Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of human senile dementia. Alzheimer’s represents a significant public health concern, having widespread social and economic implications. Consequently, protocols for early detection and therapeutic intervention (both behavioral and pharmacologic) constitute important targets for medical investigation. Furthermore, contemporary research depends upon comprehensive neurobehavioral assessment and advanced statistical and computational analytic methodologies for characterizing AD-associated sensorimotor and cognitive impairment, as well as evaluating therapeutic efficacy. This dissertation introduces data mining-based techniques (decision trees, neural networks, support vector machines) for behavioral analysis in both nontransgenic and Alzheimer’s transgenic mice, to evaluate the cognitive benefits of long-term caffeine treatment. Both treatment and transgenic effects are identified through advanced statistical (discriminant analysis) and data mining approaches. In addition, a novel mouse-based cognitive assessment paradigm, adapted from a human interference learning AD-diagnostic protocol, is implemented to evaluate both genetic (GRK5) and therapeutic (GM-CSF) effects in mice, against an Alzheimer’s transgenic background. Data mining techniques are shown to be comparable to con ventional statistical analyses, often providing complementary diagnostic information. Indeed, comparisons between data mining-based and multivariate statistical analyses, with respect to groupwise discriminability, support the use of both methodologies in neurobehavioral research. Future work involving both data mining-based and multivariate statistical analyses of cognitive-behavioral data is discussed, emphasizing the need for longitudinal studies, repeated-measure designs, and spatiotemporal modeling for evaluating the time-course of both human AD and AD-like pathology in transgenic mouse models.

Rationale: Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor (GPCR) kinase (GRK) 5 is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and upregulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium- calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of NFAT. Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts (CFs), however its pathophysiological role in these crucial cardiac cells is unknown. Objective: The aim of this study is to elucidate the role of GRK5 in the activation of cardiac fibroblasts in vitro and cardiac fibrosis after injury in vivo. Methods and Results: We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits Angiotensin II (AngII) mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice decreased fibrosis and cardiac hypertrophy after chronic AngII infusion compared to non-transgenic littermate controls (NLCs). Fibroblast-specific deletion of GRK5 was also protective in mice after ischemic injury as they presented with preserved systolic function, decreased fibrosis, and decreased hypertrophy compared to NLCs. Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. Conclusions: We present novel data demonstrating that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrates the potential beneficial effects of GRK5 inhibition in the context of cardiac disease. / Biomedical Sciences

Arginine vasopressin (AVP) is a hypothalamic nonapeptide which regulates the hypothalamic-pituitary-adrenal axis response to stress by stimulating the secretion of adrenocorticotropin (ACTH) from corticotroph cells of the anterior pituitary. This effect is mediated by binding of AVP to the pituitary vasopressin receptor (V1bR). The V1bR belongs to the G protein-coupled receptor (GPCR) super family. Repeated stimulation of anterior pituitary cells with AVP has been shown to produce a loss of responsiveness to subsequent AVP stimulation. This phenomenon appears to be mediated by desensitisation of the V1bR, and may be due to phosphorylation of the receptor by G protein-coupled receptor kinase 5 (GRK5). The aim of this research was to establish and validate methods that would allow the role of GRK5 in the desensitisation of V1bR to AVP stimulation to be investigated. As no isoform specific inhibitors for GRK5 were available, HEK293 cells transiently transfected with the rat V1bR were used as a model system for this research. This allowed RNA interference (RNAi) to be used to knockdown GRK5 expression. The protocol for RNAi-mediated knockdown of GRK5 was established as part of this research. Protocols for Western blotting and qRT-PCR were also established to allow the RNAi-mediated knockdown of GRK5 protein and mRNA to be measured. Transfection of HEK293 cells with 10nM GRK5-targeting small interfering RNAs (siRNAs) reduced the expression of GRK5 protein to 53.4% ± 3.4% (mean ± SEM) of that seen in untreated control cells at 84 hours after transfection, while GRK5 mRNA levels were reduced to 28.7% ± 1.9% (mean ± SEM) of that of control cells 48 hours after transfection. An experimental protocol was designed in this research that would coordinate the RNAi-mediated knockdown of GRK5 with transient transfection of the HEK293 cells with the rV1bR. Since, activated V1bRs couple to Gq/11 and stimulate the production of inositol phosphates (IPs), the responsiveness of the V1bR can be determined by measuring the accumulation of [H³]-IPs in cells labelled with [H³]-myo-inositol. In the protocol designed, the effect of GRK5 knockdown on V1bR desensitisation is determined by stimulating HEK293 cells expressing the rV1bR (and previously transfected with GRK5-targeting siRNA) with 0nM or 100nM AVP for 0, 5, 15, 30 or 60 minutes, and comparing the accumulation if IPs over time with that of cells that are not transfected with GRK5-targeting siRNA. This protocol can be used in future to investigate the role of GRK5 in V1bR desensitisation, and may be adapted to determine if other GRK isoforms are involved in V1bR desensitisation.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.