-α is overexpressed in a subset of AML patient blood and stem/progenitor populations. MPP-α is ubiquitously expressed in human tissues and strongly expressed in heart and brain. Furthermore, AML cell lines are differentially susceptible towards genetic knockdown of MPP-α with OCI-AML2 having the greatest decrease in cell viability and proliferation while K562 and TEX have moderate effects. These results warrant further investigation of MPP as a therapeutic target for AML.
M.Sc.
Advisors/Committee Members: Schimmer, Aaron, Medical Biophysics.
Subjects/Keywords: Acute Myeloid Leukemia; Mitochondrial Processing Peptidase; Mitochondrial protein import; NanoString; shRNA; 0992
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APA (6th Edition):
Ramakrishnan, A. (2014). Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia. (Masters Thesis). University of Toronto. Retrieved from http://hdl.handle.net/1807/68527
Chicago Manual of Style (16th Edition):
Ramakrishnan, Ashwin. “Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia.” 2014. Masters Thesis, University of Toronto. Accessed April 16, 2021.
http://hdl.handle.net/1807/68527.
MLA Handbook (7th Edition):
Ramakrishnan, Ashwin. “Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia.” 2014. Web. 16 Apr 2021.
Vancouver:
Ramakrishnan A. Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia. [Internet] [Masters thesis]. University of Toronto; 2014. [cited 2021 Apr 16].
Available from: http://hdl.handle.net/1807/68527.
Council of Science Editors:
Ramakrishnan A. Targeting the Mitochondrial Processing Peptidase as a Therapeutic Strategy for Acute Myeloid Leukemia. [Masters Thesis]. University of Toronto; 2014. Available from: http://hdl.handle.net/1807/68527
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