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You searched for +publisher:"Michigan Technological University" +contributor:("Martin Thompson"). Showing records 1 – 2 of 2 total matches.

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Michigan Technological University

1. Nikula, Chelsea. DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY.

Degree: PhD, Department of Chemistry, 2017, Michigan Technological University

Four different histone proteins comprise the octameric histone core, a key component of DNA compaction into chromatin. The N-terminal tails of each histone protein contain a variety of post-translational modifications that can help modulate gene expression. Mutations are rare in these key proteins, but when found they are often linked to very serious and lethal diseases. In 2012, mutations in histone genes HIST1H2B and H3F3A were found to be implicated in brain cancer. The protein products of these genes produced four point mutants in two proteins: H3.1K27M, H3.3K27M, and H3.3G34R/V. The positions of these mutations are located at or adjacent to known sites of post-translational modification. Trimethylation of H3K27 is a known mark of gene repression and tumors harboring the K27M mutation have been found to have globally reduced levels of this mark. G34R/V mutations have been shown to produce locally reduced levels of H3K36 trimethylation as well. H3K36 trimethylation has been tied to transcription regulation and DNA repair. While these mutations are clearly disrupting the histone post-translational landscape they may also be perturbing the nucleosome structure itself. Histone proteins interact with DNA through basic residues. As these mutations are directly changing the basic residue content of the proteins, DNA-histone interactions may be altered. Research examining these mutations thus far have focused on secondary interactions between protein complexes and the nucleosome. No studies have examined how these mutations and changes in post-translational modifications could be effecting overall nucleosome structure. The main purpose of this research was to develop a system to examine the effects that these mutations have on nucleosome structure. To address this, Aim 1 of the project involved cloning eleven genes to produce the four canonical histone proteins (H2A, H2B, H3, and H4), an H3 variant (H3.3), three H3.3 point mutants (H3.3 K27M, H3.3 G34R, H3.3 G34V), one tailless H3.1 mutant (H3.1 Δ5), and two tailless H3.3 mutants (H3.3 Δ32 and H3.3 Δ45). A new, 2-step purification method was developed for the simple and inexpensive purification of histone proteins. Purified histones were then reconstituted into nucleosomes using a salt-gradient method. Four nucleosome constructs were reconstituted, differing only in the H3 protein included (H3.1, H3.3, H3.3 K27M, or H3.3 Δ32). Atomic Force Microscopy images of the four nucleosome constructs were acquired and analyzed. The location of these histone mutations is at or adjacent to known sites of post-translational modification. Due to this, it was necessary to determine how modifications at these sites effected the nucleosome structure as well to get a full scope of the impact of the mutations. Studying individual post-translational modifications is difficult as extracting histone proteins from tissue samples produces a heterogeneous population of modifications. To generate a homogenous population of modified proteins native chemical ligation methods were… Advisors/Committee Members: Martin Thompson.

Subjects/Keywords: nucleosome; histone mutation; atomic force microscopy; histone post-translational modification; diffuse midline glioma H3K27M-mutant; Biochemistry

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Nikula, C. (2017). DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY. (Doctoral Dissertation). Michigan Technological University. Retrieved from https://digitalcommons.mtu.edu/etdr/526

Chicago Manual of Style (16th Edition):

Nikula, Chelsea. “DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY.” 2017. Doctoral Dissertation, Michigan Technological University. Accessed February 28, 2021. https://digitalcommons.mtu.edu/etdr/526.

MLA Handbook (7th Edition):

Nikula, Chelsea. “DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY.” 2017. Web. 28 Feb 2021.

Vancouver:

Nikula C. DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY. [Internet] [Doctoral dissertation]. Michigan Technological University; 2017. [cited 2021 Feb 28]. Available from: https://digitalcommons.mtu.edu/etdr/526.

Council of Science Editors:

Nikula C. DEVELOPMENT OF A SYSTEM TO STUDY THE EFFECTS OF HISTONE MUTATIONS AND POST-TRANSLATIONAL MODIFICATIONS ON NUCLEOSOME STRUCTURE VIA ATOMIC FORCE MICROSCOPY. [Doctoral Dissertation]. Michigan Technological University; 2017. Available from: https://digitalcommons.mtu.edu/etdr/526


Michigan Technological University

2. Hopson, Sarah. Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein.

Degree: PhD, Department of Chemistry, 2017, Michigan Technological University

Over the past decade, it has become apparent that the human polybromo-1 protein (BAF180) has a critical role in cancer. BAF180 is known to be a driver mutation in clear cell renal cell carcinoma, where it has been found to be mutated in approximately 40% of cases. Mutations have also been found in several other cancers, including intrahepatic cholangiocarcinomas and epithelioid sarcomas. BAF180 is the chromatin targeting subunit of the PBAF (Polybromo-associated BRG1-associated factor) chromatin remodeling complex, a role facilitated by its nine domains: six bromodomains, which recognize and bind to acetylated lysines on histones; two BAH (bromo-adjacent homology) domains, found to be critical for PCNA ubiquitination following DNA damage; and one HMG (high mobility group) box, the DNA binding component. Furthermore, proper expression of BAF180 has also been linked to cardiac development and cell cycle regulation. Despite these associations, the molecular level interactions of full-length BAF180 have yet to be studied; only the phenomenological effects of BAF180 deficiency/mutation have been studied. It is crucial that we understand the binding dynamics and specificities of wild type and mutated BAF180, since it is the recognition component of PBAF. Expression of the recombinant full-length BAF180 protein has been difficult because of the complex nature of this protein, its unusual codon usage, and size. After E. coli expression failed, other expression systems were investigated and the yeast Pichia pastoris was chosen. Pichia was chosen for several reasons: its codon usage is similar to that of BAF180 and it is a eukaryotic system possessing eukaryotic protein folding machinery and capable of performing post-translational modifications. Under control of the GAP promoter, full-length BAF180 has been successfully expressed in Pichia pastoris. This is the first time that full-length BAF180 has been cloned and expressed in a heterologous host. It was purified using anion exchange chromatography. The ability to express and purify full-length BAF180 is a huge first step towards increasing the understanding of the molecular mechanisms of this protein and its association with cancer development. Advisors/Committee Members: Martin Thompson.

Subjects/Keywords: recombinant protein expression and purification; human polybromo-1; BAF180; PBRM1; hPB1; Pichia pastoris; Biochemistry; Molecular Biology

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Hopson, S. (2017). Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein. (Doctoral Dissertation). Michigan Technological University. Retrieved from https://digitalcommons.mtu.edu/etdr/436

Chicago Manual of Style (16th Edition):

Hopson, Sarah. “Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein.” 2017. Doctoral Dissertation, Michigan Technological University. Accessed February 28, 2021. https://digitalcommons.mtu.edu/etdr/436.

MLA Handbook (7th Edition):

Hopson, Sarah. “Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein.” 2017. Web. 28 Feb 2021.

Vancouver:

Hopson S. Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein. [Internet] [Doctoral dissertation]. Michigan Technological University; 2017. [cited 2021 Feb 28]. Available from: https://digitalcommons.mtu.edu/etdr/436.

Council of Science Editors:

Hopson S. Heterologous Expression and Purification of Full-Length Human Polybromo-1 Protein. [Doctoral Dissertation]. Michigan Technological University; 2017. Available from: https://digitalcommons.mtu.edu/etdr/436

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