La maladie de Charcot-Marie-Tooth liée à l'X (CMTX), deuxième cause, en fréquence, de neuropathies héréditaire, est due à des mutations dans le gène Gjb1 codant pour la connexine 32. Afin de les utiliser comme modèle pré clinique, nous avons créé 5 lignées de souris transgéniques, ayant intégré un BAC humain portant une mutation observée dans plusieurs familles indépendantes. L'exploration de ces modèles a montré que la connexine 32 (Cx32) est impliquée dans le contrôle de la stabilité mitotique. Nous avons ensuite montré que cette instabilité implique l'activité des CaMKII et, peut être, de la kinase Pim1. Cette instabilité est corrigée par des inhibiteurs des CaMKII (KN62 et KN93). Nous avons retrouvé le même phénomène dans des cellules de malades CMTX. Nous avons également pu montrer que les animaux transgéniques montrent des anomalies du comportement locomoteur, corrigées par un traitement par des inhibiteurs de CaMKII. Finalement, nous proposons des pistes pour améliorer ces molécules, en synthétisant des analogues de KN93 / X-linked Charcot -Marie -Tooth (CMTX) disease, the second cause, in frequency, of hereditary neuropathies, is caused by mutations in the gene GJB1 encoding connexin 32. As a preclinical model, we created five lines of transgenic mice, which have integrated a human BAC contain mutation observed in several independent families. The exploration of models showed that the connexin 32 (Cx32) is involved in the control of mitotic stability. We then showed that this instability involves the activity of CaMKII and, perhaps, kinase Pim1. This instability is corrected by inhibitors of CaMKII (KN62 and KN93). We found the same phenomenon occuring in cells of CMTX patients. We also showed that transgenic animals show abnormal locomotor behavior corrected by treatment with inhibitors of CaMKII. Finally, we propose strategies to improve efficiency of these molecules by synthesizing analogues of KN93

As we learn more about immune cell subpopulations, we find an increasingly complex system of cells with diverse functions. L2pB1 cells are a PD-L2 positive B1a B lymphocyte subpopulation that has unusual properties and characteristics that are not fully understood by many. By creating and implementing a transgenic mouse model that allows for targeted depletion of this specific group of cells, we can further elucidate their physiological functions and roles in both healthy and diseased states. Here we demonstrate the depletion of L2pB1 cells utilizing a transgenic mouse model expressing Diphtheria Toxin Receptors on their surface. After a course of 4 injections of 25ng of Diphtheria Toxin per gram bodyweight, we observed a successful depletion of L2pB1 cell population. Further studies are underway investigating the effects of a high fat diet on these L2pB1 depleted mice. / 2017-04-01T00:00:00Z

A recent epidemiological study suggested that higher caffeine intake reduces the risk of Alzheimer's disease (AD). Caffeine, a widely consumed stimulatory drug, is a non-selective adenosine receptor antagonist that has been shown to increase plasma adenosine levels in rodents. To determine any long-term protective effects of caffeine in a controlled longitudinal study, caffeine was added to the drinking water of APPsw transgenic (Tg) mice between 4 and 9 1/2 months of age, with behavioral testing done during the last 6 weeks of treatment. The average daily intake of caffeine per mouse (1.5 mg) was the human equivalent of 5 cups of coffee/day. Across multiple cognitive tasks of spatial learning/reference memory, working memory, and recognition/identification, Tg mice given caffeine (Tg+Caff) performed significantly better than Tg control mice and similar to non-transgenic controls. Discriminant Function Analysis involving multiple cognitive measures clearly showed the superior overall cognitive performance of Tg+Caff mice compared to Tg controls. Analysis of Aβ in the hippocampus by ELISA revealed Tg+Caff mice had significantly less soluble Aβ1-40 and insoluble Aβ1-42. In a follow-up study involving neurochemical analysis only, caffeine was added to the drinking water of 17 month old APPsw mice for 18 days. In this study, Tg+Caff mice also showed a significant reduction of insoluble Aβ1-42 in the hippocampus. In contrast to the reduced extracellular brain levels of adenosine in Tg controls, caffeine treatment normalized brain adenosine levels in Tg mice to that of non-transgenic controls. Analysis of amyloidogenic secretase activity revealed the reduction in Αβ is likely because of a reduction in gamma secretase activity as a result of increased SAM silencing of PS1 expression. This study suggest that a modest, long-term caffeine intake of approximately 500 mg per day (5 cups of coffee) may reduce considerably the risk of AD by decreasing amyloidogenesis.

Les systèmes neuromodulateurs jouent un rôle essentiel dans la mise en place et dansla régulation des réseaux moteurs spinaux afin d’adapter finement le rythme et le patronlocomoteur aux contraintes internes et externes de l’organisme. Il a été montré que desaltérations du fonctionnement de ces systèmes étaient impliquées dans de nombreusespathologies neurologiques. La sclérose latérale amyotrophique (SLA) est une maladieneurodégénérative caractérisée par la perte des neurones moteurs corticaux et spinaux. Bienque les symptômes de la SLA n’apparaissent qu’à l’âge adulte, de plus en plus d’élémentsamènent à penser que des modifications précoces des réseaux locomoteurs spinaux ont lieudès les stades précoces du développement chez un modèle animal de la SLA, la souris SOD1.C’est dans ce cadre général que nous avons émis l’hypothèse que des altérations précoces dessystèmes neuromodulateurs pourraient intervenir dans la physiopathologie de la SLA. Dansun premier temps, nous avons comparé la modulation monoaminergique des réseaux moteursspinaux en réalisant des enregistrements extracellulaires de l’activité locomotrice générée parla préparation de moelle épinière isolée chez la souris nouveau-née sauvage et SOD1. Nousnous sommes ensuite attachés en combinant des enregistrements électrophysiologiques extraetintracellulaires avec des techniques d’immunohistochimie et de biologie cellulaire à décrirela mise en place et l’évolution avec l’âge des synapses cholinergiques reçues par lesmotoneurones en provenance d’interneurones de la lamina X : les boutons en C. Enfin, nousavons initié une approche (1) comportementale sur le long terme de l’activité motrice dessouris SOD1 et (2) des capacités plastiques des synapses glutamatergiques reçues par lesmotoneurones en culture. L’ensemble de ces travaux, nous a permis de mettre en évidence desaltérations précoces et évolutives des principaux systèmes neuromodulateurs spinaux:cholinergique, dopaminergique et noradrénergique chez les animaux SOD1. Nos résultatsmontrent pour la première fois (1) qu’une dynamique complexe des récepteurs M2 sous lesboutons en C existe et que celle-ci est perturbée chez les souris SOD1 et (2) que lesmotoneurones ne sont pas les seuls neurones à dégénérer dans la moelle de ces animaux maisque les neurones cholinergiques de la lamina X situés dans les segments lombaires L2 sontaussi la cible de processus neurodégénératifs. / Neuromodulatory systems play a crucial role in the establishment and regulation ofspinal motor networks to finely adjust the locomotor rhythm and pattern to the internal andexternal constraints. It is now well admitted that alterations in neuromodulatory functions areinvolved in diverse neurologic disorders. Amyotrophic lateral sclerosis is a neurodegenerativedisease characterized by the specific loss of cortical and spinal motor neurons. A growingbody of evidence now suggests that although ALS syndromes occur in adulthood, alterationscan be detected as early as at the embryonic stages in the spinal cord of the rodent model ofALS, the SOD1 mouse. In this context, we hypothesized that early alterations in the spinalneuromodulatory systems may be involved in the pathophysiology of ALS. To answer thisquestion, in a first step, we compared the monoaminergic modulation of spinal network byrecording extracellularly the fictive locomotion produced in the in vitro spinal cordpreparation form newborn wild-type and SOD1 mice. By combining extra- intracellularrecordings with immunohistochemical and cellular biology technics, we aimed, in a secondstep, to investigate the cholinergic synapses arising onto motoneurons and their neuronalsource, the lamina X interneurons as a function of the mouse age. Finally, we initiated (1) aninnovative behavioural study of mouse motor habits and (2) an analysis of the synapticplasticity of glutamatergic synapses imping on motoneurons in culture. Altogether, our datademonstrated early and progressive changes of the major spinal neuromodulatory systems:cholinergic, dopaminergic and noradrenergic. Our data show for the first time that: (1) M2receptors undergo a complex dynamic under C-bouton that is completely disturbed in SOD1motoneurons and (2) motoneurons are not the only cellular subtype to degenerate in SOD1mice. Indeed, we found evidence that neurodegenerative processes also target lamina Xcholinergic interneurons in the SOD1 spinal cord.

Abstract not available

Doctor of Philosophy(PhD) / Melanotransferrin or melanoma tumour antigen p97 (MTf) is a transferrin homologue that is found predominantly bound to the cell membrane via a glycosylphosphatidylinositol anchor. The molecule is a member of the transferrin super-family that binds iron through a single high affinity iron(III)-binding site. Melanotransferrin was originally identified at high levels in melanoma cells and other tumours, but at lower levels in normal tissues. Since its discovery, the function of MTf has remained intriguing, particularly regarding its role in cancer cell iron transport. In fact, considering the crucial role of iron in many metabolic pathways e.g., DNA and haem synthesis, it is important to understand the function of melanotransferrin in the transport of this vital nutrient. Melanotransferrin has also been implicated in diverse physiological processes, such as plasminogen activation, angiogenesis, cell migration and eosinophil differentiation. Despite these previous findings, the exact biological and molecular function(s) of MTf remain elusive. Therefore, it was important to investigate the function of this molecule in order to clarify its role in biology. To define the roles of MTf, six models were developed during this investigation. These included: the first MTf knockout (MTf -/-) mouse; down-regulation of MTf expression by post-transcriptional gene silencing (PTGS) in SK-Mel-28 and SK-Mel-2 melanoma cells; hyper-expression of MTf expression in SK-N-MC neuroepithelioma cells and LMTK- fibroblasts cells; and a MTf transgenic mouse (MTf Tg) with MTf hyperexpression. The MTf -/- mouse was generated through targeted disruption of the MTf gene. These animals were viable, fertile and developed normally, with no morphological or histological abnormalities. Assessment of Fe indices, tissue Fe levels, haematology and serum chemistry parameters demonstrated no differences between MTf -/- and wild-type (MTf +/+) littermates, suggesting MTf was not essential for Fe metabolism. However, microarray analysis showed differential expression of molecules involved in proliferation such as myocyte enhancer factor 2a (Mef2a), transcription factor 4 (Tcf4), glutaminase (Gls) and apolipoprotein d (Apod) in MTf -/- mice compared with MTf +/+ littermates. Considering the role of MTf in melanoma cells, PTGS was used to down-regulate MTf mRNA and protein levels by >90% and >80%, respectively. This resulted in inhibition of cellular proliferation and migration. As found in MTf -/- mice, melanoma cells with suppressed MTf expression demonstrated up-regulation of MEF2A and TCF4 in comparison with parental cells. Furthermore, injection of melanoma cells with decreased MTf expression into nude mice resulted in a marked reduction of tumour initiation and growth. This strongly suggested a role for MTf in proliferation and tumourigenesis. To further understand the function of MTf, a whole-genome microarray analysis was utilised to examine the gene expression profile of five models of modulated MTf expression. These included two stably transfected MTf hyper-expression models (i.e., SK-N-MC neuroepithelioma and LMTK- fibroblasts) and one cell type with downregulated MTf expression (i.e., SK-Mel-28 melanoma). These findings were then compared with alterations in gene expression identified using the MTf -/- mouse. In addition, the changes identified from the microarray data were also assessed in another model of MTf down-regulation in SK-Mel-2 melanoma cells. In the cell line models, MTf hyper-expression led to increased proliferation, while MTf down-regulation resulted in decreased proliferation. Across all five models of MTf down- and upregulation, three genes were identified as commonly modulated by MTf. These included ATP-binding cassette sub-family B member 5 (Abcb5), whose change in expression mirrored MTf down- or up-regulation. In addition, thiamine triphosphatase (Thtpa) and Tcf4 were inversely expressed relative to MTf levels across all five models. The products of these three genes are involved in membrane transport, thiamine phosphorylation and proliferation/survival, respectively. Hence, this study identifies novel molecular targets directly or indirectly regulated by MTf and the potential pathways involved in its function, including modulation of proliferation. To further understand the function of MTf, transgenic mice bearing the MTf gene under the control of the human ubiquitin-c promoter were generated and characterised. In MTf Tg mice, MTf mRNA and protein levels were hyper-expressed in a variety of tissues compared with control mice. Similar to the MTf -/- mice, these animals exhibited no gross morphological, histological, nor Fe status changes when compared with wild-type littermates. The MTf Tg mice were also born in accordance with classical Mendelian ratios. However, haematological data suggested that hyper-expression of MTf leads to a mild, but significant decrease in erythrocyte count. In conclusion, the investigations described within this thesis clearly demonstrate no essential role for MTf in Fe metabolism both in vitro and in vivo. In addition, this study generates novel in vitro and in vivo models for further investigating MTf function. Significantly, the work presented has identified novel role(s) for MTf in cell proliferation, migration and melanoma tumourigenesis.

Multiple checkpoints control the survival and activation of auto-reactive B cells. The discovery of the TNF family cytokine BAFF has been crucial to understanding peripheral B cell tolerance mechanisms. Homeostatic levels of BAFF are tightly regulated to maintain tolerance in the periphery. Chronically increased levels of BAFF lead to the survival of autoreactive B cells. Autoimmune patients display elevated serum BAFF levels. BAFF Tg mice model this situation with systemically high levels of BAFF and the subsequent development of two separate but related autoimmune syndromes; systemic lupus erythematosus (SLE) and Sj??gren???s syndrome (SS). The work conducted in this thesis further investigates the defects in tolerance down-stream of self-reactive B cell survival, which may contribute to autoimmune disease development in BAFF Tg mice. Expansion of the Marginal zone (MZ) B cell population correlates with the pathogenesis of several models of autoimmune disease. BAFF Tg mice are unique in that they not only display an increased splenic MZ B cell population, but also MZ B cells are found in the salivary glands of mice developing SS. The examination of genes differentially regulated between MZ and Follicular (Fo) B cells led to the investigation of sphingosine-1-phosphate receptor biology. The expression of S1P receptors was shown to be required for the positioning of MZ B cells in the spleen. Chronic BAFF stimulation alters the retention of MZ B cells through the alteration of S1P receptors and decreased integrin activation. The alteration of S1P receptors and increased ligand sensitivity leads to the accumulation of MZ B cells in the inflamed salivary glands of BAFF Tg mice. This works provides a potential mechanism for the tissue specificity seen in systemic autoimmune disease. The provision of T cell help to auto-reactive B cells is thought to underlie the development of SLE. BAFF Tg mice deficient in T cells surprisingly developed an SLE-like disease indistinguishable from that of BAFF Tg mice. Autoimmunity in BAFF Tg mice did however require signals through the toll-like receptor (TLR)-associated signalling adaptor, MyD88, which controlled the production of pathogenic autoantibodies. Therefore, autoimmunity in BAFF Tg mice results from altered B cell tolerance, which requires TLR signalling and is independent of T cell help. It is likely that autoimmune patients with elevated levels of BAFF show a similar basis for disease.

<p>Platelet-derived growth factor receptor α (PDGFRα) plays an important role during embryogenesis. After implantation, the patterns of expression of Pdgfrα and its ligand Pdgf-A undergo an "autocrine-paracrine transition", in that Pdgf-A becomes expressed in the ectoderm and epithelia, while Pdgfrα is expressed in the adjacent mesenchymal tissue. In human tumors, such as malignant glioma, both PDGF and PDGFRα are overexpressed within the same tissue, indicating that an autocrine PDGF loop is generated in the tumors. This thesis is focused on the <i>in vivo</i> functionality of the <i>PDGFR</i>α gene (<i>PDGFRA</i>) promoter, arid on the effect of autocrine PDGF-A stimulation in transgenic n-iice during embryogenesis. </p><p>To test the <i>in vivo</i> promoter function of a human PDGFRA 2.2 kb 5' flanking fragment, we generated transgenic mouse lines and found that the 2.2 kb fragment was able to promote lacZ reporter gene expression in most of the endogenous Pdgfra expressing tissues. Absence of expression and "ectopic" expression of the transgenic lacZ were also observed. To investigate the autocrine PDGF effect, we produced autocrine PDGF-As (A short-chain) transient transgenic embryos. These transgenic embryos carried a 6 kb mouse <i>Pdgfra</i> 5' flanking sequence linked to a human PDGF-As cDNA. The pattern of expression of the PDGF-As transgene mRNA was similar to that of lacZ. Some of the transgenic embryos exhibited severe abnormal phenotypes, such as midline fusion defects in the cephalic and craniofacial region and small body size, and these embryos die at mid-gestation stage. These findings indicate that a paracrine pattern of expression and the dosage of PDGF are important for sustaining normal embryo development, especially with regard to the middline fusion in craniofacial regions. </p><p>The possible signaling pathways that may be involved in regulating <i>Pdgfra</i> activity were also studied by comparison of patterns of mRNA expression of Gli, Ptc, and Paxl with that of Pdgfra. The results pointed to the possibility that the Shh signaling pathway may be involved in the regulation of <i>Pdgfra</i> expression for example during early bone and foregut development. The specific regulatory mechanisms may vary for different tissues.</p>

<p>Medulloblastoma is a highly malignant cerebellar childhood tumor. As in many other brain tumors, expression of platelet-derived growth factor (PDGF) and its receptors has been shown in medulloblastoma. To reveal the importance of this growth factor in cerebellar development and tumorigenesis, analyses were performed on human medulloblastoma cell lines and on tissue from normal mouse brain at different stages of development. The <i>in vivo</i> effect of a forced expression of PDGF-B in the cerebellar primordium was examined in transgenic mice. </p><p>In the normal mouse embryo, we found PDGF receptor-α-positive cells in the early neuroepithelium and on neuronal precursors. In the postnatal cerebellum, cells in the external germinal layer and Purkinje cells expressed the receptor. In the medulloblastoma cells, expression of all the three PDGF isoforms and PDGF receptors was seen and correlated to neuronal differentiation. Endogenously activated, <i>i.e.</i> tyrosine phosphorylated, PDGF receptors were identified. To reveal the role of PDGF in normal cerebellar development, we established transgenic mice where a PDGF-B cDNA was introduced via homologous recombination into the engrailed-1 gene. Engrailed-1 is specifically expressed at the mid-/hindbrain boundary of the early neural tube, <i>i.e.</i> in an area from which the cerebellar primordium develops. The ectopic expression of PDGF-B caused a disturbance of cerebellar development. Midline fusion of the cerebellar primordium did not occur properly, which resulted in cerebellar dysplasia in the adult mouse.</p><p>In a parallel study, the expression pattern of a glial fibrillary acidic protein (GFAP)-<i>lacZ</i> transgene was followed in the embryonic mouse central nervous system. It was shown that the human GFAP promoter was already active by embryonic day 9.5 and as development proceeded, expression occured in different, independent cell populations. Among these cell populations were the radial glial cells in the neocortex.</p>

The feasibility of the Alb-uPA/SCID/Bg chimeric mouse as a model for Hepatitis C Virus (HCV) infection was assessed experimentally by (1) the infection and treatment with another hepatotropic virus, Hepatitis B Virus (HBV) and (2) the infection of the model with HCV and the subsequent treatment of that infection with a pro-apoptotic factor (BID) targeted to infected hepatocytes. In the former, the infected mouse responded favorably, and in the manner of human patients, to a standard imunoglobulin therapy. In the latter, HCV-infected hepatocytes were successfully targeted for cell death, with repeated doses of Adenovirus-delivered BID being the most effective at inhibiting virus spread. Efficacy and toxic side-effects of BID treatment could be reconciled by modulating the timing between doses, the most effective tested being three doses of BID at 7-day intervals. Analyses of chimeric model production were undertaken to improve the quality of human hepatocyte engraftment (typically only 25-35% of mice receiving grafts are currently used experimentally). Minor variations in success rates were experienced with respect to donor age or health status, or the age of recipient mice within an operational window of 5 to 13 days from birth. The greatest obstacle to useful engraftment (aside from technical challenges) was deemed to be the genetic/cellular integrity of the recipient mouse. This conclusion was based on variable engraftment success with ‘healthy’ donor cell preparations and a consideration of variability in immune deficiency arising in mice within a SCID/Bg mouse colony. / Experimental Surgery