Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Background: Insulin is a hormone released by the pancreatic beta cells in response to elevated levels of nutrients in the blood. Insulin triggers the uptake of glucose, fatty acids and amino acids into the liver, adipose tissue and muscles. Genes regulating insulin signalling are thus of vital importance for metabolic homeostasis and for preventing the development of diabetes. This thesis aims to identify new modifiers of insulin signalling, while carrying out functional studies of a homolog to human arsenite translocating ATPase, ASNA1. ASNA1 activates the insulin signalling pathway and promotes insulin secretion in mammalian cell lines and in Caenorhabditis elegans. A second aim is to better understand how ASNA1 and its interactors regulate sensitivity to the chemotherapeutic drug, cisplatin. Results: Regulators of insulin/IGF signalling (IIS) in C. elegans were identified based on the Larval arrest arrest aspect of the asna-1 depletion phenotype. Sixty-five genes were selected by virtue of their predicted interaction with ASNA-1 and screened for asna-1-like larval arrest upon inactivation of the genes . mrps-2, mrps-10, mrpl-43 encoding mitochondrial ribosomal protein subunits, and enpl-1 encoding an ER chaperone, GRP94 homolog were identified as the genes which when inactivated caused larval arrest without any associated feeding defects. IIS was weaker and insulin secretion was defective in these knockdown animals. ENPL-1 and ASNA-1 proteins interacted with one another both ex vivo and in vitro. ASNA-1 protein and mRNA level swere greatly reduced in enpl-1 mutants and enpl-1(-);asna-1(-) double-mutant worms displayed synthetic lethality. Overexpression of the insulins INS-4 and DAF-28 caused partial rescue of the germline phenotype of enpl-1 mutants, indicating that the phenotype of enpl-1 mutants was due at least in part to insufficient insulin levels. Studies of enpl-1 mutants also helped to understand the role of asna-1 in cisplatin sensitivity. The unfolded protein response (UPR) was induced in asna-1 and enpl-1 knockdown animals. enpl-1 mutants displayed higher sensitivity to cisplatin, when compared to asna-1 mutants and this correlated to higher UPR in enpl-1 knockdown animals. Pharmacological induction of the UPR in intrinsically cisplatin resistant wildtype worms also resulted in increased cisplatin sensitivity. This suggests that manipulation of ENPL-1 levels or of the UPR could enhance the anti-tumoral effects of cisplatin based cancer therapy. With a yeast two hybridscreen 27 putative physical interactors of ASNA-1 were identified. Amongst these candidate swas smn-1, which encodes survival of motor neuron protein homolog. RNAi knockdown of smn-1 caused a larval arrest phenotype similar to asna-1 depleted animals and smn-1 positively regulated IIS, like asna-1. Defects in IIS may be at the level of insulin release because neuropeptide secretion was impaired upon smn-1 knockdown. Further in vitro binding studies showed that SMN-1 and ASNA-1 interacted and inactivation of smn-1 in asna-1 mutants resulted in decreased viability. This implies that SMN-1 is another modifier of ASNA-1 and also a new component in IIS. Conclusion: With a directed RNAi screen and a yeast two hybrid screen several interactors of ASNA-1 that are also IIS modifiers were identified. ENPL-1 and SMN-1 are both involved in insulin release. We also found that induction of the UPR in enpl-1 and asna-1 mutants is a possible mechanism for increased sensitivity to cisplatin.

No description available.

Spinal Muscular Atrophy (SMA) is a neurodegenerative, inherited disease caused by an insufficient amount of functional Survival of Motor Neurone protein (SMN), though the exact mechanism underlying this is not fully understood. The primary function of SMN is assembling a ring of Sm proteins around small nuclear RNA (snRNA) in an early, cytoplasmic stage of small nuclear ribonucleoprotein (snRNP) biogenesis, a process essential in eukaryotes. SMN, together with several mRNA binding proteins, has been linked to neural transport of mRNA towards areas of growth in Motor neurons for local translation of transcripts. Previous research in our group has found that this may involve Coatomer protein-containing vesicles transported by Dynein and requiring the Sm family protein, SmB, for maintenance. Little is known, however, about what other proteins are also present and required for correct transport and localisation of these vesicles. To further investigate this, we have produced plasmids expressing each Sm protein tagged to fluorescent proteins to help track their behaviour, in some cases for the first time, and developed a detergent-free fractionation protocol to enrich for SMN containing vesicles, providing tools that can be used to further probe behaviour and interactions in the future. Using these approaches, SmN, a neural specific Sm protein, was identified to also be present in SMN-containing vesicles similarly to SmB. Analysis of the interactomes of different Sm proteins identified a novel interactor of SMN, Neurochondrin (NCDN), that appears to be required for the correct localisation of SMN in neural cells. NCDN was found to not associate with snRNPs, indicating an snRNP-independent interaction with SMN. NCDN and SMN both independently associated and co-enriched with Rab5, indicating a potential endocytic and cell polarity role for the interaction. This interaction has the potential to be key in SMA pathology and may have therapeutic potential.

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI<23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V. Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose. Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries. / On the aetiology of ALS: A comprehensive genetic study

No description available.

The development of new high throughput technologies able to multiplex disease biomarkers as well as advances in medical treatments has lead to the recent expansion of the newborn screening panel to include DNA-based targets. Four rare disorders; deletion 22q11.2 syndrome and Spinal Muscular Atrophy (SMA), Severe Combined Immunodeficiency (SCID) and Congenital Cytomegalovirus (CMV), are potential candidates for inclusion to the newborn screening panel within the next few years. The major focus of this study was to determine whether 5’-hydrolysis assays developed for the four distinct disorders with specific detection needs and analytical ranges could be combined on the OpenArray system and in multiplexed qPCR reactions. SNP detection of homozygous SMN1 deletions in SMA, CNV detection in the 22q11.2 critical region, and quantification of the SCID biomarker, T-cell receptor excision circles (TRECs) and CMV were all required for disease confirmation. SMA and 22q11.2 gene deletions were accurately detected using the OpenArray system, a first for the technology. The medium density deletion 22q11.2 multiplex successfully identified deletion carriers having either the larger 3 Mb deletion or the smaller 1.5 Mb deletions. Both TREC and CMV targets were detected but with a decrease in sensitivity when compared to their singleplex counterparts. Lastly, copy number detection of the TBX1 was performed when multiplexed with the TREC assay, without a decrease in detection limit of either assay. Here, we provide proof of principal that qPCR multiplexing technologies are amenable to implementation with a newborn screening laboratory.