Thesis (Ph. D.)--Rutgers University, 2010. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2000. / Includes bibliographical references (leaves 138-192). Also available online through Digital Dissertations.

Thesis (M.Sc.) - Carleton University, 2007. / Includes bibliographical references (p. 131-141). Also available in electronic format on the Internet.

Thesis (Ph. D.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2006. / Nicholas V. Hud, Committee Chair ; Donald F. Doyle, Committee Member ; Rigoberto Hernandez, Committee Member ; Roger M. Wartell, Committee Member ; Loren D. Willliams, Committee Member.

Universiẗat, Diss., 2004--München.

Tierärztl. Hochsch., Diss., 2004--Hannover.

The aim of the work presented in this thesis was to establish chromatin immunoprecipitation method in our laboratory as a tool to study target genes of the nuclear receptor NHR-25 in C. elegans. Once the method is established, it will be also useful for studies of other DNA binding proteins. ChIP was performed in transiently transfected cells HEK293 and analyzed using PCR and qPCR. Although ChIP is typically used to find authentic target genes in the cell or in organisms, testing protein-DNA interactions by ChIP in transient transfection system (by transfecting both the expression vector of the protein of interest and a vector containing potential binding sequence/promoter of the protein) can be useful as it serves as a relatively quick tool to confirm the direct binding. Since the detection is by PCR, this method is sensitive yet less costly non radioactive method to analyze protein-DNA interaction. For the first step towards ChIP in C. elegans; pulling down tagged protein directly from the worm was also performed as a preparation for in vivo analysis of NHR-25 regulated genes.

Recent studies have uncovered de novo mutations of the gene encoding the chromatin remodelling protein Snf2l in patients with schizophrenia, Rett-like syndrome and intellectual disability. Snf2l and its closely related protein, Snf2h, play a critical role in embryonic and post-natal brain development. Murine models lacking functional Snf2h or Snf2l point to complementary activities of these remodelers; Snf2h cKO mice present with a significantly reduced cerebellum, while Snf2l Ex6DEL (exon 6 deleted) cerebella are larger than their wild-type counterparts. Granule neuron progenitors (GNPs) isolated from Ex6DEL cerebella display delayed cell cycle exit and hindered terminal differentiation compared to wild-type controls. Moreover, loss of Snf2l activity results in widespread transcriptome shifts which underlie the Ex6DEL GNP differentiation phenotype. In particular, key transcription factors are differentially expressed without Snf2l remodelling activity. We confirm that ERK pathway activation is misregulated in Ex6DEL GNPs, possibly in response to elevated fibroblast growth factor 8 (Fgf8) expression in these cultures. We find that Snf2l activity maintains the chromatin landscape throughout GNP differentiation, as Ex6DEL cultures have a global increase in chromatin accessibility. We suggest that Snf2l-mediated chromatin condensation is responsible for proper regulation of gene expression programs in GNP differentiation.

Impact of the chromatin remodeller SMARCAD1 on murine intestinal intraepithelial lymphocyte and white adipose tissue biology. Chromatin remodelling factors use the energy of ATP hydrolysis to drive the movement of and/or affect molecular changes to the nucleosome. One such factor, SMARCAD1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A containing DEAD/H box 1), has been previously shown to restore heterochromatin at the replication fork in vitro. This project aimed to assess the impact of SMARCAD1 on mammalian biology, utilising an animal model in which the catalytic ATPase domain of murine SMARCAD1 had been deleted using Cre/lox technology. Preliminary results had implicated SMARCAD1 in adaptive-immunity and white adipose tissue biology, and SMARCAD1 expression in these tissues/cells was confirmed by tissue-panel western blot. This project therefore aimed to build on these results to understand better the impact of SMARCAD1 on adaptive immune development and white adipose tissue biology. In addition, fewer than expected viable Smarcad1-/- homozygous offspring were produced during Smarcad1+/- x +/- matings, which both confirmed the observation from a previous knockout model of Smarcad1, and limited the number of knockout animals available for this study. Investigation of systemic B- and T-cells in the bone marrow, thymus and spleen had previously suggested there was no significant defect in adaptive immune development in Smarcad1-/- mice, however a tissue-specific and age-related loss of intra-epithelial (IEL) T-lymphocytes was found in the small intestine by flow cytometry. Analysis by qPCR of duodenal RNA suggested that differentiation rather than inflammation may underpin any loss-of-IEL phenotype, although further examination of cell-proliferation and crypt/villus anatomy by EdU incorporation and immunofluorescence revealed no overt cell-anatomical or proliferative difference in the knockout mice. The requirement for large numbers of aged mice made further investigation of the intestinal IEL phenotype logistically prohibitive. The reduction of epididymal white adipose tissue (eWAT) size had also been observed in male Smarcad1-/- mice, and serum from these mice showed elevated triglyceride (TG) and free fatty acids (FFA) levels. Transcriptomic analysis by RNA-seq of whole-WAT revealed an elevation in macrophage-related markers in knockout mice, which was confirmed by flow cytometry. As a number of reports have implicated SMARCAD1 in stem cell biology, putative adipose stem cells were isolated from +/+ and -/- mice by FACS and used for adipogenic differentiation assays ex-vivo In parallel, mouse embryonic fibroblasts from +/- and -/- mice were also assayed for adipogenic differentiation. While no significant differences in adipogenesis were observed, Smarcad1-/- mice challenged with a (60%) high fat diet did show increased weight gain over +/+ mice, and measurements of adipocyte size and cell cycle/cell proliferation analysis suggested hyperplasia rather than defects in adipogenesis may drive any WAT-related pathology in these mice.

Orientador: José Luiz Laus / Coorientador: Marcela Aldrovani / Banca: Priscila Cardoso Cristovam / Banca: Márcia Rita Fernandes Machado / Resumo: A córnea de pacientes diabéticos pode desenvolver defeitos epiteliais que comprometem a visão e causam dor. Os efeitos do diabetes sobre algumas características das células epiteliais corneais, notadamente os fenótipos nucleares, são ainda desconhecidos. A sericina é um polímero extraído do casulo do bicho-da- seda, que, aparentemente, promove reparação corneal em ratos. Com esta pesquisa estudou-se a produção lacrimal, o limiar de sensibilidade corneal (Cochet-Bonnet) e a pressão dos olhos de ratos com três semanas de diabetes induzido por aloxana (glicemia sérica ≥ 400 mg/dL). Os efeitos da instilação diária (a cada 6 horas, por quatro dias) de sericina 10% sobre a cinética de reepitelização de córneas cauterizadas (com álcali), em ratos diabéticos, foram estudados e comparados aos efeitos de placebo (solução salina balanceada) e de soro equino (uma opção terapêutica vigente). A pesquisa traz, também, informações sobre o tamanho dos núcleos (área e perímetro), o estado de compactação e a textura da cromatina, o conteúdo de DNA (ploidia) e a distribuição das regiões organizadoras de nucléolos impregnadas por íons prata (AgNORs) em células epiteliais corneais de ratos diabéticos (hiperglicêmicos) e de não diabéticos (normoglicêmicos). Os núcleos das células que reepitelizaram as córneas cauterizadas, após tratamento com sericina 10%, solução salina balanceada ou soro, foram avaliados. Os resultados mostraram que os olhos dos ratos diabéticos diferiram dos de não diabéticos ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cornea of diabetic patients can develop epithelial defects that promote vision deficit and pain. The effects of diabetes/hyperglycemia on some characteristics of corneal epithelial cells, notably the nuclear phenotypes are still unknown. The sericin is a polymer extracted from the cocoons of the silkworm, which seems to promote reepithelization of burned cornea in rats. In this study, tear production, corneal touch threshold (Cochet-Bonnet) and intraocular pressure of alloxan-induced diabetes rats with three weeks of hyperglycemia (serum glucose ≥ 400 mg/dL) were studied. In addition, the effects of daily instillation (every 6 hours for 4 days) of 10% sericin over re-epithelization kinetics of burned corneas from diabetic were also studied and compared to the effects of placebo (balanced salt solution) and horse serum (an actual therapeutic option). The research also brings information about the size of the nuclei (area and perimeter), the state of compactation and the texture of chromatin, the DNA content (ploidy) and the distribution of silver-stained nucleolar organizing regions (AgNORs) of epithelial cells in corneas of diabetic (hyperglycemic) and non-diabetic rats (normoglycemic). Nuclear phenotypes of cells that participated of the re-epithelization of the burned corneas after treatment with10%sericin, balanced salt solution or serum were studied. Results showed that the eyes of diabetic rats did not differ from the ones from non-diabetic ones, just like the tear production and intraocular pressure, but differences regarding the corneal touch threshold (2.07 ± 0.52 cm in the corneas of diabetic vs. 4.00 ± 0.56 cm in non-diabetics; p = 0.01) were found. Regarding re-epithelization kinetics, the healing time of the burns were 30 hours for corneas treated with 10% sericin or serum and 36 hours in those receiving 0balanced sa... (Complete abstract click electronic access below) / Mestre