The compound 2-(4-phenylpiperidino)cyclohexanol (AH5183), a potent inhibitor of vesicular acetylcholine (ACh) uptake, blocks the nerve impulse stimulated release of ACh from the superior cervical ganglion of the cat in a use dependent manner, but not until 14% of the initial ganglial ACh content has been released. The first objective of this work was to determine if blockade of ACh release was associated with a depletion of vesicular ACh. By using black widow spider venom (BWSV), a secretagogue which induces vesicular exocytosis, it was shown that depletion of vesicular stores did not likely occur as a result of AH5183 blockade. Furthermore, labeling the ACh synthesized in the presence of the drug, revealed that labeled ACh and pre-existing ACh were equally well released by the venom suggesting that some vesicles were insensitive to AD5183. / Thus, the second objective of these studies was to test whether an AH5183-insensitive vesicular compartment existed and to investigate which subcellular compartments played a role in the release of ACh by the various stimuli. Rat hippocampal slices were stimulated with K$ sp+$, in the presence or absence of AH5183, and then with the purified active toxin of BWSV, $ alpha$-LTX. The toxin's main effect was to decrease the ACh content of the cytoplasm. Labeling the ACh synthesized in the presence of AH5183 demonstrated that stimulation increased with specific activity of the ACh stored by the denser vesicular compartment, normally associated with a high turnover of transmitter, but not that of the lighter vesicles associated with a storage function. / To investigate how the occluded compartments recovered form AH 5183, hippocampal slices were drug-exposed, stimulated, and allowed to recover in the drug's absence. ACh synthesized during the recovery was labeled. Transmitter release and the ACh content of the occluded compartments recovered slowly from the effects of AH5183 and no quantitative relationship was found between the two events. The denser vesicular compartment recovered 1.5 times as quickly as the lighter one. The denser compartment accumulated labeled ACh while the lighter did not, suggesting that different sources of ACh exist within the cytoplasm.

No description available.

It has been proposed that contractile proteins may mediate the events of the exocytotic release of hormones or neurotransmitters because of the similarities between the process of stimulus-secretion coupling and excitation-contraction coupling. / Bovine adrenal medullary chromaffin cells were isolated and cultured and the morphological and secretory characteristics of these cells investigated. The results demonstrated that cultured chromaffin cells were a good model for developmental and secretory studies. Two isomeric forms of actin ((beta) and (gamma)), differing in isoelectric properties, were purified from chromaffin cells and characterized. The immunocytochemical localization of actin using an anti-chicken gizzard actin antibody showed that actin is distributed in a granular pattern in chromaffin cells in culture and that this distribution was similar to that of dopamine (beta)-hydroxylase. This suggested that actin might be associated with the chromaffin granules and indeed, actin was found when chromaffin granule membrane proteins were analyzed by SDS gel electrophoresis. / This evidence supports our hypothesis that actin, along with the other contractile proteins, may mediate the process of stimulus-secretion coupling.

When a new drug is introduced onto the market, patients treated with it often tend to be sicker than those who are not. Once a drug has been released onto the market, its safety and efficacy can usually only be assessed using non-experimental studies. However, these methods cannot account for the differential assignment of sicker patients to new drugs, which can make a new drug seem detrimental or even dangerous, if these systematic differences are not accounted for. This is a prototypical example of confounding by indication, which is often viewed as an insurmountable problem in pharmacoepidemiologic research. Confounding by indication consists of two components, namely channelling and the risk factor effect. / In this thesis, the index of apparent channelling (IAC) is introduced as a novel tool for the measurement of the degree of channelling. The IAC makes use of propensity scores to quantify the proportion of the variance in treatment assignment that can be accounted for by documented patient characteristics. However, the IAC can only account for channelling due to documented factors. Thus, it is always possible that there be residual channelling due to undocumented factors. Such residual channelling is of concern mostly to the extent that it leads to confounding of the treatment effect. Consequently, the index of residual channelling (IRC) is developed to estimate residual channelling and a novel approach is proposed to assess the strength of the resulting confounding bias. This model-based approach is based on estimating the interaction between treatment effect and the expected strength of residual channeling on treatment assignments of individual patients, measured by the discrepancy between their predicted treatment and the treatment they actually received. / The combination of the index of apparent channelling and the model-based approach to residual channelling provides a practical approach to the problem of assessing the impact of confounding by indication in non-experimental studies in the post-marketing evaluation of the safety and efficacy of new drugs. The application of these methods may enhance the validity of the conclusions drawn in such studies.

The opioid receptor family is comprised of three members: mu, delta and kappa, all of which are G protein coupled receptors, primarily acting through Galphai/o subunits. Clinically, mu opioid receptor (MOR) agonists are used in the treatment of moderate to severe pain. delta opioid receptor (DOR) agonists are being developed as alternative analgesics, since stimulation of this receptor results in fewer adverse side effects. Characterization of behaviourally relevant mu and delta opioid receptors, as well as interactions between them, will provide a better understanding of opioid agonist-induced analgesia. / Although the behavioural knockdown after antisense targeting of MOR has been well characterized, few studies have examined the corresponding in vitro changes. Thus, the first aim of this thesis was to determine the neuroanatomical extent of MOR knockdown after pretreatment with peptide nucleic acid antisense in rats. Antisense pretreatment completely inhibited antinociception by the mu agonist DAMGO, but produced no detectable ex vivo changes in brain or spinal MOR labelling or functional responses. This study suggests that there may be a small, critical population of MORs that mediate antinociceptive responses to agonist. / The second aim of this thesis was to compare the CNS distribution of functional DOR with radioligand binding. DOR labelling was determined autoradiographically using an agonist, ([125I]deltorphin II) and an antagonist ([ 125I]AR-M100613) radioligand. In adjacent tissue sections, functional DORs were detected using deltorphin II-induced [35S]GTPgammaS binding. Overall, radioligand binding did not strongly predict the magnitude of [35S]GTPgammaS responses, and this weak association is possibly explained by a paucity of DORs on the cell surface and/or heterogeneity in G protein receptor coupling. The highest [35S]GTPgammaS responses were found in the basal ganglia, while areas involved with pain perception (spinal cord, brain stem, and periaqueductal grey) possessed low [35S]GTPgammaS responses. / The low deltorphin II-induced [35S]GTPgammaS binding in pain-related areas could explain the moderate degree of antinociception produced by delta agonists relative to their mu counterparts. Thus, the third aim of this thesis was to investigate two pharmacological treatments (short- and long-term morphine pretreatment) that are reported to enhance behavioural responses to delta agonists. As previously observed by others, short-term exposure to morphine resulted in sensitization to spinally administered delta agonists. In contrast, long-term morphine pretreatment resulted in profound tolerance to the antinociceptive and locomotor stimulant effects of deltorphin II. After chronic morphine pretreatment, there was no detectable change in DOR labelling or [35S]GTPgammaS responses in the brain or spinal cord, suggesting that changes in downstream regulators may be responsible for this tolerance.

Gene amplification alters gene expression and can promote oncogenesis. In particular, the amplification of chromosome 19q13.1-13.2 has been found in several cancers and is known to contain the AKT2 oncogene. Two members of the SERTAD gene family of transcription factors, SERTAD1 and SERTAD3, are also located within this region. We report herein the genomic structure, regulation, and functions of SERTAD3. This gene has two transcript variants with short mRNA half-lives, and one of the variants is tightly regulated throughout G1 and S phases of the cell cycle. Overexpression of SERTAD3 induces cell transformation in vitro and tumor formation in mice, while inhibition of SERTAD3 by siRNA results in a 2-4 fold reduction in cell growth rate. Furthermore, luciferase assays based on E2F-1 binding indicate that SERTAD3 increases the activity of E2F, which can be strongly reduced by siRNA inhibition of SERTAD3. Together, our data support that SERTAD3 contributes to oncogenesis at least in part via an E2F-dependent mechanism.

Neurotrophins, NGF, NT-3, BDNF and NT4-5, are growth factor proteins key to the regulation of neuronal survival, death of differentiation. Neurotrophins bind two types of receptors, Trk tyrosine kinase receptors and the common p75 receptor. / Disturbances in Trk expression or signaling can lead to certain cancers such as neuroblastomas and melanomas (TrkA responsive) and medulloblastomas (TrkC responsive). Less is known about the role of p75 in cancer as it plays such varying roles and regulatory functions. / We characterized the in vitro and in vivo growth kinetics, tumorigenic potential and response to chemotherapeutics of PC 12-wt rat pheochromocytoma cells and variants expressing varying levels of TrkA or TrkC with or without p75. / We find that independent expression of TrkA or p75 leads to higher growth rates and tumorigenic potential whereas co-expression of Trk with p75 lowers it. Additionally, differential neurotrophin receptor expression leads to different sensitivities to chemotherapeutics. This work will help design therapies for tumors with unique neurotrophin receptor phenotypes.

Aging is accompanied by an increase in reactive oxygen species (ROS). Spermatozoa are vulnerable to the effects of ROS; these result in damage to membranes and chromatin, the former affecting their motility and the latter impeding successful progeny outcome. The epididymis is important in protecting spermatozoa by removing ROS using glutathione and antioxidant enzymes. Therefore, our goals were to investigate how aging affects (i) the redox capacity of the epididymis, spermatozoal motility and spermatozoal chromatin packaging and integrity, as well as (ii) the susceptibility of these parameters to in vivo and in vitro oxidative challenge. Caput, corpus and cauda segments of the epididymides from young and old male Brown Norway rats were collected and glutathione and antioxidant enzyme activities were measured. Spermatozoa from the cauda epididymidis were analyzed for motility; chromatin packaging and integrity were evaluated in spermatozoa from both the caput and cauda epididymides. In vivo oxidative challenge was induced by 7-day systemic administration of L-buthionine-S,R-sulfoximine (BSO), which is an inhibitor of glutathione synthesis, and in vitro oxidative challenge was induced by incubating spermatozoa in H 2O2. We found that the redox capacity of the cauda epididymidis was altered with age, where the balance shifted towards conserving glutathione. Spermatozoal chromatin become more tightly packaged and less prone to dissociation and DNA quality decreased. Additionally, spermatozoa from old rats contained fewer overall thiols and disulfide bonds, which was due to the appearance of a low-thiol population. After BSO administration glutathione recycling increased and alternative pathways became upregulated throughout the epididymis; this response was greatest in the corpus region, whereas the cauda was affected in an age-dependent manner. Spermatozoal motility became compromised after BSO, where the effect was greater in spermatozoa from older rats. Exposure to BSO and H2O2 revealed that spermatozoa from the cauda epididymidis of older rats had a unique susceptibility to their combined effects. In summary, spermatozoal quality becomes altered in an age-dependent and epididymal segment-specific manner; it is proposed that this may be attributable to the effect of aging on the cauda epididymidis. Susceptibility to oxidative challenge also increases with age, both at the level of spermatozoal chromatin and motility.

Effective therapies typically target diseased tissues without significantly affecting healthy tissues. A tumor-related glycosphingolipid (GSL), such as ganglioside GD2, distinguishes neuroectoderm tumors from their healthy counterparts and is a validated tumor target. GD2 is clinically targeted for diagnosis and immunotherapy. GD2 plays an important functional role in tumor progression and in chemoresistance. It also plays an important functional role in pain; however, the mechanisms that make GD2 important in such phenomena remain unknown.Thus, understanding the structure-activity relationship of GD2, a GSL with two sialic acids, would be helpful. However, such studies on glycolipids are challenging. We employed a chemical biology approach to elucidate the structure and function of GD2 and to further direct the rational design of GD2 ligands and vaccines for GD2-related cancer treatment.The combined use of STD NMR spectroscopy, transferred NOE experiments, and molecular modeling furnished details on the molecular recognition of the ganglioside GD2 by the clinically used anti-GD2 monoclonal antibody 3F8 that can induce apoptosis of GD2 expressing tumours. A binding model that provided the basis for a rational development of GD2 ligands and vaccines was then established. Based on the structural information of GD2-3F8 interactions, small molecule monomeric peptide ligands binding to GD2 were developed. ELISA and NMR experiments demonstrated that peptides selectively bound to GD2 via an induced-fit mechanism. Furthermore, peptidic GD2 ligands, including 3F8, mediated similar biological functions in cell-based assays of activation of NMDA receptor via Src family kinase, calcium fluxe and cAMP. These can explain at least some of the mechanisms associated with tumor progression and pain, where GD2 plays a role. However, current GD2 peptide ligands did not demonstrate any treatment effect in vivo. Hence, we turned to the design of GD2 vaccines as a therapeutic approach. The rigid nature of GD2-oligosaccharides, discovered in our structural study, makes it perfectly suited to drive a structurally convergent immune response. A novel and homogenous tetra-GD2 dendrimer was designed to mimic a clustered GD2 lipid raft. Immunization of mice with tetra-GD2 dendrimer elicited a potent anti-GD2 humoral response. The antibodies (sera or mAbs) thus generated can kill GD2-expressing cells in culture, in the absence of a complement. Tumor growth was significantly delayed in vivo in prophylactic and in therapeutic paradigms. Our research strategy may be expanded to other clinically relevant glycolipids. / Typiquement, les thérapies efficaces agissent sur les tissus malades sans toutefois nuire considérablement aux tissus en santé. Un glycosphingolipide (GSL) associé à une tumeur, tel que le ganglioside GD2, peut reconnaître selectivement les tumeurs neuroectodermes malignes et est ainsi validé comme une cible tumorale. Sur le plan clinique, on utilise le GD2 à des fins de diagnostic et d'immunothérapie. D'une part, le GD2 joue un rôle fonctionnel important dans la progression tumorale et la chimiorésistance. D'autre part, le GD2 joue un rôle fonctionnel important dans la douleur, mais les mécanismes qui expliqueraient l'importance du GD2 lors de tels phénomènes demeurent encore inconnus. C'est pourquoi il serait utile de mieux comprendre la relation structure-activité du GD2, un GSL constitué de 2 acides sialiques. Toutefois, entreprendre de telles études sur les glycolipides représente un défi de taille. Nous avons alors utilisé une approche basée sur la biologie chimique pour élucider la structure et la fonction des GD2 et pour mieux concevoir rationnellement des ligands GD2 et des vaccins pour le traitement contre le cancer lié au GD2. En combinant l'utilisation de la spectroscopie RMN DTS, des expériences NOE transférées et des modèles moléculaires, il est possible d'obtenir plus de détails sur la reconnaissance moléculaire du ganglioside GD2 au moyen du 3F8, un anticorps monoclonal anti-GD2 utilisé médicalement et qui peut induire l'apoptose de cellues cancereuses exprimant GD2. Comme point de départ pour le développement rationnel des ligands GD2 et des vaccins, nous avons établi un modèle contraignant. En nous appuyant sur l'information structurelle des interactions GD2-3F8, nous avons développé des petits ligands monomériques peptidiques liés au GD2. Des expériences RMN et ELISA ont démontré que les peptides se lient sélectivement au GD2 via un mécanisme d'ajustement induit. Par ailleurs, les ligands peptidiques GD2, dont le 3F8, deviennent médiateurs de fonctions biologiques similaires dans les essais cellulaires de l'activation des récepteurs NMDA via les kinases de la famille Src, les flux de calcium et cAMP. Ces derniers peuvent au moins expliquer certains des mécanismes associés avec la progression tumorale et la douleur, dans lesquels le GD2 jour un rôle prépondérant. Cependant, les ligands peptidiques GD2 actuels n'ont pas démontré les effets désirés au cours des traitements in vivo. C'est pourquoi nous nous sommes tournés vers le développement de nouveaux vaccins GD2 comme une approche thérapeutique. La nature rigide des oligosaccharides GD2, que nous avons découverte par le biais de notre étude structurale, devient une caractéristique parfaitement adaptée pour favoriser une réponse immunitaire structurellement convergente. Un nouveau dendrimère tetra-GD2 homogène a été conçu de manière à reproduire un radeau lipidique GD2 regroupé. L'immunisation des souris par le dendrimère tetra-GD2 a engendré une puissante réponse humorale anti-GD2. En l'absence d'un complément, les anticorps (sera ou mAbs) ainsi générés peuvent tuer les cellules exprimant le GD2 en culture. La croissance tumorale a été considérablement retardée in vivo dans les paradigmes thérapeutiques et prophylactiques. Notre stratégie de recherche pourrait ainsi être élargie pour inclure d'autres glycolipides pertinents sur le plan clinique.

The fields of Nanomedicine is rapidly expanding. Already numerous nanoparticles have entered clinical trials. However, certain nanostructures, though, therapeutically and diagnostically promising, can induce toxicity both in vitro and in vivo; cadmium telluride quantum dots fall into this category. Quantum dots (QDs) are highly fluorescent, semi-conducting nanocrystals, that consist of a metallic core. Compared to traditional fluorophores, QDs have superior optical qualities, including resistance to photobleaching, broad spectrum excitation and narrow emission. Cadmium telluride (CdTe) QDs were the first to be synthesized without organic solvents, and thus, considered suitable for biological application. However, early studies demonstrated that QDs induced cytotoxicity and oxidative stress. Although, this QD-toxicity was ascribed to cadmium (Cd) liberation from the QD-core, no empirical evidence was shown. Due to the attractiveness of QDs for biological applications, it was necessary that the mechanisms involved in QD-toxicity be understood so they may be prevented. Preliminary studies from our laboratory indicated that the antioxidant, N-acetlycysteine could prevent QD-toxicity. We, thus, hypothesized that QD-toxicity was not exclusively due to cadmium leaching from the QD. To evaluate this, an assay measuring free Cd was adapted for cellular use, to measure Cd present in both the cellular media and intracellularly. The cytotoxicity of various QDs was evaluated and correlated with the intracellular Cd. Results from this study showed no correlation between QD-toxicity and Cd release from QDs. We next questioned whether QD-toxicity could be explained as the sum of parts of the toxicity associated with core constituting metals, and whether the complexity of the model system used influences the cytotoxicity observed. We employed three model systems of the peripheral nervous system (an immortalized cell line, heterogeneous primary cultures and a three dimensional tissue model) and evaluated the toxicity of Cd, Te and QDs in each. Our findings showed that QDs are not a sum of parts and QD-toxicity is better ascribed to the induction of oxidative stress which is prevented by application of the multi-modal antioxidant, lipoic acid. Further, the model systems did not depict QD-toxicity comparably, stressing the need for standardization in nanotoxicological studies. Finally, it had been shown that Cd association with the estrogen receptor (ER) in ER expressing cells can activate estrogenic signalling. As such, Cd is considered a metalloestrogen. Given that QDs liberate Cd that can be detected in the cell, we investigated whether in ER expressing cells, QDs may act as metalloestrogens and induce estrogenic signalling. Results in vitro showed that QDs exert potent estrogenic signalling, comparable with estradiol, including cell proliferation, AKT phosphorylation, ERK phosphorylation and nuclear ER activation. These effects could be attenuated via pretreatment with the specific ER antagonist, ICI 182780, affirming that QD-induced estrogenic activity was mediated via the ER. To determine whether the estrogenic activity of QDs could also be demonstrated in vivo, ovariectomized mice were treated with QDs for two weeks, prior to being sacrificed. Subsequently, the wet weights of the mice uteruses were measured. In mice treated with QDs and estradiol a comparable 2.5 fold increase in uterine wet weight was observed. Taken together, these results indicate that CdTe QDs are both cytotoxic and endocrine disrupting, metalloestrogens. Though these nanocrystals may have valuable applications in biology the implications of QD-use are dangerous to plants, animals, humans and the environment. Therefore, it is imperative that Cd-free QDs be developed that retain the attractive qualities of QDs while preventing the detrimental side effects. Further, standardized testing of nanoparticles is imperative for the safe use of these novel tools. / La nanotechnologie et la nanomédecine sont des domaines en pleine expansion. Certaines nanoparticules sont déjà entrées dans des essais cliniques et sont deja utilisées par les patients. Par contre, certaines nanostructures, bien qu'elles soient prometteuses à des fins cliniques ou diagnostiques, sont capables d'induire de la cytotoxicité in vitro et in vivo; les boîtes quantiques de cadmium telluride (CdTe) constituent un exemple.Les boîtes quantiques (BQ) sont des nanocrystaux semi-conducteurs fluorescents qui contiennent un noyau métallique entouré d'une couche organique. Les BQ ont des propriétés optiques supérieures aux autres fluorophores traditionnelles. Par exemple, ils sont plus résistants au photoblanchiment et sont caractérisés par un spectre d'excitation large et d'émission étroit. Les BQ CdTe, les premiers à être synthétisés sans l'utilisation de solvant organique, ont été prometteurs dans certaines applications biologiques. Cependant, les premières études ont démontré que ces BQ induisent de la cytotoxicité et produisent du stress oxydatif. Le cadmium (Cd) libéré pourrait être à la base de ces effets toxiques, mais cela n'a pas encore été prouvé. Les études préliminaires au sein de notre laboratoire démontrent que le pré-traitement avec un antioxydant, N-acétyl-cystéine, était capable de diminuer le niveau de toxicité associée au BQ. On a avancé l'hypothèse que cette toxicité n'était pas exclusivement liée au Cd qui est libéré des BQ. Nous avons effectué des expériences fluorométriques où nous mesurions les niveaux de Cd libres dans la cellule et dans le milieu extracellulaire. Ces expériences indiquent qu'il n'existe aucune corrélation entre la toxicité associée au BQ et le Cd libéré. Par après, nous nous sommes demandé si les constituants métalliques du noyau des BQ et les modèles dans lesquels les BQ avaient été évalués, étaient impliqués dans la toxicité associée au BQ. Nous avons évalué la toxicité du Cd, tellurium et BQ dans trois modèles du système nerveux périphérique (lignée de cellule immortalisée, cultures primaires hétérogènes et modèle de tissue tridimensionnel). Les résultats démontrent que la toxicité des BQ est principalement attribuée à l'induction du stress oxydatif, qui peut être prévenu en appliquant un antioxydant multimodal, l'acide lipoique.Dans les cellules, Cd peut s'associer avec les récepteurs d'œstrogènes (RE) et activer les voies de signalisation reliées à ce récepteur. Par conséquent, Cd est considéré comme étant un métallo-œstrogène. Nous avons montré que les BQ libèrent du Cd et que celui-ci est internalisé et retenu dans les cellules. Les effets induits par les BQ dépendaient du modèle biologique utilisé. Les études in vitro montrent que les BQ exercent une forte signalisation oestrogénique comparable à celle de estradiol et induisent la prolifération cellulaire, la phosphorylation d'AKT et d'ERK et l'activation du RE nucléaire. Ces effets étaient atténués par un pré-traitement avec un inhibiteur du RE, ICI 182780. Ces résultats affirment, donc, que les BQ exercent leurs activités oestrogéniques via les RE. Dans nos études in vivo nous avons utilisé des souris ovariectomisées qui avaient été traitées avec des BQ ou du estradiol pendant deux semaines, et par ensuite sacrifiées. Les traitements ont fait augmenter de 2.5 fois le poids de l'utérus des souris. Dans l'ensemble, ces résultats montrent que les BQ CdTe exercent à la fois des effets cytotoxiques et métallo-eostrogéniques. Malgré leurs potentiels d'application en imagerie ou dans les procédures diagnostiques, il est clair que les BQ peuvent nuire aux plantes, aux animaux, aux humains et à l'ensemble de l'environnement. Il y a un besoin urgent de développer des BQ sans cadmium qui possèdent des qualités attrayantes de BQ mais qui sont dépourvus d'effets secondaires détrimentaux. Cet objectif pourra être atteint à l'aide d'essais plus élaborés et sophistiqués pour déceler les risques des nanoparticules.