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A holy grail in neuroscience is being able to control plasticity to facilitate recovery from insult in the adult brain. Despite success in animal models, few therapies have translated from bench to bedside. This thesis is aimed at addressing 2 major stumbling blocks in translation. The first gap is in our understanding of the mechanisms of plasticity-enhancing therapies, and the second is in our understanding the relevance of those mechanisms for human development. In chapters 2 and 3, I address the first gap by asking whether fluoxetine, a selective serotonin reuptake inhibitor, which reinstates juvenile-like plasticity in adult animals, reinstates a juvenile-like synaptic environment. We found evidence to suggest that fluoxetine is neuroprotective, as it rescued all of the MD-driven changes, but surprisingly we found no evidence that fluoxetine recreated a juvenile-like synaptic environment, with the exception of Ube3A. Ube3A is necessary for critical period plasticity, indicating that Ube3A may play a crucial in enhancing plasticity in the adult cortex. In chapter 4, I address whether D-serine, an amino acid that has similar effects to fluoxetine in terms of both plasticity and anti-depression, shares a common neurobiological signature with fluoxetine. I found that D-serine’s effects were strikingly similar to fluoxetine, with respect to markers of the E/I balance, indicating that it may be an effective alternative to fluoxetine. In chapter 5, I address the second gap by studying the development of 5 glutamatergic proteins in human V1. Some changes occurred early, as would be predicted from animals studies, while other changes were protracted, lasting into the 4th decade. These results will help guide the use of treatments, like fluoxetine, which effect glutamatergic proteins. iv Together the findings in this thesis significantly advances our understanding of the mechanisms involved in restating plasticity in the adult cortex, and their relevance to humans. / Dissertation / Doctor of Philosophy (PhD) / Neurons change to rewire, adapt, and recover. This plasticity is greatest early in development, so much research has focused on bringing it back in adults. There has been amazing progress in animal models, but this has not translated to humans. Two reasons for this are that we do not fully understand the mechanisms of these treatments in animals or whether those mechanisms are relevant for humans. My thesis addresses this by studying how 2 treatments, fluoxetine and D-serine, affect proteins that are important for plasticity, and how those proteins develop in the humans. I found that these treatments are neuroprotective, but do not recreate a younger state. One interesting standout is an increase in Ube3A, which is essential for juvenile plasticity. I also found that much of human development is similar to animals, but the time course for some proteins is uniquely prolonged in humans. These findings have implications for the use of plasticity-enhancing treatments at different ages.

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Metabolism of D-amino acids is of considerable interest due to their key importance in cellular functions. The enzymes D-serine dehydratase (DSD) and D-cysteine desulfhydrase (DCyD) are involved in the degradation of D-Ser and D-Cys, respectively. We determined the crystal structure of Salmonella typhimurium DSD (StDSD) by multiple anomalous dispersion method of phasing using selenomethione incorporated protein crystals. The structure revealed a fold typical of fold type II PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild type StDSD (WtDSD) and selenomethionine labeled StDSD (SeMetDSD), significant electron density was not observed for the co-factor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other fold type II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modeling suggested that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β-elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates. Salmonella typhimurium DCyD (StDCyD) is a fold type II PLP-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. We determined the crystal structure of StDCyD using molecular replacement method in two different crystal forms. The better diffracting crystal form obtained in presence of benzamidine illustrated the influence a small molecule in altering protein interfaces and crystal packing. The polypeptide fold of StDCyD consists of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) which resemble other fold type II PLP-dependent enzymes. X-ray crystal structures of StDCyD were also obtained in the presence of substrates, D-Cys and βCDA, and substrate analogs, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS). The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggested formation of PMP by the hydrolysis of cycloserines. Mutational studies suggested that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, we proposed a probable mechanism for the degradation of D-Cys by StDCyD. The acid-induced arginine decarboxylase (ADC) is part of an enzymatic system in Salmonella typhimurium that contributes to making this organism acid resistant. ADC is a PLP-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced StADC to 3.1 Å resolution. StADC structure revealed an 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevent inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.

Le cortex préfrontal (PFC) est le principal locus des perturbations dans l’activité des réseaux de neurones chez les schizophrènes. Ces perturbations résultent d’une dérégulation des interactions entre le système dopaminergique et le système glutamatergique dont l’origine demeure inconnue. Il est acquis que les cellules gliales détectent et intègrent les signaux synaptiques, et libèrent différentes substances neuroactives comme la D-sérine. Cet acide aminé est aujourd’hui reconnu comme le coagoniste endogène des récepteurs au glutamate de type NMDA dans de nombreuses aires cérébrales. Mon travail de thèse est centré sur le rôle de la d-sérine dans la transmission synaptique excitatrice glutamatergique dans le PFC du rongeur adulte et dans la gouvernance des interactions entre systèmes glutamatergique et dopaminergiques J’ai tout d’abord montré en utilisant des enregistrements électrophysiologiques sur tranches que la d-sérine est le coagoniste des récepteurs NMDA synaptiques dans les couches V/VI du PFC. Cet acide aminé est synthétisé par les astrocytes et contrôle l’induction de la potentialisation à long terme. D’autre part, j’ai montré que la dopamine exerce un effet biphasique sur l’activité des récepteurs NMDA synaptiques et sur l’excitabilité des neurones pyramidaux des couches V/VI du PFC et ce en contrôlant la libération de d-sérine. Une approche pharmacologique sélective a permis de mettre en évidence le rôle des récepteurs D1 dans les effets potentialisateurs et le rôle des récepteurs D2/D3 dans les effets inhibiteurs de la dopamine. Mon travail démontre que les astrocytes arborent des récepteurs à la dopamine qui contrôlent la libération de la d-sérine. / The prefontal cortex (PFC) is the main locus where dysfunctions of neuronal networks are evident in schizophrenia. These dysfunctions are caused by an impairment of cross-talk between dopaminergic and glutamatergic systems whose origin is unknown. It is now accepted that glia detect and integrate synaptic signals and then release many neuroactive substances such as D-serine. This amino acid is now considered to be the endogenous coagonist of the NMDA subtype receptors for glutamate in many brain areas. My PhD work focuses on the functions of d-serine in glutamatergic excitatory synaptic transmission in the PFC of adult rodent and in governing the interactions between dopaminergic and glutamatergic systems. First, using electrophysiological recordings on brain slices, I have shown that d-serine is the coagonist of synaptic NMDA receptors in layers V/VI of PFC. This amino acid is synthesized by glia and is crucial for the induction of long term potentiation. In addition, I have shown that dopamine has a bell-shape effect on the activity of synaptic NMDA receptors and on the excitability of excitatory pyramidal neurons by controlling the release of d-serine. The use of specific pharmacological tools allowed me to show the potentiating effects of dopamine are mediated by D1 receptors whereas the inhibitory effects are due to the activation of D2/D3 receptors. Finally, my work highlights the presence of functional dopaminergic receptors on astrocytes that modulate the release of d-serine in the PFC, thus impacting NMDA receptor activity.

The main purpose of the master thesis project is to investigate the influence of selected serine protease inhibitors (SPI) on the catalytic action of the serine proteases chymotrypsin and trypsin, in a conjugated and non-conjugated state. The inhibitors included for this study were extracted from Solanum tuberosum, i.e.common potato. The purification method included in this study consist of crude extraction by mixer, followed by a salt-out procedure with ammonium sulphate. Further purification steps were cation exchange chromatography and, finally, gel filtration to obtain SPI of high purity. The purified sample was then characterized by SDS-page and kinetic activity measurement of trypsin and chymotrypsin action on synthetic substrate derivate, N-Benzoyl-DL-arginine-4-nitroanilide hydrochloride (BAPA) and N-Succinyl-L-phenylalanine-p-nitroaniline (SFpNA) respectively. The characterization showed inhibitory inactivation of both pancreatic proteases. This would indicate successful extraction of SPI. To investigate inhibitory action in a conjugated state, either enzyme or inhibitor was immobilized onto aluminium oxide membranes. Then two different experimental setups were tested, called experiment 1 and 2. In experiment 1, the inhibitor was immobilized and the interaction was monitored from a retention shift of enzyme flow-through compared to a blank column, using detection at 280 nm of the enzyme. In experiment 2 the enzyme was instead immobilized and a mixture of inhibitor and substrate was circulated with monitoring of the catalytic activity. The main goal was thus to measure the effects on the kinetics in the conjugated state compared to enzyme and inhibitor in the free state. The result from both experiment 1 and 2 did not yield consistent and reliable result so the discussed method should be regarded as preliminary results. The study also includes investigation of inhibitor-enzyme interaction as revealed by molecular mass data to determine complex formation. This part was conducted with static light scattering analysis to determine the stoichiometry for the interaction between pancreas proteases and the inhibitor. Results from light scattering showed promising indication of many-to-one interaction between enzyme and inhibitor, which have been seen by previous studies. It should be considered a preliminary result as complex formation does not exclude aggregation of enzymes or inhibitor in the solution.

Résumé: Chaque année, les épidémies saisonnières d’influenza causent de 3 à 5 millions de cas sévères de maladie, entraînant entre 250 000 et 500 000 décès mondialement. Seulement deux classes d’antiviraux sont actuellement commercialisées pour traiter cette infection respiratoire : les inhibiteurs de la neuraminidase, tels que l’oseltamivir (Tamiflu) et les inhibiteurs du canal ionique M2 (adamantanes). Toutefois, leur utilisation est limitée par l’apparition rapide de résistance virale. Il est donc d’un grand intérêt de développer de nouvelles stratégies thérapeutiques pour le traitement de l’influenza. Le virus influenza dépend de l’activation de sa protéine de surface hémagglutinine (HA) pour être infectieux. L’activation a lieu par clivage protéolytique au sein d’une séquence d’acides aminés conservée. Ce clivage doit être effectué par une enzyme de l’hôte, étant donné que le génome du virus ne code pour aucune protéase. Pour les virus infectant l’humain, plusieurs études ont montré le potentiel de protéases à sérine transmembranaires de type II (TTSP) à promouvoir la réplication virale : TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 et matriptase, identifiée récemment par notre équipe (Beaulieu, Gravel et al., 2013), activent l’HA des virus influenza A (principalement H1N1 et H3N2). Toutefois, il existe peu d’information sur le clivage de l’HA des virus influenza B, et seulement TMPRSS2 et HAT ont été identifiées comme étant capables d’activer ce type de virus. Les travaux de ce projet de maîtrise visaient à identifier d’autres TTSP pouvant activer l’HA de l’influenza B. L’efficacité de clivage par la matriptase, hepsine, HAT et Desc1 a été étudiée et comparée entre ces TTSP. Ces quatre protéases s’avèrent capables de cliver l’HA de l’influenza B in vitro. Cependant, seul le clivage par matriptase, hepsine et HAT promeut la réplication virale. De plus, ces TTSP peuvent aussi supporter la réplication de virus influenza A. Ainsi, l’utilisation d’un inhibiteur de TTSP, développé en collaboration avec notre laboratoire, permet de bloquer significativement la réplication virale dans les cellules épithéliales bronchiques humaines Calu-3. Cet inhibiteur se lie de façon covalente et lentement réversible au site actif de la TTSP par un mécanisme slow tight-binding. Puisque cet inhibiteur cible une composante de la cellule hôte, et non une protéine virale, il n’entraîne pas le développement de résistance après 15 passages des virus en présence de l’inhibiteur dans les cellules Calu-3. L’inhibition des TTSP activatrices d’HA dans le système respiratoire humain représente donc une nouvelle stratégie thérapeutique pouvant mener au développement d’antiviraux efficaces contre l’influenza. / Abstract: Seasonal influenza epidemics cause between 3 and 5 millions severe cases of disease, leading to 250 000 to 500 000 deaths worldwide. Only two classes of drugs are currently available to treat influenza infections: neuraminidase inhibitors, such as oseltamivir (Tamiflu) and M2 channel inhibitors (adamantanes). However, the use of these antivirals is restricted by rapid emergence of viral resistance. It is therefore of great interest to develop new therapeutic strategies for the treatment of influenza disease. The influenza virus requires activation of its surface protein hemagglutinin (HA) to become infectious. This activation is achieved by proteolytic cleavage in a highly conserved amino acid sequence of the protein. Host cell proteases are responsible for this cleavage since the viral genome doesn’t encode any protease. For viruses that infect humans, many studies have shown the potential of type II transmembrane serine proteases (TTSP) to promote viral replication: TMPRSS2, TMPRSS4, HAT, MSPL, Desc1 and matriptase, recently identified by our team (Beaulieu, Gravel et al., 2013), activate HA of influenza A viruses (mainly H1N1 and H3N2). However, little is known about cleavage of influenza B virus HA, and only TMPRSS2 and HAT have been identified as being capable of activating this type of virus. This project aimed to identify other TTSPs able to activate influenza B HA. Cleavage efficacies of matriptase, hepsin, HAT and Desc1 were studied and compared. These four proteases were shown to be able to cleave influenza B HA using in vitro assays. However, only cleavage by matriptase, hepsin and HAT promoted viral replication. Moreover, these TTSPs also supported the replication of influenza A viruses. Thus, the use of a slow, tight-binding inhibitor (developed in collaboration with our laboratory) that binds to the TTSP active site, forming a covalent and reversible bond, significantly blocked viral replication in human bronchial epithelial Calu-3 cells. Since this inhibitor targets a host cell component, instead of a viral protein, viruses did not develop resistance after 15 passages in presence of the inhibitor in Calu-3 cells. Thus, inhibition of HA-activating TTSPs in the human respiratory tract represents a novel therapeutic strategy against influenza.

La capacité d'adaptation des bactéries à leur environnement repose, entre autres choses, sur des mécanismes de transduction du signal. Ces mécanismes leur permettent de percevoir la nature et les modifications du milieu dans lequel elles évoluent et d'adapter en conséquence leur métabolisme et leur physiologie. L'un de ces mécanismes identifié chez les procaryotes repose sur un processus de régulation impliquant, suite à un stimulus extérieur, une modification réversible des protéines au niveau de résidus séryles et thréonyles par phosphorylation via les sérine/thréonine protéine-kinases (STPK). Chez les bactéries pathogènes, notamment Mycobacterium tuberculosis et Staphylococcus aureus, les STPKs sont impliquées dans la régulation du métabolisme central, de la division cellulaire, de la composition de la paroi et de la virulence. Mes travaux de thèse ont eu pour objectif d'approfondir les connaissances sur la régulation post-traductionnelle des protéines via les STPKs chez ces deux pathogènes humains. Nous avons ainsi identifié de nouveaux substrats des STPKs chez M. tuberculosis et S. aureus et caractérisé l'effet de la phosphorylation sur l'activité de ces substrats. L'ensemble de mes travaux de thèse met en avant le rôle important de la régulation par les STPKs de voies métaboliques diverses chez ces deux pathogènes. / To overcome the stressful conditions imposed during host infections, pathogens have evolved various protective and offensive responses that could be achieved through cascades of phosphorylation. Many of the encountered external stimuli are transduced via sensor kinases embeded within the bacterial membrane, allowing the pathogen to adapt and survive in hostile environments. In addition to the classical two-component systems, Staphylococcus aureus and Mycobacterium tuberculosis possess eukaryotic-like Serine/Threonine Protein-Kinases (STPK). It is becoming clear that in these two human pathogens, many of the STPKs are involved in the regulation of metabolic processes, division, cell wall composition, virulence, etc. Therefore, signalling through STPK phosphorylation has recently emerged as a key regulatory mechanism in pathogenic bacteria. Thus, to investigate the mechanisms of STPK-dependent regulation in M. tuberculosis and S. aureus, we identified and characterized novel endogenous phosphorylated substrates, and analyzed the impact of phosphorylation on their specific activity. Overall, the results presented herein emphasize the important role of STPK-dependent mechanisms in various metabolic pathways in these two pathogens.

Ce travail porte sur l’étude des mécanismes de phosphorylation des protéines par les sérine/thréonine kinases chez Staphylococcus aureus. Nous avons, tout d’abord, mis en évidence et caractérisé une seconde Ser/Thr-kinase, nommée Stk2. Cette kinase présente peu d’homologies avec les autres Ser/Thr-kinases bactériennes décrites à ce jour, en particulier avec la première Ser/Thr-kinase mise en évidence précédemment chez S. aureus, Stk1. Nous avons ensuite caractérisé dix sites d’autophosphorylation de Stk2 et nous avons montré que trois sites sont nécessaires à son activité. Enfin, nous avons montré que le régulateur global de virulence, SarA, est phosphorylé à la fois par Stk1 et Stk2. La phosphorylation de SarA influence sa capacité de liaison à l’ADN. Cette étude contribue à mieux appréhender, au niveau moléculaire, le rôle des Ser/Thr-kinases dans le métabolisme des bactéries et, plus particulièrement, dans la régulation de leur virulence / We report that protein phosphorylation on serine and threonine is required for controlling staphylococcal virulence. We identified and characterized a second serine/threonine kinase, Stk2, in S. aureus. Biochemical analyses revealed that this enzyme displays autokinase activity on both threonine and serine residues. Stk2 is atypical in the sense that it exhibits a weak similarity with the first Ser/Thr-kinase previously detected, Stk1, and its undergoes a different mechanism of activation compared to the other bacterial Ser/Thr-kinases described so far. We also showed that SarA, a major transcription factor that regulates more than a hundred virulence genes, is phosphorylated by both Stk1 and Stk2. Phosphorylation of SarA leads to strong effects on its ability to bind DNA. The study of Stk1 and Stk2, at the molecular level, provides a better understanding of the role of these staphylococcal Ser/Thr-kinases in bacterial metabolism and, in particular, in the regulation of virulence