This thesis investigates the functional consequences of neutralising the negative charges on the bacteriophage T7 antirestriction protein ocr. The ocr molecule is a small highly negatively charged, protein homodimer that mimics a short DNA duplex upon binding to the Type I Restriction Modification (RM) system. Thus, ocr facilitates phage infection by binding to and inactivating the host RM system. The aim of this study was to analyse the effect of reducing the negative charge on the ocr molecule by mutating the acidic residues of the protein. The ocr molecule (117 residues) is replete with Asp and Glu residues; each monomer of the homodimer contains 34 acidic residues. Our strategy was to begin with a synthetic gene in which all the acidic residues of ocr had been neutralised. This so called ‘positive ocr’ (or pocr) was used as a template to gradually reintroduce codons for acidic residues by adapting the ISOR strategy proposed by D.S.Tawfik. After each round of mutagenesis an average of 4-6 acidic residues were incorporated into pocr. In this fashion a series of mutant libraries in which acidic residues were progressively introduced into pocr was generated. A high-throughput in vivo selection assay was developed and validated by assessing the antirestriction behaviour of a number of mutants of the DNA mimic proteins wtOcr and Orf18 ArdA. Further to this, selective screening of the libraries allowed us to select clones that displayed antirestriction activity. These mutants were purified and in vitro characterisation confirmed these mutants as displaying the minimum number of acidic residues deemed critical for the activity of ocr. This in vitro process effectively simulated the evolution of the charge mimicry of ocr. Moreover, we were able to tune the high-throughput assay to different selection criteria in order to elucidate various levels of functionality and unexpected changes in phenotype. This approach enables us to map the “in vitro” evolution of ocr to identify acidic residues that are required for protein expression, solubility and function proceeding to a fully functional antirestriction protein.

CD4⁺ T cells are a critical component of the adaptive immune compartment. Each T cell expresses a clonotypic T cell receptor (TCR) that must discriminate between self and foreign peptides presented in major histocompatibility molecules (pMHC) on the surface of antigen presenting cells to direct T cell fate decisions. Information regarding TCR-pMHC interactions must then be transmitted to the TCR-associated CD3 signaling modules, which contain ITAMs that serve as signaling substrates for Src kinases. The Src kinase, Lck, is recruited to the pMHC-bound TCR-CD3 complex via association with the CD4 coreceptor that binds MHCII. It is therefore through the coordinated interactions within the TCR-CD3-pMHC-CD4 macro-complex that productive TCR signaling can occur to inform T cell activation and fate decisions. While much is known regarding the structure of the individual subunits that make up the TCR-CD3-pMHC-CD4 macro-complex, there is little information regarding how these components come together to initiate TCR signaling and determine functional outcomes. Here, we have interrogated how interaction of these individual components leads to productive T cell activation. Specifically, we interrogated the nature of TCR-MHC interactions and provide evidence that there is intrinsic specificity of the TCR for MHCII. We have also built mouse models to determine the role of TCR-CD3 interactions and TCR dimerization in the transmission of information from the TCR to the CD3 subunits following TCR-pMHC engagement. Finally, we show that both the CD4 transmembrane and extracellular domains contribute to T cell activation in vitro. Overall, this work provides insight into how the constituents of the TCR-CD3-pMHC-CD4 macro-complex interact to initiate T cell fate and function.

No description available.

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

Healthy pregnancy requires a tightly regulated materno-fetal dialogue for processes such as embryo implantation, endometrial decidualisation (in the mouse), placentation and maternal adaptation to occur. Disruption of placental development as well as maternal adaptation can lead to fetal intrauterine growth restriction (IUGR) which increases the risk of late miscarriage/stillbirth (e.g. 53% of preterm stillbirth and 26% of term stillbirth are found to be IUGR). Furthermore, IUGR is a risk factor for neurodevelopmental conditions in childhood and for a spectrum of related adult health disorders such as cardiovascular disease and type II diabetes, often termed metabolic syndrome. Despite these pregnancy disorders being common (e.g. 1 in 200 pregnancies results in stillbirth in the UK) the molecular lesion(s) underlying their pathophysiology are poorly understood and in particular those with placental and/or maternal aetiologies most frequently remain unexplained. Here we investigate the hypothesis that poly(A)-binding protein 4 (PABP4) is required for healthy pregnancy in mice. PABP4 is an RNA-binding protein and a member of the PABP family which are central regulators of mRNA translation and stability. Using all four permutations of wild-type and knock-out crosses, we find that maternal PABP4-deficiency results in a reduced litter size and IUGR. The number of implantations at e8.5 were not reduced in Pabp4-/- females, implying that the reduced litter size was not a consequence of decreased ovulation, fertilisation or implantation frequency. Further longitudinal analysis (at e13.5, e15.5 and e18.5) reveals that fetal death primarily occurred between e18.5 and birth, suggesting these mice may provide a unique opportunity to inform on the maternal causes of stillbirth. The onset of IUGR, which was found to be symmetrical in nature, was established by e15.5 preceding the majority of fetal death. During pregnancy, a materno-fetal dialogue directs and responds to changes in gene expression to give rise to the placenta and adapt the maternal physiology. Defects in these processes may result in reduced growth and/or fetal death and were examined in Pabp4-/- mice to shed light on the mechanistic basis of these related phenotypes. Fetal to placental (F:P) weight ratio, whose changes can be indicative of placental insufficiency or placental adaptation in an attempt to aid fetal growth, was found to be increased in Pabp4-/- dams at e15.5 and e18.5 due to the presence of IUGR fetuses with placentas of normal weight. Consistent with this observation, placental volume was unchanged at e18.5. Total placental weight and volume alone fails to discriminate potential differences in the individual placental zones which include the labyrinth zone, where materno-fetal gas and nutrient exchange occur; the junctional zone, which has endocrine functions including those that promote maternal adaptation; and the decidua basalis, derived from the maternal endometrium and is the site of trophoblast invasion and maternal vascular remodelling in early pregnancy. Therefore, volumetric analysis of these zones and the maternal blood spaces, which transcend the decidua basalis and junctional zone, was undertaken. This showed no change in the maternal blood spaces or the labyrinth, the latter being the zone whose size is most frequently altered in IUGR. Critically however, the size of the maternally-derived decidua basalis was increased with a concurrent decrease in the size of the junctional zone. These morphological changes may play a causative role either through directly affecting placental function and/or by the reduced junctional zone failing to promote appropriate maternal adaptation. Alternatively, they may reflect compensatory adaptations to a primary defect elsewhere in the mother. Complementing these morphological studies, functional studies were undertaken: remodelling of maternal vasculature and the resistance index of vessels delivering blood to the fetus were assessed; as was delivery of nutrients to the fetus (measured by fetal glucose); and systemic maternal adaptations (maternal hormonal profile, circulating glucose levels and organ weights). Uterine, umbilical and decidual spiral arteries were examined, but displayed no apparent differences suggestive of normal blood supply to the fetus. However fetal blood glucose was reduced suggesting a reduced delivery of nutrients important for fetal growth. This was not due to lower circulating maternal blood glucose levels, and mRNA levels of the placental glucose transporters Glut-1 and Glut-3 were not reduced but upregulated, suggestive of an attempt to compensate for reduced fetal glucose. Furthermore, upregulation of at least one system A amino acid transporter mRNA, Snat-2, was observed. The maternal physiological state of PABP4-deficient dams showed deviations in some organ weights (e.g. spleen weight is reduced at e13.5 and e15.5) and the levels of some circulating hormones (e.g. estradiol is deceased whereas progesterone is increased at e18.5). However, future work will be required to determine which, if any, of these changes are primary defects rather than downstream consequences and to identify which mis-regulated mRNAs/pathways within in the materno-fetal dialogue underlie the phenotype. Taken together, my results suggest that the regulation of mRNA translation/stability by PABP4 is critical to achieving the correct pattern of gene expression within the materno-fetal dialogue to enable appropriate placentation and maternal adaptation. Furthermore, my results suggest that Pabp4-/- mice provide a unique opportunity to further understand the maternal causes of a spectrum of related pregnancy complications including IUGR, late miscarriage and stillbirth.

Nicotine, the major psychoactive ingredient of tobacco smoke, underlies numerous effects by activating neuronal nicotinic acetylcholine receptors. Both in vitro and in vivo studies suggest that nicotine is neuroprotective and improves cognitive performance. Epidemiology studies show that smoking is negatively correlated with the incidence of Parkinson's disease and Alzheimer's disease. Postmortem research and neuroimaging studies show that loss of nicotinic binding sites in the brain is the major feature of neurodegenerative diseases related to dementia and cognitive impairment. Caloric restriction, a regimen that extends the lifespan in all mammalian species studied so far including rodents and primates, is a highly regulated response to food deprivation. It is believed that the longevity effect of caloric restriction is mediated by SIRT1, a NAD-dependent deacetylase, and its related genes. Nicotine's effect on body weight could also lead to weight loss by decreasing caloric absorption consumption. The goal of this study was to find the possible correlation between nicotine's effects and the activation of SIRT1 and its related genes. Using beta2-/- mice that lack high affinity beta2 nicotinic acetylcholine receptors (nAChRs), we first demonstrated that beta2* nAChRs do not directly regulate expression of survival genes. However, we found that loss of beta2* nAChRs could result in augmented cellular stress, which indirectly increased expression of SIRT1, Nampt, and Ku70, possibly as an adaptive response to provide protection against neurodegeneration. We also found that loss of endogenous activation of beta2* nAChRs had less effect on synaptic connections but strongly impaired survival of hippocampal GABAergic neurons. To activate beta2* nAChRs in normal mice, we administered nicotine through drinking water. In a short-term exposure study, we determined the dose of nicotine to be used in young adult mice, and found that chronic nicotine treatment was anxiolytic, decreased caloric consumption, increased nAChR binding sites, and most importantly, increased expression of SIRT1 and its related genes. Finally, we compared long-term nicotine treatment with caloric restriction in middle-aged mice to examine their effects to brain aging, and our results indicated that in mice long term caloric restriction and nicotine treatment both tend to improve memory in aging mice, but appear to act through different mechanisms.

Animal models have yet to characterize alterations in body composition, wheel running activity, food intake, and neuroendocrine parameters, in chronic food restriction. This study investigated changes in these measures in food restricted rats, with and without access to running wheels, over four weeks. The data demonstrated that upon initiation of food restriction IGF-1 reductions paralleled loss of lean tissue while leptin levels were rapidly reduced which paralleled losses in body fat. Further, a lower limit threshold of body fat was identified, below which the correlation between leptin and fat mass was disrupted. Lastly, a decrease and plateau in body mass was mirrored by an increase and plateau in voluntary wheel activity in the food restricted population. The data suggest that there is a tight biological link between hyperactivity and body mass and that adequate nutritional support might attenuate the drive for obligate exercise even before weight is fully restored.

Animal models have yet to characterize alterations in body composition, wheel running activity, food intake, and neuroendocrine parameters, in chronic food restriction. This study investigated changes in these measures in food restricted rats, with and without access to running wheels, over four weeks. The data demonstrated that upon initiation of food restriction IGF-1 reductions paralleled loss of lean tissue while leptin levels were rapidly reduced which paralleled losses in body fat. Further, a lower limit threshold of body fat was identified, below which the correlation between leptin and fat mass was disrupted. Lastly, a decrease and plateau in body mass was mirrored by an increase and plateau in voluntary wheel activity in the food restricted population. The data suggest that there is a tight biological link between hyperactivity and body mass and that adequate nutritional support might attenuate the drive for obligate exercise even before weight is fully restored.

Since caloric restriction (CR) can modify multiple pathways central to the ischemic cascade and enhance neuroplasticity mechanisms, we hypothesized that CR should exert protective effects following brain ischemia. Previous studies have suggested benefit when CR was administered prior to ischemia. This study investigated whether prolonged CR beginning after global ischemia would result in lasting protection as assessed by performance in the open field, as a measure of functional outcome, and hippocampal CA1 neuronal counts. Adult male Mongolian gerbils were subjected to five minute bilateral carotid artery occlusion (I) or sham surgery (S) with tympanic temperature maintained at 36.5 ± 0.2ºC during the intra-ischemic period. After screening out gerbils with incomplete ischemia, each of the two surgical groups were randomly assigned to control diet (CON) or 30% CR for the duration of the study (60d). Gerbils were tested in the open field on d3, 7, 10, 30 and 60. Ischemic animals on control diet showed a significantly higher level of activity in the open field (impaired habituation) compared to SCON gerbils on all test days (p<0.001). Open field activity was decreased 9% in the ICR group versus ICON gerbils on d7 (p=0.024), suggesting a transient neuroprotective effect. Open field activity of the SCR gerbils began increasing relative to that of SCON gerbils during the last 30 days of the study (p=0.055 on d60), raising the question of suitability of the open field test for long-term studies of CR and ischemia. Brain sections obtained at d60 were stained with hematoxylin & eosin. Hippocampal CA1 neuron counts were reduced 88% by ischemia (p<0.001), and there was no sparing effect of CR. These findings suggest that prolonged CR administered beginning after global ischemia cannot diminish brain injury or enhance long-term recovery.

Since caloric restriction (CR) can modify multiple pathways central to the ischemic cascade and enhance neuroplasticity mechanisms, we hypothesized that CR should exert protective effects following brain ischemia. Previous studies have suggested benefit when CR was administered prior to ischemia. This study investigated whether prolonged CR beginning after global ischemia would result in lasting protection as assessed by performance in the open field, as a measure of functional outcome, and hippocampal CA1 neuronal counts. Adult male Mongolian gerbils were subjected to five minute bilateral carotid artery occlusion (I) or sham surgery (S) with tympanic temperature maintained at 36.5 ± 0.2ºC during the intra-ischemic period. After screening out gerbils with incomplete ischemia, each of the two surgical groups were randomly assigned to control diet (CON) or 30% CR for the duration of the study (60d). Gerbils were tested in the open field on d3, 7, 10, 30 and 60. Ischemic animals on control diet showed a significantly higher level of activity in the open field (impaired habituation) compared to SCON gerbils on all test days (p<0.001). Open field activity was decreased 9% in the ICR group versus ICON gerbils on d7 (p=0.024), suggesting a transient neuroprotective effect. Open field activity of the SCR gerbils began increasing relative to that of SCON gerbils during the last 30 days of the study (p=0.055 on d60), raising the question of suitability of the open field test for long-term studies of CR and ischemia. Brain sections obtained at d60 were stained with hematoxylin & eosin. Hippocampal CA1 neuron counts were reduced 88% by ischemia (p<0.001), and there was no sparing effect of CR. These findings suggest that prolonged CR administered beginning after global ischemia cannot diminish brain injury or enhance long-term recovery.