Mismatch repair corrects errors made during DNA replication and inactive mismatch repair is associated with Lynch Syndrome and sporadic cancer. Genome replication in eukaryotes is accompanied by chromatin formation. The first step in chromatin establishment is nucleosome assembly, that starts with histone tetramer deposition. It is not clear how three important cellular processes: genome replication, mismatch repair and nucleosome assembly are coordinated. Here we analyzed human mismatch repair in the presence of histone deposition in a reconstituted system. We showed that mismatch repair factor inhibits nucleosome assembly on the DNA region with the replicative error. Such a mechanism is important, since in this way DNA with errors remains accessible for mismatch repair system to perform the repair. The DNA synthesis step in mismatch repair is performed by DNA polymerase. Eukaryotes possess two major replicative DNA Polymerases: DNA Polymerase delta and DNA Polymerase epsilon. DNA polymerase delta is involved in mismatch repair. However, it was unknown whether DNA polymerase epsilon can also work in mismatch repair. Here we analyzed human mismatch repair with DNA Polymerase delta and DNA Polymerase epsilon in the environment of histone deposition. Our results indicated that repair activity with both polymerases was activated by histone deposition. Here it was first shown that human DNA Polymerase epsilon performs DNA synthesis during mismatch repair in vitro. Importantly, recent studies have revealed association of Polymerase epsilon mutations with cancer. Since our data showed activity of DNA Polymerase epsilon in mismatch repair, a possible tumor development mechanism may involve inactivation of mismatch repair due to Polymerase epsilon mutations. Overall, our study expanded the understanding of the mechanism of human mismatch repair in the chromatin environment.

On a montré que les PTM jouaient un rôle significatif de P. falciparum dans l'année de contrôle de la régulation transcriptionnelle, de l'expression monoaléique et de la différenciation sexuelle. Dix SET contenant des HKMTs contenant du domaine-ont été prédits; Six d'entre eux être essentiels pour le développement de stages de sang asexués. Le projet de thèse est centré sur la caractérisation biologique de PfSET7 et PfSET6. Nous avons observé l'échange de localisation cellulaire dynamique Pendant le cycle de vie: PfSET7 se trouvent dans de multiples foyers cytoplasmiques dans les stades érythrocytaires asexués et le stage de foie, et plus frappante, dans la membrane du parasite enrichie en gamétocytes. PfSET6 EXPOSÉ une localisation nucléaire dans les anneaux et un modèle ponctué dans le cytoplasme des trophozoites matures et schizontes, et est enrichi dans les structures de foyers dans le cytoplasme des gamétocytes. Pris ensemble, notre étude suggère que la méthylation non histone est beaucoup plus significative chez P. falciparum que précédemment attendu. La méthylation à médiation par PfSET7 Peut être une extension du code histone à - d'autres protéines cytosoliques; Partiellement PfSET6 s'associe à des voies de répression transcriptionnelle dans le noyau et des régulateurs post-transcriptionnels dans le cytoplasme. Une étude plus approfondie vise à identifier des cibles de domaine SET contenant des protéines dans le gène inducible knockout mutant parasite lignes. Le fait que PfSET7 et PfSET6 sont exprimés à différents stades du cycle de vie, les fait comme de nouvelles cibles pour le développement de médicaments contre cette maladie grave et de bloquer la transmission. / In P. falciparum, PTMs have been shown to play an important role in the control of transcriptional regulation, monoallelic expression, and sexual differentiation. Ten SET domain-containing HKMTs have been predicted; six of them appear to be essential for asexual blood stage development. My lab has expressed and purified two enzymatically active recombinant methyltransferase PfSET7 and PfSET6. In vitro enzyme kinetics assays shows they can methylate histones. The dissertation project is centered around the biological characterization of PfSET7 and PfSET6. We observed the dynamic changes of cellular localization during life cycle: PfSET7 are found in multiple cytoplasmic foci in asexual erythrocytic stages and liver stage, and more strikingly, enriched in parasite membrane in gametocytes. PfSET6 exhibited a nuclear localization in rings and a punctuated pattern in the cytoplasm of mature trophozoites and schizonts, and is enriched within foci-like structures in the cytoplasm of gametocytes. Taken together, our study suggests that non-histone methylation is much more important in P. falciparum than previously anticipated. PfSET7-mediated methylation may be an extension of the histone code to other cytosol proteins; PfSET6 partially associates with transcriptional repression pathways in the nucleus and post-transcriptional regulators in the cytoplasm. Further study aims to identify targets of SET domain containing proteins within the inducible gene knock out mutant parasite lines. The fact that PfSET7 and PfSET6 are expressed in different life cycle stages, makes them as novel targets for drug development that could against severe disease and to block pathogen transmission.

Ao longo dos últimos anos observou-se um aumento no número de idosos no mundo, com isso faz-se necessário buscar terapias que amenizem os danos relacionados e também elucidar os mecanismos envolvidos neste processo. O exercício físico tem sido sugerido como uma ferramenta importante, não farmacológica, para atenuar os déficits relacionados à idade. Ainda, estudos recentes sugerem uma relação entre o processo de envelhecimento cerebral e o desequilíbrio de mecanismos epigenéticos, contudo, estes dados ainda não são conclusivos. Sabe-se que o grau de neuroplasticidade varia com a idade e que as estruturas encefálicas podem responder diferentemente à exposição ao exercício. Estudos demonstram que o córtex pré-frontal está envolvido em funções de alta ordem como atenção, tomada de decisão e memória de trabalho. Portanto, o objetivo deste trabalho foi avaliar os efeitos de diferentes protocolos de exercício físico (sessão única e exercício diário moderado) sobre a modulação de marcadores epigenéticos em córtex pré-frontal de ratos Wistar de 3 e 21 meses de idade. Os animais foram submetidos ao protocolo de sessão única (20 minutos) ou o exercício diário moderado (20 minutos durante 14 dias), 1 hora após a última sessão foram eutanasiados. O córtex pré-frontal foi dissecado e a acetilação da H4, o conteúdo da DNA metiltransferase (DNMT1 e DNMT3b), assim como a atividade da histona metiltransferase H3K27 foram analisadas. Os resultados serão apresentados na versão completa desta dissertação. / Over the past few years the number of elderly people has increased in the world, therefore it is necessary to search therapies that ameliorate age-related deficits as well as elucidate the mechanisms involved in this process. Physical exercise has been suggested as an important non-pharmacological approach to alleviate the age-related decline. Furthermore, recent studies have suggested a relationship between the process of brain aging and imbalance of epigenetic mechanisms, however, these data are not conclusive. It is well described that prefrontal cortex is involved in higher functions like attention, decision making and working memory. Then, the aim of this study was to investigate the effects of two exercise protocols (single session and daily moderate exercise) on the modulation of epigenetic markers in the prefrontal cortex from Wistar rats of 3- and 21- months-old. Animals were submitted to single session protocol (20 minutes) or the daily moderate exercise (20 minutes for 14 days), and 1hour after the last exercise session animals were euthanized. Prefrontal cortex was dissected out and acetylation of H4, the content of DNA methyl transferase (DNMT1 and DNMT3B), as well as histone methyltransferase H3K27 activity were analyzed. Results will be presented in the full version.

No description available.

Le p300/CBP acétyltransférase est un co-activateur transcriptionnel très important qui est impliqué dans la régulation d'un grand nombre de processus biologiques, comme la transcription d'ADN, le développement et l'immunité innée. Jusqu'à présent, le rôle de p300/CBP dans la régulation de l'expression des gènes a été largement étudiée, mais les mécanismes qui régulent son activité enzymatique sont encore peu connus. Des études ont montré que le dysfonctionnement de p300/CBP est associé à plusieurs formes de cancer et de maladies neurodégénératives. Dés lors, chaque progrès concernant les mécanismes de régulation de p300/CBP est devenu primordial pour le développement de nouvelles thérapies. Le 'noyau' de p300/CBP contient deux domaines pour la reconnaissance des modifications post-traductionnelles (MPTs), un bromodomaine et un PHD finger (le module BP), adjacent à un domaine HAT (ou domaine histone acétyltransférase). Plusieurs enzymes, modifiant la chromatine, contiennent des domaines de reconnaissance des MPTs. Fréquemment des groupements particuliers de ces domaines sont très conservés et liés, au sein de la même protéine ou du même complexe protéique, suggérant qu'ils réalisent des fonctions coordonnées. Ces domaines adjacents peuvent agir en concertation dans la reconnaissance simultanée de différents MPTs ou peuvent exercer des fonctions différentes de celles qui sont effectuées par ces deux domaines particuliers, tels que les fonctions de régulation enzymatique. Plusieurs études suggèrent que les cycles acétylation/désacétylation dans la boucle d'auto-inhibition, à l'intérieur du domaine HAT, jouent un rôle important dans la régulation de l'activité enzymatique de p300/CBP. La proximité du module BP et du domaine HAT suggère que la spécificité de liaison, appartenant au module BP, peut être intrinsèquement liée à la régulation de l'activité du domaine HAT. L'objectif de ma thèse est de déterminer le rôle du module BP dans la régulation de l'activité du domaine HAT. Je propose que le module BP soit impliqué dans la régulation de p300/CBP de deux façons. La première consiste à établir un lien avec le domaine HAT qui stabilise la conformation auto-inhibée de l'enzyme. La deuxième exige que le module BP joue un rôle dans le choix des substrats de p300/CBP. J'ai été en mesure de montrer que BP peut se lier au domaine HAT et à la chromatine modifiée et qu'il peut reconnaître les modifications effectuées par p300/CBP lui-même. Les données obtenues indiquent que le module BP peut être impliqué dans la régulation de l'activité de p300/CBP et dans son ciblage à la chromatine. / The p300/CBP acetyltransferase is an important transcriptional co-activator which is involved in regulating a wide range of biological processes, such as DNA transcription, development and innate immunity. To date, the role of p300/CBP in gene regulation has been extensively described but little is known about the mechanisms which regulate its activity. Since misregulation of p300/CBP has been associated to the development of several forms of cancers and neurodegenerative diseases, studies directed to decipher the mechanisms of regulation of p300/CBP are of great importance for the development of new therapies. The p300/CBP 'core' contains two post-translational modifications (PTMs) recognition motifs, a bromodomain and a PHD domain (the bromo-PHD module, BP), in close proximity to a histone acetyltransferase domain (HAT). Many chromatin modifying enzymes contain recognition modules for PTMs. Frequently particular groupings of such modules are conserved and linked within the same protein or the same multisubunit complex, suggesting that they perform concerted functions. These linked modules may act combinatorially to allow recognition of multiple PTMs or display new functions that are not possessed by the single modules, such as regulatory properties. Accumulating evidence suggests that acetylation/deacetylation in a conserved autoinhibitory loop of the p300/CBP HAT domain plays an important role in regulation of HAT activity. The close apposition of the BP module and the HAT domain suggests that BP substrate recognition is intrinsically linked to regulation of HAT activity. During my thesis work, I have investigated the role of BP in HAT regulation. I propose that the BP module is involved in p300/CBP regulation by binding to the HAT domain and stabilizing the autoinhibited conformation of the enzyme. I have also investigated substrate specificity of the BP module towards modified chromatin. I could show that the BP module binds histone modifications including those that are p300/CBP dependent. Altogether, the data suggests that the BP module is involved in regulating p300/CBP HAT activity and in targeting of chromatin.

Orientador: Joaquim Mansano Garcia / Banca: Lígia Veiga Pereira / Banca: Irina Kerkis / Resumo: O estudo dos processos de diferenciação em células tronco embrionárias (CTE) representa uma importante ferramenta para o entendimento das vias moleculares que os regem, apresentando grande aplicação tanto na ciência básica quanto na engenharia de tecidos e medicina regenerativa. Pouco é conhecido sobre as marcas epigenéticas existentes na cromatina destas células, e de que forma a regulação da expressão gênica ocorre no momento da diferenciação. O presente trabalho teve como objetivo o estudo dos efeitos da hiperacetilação das histonas causada pela droga tricostatina A (TSA), uma inibidora das enzimas histona desacetilases, sobre a diferenciação destas células em estádios iniciais e avançados. Para tanto, a hiperacetilação induzida pela droga foi estimada por reações de imunocitoquímica para AcLys9H3. Os efeitos anti-proliferativos da TSA foram mensurados pelo teste de TUNEL e contagem de células. Ainda, foram conduzidos experimentos de diferenciação in vitro de CTE e análise da expressão de proteínas características de linhagens celulares diferenciadas por reações de imunocitoquímica (Oct3/4, nestina, âIII tubulina, desmina e troponina I), em cultivos tratados com TSA em diferentes concentrações e em diferentes momentos. Desta forma, foi estimada a população de tipos celulares oriundos dos folhetos embrionários ectodérmico e mesodérmico, como neurônios, e células musculares, quando foi promovida a hiperacetilação das histonas nas CTE, em diferentes momentos da diferenciação celular in vitro. A TSA induziu apoptose em níveis superiores aos do grupo controle, e retardou/inibiu a divisão celular. Promoveu hiperacetilação dose-dependente nos períodos estudados, e estimulou a diferenciação de precursores mesodérmicos (50nM d5) e ectodérmicos (15nMd0-5 e 50nMd5), cardiomiócitos (50nMd5 e 100nMd13) e neurônios (15nMd0-5, 50nMd5, 100nMd5, 100nMd13). / Abstract: Studies on embryonic stem cells (ESC) differentiation represents an important tool leading to understanding of its molecular pathways, with many applications both on basic research and tissue engineering / regenerative medicine. Little is known about epigenetic marks on ESC chromatin, and how gene expression occurs at differentiation time. The aim of this work was to study effects of histone hiperacetylation, induced by cell treatment with trichostatin A (TSA), an histone deacetylase inhibitor, on both initial and late differentiation. For that, drug-induced hyperacetylation was studied by AcLys9H3 immunocitochemistry. TSA anti-proliferative effects were analysed by TUNEL test and cell counts. Experiments on ESC in vitro differentiation and immunocitochemistry for specific cell types proteins (Oct3/4, nestin, âIII tubulin, desmin and troponin I) were performed, in treated and control groups, at different moments. This analysis showed specific cell types populations derived from embryonic ectodermal and mesodermal, such as neurons and cardiomyocytes, when histone hyperacetylation were induced, on both initial and late diferentiation. Our results showed that TSA induces apoptosis and inhibits cellular proliferation. Also, TSA promoted dose-dependent histone hyperacetylation at studied moments, and stimulated mesodermal (50nM d5) and ectodermal (15nMd0-5 e 50nMd5) precursors, cardiomyocytes (50nMd5 e 100nMd13) and neurons (15nMd0-5, 50nMd5, 100nMd5, 100nMd13) differentiation. / Mestre

Ao longo dos últimos anos observou-se um aumento no número de idosos no mundo, com isso faz-se necessário buscar terapias que amenizem os danos relacionados e também elucidar os mecanismos envolvidos neste processo. O exercício físico tem sido sugerido como uma ferramenta importante, não farmacológica, para atenuar os déficits relacionados à idade. Ainda, estudos recentes sugerem uma relação entre o processo de envelhecimento cerebral e o desequilíbrio de mecanismos epigenéticos, contudo, estes dados ainda não são conclusivos. Sabe-se que o grau de neuroplasticidade varia com a idade e que as estruturas encefálicas podem responder diferentemente à exposição ao exercício. Estudos demonstram que o córtex pré-frontal está envolvido em funções de alta ordem como atenção, tomada de decisão e memória de trabalho. Portanto, o objetivo deste trabalho foi avaliar os efeitos de diferentes protocolos de exercício físico (sessão única e exercício diário moderado) sobre a modulação de marcadores epigenéticos em córtex pré-frontal de ratos Wistar de 3 e 21 meses de idade. Os animais foram submetidos ao protocolo de sessão única (20 minutos) ou o exercício diário moderado (20 minutos durante 14 dias), 1 hora após a última sessão foram eutanasiados. O córtex pré-frontal foi dissecado e a acetilação da H4, o conteúdo da DNA metiltransferase (DNMT1 e DNMT3b), assim como a atividade da histona metiltransferase H3K27 foram analisadas. Os resultados serão apresentados na versão completa desta dissertação. / Over the past few years the number of elderly people has increased in the world, therefore it is necessary to search therapies that ameliorate age-related deficits as well as elucidate the mechanisms involved in this process. Physical exercise has been suggested as an important non-pharmacological approach to alleviate the age-related decline. Furthermore, recent studies have suggested a relationship between the process of brain aging and imbalance of epigenetic mechanisms, however, these data are not conclusive. It is well described that prefrontal cortex is involved in higher functions like attention, decision making and working memory. Then, the aim of this study was to investigate the effects of two exercise protocols (single session and daily moderate exercise) on the modulation of epigenetic markers in the prefrontal cortex from Wistar rats of 3- and 21- months-old. Animals were submitted to single session protocol (20 minutes) or the daily moderate exercise (20 minutes for 14 days), and 1hour after the last exercise session animals were euthanized. Prefrontal cortex was dissected out and acetylation of H4, the content of DNA methyl transferase (DNMT1 and DNMT3B), as well as histone methyltransferase H3K27 activity were analyzed. Results will be presented in the full version.

O desenvolvimento e a progressão do câncer bucal envolvem processos complexos de múltiplas etapas levando a modificações fenotípicas nas células epiteliais, aumento da proliferação e invasão dos tecidos subjacentes. Diversos fatores vem sendo associados à carcinogênese, dentre eles os mecanismos epigenéticos como a acetilação de histonas, que promovem mudanças na expressão de genes independente de mutações. O objetivo do presente estudo observacional transversal foi analisar a relação entre acetilação da histona 3 (acetil Histona H3) com proliferação celular e transição epitélio-mesênquima na mucosa bucal normal (MBN), leucoplasias bucais (LB) e carcinomas espinocelulares (CEC) de boca, bem como correlacioná-los com dados clínico-demográficos, graduação histopatológica e o comportamento das lesões. Foram analisados 10 casos de mucosa bucal normal (MBN), 20 casos de LB e 75 casos de CEC de boca. Todos os casos foram submetidas a análise imunoistoquímica utilizando anticorpos anti-acetil Histona H3, Ki67, vimentina e TGF-β1. A imunomarcação da acetil histona H3 foi significativamente menor nos casos de CEC quando comparados a LB (p=0.03). Não foi encontrado diferença entre os casos de MBN e LB. Paralelamente, foi observado um aumento estatisticamente significativo na proliferação durante o processo de carcinogênese (p<0.00) e o mesmo foi observado quando avaliados os marcadores da transição epitélio-mesênquima, vimentina (p=0.03) e TGF-β1 (p<0.00). A análise da associação dos marcadores com fatores clínicos-demográficos não mostrou diferença significativa. Entretanto, maior média de acetil histona H3 foi associada ao bom prognóstico (p=0.01) e também, foi observado uma tendência de uma melhor taxa de sobrevida (p=0.06). Conclui-se que os CEC de boca são hipoacetilados, exibem maior perfil proliferativo e de transição epitélio-mesênquima. Além disso, a acetil histona H3 pode ser considerada um marcador prognostico nestas lesões. / The development and progression of oral cancer involve multi-step processes leading phenotypic changes in epithelial cells, proliferation increase and invasion of adjacent tissue. Several factors have been associated with carcinogenesis, including epigenetic mechanisms such as histone acetylation, which promote changes in the expression independent of gene mutations. The aim of the present study was to analyze the association of acetylation of histone 3 (acetyl-histone H3) with cell proliferation and epithelial-mesenchymal transition in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and correlate them with data clinic-demographic, histopathological grading and the behavior of these lesions. We analyzed 10 cases of normal oral mucosa (NOM), 20 cases of OL and 75 cases of OSCC. All samples were submitted to immunohistochemical analysis using anti-acetyl histone H3, Ki67, vimentin and TGF-β1. Acetyl-histone H3 labeling was significantly lower in cases of OSCC compared to LB (p=0.03). It was not found difference between NOM and OL. In parallel, the proliferation analysis revealed a gradual increase on Ki67 labeling (p<0.00) during oral carcinogenesis with highest value detected in OSCC Also, an increase on EMT markers, vimentin (p=0.03) and TGF-β1 (p<0.00) were noted. A higher mean acetyl-histone H3 was associated with good prognosis (p= 0.01) and similarly a tendency to improved survival rate was observed (p=0.06). As conclusion, OSCC are hypoacetylated, exhibit higher proliferative profile and epithelial-mesenchymal transition characteristics. Furthermore, acetyl histone H3 can be considered a prognostic marker in OSCC.

O desenvolvimento e a progressão do câncer bucal envolvem processos complexos de múltiplas etapas levando a modificações fenotípicas nas células epiteliais, aumento da proliferação e invasão dos tecidos subjacentes. Diversos fatores vem sendo associados à carcinogênese, dentre eles os mecanismos epigenéticos como a acetilação de histonas, que promovem mudanças na expressão de genes independente de mutações. O objetivo do presente estudo observacional transversal foi analisar a relação entre acetilação da histona 3 (acetil Histona H3) com proliferação celular e transição epitélio-mesênquima na mucosa bucal normal (MBN), leucoplasias bucais (LB) e carcinomas espinocelulares (CEC) de boca, bem como correlacioná-los com dados clínico-demográficos, graduação histopatológica e o comportamento das lesões. Foram analisados 10 casos de mucosa bucal normal (MBN), 20 casos de LB e 75 casos de CEC de boca. Todos os casos foram submetidas a análise imunoistoquímica utilizando anticorpos anti-acetil Histona H3, Ki67, vimentina e TGF-β1. A imunomarcação da acetil histona H3 foi significativamente menor nos casos de CEC quando comparados a LB (p=0.03). Não foi encontrado diferença entre os casos de MBN e LB. Paralelamente, foi observado um aumento estatisticamente significativo na proliferação durante o processo de carcinogênese (p<0.00) e o mesmo foi observado quando avaliados os marcadores da transição epitélio-mesênquima, vimentina (p=0.03) e TGF-β1 (p<0.00). A análise da associação dos marcadores com fatores clínicos-demográficos não mostrou diferença significativa. Entretanto, maior média de acetil histona H3 foi associada ao bom prognóstico (p=0.01) e também, foi observado uma tendência de uma melhor taxa de sobrevida (p=0.06). Conclui-se que os CEC de boca são hipoacetilados, exibem maior perfil proliferativo e de transição epitélio-mesênquima. Além disso, a acetil histona H3 pode ser considerada um marcador prognostico nestas lesões. / The development and progression of oral cancer involve multi-step processes leading phenotypic changes in epithelial cells, proliferation increase and invasion of adjacent tissue. Several factors have been associated with carcinogenesis, including epigenetic mechanisms such as histone acetylation, which promote changes in the expression independent of gene mutations. The aim of the present study was to analyze the association of acetylation of histone 3 (acetyl-histone H3) with cell proliferation and epithelial-mesenchymal transition in oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) and correlate them with data clinic-demographic, histopathological grading and the behavior of these lesions. We analyzed 10 cases of normal oral mucosa (NOM), 20 cases of OL and 75 cases of OSCC. All samples were submitted to immunohistochemical analysis using anti-acetyl histone H3, Ki67, vimentin and TGF-β1. Acetyl-histone H3 labeling was significantly lower in cases of OSCC compared to LB (p=0.03). It was not found difference between NOM and OL. In parallel, the proliferation analysis revealed a gradual increase on Ki67 labeling (p<0.00) during oral carcinogenesis with highest value detected in OSCC Also, an increase on EMT markers, vimentin (p=0.03) and TGF-β1 (p<0.00) were noted. A higher mean acetyl-histone H3 was associated with good prognosis (p= 0.01) and similarly a tendency to improved survival rate was observed (p=0.06). As conclusion, OSCC are hypoacetylated, exhibit higher proliferative profile and epithelial-mesenchymal transition characteristics. Furthermore, acetyl histone H3 can be considered a prognostic marker in OSCC.

Bacteria are one of the largest causes of human disease, with millions of deaths every year attributed to bacterial infections, and they have become more difficult to tackle with the widespread emergence of antibiotic resistance. In this thesis, I describe my studies that pursued two approaches: one focus was on using antimicrobial histones as an alternative to treatment for antibiotic resistant bacteria; in another approach the recombinant version of an eggshell cuticle protein was expressed and purified for testing against food-safety pathogens. One major pathogen that is contributing to this challenge of antibiotic resistance is Staphylococcus aureus. The methicillin-resistant strain of S. aureus leads to increased hospital stays and increased mortality in patients. The impact of such pathogens is worsened when bacteria form surface-attached aggregates known as biofilms. Development of new approaches to eradicate antibiotic- resistant biofilms will benefit human health. This study looked at an alternative method to eradicate bacteria compared to traditional antibiotics. Histones with antimicrobial activity were extracted from chicken blood and tested against methicillin-sensitive and methicillin-resistant strains of Staphylococcus aureus biofilm (MSSA and MRSA). The histone mixture completely eradicated both strains in biofilm form at relatively low concentrations. In addition, the histone mixture also displayed fast kill kinetics against planktonic forms of the two strains. Finally, the interaction of the histone mixture with the bacterial membrane in MRSA biofilms was observed by scanning electron microscopy (SEM). Bacteria treated with the histone mixture showed clear morphological changes, including pore formation and cell collapse. Therefore, the histone mixture purified from chicken red blood cells could prove to be a good alternative to traditional antibiotics for protection against antibiotic-resistant strains of bacteria in their planktonic and biofilm forms. Reduction of food-borne illness is another important aspect in the promotion of human health. A significant contributor to food-borne illness is contaminated table eggs. The unfertilized egg can be contaminated by a variety of pathogens including Salmonella spp. and Bacillus spp. The egg is protected by the eggshell which is traversed by respiratory pores that are normally covered by a cuticle plug to restrict pathogen entry. This cuticle consists of several proteins including ovocaxlyin-32 (OCX-32). OCX-32 has a large number of naturally occurring haplotypes due non-synonymous single nucleotide polymorphisms (SNPs). In this study, the goal was to express five of the most common haplotypes of OCX-32 in Escherichia coli and purify the recombinant protein for assay of its antimicrobial activity. Five constructs that contain the cDNA of common OCX-32 haplotypes (A, B, C, D, and O) with a histidine tag at the C-terminus were generated. The constructs were subcloned into pGEX4T-1 vector which encodes Glutathione-S-transferase (GST) upstream of the multiple cloning site. My study developed methods to optimize the expression conditions, and to increase the solubility of the recombinant protein. Various expression strains of E. coli and solubility buffers were tested. In addition, the construct was subcloned into a plasmid containing the small ubiquitin-like modifier (SUMO) fusion tag; the solubility of the new SUMO-OCX-32 haplotype A recombinant fusion protein was evaluated. The best results were obtained by slow dialysis refolding of denatured SUMO-OCX-32 fusion protein. This recombinant protein showed almost complete solubility with minimal precipitation and was tested against the egg-related pathogen, Bacillus cereus. Unfortunately, the SUMO-OCX-32 recombinant protein did not inhibit growth of B. cereus. In my studies reported in this thesis, two very different approaches were taken. A histone mixture was isolated from an abundant starting material, which proved to be highly effective and promising in the eradication of S. aureus biofilms at relatively low concentrations. Alternatively, expression of a soluble recombinant protein for functional activity assay was very challenging and required the optimization of a number of methods to prepare soluble protein for testing. One of the methods tested proved effective in obtaining large amounts of soluble protein. However, further developmental work will be essential to determine if this approach is a viable strategy in acquiring functional protein.