L’hypertension artérielle pulmonaire (HTAP) est une maladie rare résultant de l’obstruction progressive des petites artères pulmonaires, via un remodelage de la paroi vasculaire, associée à une vasoconstriction entrainant une hypertrophie puis une défaillance cardiaque droite qui aboutit au décès du patient. Depuis 2013, 10 mutations, dans le gène KCNK3 (codant pour un canal potassique), ont été identifiées chez des patients atteints d’HTAP. Toutes les mutations identifiées entrainent une perte de fonction du canal. De plus, notre équipe a pu démontrer que la perte de KCNK3 est commune à toutes les formes d’HTAP : idiopathique et héritable ainsi qu’expérimentale. Durant ma thèse, mon projet principal a donc été d’étudier l’implication de la dysfonction de KCNK3 dans le développement de l’HTAP. Tout d’abord, nous avons pu démontrer que la perte d’expression/fonction de KCNK3 est également une caractéristique de l’hypertrophie / la dysfonction ventriculaire droite. Par la suite, en caractérisant un modèle unique de rats mutés pour le gène Kcnk3, nous avons pu démontrer que l'inactivation génétique de Kcnk3 chez le rat conduit à une altération vasculaire pulmonaire facilitant ainsi le développement d’une hypertension pulmonaire (HTP). Faisant de ce modèle un nouvel outil permettant de comprendre les mécanismes initiateurs de l’HTP et représenterai un outil pertinent pour développer des cibles thérapeutiques. Pour finir, nous avons montré que le développement d’une HTP due à une insuffisance cardiaque gauche (via une ligature du l’aorte) est facilitée chez nos rats mutés pour Kcnk3. / Pulmonary hypertension (PAH) is a rare disease characterized by a progressive obstruction of small pulmonary arteries, via a remodeling of arteries wall, associated with a vasoconstriction, which induce an hypertrophy of the right ventricle and then a right heart failure. Since 2013, 10 mutations have been identified in KCNK3 (coding for a potassium channel) gene of PAH patients. All mutations induce a loss of function of the channel. In previous work, our team demonstrated a loss of KCNK3 in all PAH forms: idiopathic, heritable and experimental. During my thesis, my project have been to study the involvement of KCNK3 dysfunction in the development of PAH. First, we have demonstrated that loss of KCNK3 function/expression is also a characteristic of right ventricle hypertrophy/dysfunction. Then, in our unique model of Kcnk3-mutated rats, we demonstrated that genetic inactivation of Kcnk3 in rats induce an alteration of lung vasculature facilitating pulmonary hypertension (PH) development. Making this model a new tool to understand mechanisms which induce PH and will eventually allow to develop new therapeutic targets. Finally, the development of a PH, secondary to left heart failure (via aorta stenosis) is facilitated in Kcnk3-mutated rats.

Indiana University-Purdue University Indianapolis (IUPUI) / Intervertebral discs (IVD) degenerate earlier than many other musculoskeletal tissues and will continue to degenerate with aging. IVD degeneration affects up to 80 percent of the adult population and is a major contributing factor to low back pain. Anti-sclerostin antibody is an FDA-approved treatment for osteoporosis in postmenopausal women at high-risk for fracture and, as a systemic stimulant of the Wnt/LRP5/β-Catenin signaling pathway, may impact the IVD. Stabilization of β-Catenin in the IVD increases Wnt signaling and is anabolic to the extracellular matrix (ECM), while deletion of β-catenin or LRP5 decreases Wnt signaling and is catabolic to the ECM. Here, we hypothesized that a reduction of Sost would stimulate ECM anabolism. Lumbar and caudal (tail) IVD and vertebrae of Sost KO and WT (wildtype) mice (n=8 each) were harvested at 16 weeks of age and tested by MRI, histology, immunohistochemistry, Western Blot, qPCR, and microCT. Compared to WT, Sost KO reduced sclerostin protein and Sost gene expression. Next, Sost KO increased the hydration of the IVD and the proteoglycan stain in the nucleus pulposus and decreased the expression of genes associated with IVD degeneration, e.g., heat shock proteins. However, deletion of Sost was compensated by less unphosphorylated (active) β-Catenin protein in the cell nucleus, upregulation of Wnt signaling inhibitors Dkk1 and sFRP4, and catabolic ECM gene expression. Consequently, notochordal and early chondrocyte-like cells (CLCs) were replaced by mature CLCs. Overall, Sost deletion increased hydration and proteoglycan protein content, but activated a compensatory suppression of Wnt signaling that may trigger chondrogenesis and may potentially be iatrogenic to the IVD in the long-term.

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Synapsins are primarily neuron-specific proteins critical for neurotransmission, synaptogenesis and synapse maintenance. Synapsin II has been specifically linked with increased susceptibility towards developing schizophrenia. Reduced synapsin II mRNA levels were found in the dorsolateral prefrontal cortex (PFC) of patients with schizophrenia. Moreover, synapsin II knockdown in the medial PFC (mPFC) of the adult rat was previously shown to cause schizophrenia-like behaviour and altered expression levels of vesicular proteins involved in glutamatergic and GABAergic signaling within the mPFC. The study of schizophrenia in recent years has shifted to focus on neurodevelopmental players which influence disease outcome. This study was designed to establish the link between neurodevelopmental dysregulation of synapsin II and schizophrenia. Specific knockdown of synapsin II was performed in the mPFC at postnatal day (PD) 7 and PD 17-23. Schizophrenia-like behavioural abnormalities were assessed at pre-pubertal (PD 32-35) and post-pubertal (PD 65-70) stages. Protein estimation of vesicular transporters involved in glutamate, GABA, and dopamine neurotransmitter systems were also assessed in the mPFC. Results from this study indicate (1) synapsin II knockdown during PD 17-23, but not PD 7, caused lasting schizophrenia-like abnormalities (2) abnormalities exhibited permanence at pre-pubertal and post-pubertal stages, and manifested as a function of brain development, (3) behavioural abnormalities were reminiscent of symptoms in established animal models of schizophrenia (i.e. deficits in prepulse inhibition, social withdrawal, locomotor hyperactivity), (4) neurodevelopmental synapsin II alterations induced hypoactive glutamatergic activity through decreased synapsin IIa expression levels (pre-pubertal) and decreased VGLUT-2 expression levels (post-pubertal), and (5) acute olanzapine treatment effectively attenuated schizophrenia-like abnormalities through normalized synapsin IIa expression levels (pre-pubertal) and increased GAD65/67 expression levels (post-pubertal). Results show the causal link between synapsin II expression during critical neurodevelopmental stages and schizophrenia. Additionally, evidence has been provided for the face, construct, and predictive validities of this newly developed animal model of schizophrenia. / Thesis / Doctor of Philosophy (PhD)

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Osteoactivin (OA) is a type I transmembrane glycoprotein initially identified in bone in 2002. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. To determine the role of OA in skeletal homeostasis in vivo. we utilized a mouse model with a natural mutation in the osteoactivin gene. This mutation is due to a premature stop codon, which results in the generation of a truncated 150 amino acid OA protein. This animal, which we will refer to as OA mutant, was shown by ìCT and histomorphometric analysis to have increased bone volume, trabecular thickness, and trabecular number compared to wild-type (WT) mice at 4 weeks of age, which is a time at which bone formation is most active. Histological analysis of long bones stained with TRAP (tartrate resistant acid phosphatase) and colorimetric analysis of serum TRAP 5b levels indicated that the numbers of osteoclasts are significantly increased in OA mutant samples. Interestingly, although the numbers of osteoclasts as compared to WT were higher in OA mutant mice, serum levels of C-telopeptide of type I collagen (CTX) and osteocalcin, biomarkers for bone resorption and bone formation respectively, were significantly decreased. These data suggested that in mice the presence of truncated OA protein results in increased osteoclast number, but that they are inefficient in resorbing bone and may in part contribute to the increase in bone volume in OA mutant mice in vivo. To further investigate the role of OA in osteoclast differentiation, osteoclasts were differentiated from hematopoietic stem cell progenitors ex vivo. HSCs were cultured in the presence of 50 ng/ml of M-CSF for two days and then with M-CSF and 100 ng/ml of RANKL in the presence or absence of 50 ng/ml recombinant OA. We observed a dramatic increase in multinucleated TRAP-positive osteoclasts and the number of nuclei per osteoclast in OA-treated cultures compared to control. Additionally, analysis of HSCs showed increased cell proliferation in response to exogenous OA treatment. When osteoclasts were differentiated in ex vivo cultures derived from OA mutant and WT mice, we observed decreased osteoclast number, size, and function in OA mutant compared to WT cultures. This decrease was abrogated when cultures were treated exogenously with recombinant OA. Quantitative PCR analysis of RNA isolated during osteoclast differentiation from WT and OA mutant mice reveal decreased gene expression of critical osteoclast differentiation and functional markers, which explains the osteoclast defect observed ex vivo. To investigate the role of OA in osteoblast differentiation, primary osteoblasts were derived from mesenchymal progenitors isolated from calvariae of WT and OA mutant neonatal pups. OA mutant osteoblasts were found to have decreased alkaline phosphatase (ALP) staining and activity at day 14 in culture. Furthermore when cultures were differentiated to 21 days to simulate matrix mineralization in vitro, OA mutant osteoblasts exhibited decreased Alizarin Red and Von Kossa staining. Quantitative measurement of calcium also showed decreased mineral deposition in OA mutant mice compared to WT. Electron microscopic and protein studies were able to eliminate the notion of ER stress or cell toxicity as a result of ER stress playing a role in the delayed osteoblast differentiation observed in OA mutant osteoblasts. Furthermore, OA mutant osteoblasts exhibited decreased proliferation and survival ex vivo. These data reveal an effect of osteoactivin in osteoblasts ex vivo. This study provided an in vivo tool to study the role of osteoactivin in bone cells and the regulation of bone formation and bone resorption by this molecule. Taken together, these findings suggest that the presence of truncated OA leads to increased bone volume due to defective interplay between bone-resorbing osteoclasts and bone-forming osteoblasts. Data presented here support the notion of osteoactivin as a novel molecule in modulating skeletal homeostasis in vivo. / Cell Biology

As environmental change continues and increases, understanding how species will respond to change and how these responses may affect populations will be important for conserving and managing species. Red-cockaded Woodpeckers (Picoides borealis) are well-studied and provide an ideal system in which to examine ecological and evolutionary questions related to life histories because monitoring them accurately is relatively easy, their behavior is well-described and structured. In this study, I examined the following questions using long-term data (1980-2013) from two study sites in North Carolina: 1) what traits contribute most to lifetime fitness, 2) how have traits changed over time and how is climate change influencing life history, and 3) to what extent are traits that contribute to fitness and that are changing over time heritable in this species. I found that a multitude of factors contribute to different aspects of fitness, including: parental age and hatch date affecting survival to year one and probability of attaining breeding status; and lay date, clutch size, age at first reproduction, and variance in clutch size affecting lifetime fitness. I also found that many traits were changing over time including lay date, clutch size, partial brood loss, and survival to year one. These traits were strongly influenced by local climate variables at each study site, but it is not clear that climate has changed over time at the study sites to account for all the observed changes in life history traits. Habitat improvement has also played a role as evidenced by increased fledgling production in terms of raw numbers (without accounting for covariates). I also found that lay date, clutch size, and partial brood loss had low heritabilities after accounting for other random and fixed effects. These results indicate that Red-cockaded Woodpeckers at these two study sites are shifting traits successfully in response to changing conditions, and that these changes are in the direction that increases aspects of fitness. These shifts indicate that individuals are plastic with respect to these traits, but most of the variance in traits was related to external habitat-associated factors rather than additive genetic variance or environmental × genotype interactions. / Ph. D.