Although mRNAs encoding the histone proteins are among the most abundant mRNAs in mammalian oocytes, the mechanism regulating their translation in these cells has not been identified. Most histone mRNAs are not polyadenylated but instead carry in their 3'-utr a highly conserved stem-loop structure. In somatic cells, the stem-loop binding protein (SLBP) is expressed during S-phase of the cell cycle and associates with the stem-loop of histone mRNAs promoting their processing and translation and thereby coordinating their expression to DNA replication. As histone mRNAs are abundant in immature oocytes which are in G2 of the cell cycle and in ovulated or mature oocytes which are in M-phase, I examined the expression and the regulation of histone mRNAs in immature and maturing mouse oocytes. First, I described SLBP expression during oogenesis and pre-implantation embryonic development. I showed that SLBP is present at low levels in the nucleus of the immature oocyte and accumulates significantly during maturation of the oocyte. At both stages, SLBP is the only stem-loop binding activity present. I showed that SLBP meiotic accumulation correlates with the adenylation of SLBP mRNA and is mediated by the presence of a cytoplasmic polyadenylation element in SLBP 3'-utr. Also, I demonstrated that histones are synthesized in the immature and mature oocyte and that the translation of a reporter mRNA bearing the histone 3'-utr increases dramatically during oocyte maturation consistent with the accumulation of SLBP. I specifically blocked SLBP accumulation using RNA interference and observed that both translation of the reporter mRNA and endogenous histone synthesis are significantly reduced. Moreover, SLBP-depleted eggs display a significant decrease in pronuclear size and in the total amount of histones detectable on their chromatin. Finally, I also showed that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translatio

Bibliography: leaves 113-126. / v, 126, [113] leaves, [10] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the biological significance of the mouse Listone variant H2A.Z. Describes the isolation and characterisation of H2A.Z genomic clones from different mouse genomic libraries; H2A.Z gene targeting in mouse E14 embryonic stem cells; and an attempt to generatae ES cell lines and mice which lack the functional H2A.Z protein to investigate H2A.Z function in vitro and in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995?

Thesis (Ph.D. in Biochemistry & Molecular Genetics) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 169-185). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xv, 219 p.; also includes graphics (some col.) Includes bibliographical references (p. 147-173). Available online via OhioLINK's ETD Center

Thesis (Ph.D.)--University of Florida, 2004. / Typescript. Title from title page of source document. Document formatted into pages; contains 102 pages. Includes Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xiv, 200 p.; also includes graphics (some col.). Includes bibliographical references (p. 167-197). Available online via OhioLINK's ETD Center

In eukaryotes, DNA is wrapped around a group of proteins termed histones that are required to precisely control gene expression during development. The amino acids of both the globular domains and unstructured tails of these histones can be modified by chemical moieties, such as methylation, acetylation and ubiquitination. The ‘histone code’ hypothesis proposes that specific combinations of these and other histone modifications contain transcriptional information, which guides the cell machinery to activate or repress gene expression in individual cell types. Chromatin immunoprecipitation (ChIP) experiments using undifferentiated stem cell populations have identified the genomic co-localisation of histone modifications reported to have opposing effects on transcription, which is known as bivalency. The human α-globin promoter, a well-established model for the study of transcriptional regulation, is bivalent in embryonic stem (ES) cells and this bivalency is resolved once the ES cells terminally differentiate (i.e. only activating or repressing marks remain). In a humanised mouse model, the deletion of a bone fide enhancer within the human α-globin locus results in heterogeneous expression patterns in primary erythroid cells. Notably, this correlates with an unresolved bivalent state at this promoter in terminally differentiated cells. Using this mouse model it is not feasible to ascertain whether the transcriptional heterogeneity observed in the cells lacking an α-globin enhancer is reflective of epigenetic heterogeneity (i.e. a mixed population of cells) rather than co-localisation of bivalent histone modifications within the same cells. Furthermore, the functional contribution of bivalency to development has yet to be described. To address these difficulties, I aimed to generate a fluorescent reporter system for human α-globin to facilitate the separation of transcriptionally heterogeneous erythroid cells. This model will provide material for ChIP studies on transcriptionally active and inactive populations to determine whether the epigenetic bivalency is reflective of a mixed cell population or true bivalency. In addition, I aimed to produce epigenetic editing tools to target bivalent promoters, which in combination with in vitro differentiation assays would provide an interesting framework to test the function of bivalency during development. In this study, I extensively tested gene-editing strategies for generating a fluorescent reporter knock-in in humanised mouse ES cells. I validated the suitability of humanised mouse ES cell lines for gene targeting studies and optimised a robust in vitro differentiation protocol for studying erythropoiesis. I utilised both recombineering and CRISPR/Cas9 gene editing tools in tandem with PiggyBac transposon technology, to knock-in the reporter gene. I made significant steps in gene targeting and successfully inserted the reporter downstream of the α-globin gene. I also generated a cloning system to express site-specific DNA-binding domains (TALEs) fused to epigenetic regulators with the aim to resolve bivalent histone modifications in vitro. From preliminary tests using these fusion proteins targeting Nrp1, a bivalent promoter in mES cells, I observed mild but significant changes in gene expression although histone modifications were unchanged. The various tools generated and tested in this study provide a solid foundation for future development of genetic and epigenetic editing at the human α-globin and other bivalent loci.

Made available in DSpace on 2014-06-11T19:35:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-07-06Bitstream added on 2014-06-13T18:46:30Z : No. of bitstreams: 1 oliveira_cs_dr_jabo.pdf: 1211579 bytes, checksum: 2f19bde4e0f7fbfd3f2aae7f084ceea1 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Modificações pós-translacionais de histonas são importantes componentes do código epigenético, e contribuem para o controle da transcrição gênica de cada célula. O presente trabalho descreve a participação de duas modificações de histonas no desenvolvimento embrionário pré-implantacional: a acetilação de lisinas e a citrulinização de argininas. No capítulo 1, avaliamos a presença de duas modificações de histona H3, K9ac (permissiva) e K27me3 (repressiva) em embriões bovinos no ciclo de ativação do genoma embrionário (AGE), por imunofluorescência. Ambas as marcas estão presentes e apresentam alto coeficiente de correlação, e dois perfis de embriões (com alta e baixa variação dos níveis das modificações entre blastômeros) foram descritos. A acetilação de histonas está relacionada à ativação da expressão gênica, portanto no capítulo 2 hipotetizamos que a manipulação de seus níveis em embriões bovinos poderia influenciar a ativação do genoma embrionário, o desenvolvimento de blastocistos e a inativação do cromossomo X em fêmeas. Foram testadas concentrações do inibidor das histona desacetilases tricostatina A variando de 5 a 50nM, aplicadas por 12 a 144h, iniciando 70h após a FIV. Três protocolos foram selecionados: 5nM 48h, 5nM 144h e 15nM 48h. Após, foi utilizado sêmen sexado para estudar os efeitos da TSA sobre embriões fêmeas e machos separadamente. Por imunofluorescência para H3K9ac, foi observado aumento na acetilação de histonas em ambas as concentrações (5 e 15nM), sendo 5nM mais eficaz em fêmeas do que machos. O tratamento com 15nM 48h reduziu a produção de blastocistos em machos e fêmeas, e 5nM 144h em machos. A taxa de apoptose, avaliada pelo ensaio TUNEL, foi elevada em embriões fêmeas (grupos 5nM144h e 15nM48h), e em machos (grupo 15nM48h), mas tal aumento... / Histone post translational modifications are important components of the epigenetic code, and contribute to the control of gene transcription in each cell. This work describes the participation of two histone modifications in embryonic preimplantation development: acetylation of lysines and citrullination of arginines. In chapter 1, we evaluated the presence of two modifications of histone H3, K9ac (permissive) and K27me3 (repressive) in bovine embryos in the cycle of embryonic genome activation (EGA), by immunofluorescence. Both marks are present and show a high correlation coefficient, and two profiles of embryos were described, displaying high and low variation of modifications level between blastomeres. The acetylation of histones is related to gene expression activation, so in Chapter 2 we hypothesized that the manipulation of acetylation levels in bovine embryos could influence embryonic genome activation, blastocyst development and X chromosome inactivation in females. Five concentrations of the histone deacetylase inhibitor trichostatin A (TSA), ranging from 5 to 50nM beginning 70 hours after FIV were applied per 12 to 144h. Three protocols were selected: 5nM 48h, 5nM 144h and 15nm 48h. After, sexed semen was used to study the effects of TSA on male and female embryos separately. Immunofluorescence of H3K9ac showed increased histone acetylation at both concentrations (5 and 15nM). 5nM TSA was more effective in females than in males. Treatment with 15nM 48h reduced male and female blastocyst yield, and 5nM 144h reduced male blastocyst yield. Apoptosis rate was measured by the TUNEL assay. Female 5nM144h and 15nM48h groups, and male 15nM48h group displayed higher apoptosis levels, but this increase was not observed in low quality embryos. In female embryos, TSA did not affect... (Complete abstract click electronic access below)

by Jose Victor Lopez. / Typescript. / Thesis (M.S.)--Florida State University, 1988. / Bibliography: leaves 98-109.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / The ruminant fertility is influenced by intrinsic sperm factors, like chromatin or proteins. Considering that the reproductive efficiency is dependent on a balanced and feasible nutrition, the cashew nut meal (CNM) is a low cost byproduct that must be analyzed for possible effects on sperm chromatin and proteins.Study 1:. The objectives of study 1 were to determine failures of chromatin condensation, expression levels and cellular localizations of histones; H3.3, H2B and H4, respectively in spermatozoa from low (LF) vs. high fertility (HF) bulls. The data were analyzed by t test and Pearson correlation (P < 0.05). We demonstrated that aniline blue staining was different within LF (1.73 (0.55, 0.19)) and HF Groups (0.67 (0.17, 0.06) (P < 0.0001), which was also negatively correlated with in vivo bull fertility (r = -0.90; P < 0.0001). Although those histones were consistently immune-detectable and specifically localized in bull sperm, this was not different between the two groups. Except H2B variants, H3.3 and H4 showed 100% identity and conserved among bovine, mouse and human. The H2B variants were more conserved between bovine and human than those of mouse. In conclusion, we showed that H2B, H3.3 and H4 were detectable in bull spermatozoa and that sperm chromatin condensation status, changed by histone retention, is related with bull fertility. Study 2: The objectives of study 2 were evaluate the effects of 13% of CNM inclusion in the diet of Morada Nova rams on the semen parameters, chromatin integrity and sperm proteins. Twenty rams were distributed in two equal groups: cashew nut group (CNG) and control group (COG) that received 13% and 0% of CNM in the diet for 90 days, respectively. The groups were compared for live weight, scrotal circumference, seminal parameters, chromatin integrity and sperm protein profile at 0, 45 and 90 days of the experiment. The data were evaluated by GLM for repeated measures (P < 0.05). At 90 days, CNG (69.00% (7.38; 2.33)) presented percentage of motile sperm superior than control group (60,00% (9,43; 2,98)) (P<0.05). There was not effect from the diet with CNM on chromatin integrity. But, the percentages of protein expression from ODF1 and H2B were larger in the CNG (P<0.05). The proteins: ODF1, GPX4, FTL and H2B were negatively correlated with sperm chromatin quality. In conclusion, the cashew nut meal did not affect negatively the semen quality. / A fertilidade em ruminantes à influenciada por fatores intrÃnsecos espermÃticos como a cromatina e as proteÃnas. Considerando que a eficiÃncia reprodutiva do macho depende de uma nutriÃÃo balanceada e viÃvel, o farelo de castanha de caju (FCC) à um subproduto de baixo custo que deve ser analisado quanto a possÃveis efeitos na integridade cromatÃnica e proteica dos espermatozoides. Estudo 1: O estudo 1 teve como objetivos determinar: falhas na condensaÃÃo da cromatina, nÃveis de expressÃo e localizaÃÃo celular das histonas: H3.3, H2b e H4B, respectivamente, em espermatozoides de touros de baixa (BF) e alta fertilidade (AF). Os dados foram avaliados pelo teste t e correlaÃÃo de Pearson (P < 0,05). Os resultados do teste do azul de anilina foram diferentes entre os grupos BF (1.73 (0.55, 0.19)) e AF (0,67 (0,17, 0,06) (P < 0,0001), os quais tambÃm foram negativamente correlacionados com a fertilidade in vivo de touros (r = -0,90; P < 0,0001). Apesar das histonas terem sido consistentemente imunodetectadas e localizadas nos espermatozoides, estas nÃo apresentaram diferenÃas entre os grupos. As proteÃnas H3.3 e H4 apresentaram 100% de identidade e foram conservadas entre bovinos, murinos e seres humanos. Entretanto, as variantes H2B foram mais conservadas entre touros e humanos do que entre humanos e camundongos. Em conclusÃo, as proteÃnas H2B, H3.3 e H4 foram detectÃveis em espermatozoides de touros e a condensaÃÃo da cromatina espermÃtica, alterada pela retenÃÃo de histonas, à relacionada com a fertilidade de touros. Estudo 2: O estudo 2 objetivou avaliar os efeitos da inclusÃo de 13% de FCC na dieta de carneiros Morada Nova sobre as caracterÃsticas seminais, integridade de cromatina e perfil das proteÃnas espermÃticas. Vinte carneiros foram divididos em dois grupos: castanha (GCA) e controle (GCO).que receberam na dieta 13% e 0% de FCC durante 90 dias, respectivamente. Os grupos foram comparados quanto ao peso vivo, circunferÃncia escrotal, parÃmetros seminais, integridade de cromatina e perfil das proteÃnas espermÃticas aos 0, 45 e 90 dias de experimento. Os dados foram analisados pelo mÃtodo GLM para medidas repetidas (P < 0,05). Aos 90 dias o GCA (69,00% (7,38; 2,33) apresentou porcentagem de espermatozoides mÃveis superior ao GCO (60,00% (9,43; 2,98)) (P<0.05). NÃo houve efeito da dieta contendo FCC sobre a integridade da cromatina. PorÃm, os percentuais das proteÃnas ODF1 e H2B foram mais elevados nos carneiros do GCA (P < 0,05). As proteÃnas: ODF1, GPX4, FTL e H2B foram negativamente correlacionadas com a qualidade da cromatina espermÃtica. Em conclusÃo, a inclusÃo de FCC na dieta de carneiros nÃo afetou negativamente a qualidade seminal.