Cardiovascular diseases (CVD) have been the leading cause of death worldwide for many years, making it a devasting and increasing concern across the globe. The risk factors of CVD include postnatal factors and prenatal factors. For the prenatal CVD risk factors study, we focused on maternal hypothyroidism (MH), which is a common clinical condition. Studies have shown MH progeny have increased susceptibility to both acquired cardiovascular disease in adulthood and congenital heart disease, but the underlying mechanisms are not well understood. The goal of the present experiments was to test the hypothesis that MH reduces early postnatal cardiac myocyte proliferation in the progeny so that their adult hearts have a smaller complement of cardiac myocytes, which leads to adverse cardiac disease responses. MH model was induced by thyroidectomy (TX) with total thyroxine (TT4) under 1ng/dl after surgery. The progeny from mice that underwent Sham or TX surgery was termed WT (wild type) or MH (maternal hypothyroidism) progeny, respectively. Hearts were collected from WT and MH progeny to determine heart weight (HW), CM size, CM proliferation, and cell culture. RNA-seq was performed on heart tissue at postnatal day 5 (P5) and P60. Transverse Aortic Constriction (TAC) was performed to cause pressure overload-induced cardiac hypertrophy and/or heart failure (HF) in adult WT and MH progeny. ECHO (in-vivo) and histological (ex-vivo) studies were performed at specific times after TAC. Thyroid hormone treatment (levothyroxine, T4) for MH mother was administered. The results showed that the Heart weight (HW) to body weight (BW) ratio at P60 was no difference between groups, but the MH progeny had a larger CM size, consistent with fewer CM numbers. MH progeny had lower EdU+, Ki67+, and PH3+ CMs, and fewer mononucleated CMs, which shows they had a decreased CM proliferation capacity. RNA-seq data showed that genes related to DNA replication were downregulated in P5 MH progeny, including Bmp10. Both in vivo and in vitro studies showed Bmp10 treatment increased CM proliferation in the presence of thyroid hormone. In adult progeny, RNA-seq data showed that MH mice had genes upregulated in the inflammatory response before TAC surgery. Six weeks after TAC, the MH progeny had a greater HW/BW ratio, larger CM size, and more severe LV fibrosis consistent with more severe cardiac pathological remodeling compared with WT progeny. T4 supplemented treatment for MH mothers preserved progeny’s early postnatal CM proliferation capacity and the excessive pathological remodeling after TAC. Concluding, CM proliferation during the early postnatal development stage was significantly attenuated in MH progeny, which results in fewer CMs and CM hypertrophy in adult MH progeny. These changes are associated with worse cardiac disease responses under pressure overload in adult MH progeny. For the postnatal CVD risk factors study, we focused on calcium overload and metabolic disorder, which play a critical role in heart failure with preserved ejection fraction (HFpEF). HFpEF is defined as HF with an EF ≥50% and elevated cardiac diastolic filling pressures. The underlying causes of HFpEF are multifactorial and not well-defined. A transgenic mouse with low levels of cardiomyocyte (CM)-specific inducible Cavβ2a expression (β2a-Tg mice) showed increased cytosolic CM Ca2+, and modest levels of CM hypertrophy and fibrosis. This study aimed to determine if β2a-Tg mice develop an HFpEF phenotype when challenged with two additional stressors, a high-fat diet (HFD) and L-NAME (LN). Four-month-old wild-type (WT) and β2a-Tg mice were given either normal chow (WT-N, β2a-N) or HFD and/or L-NAME (WT-HFD, WT-LN, WT-HFD-LN, β2a-HFD, β2a-LN, and β2a-HFD-LN). Some animals were treated with the HDAC (hypertrophy regulators) inhibitor suberoylanilide hydroxamic acid (SAHA) (β2a-HFD-LN-SAHA). Echocardiography was performed monthly. After four months of treatment, terminal studies were performed, including invasive hemodynamics and organ weight measurements. Cardiac tissue was collected. Our results showed that four months of HFD plus L-NAME treatment did not induce a profound HFpEF phenotype in FVB WT mice. β2a-HFD-LN (3-Hit) mice developed features of HFpEF, including increased natriuretic peptide (ANP) levels, preserved EF, diastolic dysfunction, robust CM hypertrophy, increased M2 macrophage population, and myocardial fibrosis. SAHA reduced the HFpEF phenotype in the 3-Hit mouse model by attenuating these effects. Concluding, the 3-Hit mouse model induced a reliable HFpEF phenotype with CM hypertrophy, cardiac fibrosis, and an increased M2 macrophage population. This model could be used for identifying and preclinical testing of novel therapeutic strategies. / Biomedical Sciences

Micro and nanofabrication technologies have been used extensively in many biomedical and biological applications. Integration of MEMS technology and biology (BioMEMS) enables precise control of the cellular microenvironments and offers high throughput systems. The focus of this research was to develop three dimensional (3-D) isotropic microstructures for comprehensive analysis on cell-substrate interactions. The aim was to investigate whether the normal and cancerous cells differentially respond to their underlying substrate and whether the differential response of the cells leads to a novel label-free technique to distinguish between normal and cancerous cells. Three different generations of 3-D isotropic microstructures comprised of curved surfaces were developed using a single-mask, single-etch step process. Our experimental model included HS68 normal human fibroblasts, MCF10A normal human breast epithelial cells and MDA-MB-231 metastatic human breast cancer cells. Primary findings on the first generation of silicon substrates demonstrated a distinct adhesion and growth behavior in HS68 and MDA-MB-231 cells. MDA-MB-231 cells deformed while the fibroblasts stretched and elongated their cytoskeleton on the curved surfaces. Unlike fibroblasts, MDA-MB-231 cells mainly trapped and localized inside the deep microchambers. Detailed investigations on cytoskeletal organization, adhesion pattern and morphology of the cells on the second generation of the silicon substrates demonstrated that cytoskeletal prestress and microtubules organization in HS68 cells, cell-cell junction and cell-substrate adhesion strength in MCF10A cells, and deformability of MDA-MB-231 cells (obtained by using AFM technique) affect their behavior inside the etched cavities. Treatment of MDA-MB-231 cells with experimental breast cancer drug, SAHA, on the second generation of substrates, significantly altered the cells morphology, cytoarchitecture and adhesion pattern inside the 3-D microstructures. Third generation of silicon substrates was developed for comprehensive analysis on behavior of MDA-MB-231 and MCF10A cells in a co-culture system in response to SAHA drug. Formation of colonies of both cell types was evident inside the cavities within a few hours after seeding the cells on the chips. SAHA selectively altered the morphology and cytoarchitecture in MDA-MB-231 cells. Most importantly, the majority of MDA-MB-231 cells stretched inside the etched cavities, while the adhesion pattern of MCF10A cells remained unaltered. In the last part of this dissertation, using AFM analysis, we showed that the growth medium composition has a pronounced effect on cell elasticity. Our findings demonstrated that the proposed isotropic silicon microstructures have potential applications in development of biosensor platforms for cell segregation as well as conducting fundamental biological studies. / Ph. D.

L'épilepsie du lobe temporal (ELT) est la forme la plus fréquente de l'épilepsie chez l'adulte. Elle se traduit par des crises spontanées et récurrentes, qui sont résistantes à tout traitement dans 90% des cas. Une agression initiale du cerveau (traumatisme crânien, méningite, convulsions fébriles etc.), est souvent à l'origine de la transformation d'un cerveau « sain » en cerveau épileptique. L'ensemble des processus responsables de cette transition s'appelle l'épileptogenèse. Pouvoir bloquer et/ou retarder l'épileptogenèse chez les patients à risque est une question de santé majeure. En plus des crises, l'ELT soulève d'autres questions. Elle est souvent associée à des déficits cognitifs, qui sont la conséquence de la réorganisation des circuits neuronaux. Ces déficits pourraient être traités de façon indépendante de l'épilepsie elle-même. Le projet de recherche de cette thèse s'inscrit dans ce cadre général. / Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. It translates into spontaneous and recurrent seizures, which are resistant to any treatment in 90% of cases. An initial brain insult (head injury, meningitis, febrile seizures etc.), is often the cause of the transformation of a "healthy" brain into an epileptic one. The process responsible for this transition is called epileptogenesis. Blocking and/or delaying epileptogenesis in at-risk patients is a key issue for public health. In addition to the seizures, TLE raises other problems. It is often associated with cognitive deficits, which are the result of the reorganization of neuronal circuits. These deficits may be treated independently of epilepsy itself. The work presented here fits into this general framework.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

Pubertal timing displays wide, normally distributed variation in a healthy population of sexually maturing adolescents. However, like many complex traits, factors contributing to the variation are not well understood. Epigenetic regulation may contribute to some of the population variation. The role that epigenetics, specifically DNA methylation and histone acetylation, may play in regulating pubertal timing was investigated in C57BL/6 female mice: investigating whether population variation in pubertal timing among inbred mice could be explained by environmental factors; whether perturbing the epigenome using a histone deacetylase inhibitor or methyl-donor would alter pubertal timing; and examining genome-wide methylation patterns in hypothalami of early versus late maturing mice. Results demonstrate that measurable micro-environmental factors have only negligible effects on pubertal timing; pubertal timing was significantly altered by administration of epigenetic modifying agents; differences in methylation patterns are subtle. This initial evidence supports the involvement of epigenetic mechanisms in regulating pubertal timing.

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