Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Noch immer ist die Behandlung von Neuropathien mit den gängigen therapeutischen Mitteln für viele Patienten sehr unbefriedigend. Als erfolgsversprechender therapeutischer Ansatz werden zur Zeit Wege erforscht, welche direkt in die molekularen Entstehungsmechanismen pathologischer Veränderungen und regenerationsfördernder Mechanismen eingreifen, um dadurch eine Heilung von Nervenschäden zu ermöglichen. Bisher sind die Erkenntnisse über diese Mechanismen nicht vollständig genug, um daraus eine sichere Behandlungsmöglichkeit abzuleiten. Wegweisende Erkenntnisse deuten sich allerdings durch Studien von unterschiedlichen Vertretern des Zytokinnetzwerkes an - darunter auch TNF-alpha - welche als molekulare Ursache neuropathischer Veränderungen diskutiert werden. In dieser Studie wurde an Knockoutmäusen der Einfluss des jeweiligen TNF-alpha-Rezeptors auf morphologische Veränderungen nach CCI (Chronic constriction injury) und Crush-Verletzung des N. ischiadicus untersucht. Nach 3,7,15 und 36 Tagen (CCI) bzw. 3,7 und 28 Tagen (Crush) wurden in Methylenblau gefärbten Semidünnschnitten intakte und degenerierte Nervenfasern, Makrophagen, Angioproliferation, Ödembildung udn Veränderung des Anteils nicht neuronaler Zellen lichtmikroskopisch beurteilt. Zusätzlich wurden Mac-1+ Makrophagen immunzytochemisch erfasst. Die Ergebnisse zeigten in beiden Modellen und bei beiden Knockouttypen eine starke axonale Schädigung, die von einer großen endoneuroalen Makrophagenansammlung begleitet war. Bei TNF-R1-/- Mäusen war eine stärkere und verlängerte Degeneration mit entsprechend höheren Makrophagenzahlen sichtbar. In den Immunzytochemischen Färbungen wiesen die TNF-R1-/- Mäuse hingegen den geringsten Makropahgenanteil auf.Trotz der starken Schädigung war die anschließende Regeneration im Gegensatz zu WT und TNF-R2-/- Mäusen besser. Die Ödembildung war bei den TNF-R2-/- nach CCI besonders stark ausgeprägt und von einer schlechten Regeneration gefolgt. Während die gefundenen Daten auf eine Beteiligung beider Rezeptoren während degenerativer Prozesse hindeuten, scheint insbesondere TNF-R2 regenerationsfördernde Effekte zu vermitteln. / Current Treatment of neuropathic disorders is still dissatisfactory for many patients. A promising approach is the investigation of agents that directly interfere with molecular development of pathologic changes and regeneration. Up to now, consolidated findings of the underlying mechanisms are not yet sufficent to allow therapeutic intervention. Pathbreaking findings come from studies investigating different agents of the cytokine network - as e.g. TNF-alpha - that are discussed as molecular cause of neuropathic changes. This study investigated the influence of both TNF-alpha-receptors on morphologic changes after CCI (chronic constriction injury) and crush-injury of the sciatic nerve of TNF-R-knockoutmice. After 3,7,15 and 36 days (CCI), and 3,7 and 28 respectively (Crush),intact and degenerating nerve fibers, macrophages, angioproliferation, development of edema and changes in the amount of non-neuronal cells were acquired by light microscopy of toluidin-stained semithin sections. Additionally Mac-1+ macrophages were acquired via immuncytochemically stained sections. The results showed strong axonal damage in both knockout-types accompanied by large amounts of endoneurial macrophages. TNF-R1-/-mice showed a longer degeneration phase including respectively higher amounts of macrophages. In contrast the TNF-R1-/-mice revealed the fewest amount of macrophages in immunocytochemical sections. Despite the strong damage better nerve regeneration was observed compared to WT and TNF-R2-/-mice. Formation of edema was pronounced in TNF-R2-/- after CCI and followed by poorly regeneration. Whereas these findings point to a participation of both receptors in degeneration, TNF-R2 seems to support regeneration.

A insuficiência cardíaca (IC) é uma síndrome clínica de alta incidência e mau prognóstico, caracterizada por fadiga, dispnéia e grande limitação aos esforços físicos. Essas alterações não estão apenas limitadas ao comprometimento cardíaco, mas em parte, são decorrentes também de alterações morfo-funcionais da musculatura esquelética. Para a dissertação foram utilizados camundongos com deleção dos genes para os receptores ?2A e ?2C adrenérgicos (KO) que desenvolvem cardiomiopatia induzida por hiperatividade simpática, associada à sinais clínicos de IC e 50% de mortalidade aos sete meses de idade. Foi objetivo desse estudo realizar a caracterização fenotípica do músculo esquelético por meio de avaliações funcionais e morfológicas em camundongos KO previamente ao desenvolvimento da IC (três meses de idade), e ao longo de sua progressão (cinco e sete meses de idade). Somente na faixa etária de sete meses de idades foi constatado o estabelecimento da miopatia muscular esquelética. Nessa fase, observou-se rarefação vascular, atrofia muscular, aumento na porcentagem de fibras glicolíticas e redução na atividade máxima da enzima citrato sintase, que em conjunto, contribuem para a antecipação da fadiga observada nesse modelo, e como conseqüência, para a redução da tolerância aos esforços. Os resultados sugerem que os camundongos KO apresentam alterações morfo-funcionais da musculatura esquelética semelhantes às observadas nos demais modelos de IC e em indivíduos portadores dessa síndrome. Portanto, sendo um ótimo modelo experimental para estudos de futuras estratégias terapêuticas que visem minimizar as alterações na musculatura esquelética decorrentes da IC / Heart failure (HF) is a clinical syndrome with high incidence and bad prognostic, characterized by fatigue, dyspnea, and increased intolerance to exercise. These changes are not only related to the cardiovascular tissue, but are at least in part, consequence of morphofunctional alterations in skeletal muscles. To the present study it was used mice lacking both ?2A/?2C AR subtypes (KO) which develop cardiomyopathy induced by sympathetic hyperactivity, associated to clinical signals of HF and 50% of mortality at seven months of age. The aim of the present study was characterize the phenotype of skeletal muscle by functional and morphological evaluations in KO before (three months of age) and during the HF progression (five and seven months of age). Skeletal muscle alterations due HF were observed only at seven months of age. The alterations were characterized by vascular rarefaction, muscular atrophy, increase in glycolitic fibers percentage and reduction of maximal activity of citrate synthase and contributed with early fatigue observed in this model, and consequently, exercise intolerance. The results of present study suggest that KO mice present morphofunctional changes in skeletal muscles in a similarly to others models of HF and in patients that have this syndrome. Therefore, consisting in an excellent experimental model to future studies related to therapeutic strategies to minimize the skeletal muscle changes due HF

First study determined the effects of genetic deletion of nNOS on the levels of spontaneous apoptosis in brain of young-adult (2-3 months) and aged (12-18 months) mice, using nNOS-knockout mice with age-matched B6129SF2/J mice as wild-type control. The results indicate that aging resulted in 11-fold increase in levels of apoptotic-DNA-fragmentation in B6129SF2/J mouse brain. nNOS-knockout mice demonstrated dramatic (72-fold) increases in levels of apoptotic-DNA-fragmentation in young-adult, but not aged, brains. Aging resulted in decreased number of nNOS-positive cells, increased number of iNOS-positive cells and no change of eNOS-positive cells in control mice. The data suggest that nNOS may serve an anti-apoptotic/neuroprotective role in young-adult mouse brain. However, because of diminished nNOS and increased iNOS with aging, this neuroprotective effect may become less effective in aged mice. / Fourth study showed that new microchip-electrophoresis-technology can be successfully used to identify and quantify levels of apoptosic-DNA-fragments in brain slice cultures, similar to our previous studies with CE-LIF. Because of the much greater throughput of microchip-electrophoresis-system, compared to CE-LIF, this new technology should help accelerate the progress of apoptosis research. / In second study, apoptotic effects of genetic deletion of either eNOS or iNOS were studied using young-adult (1-4 months) and aged (12-24 months) eNOS- or iNOS-knockout mice with age-matched C57BL/6J wild-type control mice. The data show that both young-adult and aged iNOS-knockout mice had dramatically (8- to 36-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially noticeable in hippocampus and medulla oblongata. Both young-adult and aged eNOS-knockout mice also had dramatically (18- to 35-fold) higher levels of apoptotic-DNA-fragmentation compared to control, especially in cerebral cortex, hippocampus and medulla oblongata. The data suggest that both iNOS and eNOS provide neuroprotective effects, helping to limit the extent of spontaneous apoptosis in brain of young-adult and aged mice. / Nitric oxide (NO) has either pro-apoptotic or anti-apoptotic effects on neuronal cells, depending on concentration of NO produced by different source of NO synthases (NOSs) including neuronal-NO-synthase (nNOS/NOS-1), inducible-NO-synthase (iNOS/NOS-2) or endothelial-NO-synthase (eNOS/NOS-3) and possibly age of the individual. The present study determines if genetic deletion of nNOS, iNOS or eNOS alters levels of aging-induced apoptosis in vivo and hydrogen peroxide (H2O2)-induced-apoptosis in organotypic brain slice cultures using NOS-knockout mice. The quantitative ultrasensitive techniques using capillary-electrophoresis with laser-induced-fluorescent detector (CE-LIF) and Cell-Death---Detection-ELISA were used as novel ways to accurately measure the levels of apoptotic-DNA-fragmentation. Expressions of different forms of NOSs were determined by immunohistochemical-staining. / Third study determined H2O2-induced apoptosis in hippocampal and cerebellar slices from young-adult (8-10 weeks) and aged (12-24 months) C57BL/6J control mice, as well as iNOS- and eNOS-knockout mice (determined by Cell-Death-Detection-ELISA measuring levels of apoptotic-DNA-fragmentation). The data show spontaneous onset of apoptosis occurred in both hippocampal and cerebellar slices during culturing, beginning at 24 hours and progressively increasing for 48--72 hours. Staurosporine (positive-control) and H2 O2 both caused time-dependent increases in apoptosis in both hippocampal and cerebellar slices, compared to time-matched controls. Lastly, genetic deletion of iNOS greatly reduced levels of spontaneous apoptosis in young hippocampus and aged cerebellum, suggesting iNOS had contributed to induction of spontaneous apoptosis. / Chow Wing Han Vivian. / "Dec 2005." / Advisers: Siew Boon Chew Cheng; Ray Ronald Fiscus. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6218. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 144-153). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Macrophages belong to the mononuclear phagocyte system and they perform fundamental roles to maintain homeostasis in the organism. Macrophage development, survival, proliferation and functionality depend upon the colony stimulating factor 1 (CSF1) and interleukin-34 (IL-34), which signal through the CSF1 receptor (CSF1R). CSF1R is a type III tyrosine kinase receptor that is present in the plasma membrane of monocytes and macrophages. Mutations in Csf1r in mice produce the loss of many tissue macrophage populations and multiple developmental abnormalities. In humans, abnormal enhancement of CSF1R expression has been correlated to adverse prognosis in a subset of carcinomas; and mutations in the human CSF1R are associated with an autosomal-dominant neurodegenerative disease. CSF1R is encoded by the c-fms proto-oncogene and its expression is partially controlled by the fms-intronic regulatory element (FIRE). The FIRE sequence is highly conserved across species and contains binding motifs for multiple transcription factors, which are relevant for haematopoiesis. Previous results from murine Csf1r transgenes showed that FIRE is essential for driving Csf1r expression, and that interactions between FIRE and multiple myeloid transcription factors contribute to maximal regulatory activity. This project aimed to study the role of FIRE in its normal chromatin context, in vivo. A FIRE knockout (FIRE-/-) mouse model was generated using the CRISPR/Cas9 technology in mouse embryonic stem cells (ESCs) and in mice. In ESCs, the deletion severely compromised the differentiation of macrophages from embryoid bodies generated in vitro. In mice, the frequency of the FIRE- /- genotype in the progeny does not follow a Mendelian distribution and about 5% of the offspring developed hydrocephalus. Unlike Csf1r -/-mice, which die before weaning, most surviving FIRE-/- mice grew normally and were fertile. The impact of the mutation on macrophage populations is selective. FIRE-/- mice are not monocyte deficient (identified as F4/80+ Csf1r+ cells in peripheral blood), although these cells have reduced levels of Csf1r mRNA and do not bind porcine CSF1 Fc fusion protein. The development of peritoneal macrophages and Iba-1+ microglia was abolished, but Adgre1+ (F4/80+) macrophage populations in liver and spleen were unaffected. Csf1r was greatly reduced in bone marrow progenitors, but about 30% of these cells were able to differentiate into macrophages in vitro, upon exposure to recombinant human CSF1 (rhCSF1). This study shows that FIRE is essential for the development of a subset of tissue-resident macrophage populations. In FIRE-/- mice, potential compensation from additional regulatory elements within Csf1r might underlie the development of unaffected tissue-resident macrophages.

Transmembrane signal transduction by ligand-activated G protein-coupled receptors (GPCRs) controls virtually every aspect of mammalian physiology, and this receptor class is the target of 40-50% of currently marketed pharmaceuticals. In addition to the clinical use of direct GPCR agonists and antagonists, it is now believed that GPCR effectors and regulators may also be viable drug targets with improved therapeutic efficacy and specificity. The prototypic role of Regulator of G protein Signaling (RGS) proteins is inhibition of G protein signaling through acceleration of GTP hydrolysis by GΑ, which promotes re-association of GΑ and GΒΓ subunits with the receptor at the cell membrane. In this way, RGS proteins determine the magnitude and duration of the cellular response to GPCR stimulation. Though RGS protein biochemistry has been well elucidated in vitro, the physiological functions of each RGS family member remain largely unexplored. RGS6 belongs to the R7 subfamily of RGS proteins originally identified in brain. Our acquisition of an RGS6-/- mouse allowed us to survey RGS6 expression in all tissues of the body revealing the greatest expression of RGS6 in brain. Despite robust neural RGS6 expression, little is known regarding functional roles of RGS6 in the brain and spinal cord. In addition, we identified several novel, higher molecular weight RGS6 immunoreactive bands specifically present in the nervous system. The plan of this thesis work was multifaceted. We sought to elucidate novel GPCR signaling cascades modulated by RGS6 in brain while simultaneously characterizing the expression patterns and identity of the novel RGS6 species specifically detected in the nervous system. Considering the large diversity of RGS6 isoforms present in brain, the abundance of potential RGS6 binding partners, and the possibility of discovering new mechanisms involved in RGS6 regulation, elucidation of the novel RGS6 molecular species is of paramount importance. Utilizing RGS6-/- mice we identified RGS6 as a critical modulator of two GPCRs in brain. First, by inhibiting the serotonin receptor 1A (5-HT1AR)-adenylyl cyclase (AC) axis, RGS6 functions to promote anxiety- and depression-related behaviors in mice. As a result, RGS6-/- mice exhibit a robust anxiolytic and antidepressant phenotype remarkably similar to that of animals treated chronically with therapeutic doses of selective serotonin reuptake inhibitors (SSRIs). RGS6 also inhibits GABAB receptor (GABABR)-G protein- activated inwardly rectifying potassium (GIRK) channel current in cerebellar granule cells, and loss of RGS6 results in cerebellar ataxia and gait abnormalities reversible by GABABR blockade. Furthermore, evaluation of voluntary alcohol drinking behaviors in WT versus RGS6-/- mice revealed a striking reduction in alcohol intake resulting from RGS6 loss in both acute and chronic alcohol consumption paradigms due, at least in part, to potentiation of GABABR signaling. Thus, RGS6 inhibitors have potential clinical utility in the treatment of mood disorders and alcoholism. We have shown that one novel RGS6 immunoreactive band expressed in the brain and spinal cord is a phospho-protein sensitive to Λ phosphatase-mediated dephosphorylation. Further, new information acquired from PCR amplification of RGS6 mRNA species from human brain cDNA libraries has necessitated substantial revisions to the RGS6 splicing scheme devised by the Fisher laboratory in 2003. To the 36 isoforms generated from two alternate transcription start sites (RGS6L vs. RGS6), the inclusion or exclusion of exons 14 and 17, and variable splicing to one of 7 different 3' terminal exons, we have added the possible insertion of three novel internal exons (A1, A2, A3), a retained intron, and two new 3' terminal exons. As a result, the number of RGS6 mRNAs present in brain could be as many as 248 unique species, an astonishing diversity unprecedented in the RGS protein family.

Um eine mögliche elektrophysiologische, kardiale Ursache für den plötzlichen Tod von STIM2 Knock-Out Mäusen zu prüfen, wurde eine elektrophysiologische Charakterisierung mittels Ruhe- und Stress-EKG, telemetrischem Langzeit-EKG sowie Elektrophysiologischer Untersuchung durchgeführt. Hierbei konnte keine kardial-elektrophysiologische Grundlage für den plötzlichen Tod dieser Tiere gefunden werden. / For testing a possible electrophysiological, cardiac reason for the sudden death of STIM2 knockout mice, a electrophysiological characterization was performed by use of resting and stress ecg, telemetric long-term ecg and electrophysiology study. In this study couldn't be found any cardiac electrophysiological cause for the sudden death of these animals.

Mon travail de doctorat a consisté en l'étude de deux gènes, TSAP6 et TCTP, qui sont régulés de manière différentielle dans la réversion tumorale. Les protéines encodées par ces deux gènes interagissent et forment un complexe tant in vitro que in vivo. Mon objectif était de mieux comprendre la fonction biologique de ces deux gènes. Différentes approches ont été employées, notamment la cristallographie (TCTP), souris knockout (TSAP6 et TCTP) et études par mutagénèse dirigée (TSAP6 et TCTP). <br />Les souris knockout pour le gène tsap6 présentent une anémie microcytaire. Nous démontrons que cette anémie est due à un retard de maturation des réticulocytes ainsi qu'à une sécrétion défectueuse du récepteur à la Transferrine par les exosomes. Étant donné que le gène TSAP6 est une cible transcriptionnelle directe de la p53, nous avons voulu analyser la sécrétion des exosomes suite à une stimulation de p53 (par rayon X ou Actinomicine D). Nous montrons qu'en l'absence de TSAP6, on ne parvient pas à stimuler une sécrétion des exosomes. De manière plus générale ces expériences ont démontré que le gène TSAP6 avait in vivo un rôle prédominant dans la sécrétion des exosomes, y compris suite à l'activation de la p53. <br />Nous avons surtout étudié le rôle que joue TCTP dans l'apoptose. La délétion du gène tctp dans les souris knockout est létale en provoquant une augmentation de l'apoptose au cours de l'embryogenèse. Cette mort embryonnaire se produit entre les 6,5ème et 9,5ème jours du développement. La détermination de la structure de la protéine TCTP par cristallographie avec une résolution de 2A montre que la protéine humaine est très homologue à celle de s.pombe. De plus, nous avons observé suite à une analyse informatique une homologie de structure entre les hélices H2 et H3 de TCTP et les hélices H5 et H6 de BAX, une protéine pro-apoptotique impliquée dans la perméabilité de la membrane mitochondriale. On a démontré que grâce à ces deux hélices, TCTP effectue son action anti-apoptotique et inhibe la dimérisation de BAX à la membrane des mitochondries, empêchant ainsi l'apoptose.<br />Nous montrons également que la Sertraline et la Thioridazine, qui induisent la mort des cellules tumorales et une baisse concomitante de TCTP, se lient directement à TCTP et empêchent ainsi la formation du complexe TSAP6-TCTP.

Les facteurs spectroscopiques nous renseignent sur l'occupation des couches nucléaires et peuvent être extraits par des réactions directes comme le transfert à basse énergie et le knockout aux énergies intermédiaires. L'étude récente de noyaux radioactifs de la couche sd ayant une large différence d'énergie de séparation, DeltaS=Sp-Sn~20 MeV, montre que les sections efficaces d'arrachage d'un nucléon très lié sont considérablement réduites par rapport aux prédictions théoriques. Cette tendance n'est pas observée pour des noyaux moins exotiques, jusqu'à DeltaS~12 MeV, par réaction de transfert (d,p).Pour comprendre l'origine de cette réduction, nous avons réalisé deux expériences complémentaires sur un noyau présentant une large différence d'énergie de séparation l'14O : (i) le knockout d'un nucléon au NSCL, 14O (53 MeV/n) et 16C (75 MeV/n), sur une cible de 9Be; (ii) le transfert d'un nucléon avec le faisceau d'14O à 18 MeV/n de SPIRAL, 14O(d,t)13O et 14O(d,3He)13N, étudié avec le dispositif MUST2. L'analyse des données présentée en détails dans ce manuscrit conduit à des facteurs spectroscopiques incompatibles entre ces deux expériences lorsqu'un neutron fortement lié est enlevé ou transféré de l'14O. Dans le cas du knockout, la section efficace mesurée est fortement réduite par rapport aux prédictions basées sur un modèle eikonal et le modèle en couches. La distribution en moment parallèle de l'13O mesurée après l'arrachage d'un neutron présente par ailleurs une forme qui n'est pas reproduite par le modèle eikonal. Dans le cas du transfert, une telle réduction n'est pas observée et le rapport entre expérience et théorie est compatible avec ce qui est obtenu pour les noyaux stables. Ces résultats suscitent de nouveaux développements théoriques pour la modélisation du mécanisme de réaction dans ces cas extrêmes où le nucléon enlevé est fortement lié.

<p>Antibodies have the ability to influence the antibody response against the very antigen they are specific for, in a process called antibody feedback regulation. Depending on the nature of the antigen, the antibody response can be either enhanced or almost completely inhibited. This thesis focuses on the underlying mechanisms of antibody feedback regulation <i>in vivo</i>. </p><p>Antigen-specific IgG can inhibit the antibody response to a particulate antigen. Based on its ability to inhibit B cell activation, the inhibitory FcγRIIB (low affinity receptor for IgG) has been suggested to be involved. Here we show that although FcγRIIB is required for efficient suppression<i> in vitro, </i>it is not required <i>in vivo</i>. Therefore, even though FcγRIIB can inhibit antibody responses, other mechanisms (such as epitope masking and enhanced antigen clearance) play a more dominant role<i> in vivo</i>.</p><p>The antibody response to soluble antigen is greatly enhanced when it is introduced to the immune system in complex with antigen-specific IgG or IgE. We found that FcγRIIB attenuates the magnitude of IgG-mediated enhancement. In mice lacking FcγRIIB, IgG enhanced the antibody response much more efficiently than in normal mice.</p><p>Since B cells require CD4⁺ T cell help in order to become antibody-producing cells, we examined the CD4⁺ T cell response to immune complexes <i>in vivo</i>. Using an adoptive transfer strategy with transgenic ovalbumin (OVA)-specific CD4⁺T cells, we could show that the enhanced OVA-specific IgG response to IgG2a/OVA and IgE/OVA complexes was preceded by a potent OVA-specific CD4⁺ T cell response. IgG2a-mediated enhancement was dependent on activating Fcγ receptors, whereas IgE-mediated enhancement was dependent on CD23, the low affinity receptor for IgE. We identified CD23⁺ B cells as the responsible effector cells for IgE-mediated enhancement<i> in vivo</i>. Taken together, these results show that Fc receptor-mediated antigen presentation is a major mechanism underlying antibody feedback enhancement. </p>