Dept: Biochemistry ❌
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University of Alberta
1.
Robertson, Ian Michael.
NMR investigation into the therapeutic potential of
troponin.
Degree: PhD, Biochemistry, 2011, University of Alberta
URL: https://era.library.ualberta.ca/files/kd17ct46z 
► The pumping of the heart is controlled at the molecular level by the calcium dependent interaction between troponin C (cTnC) and troponin I (cTnI). The…
(more)
▼ The pumping of the heart is controlled at the
molecular level by the calcium dependent interaction between
troponin C (cTnC) and troponin I (cTnI). The central role this
protein-protein interaction plays in the muscle contraction cascade
makes it a prime target for the development of drugs for the
treatment of heart disease. In Chapters 2 and 3, we show that the
natural products, EGCg and resveratrol, bind preferentially to the
C-terminal domain of cTnC (cCTnC). NMR structures reveal that EGCg
binds to the surface of the hydrophobic pocket of cCTnC, whereas
resveratrol binds deeper in the protein, akin to the
Ca2+-sensitizer, EMD 57033. The comparisons between the two
structures highlight specific interactions between the compounds
and cCTnC that define differences in their binding poses. The next
section (Chapters 4, 5, and 6) is devoted to understanding the
mechanism of drugs that target the N-terminal domain of cTnC
(cNTnC). Specifically, the modulation of cTnI binding to cNTnC is
entertained as the mechanism by which molecules that bind to cNTnC
modulate contraction. In Chapter 4 some pharmacophores are
identified and an ideal cNTnC-cTnI construct for the design of
drugs is described. Chapter 5 explores the structure and function
of a novel Ca2+-sensitizer, dfbp-o. We find that dfbp-o enhances
cTnI binding in vitro and increases contractility in situ. This
enhanced cTnI binding is postulated to originate from an
electrostatic attraction between R147 of cTnI and the carboxylate
moiety of dfbp-o. In Chapter 6 the synthesis and activity of some
novel analogs of the inhibitor, W7, is outlined. The results
support the electrostatic mechanism outlined in Chapter 5. In
Chapters 7 and 8 we investigate how one can modify calcium
sensitivity by changing residues on either cNTnC or cTnI. We show
that the mutation L48Q stabilizes the open state of cNTnC thereby
enhancing cTnI binding and contractiltity. A specific histidine on
skeletal TnI has been shown to increase the calcium sensitivity of
a myofilament when compared to cTnI, at low pH. In Chapter 8, we
show that under acidic conditions, this histidine is protonated and
its binding to cNTnC is enhanced by the appearance of an
electrostatic interaction with E19 of cNTnC.
Subjects/Keywords: cardiomyopathy; levosimendan; troponin C; inotrope; NMR spectroscopy
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APA (6th Edition):
Robertson, I. M. (2011). NMR investigation into the therapeutic potential of
troponin. (Doctoral Dissertation). University of Alberta. Retrieved from https://era.library.ualberta.ca/files/kd17ct46z
Chicago Manual of Style (16th Edition):
Robertson, Ian Michael. “NMR investigation into the therapeutic potential of
troponin.” 2011. Doctoral Dissertation, University of Alberta. Accessed December 15, 2019.
https://era.library.ualberta.ca/files/kd17ct46z.
MLA Handbook (7th Edition):
Robertson, Ian Michael. “NMR investigation into the therapeutic potential of
troponin.” 2011. Web. 15 Dec 2019.
Vancouver:
Robertson IM. NMR investigation into the therapeutic potential of
troponin. [Internet] [Doctoral dissertation]. University of Alberta; 2011. [cited 2019 Dec 15].
Available from: https://era.library.ualberta.ca/files/kd17ct46z.
Council of Science Editors:
Robertson IM. NMR investigation into the therapeutic potential of
troponin. [Doctoral Dissertation]. University of Alberta; 2011. Available from: https://era.library.ualberta.ca/files/kd17ct46z
2.
Anand, A Vijaya.
Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -.
Degree: Biochemistry, 2012, Bharathidasan University
URL: http://shodhganga.inflibnet.ac.in/handle/10603/4764
► Cardio and cerebrovascular diseases (CVDs) are multifactorial in etiology, and share some common risk factors. The death rates of these diseases are incredibly increasing due…
(more)
▼ Cardio and cerebrovascular diseases (CVDs) are
multifactorial in etiology, and share some common risk factors. The
death rates of these diseases are incredibly increasing due to the
fact, the markers that are in existence fail to introspect the
situation in detail. The Framingham Study showed that 35% of cases
of coronary heart disease (CHD) were in people with normal total
cholesterol (TC) levels, although the impact of elevated
cholesterol on stroke risk has been disputed. These findings point
out the need for markers that better predict cardiovascular risk.
In the most recent years, special importance is being laid on the
role of inflammation in the pathogenesis of atherosclerosis and its
complications. A number of studies have examined various
circulating markers of inflammations (e.g., serum amyloid A,
interleukin etc.,), out of those so far studied, high-sensitivity
C-reactive protein (hsCRP) seems to have the most consistent
relation to the risk of CVDs in a variety of clinical settings. The
objective of the present study, therefore, was to assess the
prognostic value of hsCRP alone, as well as in combination with
various blood lipids in patients with CVDs. The results of the
present study, along with the other analyses of large
population-based cohorts, confirmed the inclusion of hsCRP as a
risk marker for CVDs to have important implications. The most
important role of the drug statins in the reduction of serum lipids
has been well documented in both primary and secondary prevention
studies. However, these agents remain underutilized in several
settings. More recently, evidence suggesting that statins may
positively impact many organ systems and disease states independent
of lipid reduction, has emerged and their anti-inflammatory
properties have also been investigated. Hence, the present study
was also designed to determine the effect of atorvastain on hsCRP
and various other biomarkers in patients with
CVDs.
References given
Advisors/Committee Members: Kalavathy, S.
Subjects/Keywords: Cardiovascular disease; Stroke; C-reactive protein; Biochemistry
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APA (6th Edition):
Anand, A. V. (2012). Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -. (Thesis). Bharathidasan University. Retrieved from http://shodhganga.inflibnet.ac.in/handle/10603/4764
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):
Anand, A Vijaya. “Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -.” 2012. Thesis, Bharathidasan University. Accessed December 15, 2019.
http://shodhganga.inflibnet.ac.in/handle/10603/4764.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Anand, A Vijaya. “Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -.” 2012. Web. 15 Dec 2019.
Vancouver:
Anand AV. Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -. [Internet] [Thesis]. Bharathidasan University; 2012. [cited 2019 Dec 15].
Available from: http://shodhganga.inflibnet.ac.in/handle/10603/4764.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Anand AV. Role of high sensitivity C - reactive protein (hsCRP) as
a risk marker in cardio and cerebrovascular diseases and the effect
of atorvastatin therapy on hsCRP; -. [Thesis]. Bharathidasan University; 2012. Available from: http://shodhganga.inflibnet.ac.in/handle/10603/4764
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Illinois – Urbana-Champaign
3.
Frimel, Aaron Michael.
The C-terminal domains of leucyl-tRNA synthetases.
Degree: MS, Biochemistry, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/95620
► The mitochondrial Leucyl-tRNA synthetase (mtLeuRS) of certain Caenorhabditis species contains an idiosyncratic C-terminal addition. Bioinformatic analyses identified this domain in the mtLeuRS and also four…
(more)
▼ The mitochondrial Leucyl-tRNA synthetase (mtLeuRS) of certain Caenorhabditis species contains an idiosyncratic
C-terminal addition. Bioinformatic analyses identified this domain in the mtLeuRS and also four putative nuclear localization sequences (NLSs) encoded within the domain (Ezak, Hong, Chaparro-Garcia, & Ferkey, 2010). This
C-terminal extension of Caenrhabditis mitochondrial LeuRS, or CECL domain, is highly positively charged and shows no obvious homologs in protein databases. Bioinformatic and computational modeling suggest that CECL is an added domain rather than being a part of the canonical
C-terminal domain. An annotated splice-variant of the mitochondrial LeuRS hints at a mechanism for subcellular localization. Deletion of CECL from the chromosome prevents homozygous worms from surviving fertilization in most cases. From these observations, we hypothesize that the mitochondrial LeuRS of
C. elegans performs an alternate nuclear function, perhaps carried out specifically in the neurons of the animal.
Advisors/Committee Members: Martinis, Susan A (advisor).
Subjects/Keywords: Leucyl-tRNA synthetase; Mitochondria; C. elegans
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APA (6th Edition):
Frimel, A. M. (2016). The C-terminal domains of leucyl-tRNA synthetases. (Thesis). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/95620
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):
Frimel, Aaron Michael. “The C-terminal domains of leucyl-tRNA synthetases.” 2016. Thesis, University of Illinois – Urbana-Champaign. Accessed December 15, 2019.
http://hdl.handle.net/2142/95620.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Frimel, Aaron Michael. “The C-terminal domains of leucyl-tRNA synthetases.” 2016. Web. 15 Dec 2019.
Vancouver:
Frimel AM. The C-terminal domains of leucyl-tRNA synthetases. [Internet] [Thesis]. University of Illinois – Urbana-Champaign; 2016. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2142/95620.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Frimel AM. The C-terminal domains of leucyl-tRNA synthetases. [Thesis]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/95620
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Virginia Tech
4.
Ojani, Reyhaneh.
Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway.
Degree: PhD, Biochemistry, 2016, Virginia Tech
URL: http://hdl.handle.net/10919/80342
► Juvenile hormone (JH) is an important insect hormone that controls diverse biological processes in postembryonic development and adult reproduction. JH exerts its effects through the…
(more)
▼ Juvenile hormone (JH) is an important insect hormone that controls diverse biological processes in postembryonic development and adult reproduction. JH exerts its effects through the nuclear receptor Methoprene-tolerant (MET). MET is a transcription factor of the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family. In the presence of JH, MET forms a heterodimer with its DNA-binding partner Taiman (TAI). The MET-TAI complex directly binds to the regulatory regions of some JH target genes and regulates their transcription. However many questions remain unanswered regarding the JH-regulated gene expression. The work in this report aims to determine the role of protein kinase
C (PKC) in JH signaling in adult mosquitoes and to find the direct target genes of Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor encoded by a JH early response gene.
We discovered that PKC is an essential component of a membrane-initiated JH signaling pathway. PKC was activated by JH in a phospholipase
C (PLC)-dependent manner. Inhibition of PKC activity dramatically decreased the JH-induced gene expression. RNAi experiment indicated that several PKC isoforms were involved in the JH action in adult female mosquitoes. We showed that PKC modulated the transactivation activity of MET by enhancing the binding of MET and TAI to the promoters of JH target genes.
Kr-h1 is rapidly upregulated by JH in newly emerged mosquitoes. RNAi-mediated depletion of AaKr-h1 caused a substantial decrease in oviposited eggs, indicating that this protein plays an essential role in mosquito reproduction. We combined chromatin immunoprecipitation (ChIP) with cloning of the generated DNA and have identified chromatin binding sites of AaKr-h1 in Aedes aegypti. After adult emergence, binding of AaKr-h1 to its in vivo targets increased with the JH-induced increase in AaKr-h1. Interestingly, depletion of AaKr-h1 in newly emerged mosquitoes led to considerable upregulation of some AaKr-h1 target genes but downregulation of other target genes. The results suggest that AaKr-h1 acts downstream of AaMET to regulate gene expression in response to JH and that AaKr-h1 can activate or repress the expression of individual target gene.
Advisors/Committee Members: Zhu, Jinsong (committeechair), Mackey, Zachary Byron (committee member), Sharakhov, Igor V. (committee member), Gillaspy, Glenda E. (committee member).
Subjects/Keywords: Juvenile hormone; Protein kinase C; Gene regulation
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APA (6th Edition):
Ojani, R. (2016). Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway. (Doctoral Dissertation). Virginia Tech. Retrieved from http://hdl.handle.net/10919/80342
Chicago Manual of Style (16th Edition):
Ojani, Reyhaneh. “Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway.” 2016. Doctoral Dissertation, Virginia Tech. Accessed December 15, 2019.
http://hdl.handle.net/10919/80342.
MLA Handbook (7th Edition):
Ojani, Reyhaneh. “Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway.” 2016. Web. 15 Dec 2019.
Vancouver:
Ojani R. Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway. [Internet] [Doctoral dissertation]. Virginia Tech; 2016. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/10919/80342.
Council of Science Editors:
Ojani R. Molecular mechanisms underlying Juvenile hormone (JH) signaling pathway. [Doctoral Dissertation]. Virginia Tech; 2016. Available from: http://hdl.handle.net/10919/80342
Case Western Reserve University
5.
Young, Duprane Pedaci.
From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines.
Degree: PhD, Biochemistry, 2008, Case Western Reserve University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=case1201365244
► Expression of the acute phase protein C-reactive protein (CRP) is tightly regulated in hepatocytes. While very little CRP mRNA is transcribed normally, inflammatory stimuli…
(more)
▼ Expression of the acute phase protein
C-reactive protein (CRP) is tightly regulated in hepatocytes. While
very little CRP mRNA is transcribed normally, inflammatory stimuli
are followed by a dramatic increase in mRNA synthesis and
accumulation. Interleukins -6 and 1ß (IL-6 and IL-1ß) are believed
to be the major cytokines responsible for induction of CRP and
other acute phase proteins. We previously
demonstrated that <i>in vitro</i>
c-Rel plays a novel
regulatory role by forming a complex with
C/EBPß when
C/EBPß is
bound to the CRP gene promoter following cytokine stimulation.
c-Rel does not by itself bind to the DNA. In these studies we found
that recombinant
c-Rel
(1-300) (lacks
transactivation domain) increased the affinity of recombinant
C/EBPß for a CRP-derived
C/EBP site (-53) at least 10 fold.
C/EBPß
and
c-Rel
(1-300) were found to physically
interact in solution, and overexpression of
c-Rel in the presence
of overexpressed
C/EBPß stimulated CRP transcription. We concluded
that
c-Rel(1-300) binding to
C/EBPß increased the affinity of
C/EBPß for the CRP-
C/EBP(-53) site, and that the transactivation
domain of
c-Rel is not necessary for this effect, which depends on
protein: protein contacts with
C/EBPß. We also
employed chromatin immunoprecipitation (ChIP) assays to determine
the kinetics of transcription factor occupancy of these
transcription factors on the endogenous CRP promoter.
C/EBPß,
STAT3, p50, and
c-Rel were found bound to the endogenous CRP
promoter in the absence of cytokines, and cytokine treatment
markedly increased binding of only
C/EBPß. In addition,
c-Rel and
TBP appeared to occupy the promoter in parallel in the presence of
cytokines. In the absence of cytokines, CRP mRNA accumulation was
not measurable but began to increase by 3 h after exposure of cells
to IL-1ß + IL-6, peaking at 12 h with secondary peaks at 18 h and
24 h. The secondary peaks in mRNA expression paralleled the pattern
of binding of
c-Rel and TBP to the CRP promoter. We conclude that
the CRP promoter has a low level of transcription factor occupancy
in the absence of cytokines and induction occurs with binding of
C/EBP, and that
c-Rel and TBP are important for modulating CRP
expression.
Advisors/Committee Members: Samols, David (Advisor).
Subjects/Keywords: Biology, Molecular; APR; acute phase response; APP; acute phase protein; CRP; C-reactive protein; cytokines; IL-6; IL-1; C/EBP; STAT3; p50; c-Rel; ChIP; Chromatin Immunoprecipitation Assay
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APA ·
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Manager
APA (6th Edition):
Young, D. P. (2008). From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1201365244
Chicago Manual of Style (16th Edition):
Young, Duprane Pedaci. “From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines.” 2008. Doctoral Dissertation, Case Western Reserve University. Accessed December 15, 2019.
http://rave.ohiolink.edu/etdc/view?acc_num=case1201365244.
MLA Handbook (7th Edition):
Young, Duprane Pedaci. “From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines.” 2008. Web. 15 Dec 2019.
Vancouver:
Young DP. From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines. [Internet] [Doctoral dissertation]. Case Western Reserve University; 2008. [cited 2019 Dec 15].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1201365244.
Council of Science Editors:
Young DP. From <i>In Vitro</i> to <i>In
Vivo:</i> Control of C-Reactive Protein Gene Expression by
Cytokines. [Doctoral Dissertation]. Case Western Reserve University; 2008. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1201365244
University of Illinois – Urbana-Champaign
6.
Luthra, Abhinav.
Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1).
Degree: PhD, Biochemistry, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/78708
► CYP19A1 or aromatase, is a human steroidogenic P450 important for estrogen biosynthesis in humans. Over activation of aromatase results in malignancies of the breast tissue,…
(more)
▼ CYP19A1 or aromatase, is a human steroidogenic P450 important for estrogen biosynthesis in humans. Over activation of aromatase results in malignancies of the breast tissue, especially in post menopausal women. In fact, aromatase inhibitors constitute the front line therapy for estrogen receptor positive (ER+) breast cancer in post-menopausal women which accounts for over 70% of all breast cancer cases in the United States.
Starting with its androgenic substrates, testosterone and androstenedione, CYP19A1 forms estradiol and estrone utilizing one molecule of atmospheric oxygen and two reducing equivalents in the form of NADPH. This is accomplished in a three-step process one of which involves a carbon-carbon bond scission and aromatization. The catalytic mechanism of P450s has been long studied and it is well known that an oxo-ferryl π-cation radical, known as “Compound 1” in P450 chemistry is the reactive intermediate that catalyzes most of the reactions of P450s. The identity of the reaction intermediate that catalyzes the terminal step estrogen biosynthesis by CYP19A1 is still a mystery. There is evidence in the literature suggesting the involvement of Compound 1 via a hydrogen abstraction that initiates deformylation and subsequent aromatization. There is also suggestion of the peroxo-anion or “Compound 0” acting as a nucleophile, attacking the electrophilic carbonyl carbon of 19-oxo-androstenedione forming a peroxide adduct that then fragments to produce acyl-carbon cleavage.
Owing to the interesting chemistry CYP19A1 catalyzes and its role in human health I focused my attention towards elucidating the mechanism of this critical enzyme with the hope that a detailed picture of the workings of CYP19A1 will help guide efforts to make more specific inhibitors and improve breast cancer prognosis.
CYP19A1 is a membrane-bound hemeprotein with a rich spectroscopic landscape thus affording an opportunity to apply a repertoire of biophysical approaches to help piece together a reaction mechanism. I used the Nanodisc technology to stabilize CYP19A1 in its native membrane-like environment to obtain a mono-disperse, stable and homogenous enzyme preparation that is amicable to the optical, resonance Raman (rR) and electron paramagnetic resonance (EPR) spectroscopy and also, cryoradiolysis and cryospectroscopy. The approach I have applied in this project has been that of characterizing the individual fate of reaction intermediates on their way from substrates to products thereby catching them ‘in action’.
My cryospectroscopy, EPR, rR and steady state kinetics efforts outlined in this doctoral thesis all implicate “Compound 1” as the reactive intermediate that is responsible for the carbon-carbon scission reactivity of CYP19A1.
Advisors/Committee Members: Sligar, Stephen G. (advisor), Sligar, Stephen G. (Committee Chair), Martinis, Susan A. (committee member), Hergenrother, Paul J. (committee member), Kranz, David M. (committee member).
Subjects/Keywords: Conformational Substates of P450s; Temperature Derivative Spectroscopy; Compound 1; Peroxo-anion; Aromatization; C-C Scission; P450; Human Aromatase; CYP19A1
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APA ·
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MLA ·
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to Zotero / EndNote / Reference
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APA (6th Edition):
Luthra, A. (2015). Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1). (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/78708
Chicago Manual of Style (16th Edition):
Luthra, Abhinav. “Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1).” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed December 15, 2019.
http://hdl.handle.net/2142/78708.
MLA Handbook (7th Edition):
Luthra, Abhinav. “Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1).” 2015. Web. 15 Dec 2019.
Vancouver:
Luthra A. Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1). [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2142/78708.
Council of Science Editors:
Luthra A. Spectroscopic characterization of iron-oxygen intermediates in human aromatase (CYP19A1). [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/78708
Virginia Tech
7.
Mercedes-Camacho, Ana Yokayra.
Pin1: WW domain ligands, catalytic inhibitors, and the mechanism.
Degree: PhD, Biochemistry, 2011, Virginia Tech
URL: http://hdl.handle.net/10919/77093
► The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an…
(more)
▼ The peptidyl prolyl cis/trans isomerase, PPIase, has been the focus of numerous studies in the field of cell cycle regulation since proline-directed phosphorylation is an essential signaling mechanism that might arrest cancer proliferation. Pin1 is the first phosphorylation-dependent PPIase enzyme to be discovered. The Pin1 regulatory mechanism, acting on other mitotic proteins in vivo and in vitro, remains largely unknown. For the study of Pin1 function, two types of assays were used to identity ligands for Pin1: (1) The Enzyme-Linked Enzyme Binding Assay (ELEBA) for the identification of WW domain ligands, (2) a catalytic assay to identified inhibitors of Pin1 catalytic activity. The ELEBA offers a selective approach for detecting ligands that bind to the Pin1 WW domain from chemical libraries. By using the ELEBA, a pSer-Pro peptidomimetic library of 315 ligands was screened, identifying three promising ligands cis-D2, O2, and M18. Competitive Kd values for cis-D2, O2, and M18 were determined to be 263 ± 6.4, 206 ± 3.4, and 130 ± 3.0μM, respectively. Furthermore, we screened the pSer-Pro peptidomimetic library using a Pin1 discontinuous-catalytic assay to identify inhibitors of Pin1. Ligands D20 and K7 were identified to decrease more than 90% of the Pin1 catalytic activity.
To investigate the nature of the Pin1 interaction with
c-Myc, we synthesized and characterized four peptides corresponding to the
c-Myc sequence. These peptides were used in NMR isomerization studies of Pin1 by our collaborator Dr. Jeffry Peng (University of Notre Dame). Preliminary work shows that Pin1 binds and isomerizes the Ac–LLPpTPPLSPS–NH₂ peptide at the cMyc pThr58 position.
Finally, we measured a secondary kinetic isotope effect (2º KIE) to study the Pin1 catalytic mechanism of proline isomerization. The ratio of kH/kD for unlabeled and [d₃]Ser-labeled substrate gave a SKIE value of 1.34 ± 0.01. The normal 2º KIE value indicates that carbonyl-serine hybridization is not changing from sp² to sp³. This result supports substrate analogue inhibitor studies, and previous solvent and SKIE results on Pin1, that suggest a twisted amide mechanism assisted by a transient hydrogen bond in the transition state.
Advisors/Committee Members: Etzkorn, Felicia A. (committeechair), Bevan, David R. (committee member), Kennelly, Peter J. (committee member), Li, Jianyong (committee member).
Subjects/Keywords: PPIases; Pin1 inhibitors; WW domain ligands; and KIE; c-Myc; ELEBA
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APA (6th Edition):
Mercedes-Camacho, A. Y. (2011). Pin1: WW domain ligands, catalytic inhibitors, and the mechanism. (Doctoral Dissertation). Virginia Tech. Retrieved from http://hdl.handle.net/10919/77093
Chicago Manual of Style (16th Edition):
Mercedes-Camacho, Ana Yokayra. “Pin1: WW domain ligands, catalytic inhibitors, and the mechanism.” 2011. Doctoral Dissertation, Virginia Tech. Accessed December 15, 2019.
http://hdl.handle.net/10919/77093.
MLA Handbook (7th Edition):
Mercedes-Camacho, Ana Yokayra. “Pin1: WW domain ligands, catalytic inhibitors, and the mechanism.” 2011. Web. 15 Dec 2019.
Vancouver:
Mercedes-Camacho AY. Pin1: WW domain ligands, catalytic inhibitors, and the mechanism. [Internet] [Doctoral dissertation]. Virginia Tech; 2011. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/10919/77093.
Council of Science Editors:
Mercedes-Camacho AY. Pin1: WW domain ligands, catalytic inhibitors, and the mechanism. [Doctoral Dissertation]. Virginia Tech; 2011. Available from: http://hdl.handle.net/10919/77093
North Carolina State University
8.
Tran, Elizabeth Jane.
Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex.
Degree: PhD, Biochemistry, 2004, North Carolina State University
URL: http://www.lib.ncsu.edu/resolver/1840.16/5037
► Box C/D ribonucleoprotein complexes (RNPs) are evolutionarily ancient nucleotide modification machines found in both Eukarya and Archaea. The box C/D RNAs are essential for ribosome…
(more)
▼ Box
C/D ribonucleoprotein complexes (RNPs) are evolutionarily ancient nucleotide modification machines found in both Eukarya and Archaea. The box
C/D RNAs are essential for ribosome biogenesis and primarily function by guiding 2'-O-methylation of ribosomal RNA (rRNA). The site of modification is determined by base-pairing between the target RNA and the box
C/D RNA through a region of complementarity. The box
C/D RNAs possess terminal box
C/D and internal
C'/D' motifs that fold to form K-turn RNA elements. In eukaryotes, the box
C/D RNAs associate with a common set of four core proteins to form an RNP. The core proteins, 15.5kD, Nop56p, Nop58p and Fibrillarin, are differentially distributed on eukaryotic box
C/D RNAs to form an asymmetric RNPs.
We have characterized the structure and function of the archaeal box
C/D RNP using Methanocaldococcus jannaschii sR8 RNP as a model box
C/D complex. Archaeal genomes contain genes for a Fibrillarin homolog and a single homolog for both Nop56p and Nop58p termed Nop56/58p. Our initial investigations identified ribosomal protein L7 as the archaeal homolog of the eukaryotic 15.5kD protein. Strikingly, L7 has a dual role as a component of both the ribosome and the box
C/D RNP. A methylation-competent sR8 RNP was assembled in vitro using the three recombinant M. jannaschii box
C/D RNA core proteins. This reconstituted complex is symmetric with respect to core protein binding and guides nucleotide modification from both the box
C/D and
C'/D' RNPs. Additionally, efficient RNA 2'-O-methylation requires juxtaposed box
C/D and
C'/D' motifs on the same box
C/D RNP complex. Finally, the identification of box
C/D RNPs in both Archaea and Eukarya led us to question the evolutionary origins of these ancient modification complexes. Based on the demonstration of a common RNP element (L7: K-turn motif) in both the archaeal large ribosomal subunit and the box
C/D RNP complex, we propose that the trans-acting nucleotide modification machines evolved elements in the primitive translational apparatus.
Advisors/Committee Members: Steven Spiker, Committee Member (advisor), Janes W. Brown, Committee Member (advisor), Paul Wollenzien, Committee Member (advisor), Dennis Brown, Committee Member (advisor), E. Stuart Maxwell, Committee Chair (advisor).
Subjects/Keywords: snoRNA; box C/D; archaea
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APA (6th Edition):
Tran, E. J. (2004). Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex. (Doctoral Dissertation). North Carolina State University. Retrieved from http://www.lib.ncsu.edu/resolver/1840.16/5037
Chicago Manual of Style (16th Edition):
Tran, Elizabeth Jane. “Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex.” 2004. Doctoral Dissertation, North Carolina State University. Accessed December 15, 2019.
http://www.lib.ncsu.edu/resolver/1840.16/5037.
MLA Handbook (7th Edition):
Tran, Elizabeth Jane. “Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex.” 2004. Web. 15 Dec 2019.
Vancouver:
Tran EJ. Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex. [Internet] [Doctoral dissertation]. North Carolina State University; 2004. [cited 2019 Dec 15].
Available from: http://www.lib.ncsu.edu/resolver/1840.16/5037.
Council of Science Editors:
Tran EJ. Structure and Function of the Archaeal Box C/D Ribonucleoprotein Complex. [Doctoral Dissertation]. North Carolina State University; 2004. Available from: http://www.lib.ncsu.edu/resolver/1840.16/5037
Queens University
9.
Duan, Da.
Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
.
Degree: Biochemistry, 2013, Queens University
URL: http://hdl.handle.net/1974/8118
► The kinesin-14 Kar3 from Saccharomyces cerevisiae (Sc) is a C-terminal motor that forms a heterodimer with the kinesin-accessory protein Vik1. Although Vik1 possesses a typical…
(more)
▼ The kinesin-14 Kar3 from Saccharomyces cerevisiae (Sc) is a C-terminal motor that forms a heterodimer with the kinesin-accessory protein Vik1. Although Vik1 possesses a typical kinesin motor domain (MD) fold, it lacks a nucleotide-binding site. However, it binds microtubules with affinities that can be regulated Kar3’s nucleotide state. This implies intermolecular communication between its subunits. This thesis aimed to understand this communication by studying the structures and functions of Kar3Vik1 orthologs.
First, we biochemically characterized Kar3 from Ashbya gossypii (Ag) and determined the crystal structure of its MD. It was shown that the active site features of the AgKar3MD are similar to that of the ScKar3 R598A mutant, and that the β1 lobe at the edge of the MD was unique in structure and amino acid content. These results may provide a rationale for the unique enzymatic properties of this motor that could be relevant to its interaction with AgVik1 and function in Ashbya gossypii.
We also determined the crystal structures of Kar3 and Vik1 orthologs from Candida glabrata (Cg). While the CgKar3MD structure was very similar to that of ScKar3MD, crystals of CgVik1 captured three novel conformations of the Vik1 motor homology domain (MHD). We observed that when the N-terminal neck helix docks against the MHD core in two unique positions, the C-terminus resembling neck mimics of kinesin-14 motors also docks against the neck-core junction. However, when the neck is non-helical and disengaged from the MHD, the C-terminus is undocked and disordered.
To assess the functional importance of these N- and C-terminal segments of Vik1 MHD, we created CgKar3Vik1 constructs whose Vik1 subunit contained either a point mutation or complete truncation of the C-terminus (neck mimic), and analyzed their biophysical properties. All mutants showed defective ATPase activity and microtubule-gliding ability. Characterization of the mutations in CgVik1MHD by molecular dynamics simulations showed that residues Ile578 and Asn580 are not only involved in stabilizing interactions between the neck and neck mimic but they also influence and respond to conformational changes of the neck. These observations implicate the N- and C-termini of Vik1 as a key element of Kar3Vik1 function and communication.
Subjects/Keywords: Motility;
Candida Glabrata;
Ashbya Gossypii;
N-C Termini Interaction;
Saccharomyces Cerevisiae;
c-terminal kinesin;
Heterodimer;
Minus-End Directed Motility;
Kinesin;
Microtubules;
Mitosis
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CSE |
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Manager
APA (6th Edition):
Duan, D. (2013). Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/8118
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):
Duan, Da. “Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
.” 2013. Thesis, Queens University. Accessed December 15, 2019.
http://hdl.handle.net/1974/8118.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Duan, Da. “Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
.” 2013. Web. 15 Dec 2019.
Vancouver:
Duan D. Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
. [Internet] [Thesis]. Queens University; 2013. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/1974/8118.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Duan D. Understanding the Mechanism of Motility of the Heterodimeric Kinesin-14 KAR3VIK1
. [Thesis]. Queens University; 2013. Available from: http://hdl.handle.net/1974/8118
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
University of Utah
10.
Ferris, Elliott Carter.
The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly.
Degree: MS, Biochemistry, 2011, University of Utah
URL: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/214/rec/2651
► Proper assembly of Cytochrome c Oxidase (CcO) is vital to mitochondrial respiration. The metallation and incorporation of CcO subunit 2 (Cox2) are important assembly steps…
(more)
▼ Proper assembly of Cytochrome c Oxidase (CcO) is vital to mitochondrial respiration. The metallation and incorporation of CcO subunit 2 (Cox2) are important assembly steps that remain poorly understood. The Cox2 assembly factor Cox20 may provide a unique window on CcO assembly. Cox20 is known to bind Cox2 before the latter is incorporated into the larger CcO assembly in S. cerevisiae. Conservation of Cox20 in higher organisms and the lethality of its deletion in Drosophila melanogaster may suggest Cox20 plays a conserved role in CcO assembly. In an attempt to understand the nature of the hypothesized conserved role of Cox20, conserved residues within Cox20 were mutated and the resulting mutants studied. Mutation of a conserved cysteine pair impacted mitochondrial respiration, but did not block CcO assembly. The mutant Cox20 C87A forms a mixed disulfide species. Identifying the partner molecule may shed light on the role of Cox20. The present study finds Cox20, a 23 kD protein, in high molecular weight complexes that are unlikely to be homo-oligomeric. Identifying any other proteins in these complexes may shed light on the role Cox20 plays in CcO assembly. To this end, Cox20 was purified and analyzed by mass spectrometry (MS) to identify any accompanying proteins. Small quantities of the known CcO assembly factors Mss2, Coa1, and Mss51 were identified.
Subjects/Keywords: Cox2; Cox20; Cyclooxygenase 2; Cyclooxygenases; Cytochrome oxidase; C oxidase assembly; Mitochondrial respiration; Cox2 assembly factor
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APA (6th Edition):
Ferris, E. C. (2011). The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly. (Masters Thesis). University of Utah. Retrieved from http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/214/rec/2651
Chicago Manual of Style (16th Edition):
Ferris, Elliott Carter. “The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly.” 2011. Masters Thesis, University of Utah. Accessed December 15, 2019.
http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/214/rec/2651.
MLA Handbook (7th Edition):
Ferris, Elliott Carter. “The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly.” 2011. Web. 15 Dec 2019.
Vancouver:
Ferris EC. The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly. [Internet] [Masters thesis]. University of Utah; 2011. [cited 2019 Dec 15].
Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/214/rec/2651.
Council of Science Editors:
Ferris EC. The role of Cox20 in Cox2 maturation and cytochrome C oxidase assembly. [Masters Thesis]. University of Utah; 2011. Available from: http://content.lib.utah.edu/cdm/singleitem/collection/etd3/id/214/rec/2651
Florida International University
11.
Gu, Cong.
Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum.
Degree: PhD, Biochemistry, 2018, Florida International University
URL: https://digitalcommons.fiu.edu/etd/3887
;
FIDC007020
► Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and…
(more)
▼ Macropinocytosis and phagocytosis, two actin-dependent and clathrin independent events of endocytosis, enable the cells such as macrophages and neutrophils to either internalize pathogens and initiates the human innate immune response or serve as a direct entry route for productive infection of pathogen.
Dictyostelium discoideum, soil-living amoeba, a unicellular eukaryote that could professionally internalize fluid phase or particles several folds more than that of macrophages and neutrophils. Additionally, multiple key signaling pathways are conserved between
Dictyostelium and mammalian cells, including pathways affecting small GTPases Ras and Rac and their downstream effectors, and F-Actin remodeling. All these traits makes
Dictyostelium an excellent model organism to study the process pf macropinocytosis and phagocytosis.
Upon internalization of the prey, these macropinocytes and phagocytes are often in an environment of increased production of superoxide radicals in the prey-containing vesicles, which helps stimulates the downstream signaling pathways to digest the prey inside. However, the mechanism of how superoxide regulates the process of macropinocytosis and phagocytosis is not fully understood. We had previously reported that
Dictyostelium cells lacking
Superoxide dismutase C (
SodC) exhibited aberrantly high level of active RasG, high basal level of Phosphatidylinositol-3,4,5-triphosphate (PIP3), and severe chemotaxis defects. Now we report that <em>sodC
-</em> cells displayed aberrant endosomal vesicle trafficking, significantly compromised particle uptake and defective cell to substratum matrix adhesion compared to that of wild type cells. By using high resolution live imaging microscope we also show that <em>sodC
-</em> cells have defects in F-Actin remodeling at the phagocytic rim extension and F-Actin depolymerization of the nascent phagosome. Interestingly, the introduction of overexpressing of cytoplasmic superoxide dismutase (SodA), redox insensitive RasG (C118A) or treatment of PI3K inhibitor LY294002 in <em>sodC
-</em> cells significantly rescued the defects of endosomal vesicle trafficking, particle uptake and adhesion. This project suggests that superoxide dismutase
C regulates the endosomal vesicle trafficking, phagocytosis and cell to substratum matrix adhesion through the RasG/PI3K signaling axis in
Dictyostelium cells.
Advisors/Committee Members: Lou Kim, Xiaotang Wang, Lidia Kos, Jessica Liberles.
Subjects/Keywords: Superoxide Dismutase C; Macropinocytosis; Phagocytosis; Dictyostelium Discoideum; Biochemistry; Cell Biology; Molecular Biology
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APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Gu, C. (2018). Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum. (Doctoral Dissertation). Florida International University. Retrieved from https://digitalcommons.fiu.edu/etd/3887 ; FIDC007020
Chicago Manual of Style (16th Edition):
Gu, Cong. “Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum.” 2018. Doctoral Dissertation, Florida International University. Accessed December 15, 2019.
https://digitalcommons.fiu.edu/etd/3887 ; FIDC007020.
MLA Handbook (7th Edition):
Gu, Cong. “Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum.” 2018. Web. 15 Dec 2019.
Vancouver:
Gu C. Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum. [Internet] [Doctoral dissertation]. Florida International University; 2018. [cited 2019 Dec 15].
Available from: https://digitalcommons.fiu.edu/etd/3887 ; FIDC007020.
Council of Science Editors:
Gu C. Superoxide Dismutase C Modulates Macropinocytosis and Phagocytosis in Dictyostelium Discoideum. [Doctoral Dissertation]. Florida International University; 2018. Available from: https://digitalcommons.fiu.edu/etd/3887 ; FIDC007020
University of Illinois – Urbana-Champaign
12.
Mattis, Daiva Maria.
Engineering of bacterial exotoxin and endotoxin antagonists.
Degree: PhD, Biochemistry, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/88242
► Gram negative and positive bacteria have evolved toxins to aid in their ability to colonize host organisms. Some gram-positive bacteria produce exotoxins called superantigens that…
(more)
▼ Gram negative and positive bacteria have evolved toxins to aid in their ability to colonize host organisms. Some gram-positive bacteria produce exotoxins called superantigens that hyper-stimulate the immune system by crosslinking the variable region of the beta chain (Vβ) of T cell receptors with the antigen presenting major histocompatibility complex II molecule on the surface of antigen presenting cells. This hyper-stimulation leads to overproduction of cytokines, which can result in toxic shock. In addition, the action of the superantigens has been implicated in many diseases including necrotizing pneumonia and endocarditis. Gram-negative bacteria produce lipopolysaccharide (LPS), also called endotoxin, as a major constituent in their outer cell walls. LPS binds to the host protein called MD-2 and the LPS:MD-2 complex associates with cell surface homodimeric Toll-like receptor 4 (TLR4). This tri-molecular interaction can lead to massive stimulation of cytokines from TLR4+ antigen presenting cells, resulting in endotoxic-mediated septic shock. This process has also been suggested to play a role in asthma. A lack of therapeutics for both exotoxin and endotoxin induced shock and implicated diseases, as well as an interest in further understanding these molecular interactions, guided my studies and development of high affinity agents to neutralize these toxins.
In chapter two, directed evolution was used to engineer a high affinity antagonist against the superantigen Staphylococcal enterotoxin C3 (SEC3). I used a previously error-prone engineered Vβ against SEC3 as a starting template for further engineering. Yeast display was used to create two libraries in two different regions of the previously engineered Vβ to improve its affinity for SEC3. The mutations from the highest affinity mutant selected from each library were combined to create a single mutant that had improved binding to SEC3 over either mutant. The highest affinity Vβ antagonist was tested and found to be effective in various rabbit models with SEC3 by the Schlievert laboratory.
Chapter three describes the cross reactivity of the high affinity SEC3 antagonist described in chapter two, with allelic variants of SEC3 (SEC1, SEC2, and SEC4), as well as the highly homologous superantigen, Staphylococcal enterotoxin B (SEB). Residues potentially responsible for the cross reactivity with SEB were mutated and tested for binding to SEB and SEC3. The SEC4 secreting bacteria strain MW2 was used in necrotizing pneumonia and infective endocarditis rabbit models by the Schlievert laboratory that confirmed the in vivo ability of the antagonist to effectively neutralize more than one strain of SEC.
In chapter four, MD-2 was expressed on the surface of yeast and shown to bind MD-2 specific monoclonal antibodies and to its ligands, LPS and TLR4. To test the platform, alanine mutants were engineered at residues identified from previous studies that tested for binding to LPS as well as TLR4. The alanine mutants behaved as anticipated based on the previously…
Advisors/Committee Members: Kranz, David M. (advisor), Kranz, David M. (Committee Chair), Gerlt, John A. (committee member), Lu, Yi (committee member), Chen, Lin-Feng (committee member).
Subjects/Keywords: Yeast-display; Superantigen; Staphylococcal Enterotoxin C; MD-2; Toll-like receptor 4 (TLR4); Lipopolysaccharide (LPS)
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APA ·
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MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Mattis, D. M. (2015). Engineering of bacterial exotoxin and endotoxin antagonists. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/88242
Chicago Manual of Style (16th Edition):
Mattis, Daiva Maria. “Engineering of bacterial exotoxin and endotoxin antagonists.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed December 15, 2019.
http://hdl.handle.net/2142/88242.
MLA Handbook (7th Edition):
Mattis, Daiva Maria. “Engineering of bacterial exotoxin and endotoxin antagonists.” 2015. Web. 15 Dec 2019.
Vancouver:
Mattis DM. Engineering of bacterial exotoxin and endotoxin antagonists. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2142/88242.
Council of Science Editors:
Mattis DM. Engineering of bacterial exotoxin and endotoxin antagonists. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/88242
Queens University
13.
Podzelinska, Kateryna.
Structure-Guided Studies of Bacterial Competition Mechanisms
.
Degree: Biochemistry, 2015, Queens University
URL: http://hdl.handle.net/1974/13888
► Microorganisms have evolved a stunning array of strategies for nutrient competition, ranging from concerted effort of antibiotic release to kill off competing species, to evolving…
(more)
▼ Microorganisms have evolved a stunning array of strategies for nutrient competition, ranging from concerted effort of antibiotic release to kill off competing species, to evolving complex enzymatic pathways that are capable of scavenging nutrients from sources not utilizable by other organisms. The carbon-phosphorous (C-P) lyase pathway is a survival mechanism that is activated during phosphate limitation in certain species of bacteria and enables cleavage of the extremely stable C-P bond in order to obtain phosphorous from organophosphonates. The structure and biochemical characterization of PhnP, a critical accessory protein from C-P lyase pathway of E. coli is presented in this thesis. The structure of PhnP revealed a conserved metal-dependent hydrolase active site with two Mn2+ ions, and another unique mononuclear Zn2+ site that appears to have a structural role. A non-physiological ligand that fortuitously co-crystallized with the enzyme provided insights into the catalytic features of the active site. We were able to demonstrate hydrolytic activity towards a number of phosphodiesterase substrates, and propose a plausible physiological role for PhnP. These results contribute to deciphering the mechanism of phosphonate utilization, which would allow design of bioremediation programs to remove toxic phsophonates from the environment. Antibiotic production is another mechanism for resource competition. Structural characterization of CmlS, a halogenase from the chloramphenicol biosynthesis pathway of S. venezuelae is presented. The crystal structure revealed a novel covalent modification of its FAD cofactor, which was confirmed through ESI-MS and chemical denaturation studies. The unique C-terminal domain, active site architecture, and the position of the C-terminus suggest that halogenation mechanism of CmlS may differ from the currently proposed mechanism for structurally related halogenases. This work provides early steps towards understanding mechanisms of enzymatic halogenations, which is of great scientific, as well as pharmaceutical interest.
Subjects/Keywords: Phosphonate Utilization;
Chloramphenicol Biosynthesis;
C-P Lyase;
Flavin-Dependent Halogenase;
PhnP;
CmlS;
Phosphodiesterase
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APA ·
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MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Podzelinska, K. (2015). Structure-Guided Studies of Bacterial Competition Mechanisms
. (Thesis). Queens University. Retrieved from http://hdl.handle.net/1974/13888
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):
Podzelinska, Kateryna. “Structure-Guided Studies of Bacterial Competition Mechanisms
.” 2015. Thesis, Queens University. Accessed December 15, 2019.
http://hdl.handle.net/1974/13888.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
MLA Handbook (7th Edition):
Podzelinska, Kateryna. “Structure-Guided Studies of Bacterial Competition Mechanisms
.” 2015. Web. 15 Dec 2019.
Vancouver:
Podzelinska K. Structure-Guided Studies of Bacterial Competition Mechanisms
. [Internet] [Thesis]. Queens University; 2015. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/1974/13888.
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Council of Science Editors:
Podzelinska K. Structure-Guided Studies of Bacterial Competition Mechanisms
. [Thesis]. Queens University; 2015. Available from: http://hdl.handle.net/1974/13888
Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation
Vanderbilt University
14.
Crooke, Cornelia Elizabeth.
Regulation of Fibronectin Assembly by PLC-gamma1.
Degree: PhD, Biochemistry, 2009, Vanderbilt University
URL: http://etd.library.vanderbilt.edu//available/etd-04012009-100228/
;
► Phospholipase C-γ1 (PLC-γ1) mediates cell adhesion and migration through an undefined mechanism. Here, we examine the role of PLC-γ1 in cell-matrix adhesion in a hanging…
(more)
▼ Phospholipase
C-γ1 (PLC-γ1) mediates cell adhesion and migration through an undefined mechanism. Here, we examine the role of PLC-γ1 in cell-matrix adhesion in a hanging drop assay of cell aggregation. Plcg1 Null (-/-) mouse embryonic fibroblasts formed aggregates that were larger and significantly more resistant to dissociation than cells in which PLC-γ1 is re-expressed (Null + cells). Aggregate formation could be disrupted by inhibition of fibronectin interaction with integrins, indicating that fibronectin assembly may mediate aggregate formation. Fibronectin assembly was mediated by integrin α5β1 in both cell lines, while assays measuring fibronectin assembly revealed increased assembly in the Null cells. Null and Null + cells exhibited equivalent fibronectin mRNA levels and equivalent levels of fibronectin protein in pulse-labeling experiments. However, levels of secreted fibronectin in the conditioned medium were increased in Null cells. The data implicates a negative regulatory role for PLC-γ1 in cell aggregation by controlling the secretion of fibronectin into the media and its assembly into fibrils.
Advisors/Committee Members: David Cortez (committee member), Bruce Carter (committee member), Graham Carpenter (chair), Ambra Pozzi (committee member), Roy Zent (committee member).
Subjects/Keywords: integrins; extracellular matrix; phospholipase C gamma 1
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Export
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APA (6th Edition):
Crooke, C. E. (2009). Regulation of Fibronectin Assembly by PLC-gamma1. (Doctoral Dissertation). Vanderbilt University. Retrieved from http://etd.library.vanderbilt.edu//available/etd-04012009-100228/ ;
Chicago Manual of Style (16th Edition):
Crooke, Cornelia Elizabeth. “Regulation of Fibronectin Assembly by PLC-gamma1.” 2009. Doctoral Dissertation, Vanderbilt University. Accessed December 15, 2019.
http://etd.library.vanderbilt.edu//available/etd-04012009-100228/ ;.
MLA Handbook (7th Edition):
Crooke, Cornelia Elizabeth. “Regulation of Fibronectin Assembly by PLC-gamma1.” 2009. Web. 15 Dec 2019.
Vancouver:
Crooke CE. Regulation of Fibronectin Assembly by PLC-gamma1. [Internet] [Doctoral dissertation]. Vanderbilt University; 2009. [cited 2019 Dec 15].
Available from: http://etd.library.vanderbilt.edu//available/etd-04012009-100228/ ;.
Council of Science Editors:
Crooke CE. Regulation of Fibronectin Assembly by PLC-gamma1. [Doctoral Dissertation]. Vanderbilt University; 2009. Available from: http://etd.library.vanderbilt.edu//available/etd-04012009-100228/ ;
University of Illinois – Urbana-Champaign
15.
Hosseinzadeh, Parisa.
Isolating, characterizing, and engineering novel Cu-proteins and peroxidases.
Degree: PhD, Biochemistry, 2015, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/89172
► Metalloproteins are a fascinating class of proteins that function at the heart of several important biological processes including photosynthesis, respiration, and nitrogen fixation. It is…
(more)
▼ Metalloproteins are a fascinating class of proteins that function at the heart of several important biological processes including photosynthesis, respiration, and nitrogen fixation. It is even more amazing, considering that nature uses a small set of tertiary structures and metal centers to perform all these different functions with efficiency and selectivity. How nature tunes the activity within these scaffolds has been the area of research for many years. The goal of this work is to understand the underlying mechanisms of such tuning with a special focus on the role of subtle changes of residues in the secondary coordination sphere of the metal ion, an underexplored area of study. I use protein engineering techniques not only to shed light on the mechanisms underlying such changes, but also to design new functionalities within our scaffold proteins and to enhance their properties for specific purposes, such as fuel generation.
This work is divided into three main sections. In the first, I focus on characterizing a novel metalloprotein, N. mar_1307, from the organism Nitrosopumilus maritimus. While the protein shares a protein fold and Type 1 copper coordination site with other common electron transfer cupredoxins, the lack of an axial residue creates an open binding position in the Cu center, leading to a novel enzymatic function, NO oxidation. The purification, characterization, and activity assays of the protein are described in detail in chapter 2.
The second and major focus of this work is on tuning the reduction potential of azurin, a common electron transfer protein. In chapter 3 I demonstrate that how by making mutations around the Cu site, and replacing Cu with Ni I can obtain an azurin variant with a reduction potential of nearly 1V, the highest potential that can be observed under physiological conditions, along with other variants with negative potentials. Chapter 4 describes the characterization of a series of Phe114 mutants that were used to understand the role of this critical secondary sphere residue in tuning the reduction potential of the Cu site. Chapter 5 demonstrates the Marcus inverted region of electron transfer in a series of azurin variants with different reduction potentials. Finally, I show my initial attempts toward the design of a high-throughput screening platform for the directed evolution of azurin in chapter 6.
In chapters 7 and 8, I focus on the design of novel functionalities in one of our model scaffolds, cytochrome
c peroxidase (CcP). Chapter 7 describes the work done to enhance the Mn(II) oxidation activity in a designed model of manganese peroxidase within the CcP scaffold based on modifications of the second coordination sphere around the Mn(II) binding site. In chapter 8 I report the design and characterization of a novel CcP variant that shows catalase-like activity in “as-purified” form and forms a heme-protein crosslink in the heme-bound form.
Advisors/Committee Members: Lu, Yi (advisor), Lu, Yi (Committee Chair), Gennis, Robert R (committee member), Tajkhorshid, Emad (committee member), Zhao, Humin (committee member).
Subjects/Keywords: Protein design; Redox Potential tuning; Secondary coordination sphere; metalloprotein; azurin; cytochrome c peroxidase; N-mar 1307
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APA (6th Edition):
Hosseinzadeh, P. (2015). Isolating, characterizing, and engineering novel Cu-proteins and peroxidases. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/89172
Chicago Manual of Style (16th Edition):
Hosseinzadeh, Parisa. “Isolating, characterizing, and engineering novel Cu-proteins and peroxidases.” 2015. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed December 15, 2019.
http://hdl.handle.net/2142/89172.
MLA Handbook (7th Edition):
Hosseinzadeh, Parisa. “Isolating, characterizing, and engineering novel Cu-proteins and peroxidases.” 2015. Web. 15 Dec 2019.
Vancouver:
Hosseinzadeh P. Isolating, characterizing, and engineering novel Cu-proteins and peroxidases. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2015. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2142/89172.
Council of Science Editors:
Hosseinzadeh P. Isolating, characterizing, and engineering novel Cu-proteins and peroxidases. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2015. Available from: http://hdl.handle.net/2142/89172
Arizona State University
16.
Lawrence, Robert Michael.
Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring.
Degree: PhD, Biochemistry, 2011, Arizona State University
URL: http://repository.asu.edu/items/9091
► ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation…
(more)
▼ ATP synthase is a large multimeric protein complex
responsible for generating the energy molecule adenosine
triphosphate (ATP) in most organisms. The catalysis involves the
rotation of a ring of c-subunits, which is driven by the
transmembrane electrochemical gradient. This dissertation reports
how the eukaryotic c-subunit from spinach chloroplast ATP synthase
has successfully been expressed in Escherichia coli and purified in
mg quantities by incorporating a unique combination of methods.
Expression was accomplished using a codon optimized gene for the
c-subunit, and it was expressed as an attachment to the larger,
more soluble, native maltose binding protein (MBP-c1). The fusion
protein MBP-c1 was purified on an affinity column, and the c1
subunit was subsequently severed by protease cleavage in the
presence of detergent. Final purification of the monomeric c1
subunit was accomplished using reversed phase column chromatography
with ethanol as an eluent. Circular dichroism spectroscopy data
showed clear evidence that the purified c-subunit is folded with
the native alpha-helical secondary structure. Recent experiments
appear to indicate that this monomeric recombinant c-subunit forms
an oligomeric ring that is similar to its native tetradecameric
form when reconstituted in liposomes. The F-type ATP synthase
c-subunit stoichiometry is currently known to vary from 8 to 15
subunits among different organisms. This has a direct influence on
the metabolic requirements of the corresponding organism because
each c-subunit binds and transports one H+ across the membrane as
the ring makes a complete rotation. The c-ring rotation drives
rotation of the gamma-subunit, which in turn drives the synthesis
of 3 ATP for every complete rotation. The availability of a
recombinantly produced c-ring will lead to new experiments which
can be designed to investigate the possible factors that determine
the variable c-ring stoichiometry and structure.
Subjects/Keywords: Biochemistry; Biophysics; Biology, Plant Physiology; ATP synthase; chloroplast; coupling ratio; recombinant expression; ring stoichiometry; subunit c III ring
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Export
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APA (6th Edition):
Lawrence, R. M. (2011). Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring. (Doctoral Dissertation). Arizona State University. Retrieved from http://repository.asu.edu/items/9091
Chicago Manual of Style (16th Edition):
Lawrence, Robert Michael. “Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring.” 2011. Doctoral Dissertation, Arizona State University. Accessed December 15, 2019.
http://repository.asu.edu/items/9091.
MLA Handbook (7th Edition):
Lawrence, Robert Michael. “Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring.” 2011. Web. 15 Dec 2019.
Vancouver:
Lawrence RM. Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring. [Internet] [Doctoral dissertation]. Arizona State University; 2011. [cited 2019 Dec 15].
Available from: http://repository.asu.edu/items/9091.
Council of Science Editors:
Lawrence RM. Recombinant Expression, Purification, and Reconstitution of
the Chloroplast ATP Synthase c-subunit Ring. [Doctoral Dissertation]. Arizona State University; 2011. Available from: http://repository.asu.edu/items/9091
Case Western Reserve University
17.
Misra-Press, Anita.
Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas.
Degree: PhD, Biochemistry, 1991, Case Western Reserve University
URL: http://rave.ohiolink.edu/etdc/view?acc_num=case1059152679
► PDGF-A mRNA was expressed in all glioblastoma cell lines and in normal human glial cells. In contrast, PDGF-B mRNA was absent from normal glial cells…
(more)
▼ PDGF-A mRNA was expressed in all glioblastoma cell
lines and in normal human glial cells. In contrast, PDGF-B mRNA was
absent from normal glial cells but was expressed in the majority of
glioblastoma cell lines. These observations supported a role for
PDGF-B in the autocrine growth stimulation of glioblastomas. The
expression of both the PDGF genes was stimulated by phorbol
12-myristate 13-acetate (PMA), transforming growth factor beta
(TGF-ß), or increases in intracellular calcium. Previous results
had shown the TGF-ß mediated pathway towards PDGF gene expression
to be independent of the PKC pathway. dB-cAMP was able to abolish
the stimulation of both these genes by PMA or TGF-ß. aThus, the
protein kinase A (PKA) transduction pathway had a dominant
inhibitory effect on the PKC and TGF-ß mediated pathways toward
induction of the PDGF genes. dB-cAMP exhibited a strong inhibitory
effect on basal levels of PDGF-B expression while its effect on
PDGF-A was weak. dB-cAMP caused an exponential decay of PDGF-B mRNA
with a half life of decay similar to its half life with actinomycin
D. The primary mechanism by which dB-cAMP exerted this negative
effect was by an inhibition of PDGF-B gene transcription. The
ability of glioblastoma cells to differentiate in response to
dB-cAMP appeared to correlate with inhibition of PDGF-B
transcription. PKC isotypes expressed in human glioblastoma cells
were also examined. PCK-a mRNA was expressed in all 8 glioblastoma
cell lines and in normal glial cells, unlike PKC-ß which was not
expressed in any. aPMA translocated PKC-a from its normal cytosolic
location to both the plasma membrane and the nucleus in A172 cells
and preferentially to the nucleus in A2781 cells. The translocation
to the nucleus and plasma membrane occurred within 10 minutes of
PMA exposure. Down-regulation of PKC-a was observed on prolonged
PMA treatment. The PMA-induced nuclear translocation of PKC-a was
accompanied by an increased phosphorylation of nuclear envelope
proteins. TGF-ß or dB-cAMP were also able to cause changes in the
amounts and distribution of PKC-a and ¿ in the different
subcellular locations. These results demonstrate "cross-talk"
between separate signaling systems
Advisors/Committee Members: Goldthwait, David (Advisor).
Subjects/Keywords: Biology, Molecular; Platelet-Derived Growth Factor; Glioblastomas; Protein Kinase C
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APA (6th Edition):
Misra-Press, A. (1991). Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas. (Doctoral Dissertation). Case Western Reserve University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=case1059152679
Chicago Manual of Style (16th Edition):
Misra-Press, Anita. “Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas.” 1991. Doctoral Dissertation, Case Western Reserve University. Accessed December 15, 2019.
http://rave.ohiolink.edu/etdc/view?acc_num=case1059152679.
MLA Handbook (7th Edition):
Misra-Press, Anita. “Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas.” 1991. Web. 15 Dec 2019.
Vancouver:
Misra-Press A. Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas. [Internet] [Doctoral dissertation]. Case Western Reserve University; 1991. [cited 2019 Dec 15].
Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1059152679.
Council of Science Editors:
Misra-Press A. Regulation of the PDGF genes and translocation patterns of
protein kinase C isotypes in human glioblastomas. [Doctoral Dissertation]. Case Western Reserve University; 1991. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=case1059152679
Virginia Tech
18.
Allen, William Joseph.
Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease.
Degree: PhD, Biochemistry, 2011, Virginia Tech
URL: http://hdl.handle.net/10919/78000
► Molecular modeling is a term referring to the study of proteins, nucleic acids, lipids, and other bio- or macro- or small molecules at the atomistic…
(more)
▼ Molecular modeling is a term referring to the study of proteins, nucleic acids, lipids, and other bio- or macro- or small molecules at the atomistic level using a combination of computational methods, physico-chemical principles, and mathematical functions. It can be generally sub-divided into two areas: molecular mechanics, which is the treatment of atoms and bonds as Newtonian particles and springs, and quantum mechanics, which models electronic behaviors using the Schrödinger equation and wavefunctions. Each technique is a powerful tool that, when used alone or in combination with wet lab experiments, can yield useful results, the products of which have broad applications in studying human disease models, oxidative damage, and other biomolecular processes that are otherwise not easily observed by experiment alone. Within this document, we study seven different such systems. This includes the mode of inhibitor binding to the enzyme monoamine oxidase B, the active site mechanism of that same enzyme, the dynamics of the unstructured p53
C-terminal domain in complex with globular, structured proteins, the process of the viral protein B2 unbinding from double-stranded RNA, and a focus on the dynamics of a variable loop in the antigenic peanut protein Ara h 2. In addition to those conventional molecular modeling studies, several of which were done in tandem with wet lab experiment, we also discuss the validation of charges and charge group parameters for small molecules used in molecular mechanics, and the development of software for the analysis of lipid bilayer systems in molecular mechanics simulations. As computational resources continue to evolve, and as more structural information becomes available, these methods are becoming an integral part of the study of biomolecules in the context of disease.
Advisors/Committee Members: Bevan, David R. (committeechair), Li, Jianyong (committee member), Smith, Edward J. (committee member), Tanko, James M. (committee member), Helm, Richard Frederick (committee member).
Subjects/Keywords: Arachis Ara h 2 protein; molecular modeling; monoamine oxidase B; p53 C-terminal domain; B2 suppressor of RNA silencing; lipid bilayer analysis
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APA (6th Edition):
Allen, W. J. (2011). Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease. (Doctoral Dissertation). Virginia Tech. Retrieved from http://hdl.handle.net/10919/78000
Chicago Manual of Style (16th Edition):
Allen, William Joseph. “Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease.” 2011. Doctoral Dissertation, Virginia Tech. Accessed December 15, 2019.
http://hdl.handle.net/10919/78000.
MLA Handbook (7th Edition):
Allen, William Joseph. “Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease.” 2011. Web. 15 Dec 2019.
Vancouver:
Allen WJ. Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease. [Internet] [Doctoral dissertation]. Virginia Tech; 2011. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/10919/78000.
Council of Science Editors:
Allen WJ. Practical Applications of Molecular Modeling Pertaining to Oxidative Damage and Disease. [Doctoral Dissertation]. Virginia Tech; 2011. Available from: http://hdl.handle.net/10919/78000
Texas A&M University
19.
Yang, Yuan.
Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation.
Degree: PhD, Biochemistry, 2016, Texas A&M University
URL: http://hdl.handle.net/1969.1/158924
► Protein kinase C (PKC) isoenzymes sit in the crossroad of numerous signaling pathways involved in cellular functions such as proliferation, differentiation, migration and survival. The…
(more)
▼ Protein kinase
C (PKC) isoenzymes sit in the crossroad of numerous signaling pathways involved in cellular functions such as proliferation, differentiation, migration and survival. The dysregulation of PKCs have been shown to associate with human diseases including cancers, cardiovascular diseases and neurodegenerative diseases. However, the knowledge of PKC regulation is still limited. The objective of this dissertation is to determine the roles of the PKCa
C-terminal tail and the C2 regulatory domain in its maturation, activation and down-regulation.
The
C-terminal V5 domain of PKC contains the least conserved sequence among the isoenzymes. In this study, nuclear magnetic resonance (NMR) and circular dichroism are used to show that the isolated V5 domain is intrinsically disordered in solution. A detailed characterization is provided for the V5 domain’s secondary structural preference, dynamic properties and propensity to interact with a hydrophobic environment.
NMR techniques are used to demonstrate that the PKCa C2 domain interacts with the V5 domain. A structural model for the C2–V5 complex is determined. In addition, NMR-detected binding studies reveal that V5 and calcium interact with C2 cooperatively. Mutations that disrupt the C2–V5 interface altered both the conformation of full-length PKCa and the kinetics of membrane translocation. These results indicate that C2-V5 interaction plays an essential role in PKC regulation, through its contribution to both autoinhibition and activation. Furthermore, the V5 domain is shown to directly interact with the peptidyl-prolyl isomerase Pin1. The V5–Pin1 interaction is observed to be highly specific, non-catalytic, and is enhanced by the avidity from bivalent binding. These data provide insights into a novel mechanism for the Pin1-mediated down-regulation process of PKCs.
The metal-dependent membrane interactions of the C2 domain are studied with cadmium as structural surrogate and the results compared to other divalent metals. Conformational dynamics change induced by calcium binding is detected for regions connecting to other PKC domains. These results reveal specific roles of calcium ion during membrane interaction and conformational rearrangement of PKC.
Together, these data contribute to our understanding of PKC regulation with concerted intramolecular contacts and complex intermolecular interactions, which can help to develop isoenzyme-specific agents to modulate PKC activity.
Advisors/Committee Members: Igumenova, Tatyana I (advisor), Li, Pingwei (committee member), Rye, Hays S (committee member), Sachs, Matthew S (committee member).
Subjects/Keywords: Protein kinase C; intrinsically disordered protein (IDP); V5 domain; nuclear magnetic resonance (NMR); structure; dynamics; peptidyl-prolyl isomerase Pin1; C2 domain; metal; membrane binding
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APA ·
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MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
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APA (6th Edition):
Yang, Y. (2016). Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation. (Doctoral Dissertation). Texas A&M University. Retrieved from http://hdl.handle.net/1969.1/158924
Chicago Manual of Style (16th Edition):
Yang, Yuan. “Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation.” 2016. Doctoral Dissertation, Texas A&M University. Accessed December 15, 2019.
http://hdl.handle.net/1969.1/158924.
MLA Handbook (7th Edition):
Yang, Yuan. “Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation.” 2016. Web. 15 Dec 2019.
Vancouver:
Yang Y. Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation. [Internet] [Doctoral dissertation]. Texas A&M University; 2016. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/1969.1/158924.
Council of Science Editors:
Yang Y. Investigating the Roles of the C-terminal Tail and the Metal-dependent C2 Domain in PKCa Regulation. [Doctoral Dissertation]. Texas A&M University; 2016. Available from: http://hdl.handle.net/1969.1/158924
University of Illinois – Urbana-Champaign
20.
Subramanyam, Shyamal.
Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins.
Degree: PhD, Biochemistry, 2016, University of Illinois – Urbana-Champaign
URL: http://hdl.handle.net/2142/90879
► RAD51 protein plays an important role in homologous genetic recombination (HR), an essential DNA metabolic process used by cells to faithfully repair the most deleterious…
(more)
▼ RAD51 protein plays an important role in homologous genetic recombination (HR), an essential DNA metabolic process used by cells to faithfully repair the most deleterious forms of DNA damage and maintain genomic integrity. RAD51 along with its bacterial counterpart RecA, bacteriophage UvsX and archaeal RadA have been subjected to genetic and biochemical scrutiny resulting in a plentitude of mechanistic and functional information on formation, regulation and activities of these recombinases. An important disconnect between these two lines of investigation still exists because the recombinase functions of RAD51 are highly regulated through mediator proteins like the BRCA2 recombination mediator, and a host of post translational modifications, namely phosphorylation. The mechanism and biochemical implications of these regulatory processes have not been satisfactorily evaluated in-vitro.
This work characterizes the interaction between RAD51 and the BRCA2 recombination mediator protein using computational methods to generate homology models for this interaction which are validated through experimental data. Using the knowledge gained from our structural model for the RAD51 recombinase, I developed a novel strategy to understand several key mechanisms for the regulation of RAD51 by phosphorylation. RAD51 is phosphorylated by the cABL tyrosine kinase. The mechanistic and functional significance of this event is largely disputed. Using biochemical and single molecule assays reconstituting major activities of RAD51, I have successfully dissected the biochemical mechanism of regulation of RAD51 by the
c-Abl kinase. The results of this work strongly correlate with observations made in previous cell based analysis.
Advisors/Committee Members: Spies, Maria (advisor), Schuler, Mary A (Committee Chair), Tajkhorshid, Emad (committee member), Gennis, Robert B (committee member).
Subjects/Keywords: RAD51 Recombinase; DNA Repair; DNA Damage; Protein-Protein Interactions; Post Translational Modifications; Phosphorylation; Recombination Mediator; c-Abl Kinase; BRCA2; Single Molecule Total Internal Reflection Microscopy; Homology Modeling
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APA ·
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Export
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APA (6th Edition):
Subramanyam, S. (2016). Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins. (Doctoral Dissertation). University of Illinois – Urbana-Champaign. Retrieved from http://hdl.handle.net/2142/90879
Chicago Manual of Style (16th Edition):
Subramanyam, Shyamal. “Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins.” 2016. Doctoral Dissertation, University of Illinois – Urbana-Champaign. Accessed December 15, 2019.
http://hdl.handle.net/2142/90879.
MLA Handbook (7th Edition):
Subramanyam, Shyamal. “Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins.” 2016. Web. 15 Dec 2019.
Vancouver:
Subramanyam S. Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins. [Internet] [Doctoral dissertation]. University of Illinois – Urbana-Champaign; 2016. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2142/90879.
Council of Science Editors:
Subramanyam S. Mechanism of regulation of human RAD51 recombinase through post translational modifications & mediator proteins. [Doctoral Dissertation]. University of Illinois – Urbana-Champaign; 2016. Available from: http://hdl.handle.net/2142/90879
21.
-8603-1251.
Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase.
Degree: PhD, Biochemistry, 2017, University of Texas – Austin
URL: http://hdl.handle.net/2152/47432
► Combination therapy with direct-acting antivirals including nucleotide analogs (NAs) and non-nucleoside inhibitors (NNIs) targeting the RNA-dependent RNA polymerase NS5B have seen recent advancements and have…
(more)
▼ Combination therapy with direct-acting antivirals including nucleotide analogs (NAs) and non-nucleoside inhibitors (NNIs) targeting the RNA-dependent RNA polymerase NS5B have seen recent advancements and have dramatically improved the potency of Hepatitis
C Virus (HCV) treatment. However, other than the identification of their site of action, very little is known about the inhibition mechanisms of these clinically relevant drugs. Lately, our lab has developed robust kinetic assays to characterize de novo RNA synthesis catalyzed by HCV NS5B, and then applied the assays to examine the mechanistic basis of action and to establish kinetic parameters governing the efficacy of various clinically relevant NAs and NNIs provided by three pharmaceutical companies (Gilead Sciences, Inc., Alios Biopharma and AbbVie Inc.). In addition, to probe the enzyme conformational dynamics of NS5B from de no initiation to elongation in the presence and absence of allosteric inhibitors, we collaborated with Dr. Patrick Wintrode on Hydrogen Deuterium exchange kinetics and Dr. Serdal Kirmizialtin on Molecular Dynamics simulations. Together, our collaborative efforts have provided a fundamental understanding of RNA-dependent RNA replication catalyzed by the HCV viral polymerase NS5B, and have established the inhibition mechanisms of anti-HCV agents. This work offers significant insights to aid the development of more effective drugs against HCV.
Advisors/Committee Members: Johnson, Kenneth A. (Kenneth Allen) (advisor), Lambowitz, Alan (committee member), Whitman, Christian P (committee member), Finkelstein, Ilya J (committee member), Zhang, Yan (committee member).
Subjects/Keywords: HCV; NS5B; Viral polymerase; RNA replication; Hepatitis C treatment; Hepatitis C virus; Nucleotide analogs; Non-nucleoside inhibitors; Inhibition mechanisms
…1
1.2 Significance of Hepatitis C Viral Infection… …5
1.5 De novo RNA synthesis for Hepatitis C Viral Replication.......................7
1.6… …inhibitors on Hepatitis C viral polymerase NS5B.........................................20
2.1… …NNI2) on Hepatitis C viral polymerase NS5B… …x29; by
Hepatitis C viral polymerase NS5B…
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APA ·
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Export
to Zotero / EndNote / Reference
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APA (6th Edition):
-8603-1251. (2017). Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase. (Doctoral Dissertation). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/47432
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Chicago Manual of Style (16th Edition):
-8603-1251. “Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase.” 2017. Doctoral Dissertation, University of Texas – Austin. Accessed December 15, 2019.
http://hdl.handle.net/2152/47432.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
MLA Handbook (7th Edition):
-8603-1251. “Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase.” 2017. Web. 15 Dec 2019.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Vancouver:
-8603-1251. Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase. [Internet] [Doctoral dissertation]. University of Texas – Austin; 2017. [cited 2019 Dec 15].
Available from: http://hdl.handle.net/2152/47432.
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Council of Science Editors:
-8603-1251. Structural dynamics and inhibition of Hepatitis C RNA-dependent RNA polymerase. [Doctoral Dissertation]. University of Texas – Austin; 2017. Available from: http://hdl.handle.net/2152/47432
Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
22.
Seashols, Sarah.
Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids.
Degree: PhD, Biochemistry, 2013, Virginia Commonwealth University
URL: https://scholarscompass.vcu.edu/etd/3258
► Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs…
(more)
▼ Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs are small, single-stranded RNA molecules that affect protein expression at the translational level, and dysregulation can dramatically affect cell metabolism. Comparison of 736 microRNA expression levels between the poorly metastatic SV40T immortalized prostate epithelial cell line P69 to its highly tumorigenic and metastatic subline M12 identified 231 miRs that were overexpressed and 150 miRs that showed loss of expression in the M12 cell line. Further evaluation of fourteen identified miRs was accomplished using other prostate cell lines as well as laser-capture microdissected prostate samples. Inhibition of miR-147b was found to affect proliferative, migratory and invasive capabilities of M12 cells, and reduced tumour growth in nude athymic mice. AATF, an activator of the cell-cycle inhibitor p21, was identified as a target. Overexpression of miR-9 was found to affect the epithelial to mesenchymal transition through suppression of e-cadherin, a protein characterized as lost in EMT, as well as suppression of SOCS5, an attenuator of JAK-STAT signaling. Inhibition of miR-9 resulted in reduction of migratory and invasive potential, and significant reduction of tumorigenesis and metastases in male nude athymic mice.
miR-17-3p was previously identified as down-regulated in prostate cancer and loss of miR-17-3p shown to cause vimentin transcriptional activation. Reverse phase microarray analysis (RPMA) identified
c-KIT as a potential second mRNA target for miR-17-3p. miR-17-3p was shown to modulate not only protein levels, but also messenger RNA levels of
c-KIT. Four miR-17-3p binding sites in the
c-KIT mRNA were identified. Thus, a number of microRNAs involved in prostate cancer were identified, and their targets found to be highly relevant to tumour progression and could potentially be used as targets for therapy or diagnostics.
Stability of microRNAs in forensically relevant biological fluids was evaluated through heat treatment, ultraviolet radiation, and chemical treatment. The dried body fluids showed some susceptibility to harsh treatment, but in most cases microRNAs were still detectable in the samples. microRNAs could represent a highly stable species for body fluid identification methods in forensic science.
Advisors/Committee Members: Zendra Zehner.
Subjects/Keywords: microRNA; prostate cancer; forensic body fluid identification; c-KIT; AATF/Che-1; e-cadherin; SOCS5; Biochemistry, Biophysics, and Structural Biology; Life Sciences
…25
Figure 2-2: Re-Introduction of miR-17-3p into M12 cells ablates p-c-KIT levels… …29
Figure 2-3: c-KIT activation induces signal transduction in a variety of proliferative… …31
Figure 2-4: c-KIT protein mRNA expression and protein levels are significantly reduced… …40
Figure 2-5: The top two miR-17-3p binding sites in the c-KIT 3’-UTR region are highly… …42
xi
Figure 2-6: Predicted binding of miR-17-3p to 2 c-KIT 3’-UTR region binding sites…
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APA (6th Edition):
Seashols, S. (2013). Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids. (Doctoral Dissertation). Virginia Commonwealth University. Retrieved from https://scholarscompass.vcu.edu/etd/3258
Chicago Manual of Style (16th Edition):
Seashols, Sarah. “Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids.” 2013. Doctoral Dissertation, Virginia Commonwealth University. Accessed December 15, 2019.
https://scholarscompass.vcu.edu/etd/3258.
MLA Handbook (7th Edition):
Seashols, Sarah. “Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids.” 2013. Web. 15 Dec 2019.
Vancouver:
Seashols S. Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids. [Internet] [Doctoral dissertation]. Virginia Commonwealth University; 2013. [cited 2019 Dec 15].
Available from: https://scholarscompass.vcu.edu/etd/3258.
Council of Science Editors:
Seashols S. Variation and Modulation of microRNAs in Prostate Cancer and Biological Fluids. [Doctoral Dissertation]. Virginia Commonwealth University; 2013. Available from: https://scholarscompass.vcu.edu/etd/3258
23.
Schiavi, Susan C.
MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis.
Degree: Biochemistry and Molecular Pharmacology, Biochemistry, 1988, U of Massachusetts : Med
URL: https://escholarship.umassmed.edu/gsbs_diss/259
► PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse c-myc and adenovirus E1A genes were…
(more)
▼ PC12 rat pheochromocytoma cells respond to nerve growth factor (NGF) by neuronal differentiation and partial growth arrest. Mouse
c-myc and adenovirus E1A genes were introduced into PC12 cells to study the influence of these nuclear oncogenes on neuronal differentiation. Expression of myc and E1A blocked morphological differentiation and caused NGF to stimulate rather than inhibit cell proliferation. NGF binding to cell surface receptors, activation of ribosomal S6 kinase, and ornithine decarboxylase induction were similar in myc and E1A expressing clones compared with wild-type PC12 cells, suggesting that changes in the cellular response to NGF were at a post-receptor level. The ability of myc and E1A expression to block the transcription-dependent induction of microtubule associated proteins by NGF further suggested that these genes may inhibit differentiation by interfering with NGP's ability to regulate transcription. These results illustrate that NGF can promote either growth or differentiation of PC12 cells, and that myc or E1A alter the phenotypic responses to growth factors.
Advisors/Committee Members: Dr. Gary L. Johnson.
Subjects/Keywords: Nerve Growth Factor; Proto-Oncogene Proteins c-myc; Adenovirus E1A Proteins; PC12 Cells; Cell Differentiation; Neurons; Transcription Factors; Mice; Amino Acids, Peptides, and Proteins; Animal Experimentation and Research; Cells; Genetic Phenomena; Nervous System; Viruses
Record Details
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Record Details
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❌
APA ·
Chicago ·
MLA ·
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CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Schiavi, S. C. (1988). MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis. (Doctoral Dissertation). U of Massachusetts : Med. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/259
Chicago Manual of Style (16th Edition):
Schiavi, Susan C. “MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis.” 1988. Doctoral Dissertation, U of Massachusetts : Med. Accessed December 15, 2019.
https://escholarship.umassmed.edu/gsbs_diss/259.
MLA Handbook (7th Edition):
Schiavi, Susan C. “MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis.” 1988. Web. 15 Dec 2019.
Vancouver:
Schiavi SC. MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis. [Internet] [Doctoral dissertation]. U of Massachusetts : Med; 1988. [cited 2019 Dec 15].
Available from: https://escholarship.umassmed.edu/gsbs_diss/259.
Council of Science Editors:
Schiavi SC. MYC and E1A Oncogenes Alter the Response of PC12 Cells to Nerve Growth Factor and Block Differentiation: A Thesis. [Doctoral Dissertation]. U of Massachusetts : Med; 1988. Available from: https://escholarship.umassmed.edu/gsbs_diss/259
. 
Open Access Theses and Dissertations
