Administration of cyclophosphamide to males targets the germ cells and causes DNA damage including single strand breaks and DNA-DNA cross links. When males are treated with a chronic low dose of cyclophosphamide and then mated to normal females, progeny loss is manifested at the pre- and post-implantation stages of development. The earliest events that lead to embryonic loss were traced to day 2 of gestation when embryos had a decreased DNA synthesis profile and lower cell numbers than control litters. I investigated the hypothesis that chronic exposure of male rats to cyclophosphamide alters zygotic gene expression thus leading to embryonic loss. To assess DNA damage in the embryo, the Comet Assay was performed on 1-cell stage embryos. A significant number of embryos sired by cyclophosphamide-treated males showed the appearance of the Comet indicative of the effect of damaged sperm on the embryos starting from the 1-cell stage. Using a candidate gene approach, the antisense RNA (aRNA), I described the presence of several DNA repair gene families in normal embryos. Progeny sired by cyclophosphamide-treated males manifested a differential expression profile for several of these genes when compared to controls, suggestive of the ability of the embryo to respond to damaged sperm through the major DNA repair systems. To study the functional capacity of progeny sired by cyclophosphamide-treated males, I assessed total RNA synthesis in both groups; while control litters showed a peak of RNA synthesis at the 4-cell stage, the treatment group showed constant low expression throughout the stages examined. I mapped the profile of a number of gene families whose roles are essential for early development in both control and cyclophosphamide-treated groups. While control embryos showed a peak of expression for the majority of genes at the 8-cell stage, that of the cyclophosphamide-group showed an early induction at the 2-cell stage, indicative of loss of the tightly regulated

No description available.

CD45 phosphatase is expressed ubiquitously on cells of lymphoid origin, and has long been considered a drug target for immunosuppression. CD45 is a key component of the signaling pathway in immune cells, regulating activation and development of these cells. Drug intervention could suppress growth and provide a therapeutic avenue for treatment of transplant rejection, auto-immune diseases such as rheumatoid arthritis, and various cancers of the immune system. The intracellular, catalytic portion of CD45 is comprised of the D1 and D2 domains. The active site is located in the D1 domain, and the D2 domain is catalytically inactive. We decided to target the inter-domain region between D1 and D2 for the development of a small molecule allosteric inhibitor. The linker region between CD45 D1 and D2 was modeled in silico, and NCI database was screened for compounds that dock in this region. Hits were tested in vitro and we detected two CD45 inhibitors, one of which was demonstrated to be highly selective for CD45 in tyrosine phosphatase counter-assays. Analogs were generated which increased in vitro potency, and cell membrane permeability. The most active inhibitor, 211, was pursued further. The kinetics of CD45 inhibition by 211 indicated a non-competitive and irreversible mechanism of action, as well as some degree of substrate specificity. Site-directed mutations in CD45 enzyme at the predicted 211 binding sites revealed a loss of inhibition by 211 in the mutant CD45, consistent with the in silico prediction of allosteric inhibition. Circular dichroism studies indicated a conformational change in CD45 enzyme, in the presence of 211. We first validated that CD45 was the target of 211 in cells, by evidence of a change in the phosphorylation of its in vivo substrate Lck in Jurkat cells. As a control, the phosphorylation status of Lck was not affected in J45.01 (CD45-negative) cells with 211 treatment. 211 was shown to suppress the activation of T cell receptor signaling in primary T cells. In vivo, 211 inhibits the antigen-induced immune response in the mouse footpad.CD45 inhibitor 211 was assayed for its anti-cancer properties in the EL4 mouse thymoma cell line. 211 inflicted dose-dependent toxicity on these cells via cell cycle rest and apoptosis. EL4 cells injected into mice were delayed in developing a solid tumor when mice were treated with 211, and metastasis to the lymph nodes was reduced in these animals. Our work has shown the therapeutic potential of targeting CD45 by revealing a novel allosteric binding site for a CD45 inhibitor, and this approach may be useful for targeting other two-domain tyrosine phosphatases. / La phosphatase CD45 est exprimée de manière ubiquitaire dans les cellules d'origine lymphoïde, et a longtemps été considérée comme une cible thérapeutique pour l'immunosuppression. CD45 est une composante clé de la voie de signalisation dans les cellules immunitaires, dans la régulation de l'activation et dans le développement de ces cellules. L'intervention médicale pourrait bloquer la croissance et fournir une avenue thérapeutique pour le traitement du rejet de greffe, les maladies auto-immunes telle que la polyarthrite rhumatoïde, et de divers cancers du système immunitaire. La partie intracellulaire catalytique de CD45 comprend les domaines D1 et D2. Le site actif est situé dans le domaine D1, et D2 est le domaine catalytiquement inactif. Nous avons décidé de cibler la région inter-domaine entre D1 et D2 pour développer des possibles inhibiteurs allostériques de petit poids moléculaires. La région de liaison entre CD45 D1 et D2 a été modélisée in silico. Nous avons d'abord analysé une base de données NCI pour des composés qui s'amarrent dans cette région. Puis, nous avons testé in vitro plusieurs composés obtenus, et nous avons détecté deux inhibiteurs de CD45, dont l'un a été démontré pour être très sélectif pour CD45 dans des contre-essais avec autres phosphatases tyrosine. Des analogues ont été produit, et ceux-ci avaient une plus grande puissance in vitro et une meilleure perméabilité de la membrane cellulaire. L'inhibiteur le plus actif, 211, a été poursuivi comme tête de série.La cinétique de l'inhibition de CD45 par 211 indique un mécanisme d'action non compétitif et irréversible, ainsi qu'un certain degré de spécificité de substrat. Des mutations du CD45 sur les sites de liaisons du 211 ont montré une perte de l'inhibition du 211 sur le CD45 mutant, confirmant ainsi les résultats obtenus par l'inhibition allostérique in silico. Des études de dichroïsme circulaire ont indiqué un changement de conformation de l'enzyme CD45, en présence de 211. Nous avons d'abord validé que CD45 est la cible de 211 dans les cellules, par des preuves d'un changement de phosphorylation Lck dans les cellules Jurkat, mais pas J45.01 (CD45 négatives), avec traitement de 211. Nous avons montré que 211 est capable d'inhiber l'activation de signalisation induite par l'anticorps de récepteur cellule T dans les cellules T primaires, et témoignent d'une inhibition de la réponse immunitaire dans le coussinet plantaire du souris.L'inhibiteur de CD45 211 a été testé pour ses propriétés anti-cancer dans la lignée de cellules du thymus du souris EL4. Une toxicité dose-dépendante induite par 211 a été jugée de nature apoptotique, et déclenché par un arrêt du cycle cellulaire après 16 heures de traitement. Des cellules EL4 injectées dans des souris ont été retardés dans le développement d'une tumeur solide lorsque les souris ont été traitées avec 211, et des métastases dans les ganglions lymphatiques ont été réduites chez ces animaux.Nos travaux ont révélé un nouveau site de liaison d'un inhibiteur de CD45, et cette approche peut être utile pour cibler d'autres tyrosine phosphatases de deux domaines. Nous avons également affiché le potentiel thérapeutique du ciblage CD45 allostérique.

No description available.

The interaction of naloxonazine, a putative long lasting or "irreversible" mu$ sb1$ receptor antagonist, with morphine, morphine-6-glucuronide (M6G) and sufentanil was studied in two nociceptive tests using rats, the formalin test and the tail-immersion test. Also, the displacement of ($ sp3$H) naloxone binding by selective opioid agonists in the rat brain membrane was performed after naloxonazine pretreatment in vivo. / The specificity of naloxonazine was dependant on the nociceptive test used. In the tail-immersion test, intracranial naloxonazine (1 ug 4 hours before testing) produced a nonparallel right shift of the dose effect relations of all three agonists studied, consistent with long lasting "irreversible" antagonist properties of naloxonazine. In the formalin test, the same naloxonazine pretreatment regimen produced parallel right shift of the morphine dose effect relation but failed to alter the effects of M6G and sufentanil, suggesting either "reversible" antagonist properties or a more complex mechanism. Displacement binding assays suggest that naloxonazine interacts with mu and delta opioid receptor sites. / The data imply that naloxonazine interacts in a long lasting manner with more than one opioid receptor subtype. An allosteric interaction between opioid receptor subtypes is proposed to explain the effects in the formalin test.

The effects of glucocorticoids and agents affecting membrane phospholipid metabolism on $ beta$-adrenergic receptors ($ beta$AR) have been studied in A549 cultured human lung tumor cells and in IM-9 cultured human lymphocytes. Various glucocorticoids increased the density of $ beta$AR in A549 lung cells but not in IM-9 lymphocytes, although they did increase insulin receptor density in IM-9 lymphocytes. In contrast, bee venom phospholipase A$ sb2$ (0.05-0.2 units/ml), but not phospholipases C or D., downregulated $ beta$AR in IM-9 lymphocytes, but did not affect $ beta$AR in A549 lung cells. Upregulation of $ beta$AR by glucocorticoids was partially reversed by arachidonic acid (10 $ mu$M) but not by lysophospatidylcholine. These results suggest that glucocorticoids upregulate pulmonary $ beta$AR partly through their inhibition of membrane phospholipase A2 and the subsequent generation of arachidonic acid and/or its metabolites, which may act by modulating a direct effect of glucocorticoids on the synthesis of $ beta$AR. Coculturing IM-9 lymphocytes and A549 lung cells results in a 2-3-fold increase in the density of $ beta$AR in A549 lung cells. Lymphocyte conditioned medium (LCM) has the same effect, which is moderately sensitive to heat, is retained by ultrafiltration over a 10,000 m.w. cut-off filter, and is reduced by trypsin treatment or by preincubation of lymphocytes with cycloheximide (1$ mu$m). Treatment of lung cells with cycloheximide also prevents the effect of LCM. Glucocorticoids, which also increase $ beta$AR density in A549 lung cells, markedly potentiate the effects of LCM. Gel permeation HPLC of LCM yields 3 peaks of biological activity with M.W. 70,000, 35,000 and 15,000. Monocytic Interleukin-1 (IL-1) mimics the effect of LCM in that it increases $ beta$AR density in A549 lung cells and its effect is potentiated by cortisol. Recombinant IL-1$ alpha$ is somewhat more potent then IL-1$ beta$, while Interleukin-2 and interferon-$ alpha$ are inef

No description available.

It is widely believed that nicotine is the main reason for acquiring and maintaining tobacco addiction. However, animal studies suggest that nicotine is only a weak reinforcer compared to other drugs of abuse. For example, nicotine does not consistently produce a conditioned place preference (CPP), a standard measure of reward in rats. We attempted to examine the reason(s) for this discrepancy by manipulating several procedural variables in this paradigm. In addition, we hypothesized that partial monoamine oxidase (MAO) inhibition, as observed in smokers, may potentiate the rewarding effects of nicotine in the CPP paradigm. Overall, we were not able to obtain reliable nicotine CPP and none of the procedural variables tested (e.g. speed of injection, nicotine pre-treatment) proved to play an important role in acquisition of nicotine CPP. Possible reasons for the failure of our experiments and of other nicotine CPP studies are discussed.

Male reproductive health is of growing concern, as male toxicant exposure can affect progeny outcome; sperm chromatin structure integrity may be a contributing factor. The formation of mature sperm involves the expression of numerous proteins involved in organizing and packaging the chromatin in a specific manner; this ensures transmission and participation of the paternal genome in embryogenesis. Exposure of male rats to cyclophosphamide as spermatid chromatin is remodeled has an adverse effect on embryo development. The hypothesis of this thesis is that cyclophosphamide exposure causes genetic damage and alters the sperm proteome, thus disrupting components of chromatin condensation and organization during spermiogenesis. The first objective was to assess the phase specificity of the susceptibility of spermiogenic germ cells to cyclophosphamide-induced DNA damage. Spermatozoa were analyzed for DNA strand breaks using the comet assay. Cyclophosphamide-induced DNA damage was dose-related and accumulated over time. Germ cell phase-specific damage was maximal during midspermiogenesis; this reflects an increased susceptibility of step 9-14 spermatids at a key point in sperm chromatin remodeling, the histone-protamine exchange. The second objective was to examine the sperm chromatin structure and basic proteome. Multiple assays demonstrated that the effects of cyclophosphamide on sperm chromatin structure were also germ cell-phase specific; midspermiogenic spermatids were most sensitive. Sperm were less condensed with reduced total thiol and protamine contents. The sperm basic proteome was also altered; identified proteins are involved in a variety of spermiogenic and fertilization events. The nuclear matrix organizes chromatin into loop domains, and various components of somatic cell matrices are targets for chemotherapeutic agents. Therefore the last objective of this study was to assess the effect of cyclophosphamide exposure on the protein profile of the sperm nuclear matrix. The expression of several nuclear matrix protein components, a number of which were identified for the first time, was altered following drug exposure. Together these results show that cyclophosphamide alters male germ cell chromatin remodeling at both the DNA and protein level; this could alter sperm function and thus explain the adverse effects on early embryo development.

Neuropathic pain presents itself with many faces. It can be devastatingly painful leading to a person's inability to lead a productive life or it can be mild. In this thesis I discuss one of the countless possible mechanisms which could underlie the development and maintenance of this disorder; namely the involvement of peripheral changes in innervation. The approach taken in this thesis was mainly based on protein analyses, based on the assumption that protein expression (or lack thereof) is indicative of changes in the physiological properties of structures that either express or secrete proteins. I also address the models which are commonly used by researchers to elucidate the mechanisms underlying chronic neuropathic pain in hopes of developing viable treatments. The focus of this work is on the periphery, more specifically involving the glabrous skin of the rat hind paw. This skin is innervated by branches of the sciatic nerve which are comprised of, in part, sensory fibres. These are sub-classified into the thickly and thinly myelinated A-beta and A-delta afferents, respectively, as well as unmyelinated C-fibres. The latter population can be divided into two subgroups based on their peptide content where the peptide rich are termed peptidergic and the peptide-poor, non-peptidergic. This area also receives innervation from post-ganglionic sympathetic efferents. This thesis comprises three experimental chapters of my independent work into further exploring the models and mechanisms underlying the maintenance and generation of neuropathic pain. The first experimental chapter addresses the long term changes of skin innervation which occur following the application of a commonly used model of neuropathic pain, namely the chronic constriction injury (CCI) of the sciatic nerve model. I examined the myelinated afferent population as a whole, the unmyelinated peptidergic afferents as well as the non-peptidergic C-fibres from three days through 1.5 years following the application of the nerve injury. There was a persistent loss of myelinated afferents, loss and delayed sprouting of non-peptidergic C-fibres as well as a brief loss and permanent sprouting of peptidergic afferents within the upper dermis. The second study is a comparative examination of the morphological and behavioural changes following application of two very similar models of neuropathic pain. The CCI model is based on the application of loose chromic gut ligatures and the variation of this is the cuff model in which the chromic gut ligatures are replaced by a fixed diameter polyethylene cuff. The application of the cuff model resulted in a persistent mechanical hypersensitivity in contrast to the CCI in which the mechanical hypersensitivity resolved within about a month. However, the application of either model resulted in a transient thermal hypersensitivity. Innervation changes following application of the cuff model included a reduction in myelinated afferent density within the upper dermis in both models, the unmyelinated, non-peptidergic afferents declined in fibre density followed by a delayed recovery in the cuff model and the peptidergic afferents gradually declined and remained so following application of the cuff in contrast to the application of the CCI. The third chapter addresses the changes in the expression of nerve growth factor (NGF) and its receptors following CCI. The precursor form of NGF, proNGF was localized to mast cells, vascular endothelium and keratinocytes in the epidermis in naïve skin. Following injury, proNGF increased. After lesion, Schwann cells expressed high levels of the NGF receptor p75. The two receptors for NGF, TrkA and p75, were differentially regulated during the progression of the nerve injury. The work of this thesis has provided new findings documenting the morphological changes underlying the generation and maintenance of neuropathic pain as well as the molecular generators of these changes. / La douleur neuropathique peut se présenter sous plusieurs formes. Dans cette thèse, j'analyse les mécanismes périphériques pouvant déclencher et maintenir la douleur neuropathique. L'approche adoptée dans cette thèse est surtout basée sur une analyse de la concentration et distribution anatomique de protéines, avec l'idée sous-jacente que son niveau d'expression (ou absence d'expression) est révélatrice de changements physiologiques dans les structures qui produisent ou sécrètent ces protéines. Le focus de ce travail se déroule dans la périphérie, en particulier la peau non couverte de poils de la surface plantaire du membre postérieur du rat. Cette peau est innervée par des fibres nerveuses sensorielles provenant du nerf sciatique. Ces fibres sont normalement classées comme afférences myélinisées épaisses et finement myélinisées (A-bêta et A-delta respectivement), ainsi que comme fibres amyéliniques (C). La dernière population peut être divisée en deux sous-groupes en fonction de présence ou absence de neuropeptides en peptidergiques et non-peptidergiques. Cette région reçoit également une innervation par des fibres post-ganglionnaires sympathiques. Cette thèse est composée de trois chapitres expérimentaux. Le premier chapitre se penche sur les changements à long terme de l'innervation cutanée qui se produisent après l'application d'un modèle de douleur neuropathique couramment utilisé, à savoir la blessure par constriction chronique du nerf sciatique (CCI). J'ai examiné les changements des fibres myélinisées, des fibres amyéliniques peptidergiques, ainsi que des fibres non-peptidergiques dès trois jours après l'application de la lésion jusqu'à un an et demi après la lésion. Il y avait une perte persistante des afférences myélinisées, une perte suivie d'une récupération tardive des fibres C non-peptidergiques ainsi qu'une brève perte suivie de récupération permanente des afférences peptidergiques dans la partie supérieure du derme. Le second chapitre expérimental est un examen comparatif des changements morphologiques après l'application de deux modèles très similaires de douleur neuropathique. Le premier est la constriction chronique du nerf sciatique (CCI) suivant l'application de 4 ligatures; dans le deuxième la constriction du nerf sciatique est obtenue par l'application autour du nerf d'un segment de tube polyéthylène (cuff). L'application du « cuff » résultait en une hypersensibilité mécanique persistante, contrairement au CCI dans lequel l'hypersensibilité mécanique disparaissait dans un mois environ. Toutefois, les résultats de l'application des deux modèles sur la sensibilité thermique étaient similaires, avec une hyper-sensitivité à la chaleur qui durait environ un mois. Les afférences myélinisées étaient réduites en densité dans la partie supérieure du derme dans les deux modèles et cette baisse était permanente. Cependant, les afférences non-peptidergiques ont souffert une baisse de densité suivie d'un retour retardé à des valeurs des contrôles dans le modèle « cuff ». Dans ce dernier modèle, les afférences peptidergiques ont diminué progressivement en densité et n'ont pas récupéré, à la différence de l'application de la CCI. Le troisième chapitre expérimental concerne les changements dans l'expression du facteur de croissance nerveuse (NGF) et de ses récepteurs dans des animaux avec une lésion CCI. Le précurseur du NGF, proNGF a été localisé dans l'épiderme de la peau naïve. À la suite de la lésion CCI, nous avons détecté une augmentation du proNGF. Après la lésion, les cellules de Schwann avaient une expression très augmentée du récepteur du NGF p75. Les récepteurs au NGF, TrkA et p75 sont différentiellement régulés dans la période après la lésion du nerf. Le travail de cette thèse a eu comme objectif de clarifier les changements morphologiques contribuant au déclanchement et la manutention de la douleur neuropathique, ainsi que les mécanismes moléculaires de ces changements.