Understanding the molecular composition of the synapse is one of the ongoing problems in neuroscience. The synaptic proteome consists of about 1100 proteins, most of which are organised into complexes. Understanding the composition and function of these complexes is important for understanding the mechanisms and pathways of diseases which these protein complexes are involved in determining the sub-cellular localisation of these proteins and also their spatial distribution in the nervous system may lead to important insights into the function of such protein complexes thus furthering our understanding of the function and diseases of the nervous system. Neuro-proteomics has led to identification of an NMDA receptor complex (also called MAGUK-associated signalling complex (MASC)) which has been extensively studied in mouse. It consists of ~186 proteins in mouse and represents 10 different functional classes of proteins. Drosophila offers an opportunity to study this complex in more detail because it is 50% less complex while still maintaining all the functional classes of proteins. Since the fundamental structure and principles of neuronal function are conserved between vertebrates and Drosophila the physiology and molecular biology of disease can be better resolved in this simple organism. Here I present an effort to characterise the MASC complex in Drosophila and present the molecular composition of the MASC complex in Drosophila. I will present an anatomical map of the complex in the fly brain and also show that there is significantly high conservation of this complex between Drosophila and mouse. Two different approaches have been used in this thesis to study the molecular composition of this complex give results which are in partial agreement with each other. Importantly it provides an evaluation of these two methods and indicates that they can be used to study molecular complexes in Drosophila.

While most Human papillomavirus (HPV) infections are asymptomatic and self-resolved, high-risk types, such as HPV-16 and HPV-18 are responsible for 99% of cervical cancers worldwide, whereas low-risk types, such as HPV-6 and HPV-11, are responsible for 90% of genital warts. While the different types of HPV and their varying oncogenicities have been studied extensively, it is still not clear what features of a HPV type make it more oncogenic than another. Two aspects which could affect the oncogenicity of HPV were studied: HPV variants and the E6 protein’s interaction with membrane associated guanylate kinase (MAGUK) proteins. Previous studies have shown that some HPV-16 variants may be more oncogenic than others. The first goal of this work was to characterise the HPV-16 variants in a Manitoba cervical cancer sample population to possibly identify mutations which could be associated with an increased risk of developing cervical cancer. Seventy-five samples from different individuals were sequenced in three distinct regions: the long control region and the E6 and E7 open reading frames. The DNA sequences obtained from these genomic regions were then compared between HPV-16 cervical cancer samples and Manitoba HPV-16 non cancer samples to identify any mutations that were exclusive to the cervical cancer samples. No specific mutations in any of the regions could be associated with cervical cancer. It is also proposed that HPV16 E6 protein’s interaction with MAGUK proteins contributes to its oncogenicity since low-risk E6 proteins lack this ability. The second goal of this work was to investigate which regions of high-risk HPV E6 proteins are needed in order to achieve MAGUK protein degradation, more specifically MAGI-1 degradation. Wild-type high-risk HPV16E6, low-risk HPV6E6, as well as mutants, were synthesized and cloned into vectors. In vitro translated proteins were used in MAGI-1 degradation assays. The ability of both wild-type HPV6 and HPV16 E6 proteins to degrade MAGI-1 was confirmed. Based on the performance of the different mutants in these degradation assays, it was determined that the PDZ-binding domain is necessary but not sufficient to induce E6-induced MAGI-1 degradation. In conclusion, it was determined that the entire HPV16 E6 protein is needed for the induction of MAGI-1 degradation.

L'objectif de ce travail a été de comprendre comment les canaux ioniques sont adressés, organisés et régulés dans des domaines spécialisés de la membrane plasmique des cardiomyocytes. Parmi les partenaires des canaux, les protéines MAGUK (Membrane Associated GUanylate Kinase) sont spécialisées dans l'ancrage, l'agrégation et la formation de complexes macromoléculaires à la membrane. J'ai caractérisé pour la première fois au niveau cardiaque une de ces protéines MAGUK, la protéine CASK. CASK est localisée à la membrane latérale des cardiomyocytes et exclue des disques intercalaires, zones de conduction privilégiée dans l'axe longitudinal. À la membrane latérale, la protéine CASK est exprimée au sein du complexe costamérique dystrophine/glycoprotéines. L'inhibition de CASK entraîne l'augmentation du courant sodique INa dans les HEK293 et les myocytes cardiaques. Dans les HEK293, la microscopie à onde évanescente (TIRF) et des expériences de biotinylation ont mis en évidence que cette augmentation du courant INa est associée à une augmentation du nombre de canaux NaV1.5 à la membrane. La microscopie de conductance ionique (SICM) couplée au patch clamp en configuration cellule attachée a permis de montrer que CASK retient les canaux sodiques au niveau des crêtes et prévient leur agrégation en clusters dans les T-tubules. Enfin, l'inhibition de CASK in vivo par une stratégie reposant sur l'utilisation de virus adéno-associés (AAV) est responsable d'un allongement de la durée de dépolarisation ventriculaire et de l'apparition d'une cardiopathie dilatée. / The aim of the thesis was to understand how ion channels are addressed, organized and regulated in specialized domains of the plasma membrane of cardiac myocytes. Among these partners, the MAGUK proteins (Membrane Associated GUanylate Kinase) are specialized in anchoring, aggregation and clustering of macromolecular complexes at the plasma membrane. In particular, characterized for the first time at the level of the hearth, one of these MAGUK proteins is the CASK protein. CASK is localized at the lateral membrane of cardiomyocytes, but excluded from intercalated disks which are privilege zones of the longitudinal axial conduction. At the lateral membrane, CASK protein is expressed among the costameric dystrophin/glycoproteins complex. CASK inhibition leads to the increase in sodium current density in HEK293 cells and in cardiomyocytes. In HEK293, evanescent wave microscopy (TIRF) and biotinylation experiments pointed out that the INa increase is associated to an increase in the number of NaV1.5 channels at the plasma membrane. Scanning ion conductance microscopy (SICM) coupled to cell-attached patch-clamp has demonstrated that CASK holds together sodium channels at the crest level and prevents their aggregation into clusters in the T-tubules. Finally, inhibition of CASK, in vivo, using an adeno-associated virus strategy resulted to an increase in duration of ventricular depolarization and to the appearance of dilated cardiomyopathy.

In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome. Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system. Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition. My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.

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Signaling pathways are complex and function to transmit signals from the extracellular environment into the cell. Analysis of results obtained from a high throughput siRNA screen led to the identification of Membrane protein palmitoylated 3 (MPP3) and Leukocyte Tyrosine Kinase (LTK) as novel negative regulators of the Wnt pathway. MPP3 is a MAGUK family protein and domain mapping studies indicated that the Guk domain plays a role in the negative regulation of the pathway. LTK, a receptor tyrosine kinase, has several transcript variants one of which lacks the entire kinase domain (LTK∆KD). While LTK∆KD interacted with the Wnt receptor Frizzled7, the full length LTK did not, suggesting distinct modes of pathway regulation. Analysis of neuronal cells, NIE115 and Neuro2a, demonstrated LTK is expressed and that cells are Wnt3a responsive, thereby providing a neuronal model system appropriate for further studies on the mechanism and biological role of LTK as a negative regulator of the Wnt pathway

Signaling pathways are complex and function to transmit signals from the extracellular environment into the cell. Analysis of results obtained from a high throughput siRNA screen led to the identification of Membrane protein palmitoylated 3 (MPP3) and Leukocyte Tyrosine Kinase (LTK) as novel negative regulators of the Wnt pathway. MPP3 is a MAGUK family protein and domain mapping studies indicated that the Guk domain plays a role in the negative regulation of the pathway. LTK, a receptor tyrosine kinase, has several transcript variants one of which lacks the entire kinase domain (LTK∆KD). While LTK∆KD interacted with the Wnt receptor Frizzled7, the full length LTK did not, suggesting distinct modes of pathway regulation. Analysis of neuronal cells, NIE115 and Neuro2a, demonstrated LTK is expressed and that cells are Wnt3a responsive, thereby providing a neuronal model system appropriate for further studies on the mechanism and biological role of LTK as a negative regulator of the Wnt pathway