Indiana University-Purdue University Indianapolis (IUPUI) / The non-hematopoietic, stationary stromal cells located inside and surrounding skin-draining lymph nodes play a key role in regulating immune responses. We studied distinct populations of lymph node stromal cells from both human subjects and animal models in order to describe their phenotype and function. In the mouse model, we studied two distinct populations: an endothelial cell population expressing Ly51 and MHC-II, and an epithelial cell population expressing the epithelial adhesion molecule EpCAM. Analysis of intra-nodal and extra-nodal lymph node (CD45-) stromal cells through flow cytometry and qPCR provides a general phenotypic profile of the distinct populations. My research focused on the EpCAM+ epithelial cell population located in the fat pad surrounding the skin draining lymph nodes. The EpCAM+ population has been characterized by surface marker phenotype, anatomic location, and gene expression profile. This population demonstrates the ability to inhibit the activation and proliferation of both CD4 and CD8 T cells. This population may play a role in suppressing overactive inflammation and auto-reactive T cells that escaped thymic deletion. The other major arm of my project consisted of identifying a novel endothelial cell population in human lymph nodes. Freshly resected lymph nodes were processed into single cell suspensions and selected for non-hematopoietic CD45- stromal cells. The unique endothelial population expressing CD34 HLA-DR was then characterized and analyzed for anatomic position, surface marker expression, and gene profiles. Overall, these studies emphasize the importance of stationary lymph node stromal cells to our functioning immune systems, and may have clinical relevance to autoimmune diseases, inflammation, and bone marrow transplantation.

Indiana University-Purdue University Indianapolis (IUPUI) / It has been proposed that the GABAB receptor subtype plays a role in alcoholism and alcohol use disorders (AUDs) (Cousins et al., 2002; Agabio et al., 2012). Specifically, the GABAB agonist baclofen has been looked at extensively in clinical and pre-clinical studies. In various animal models of chronic and intermittent consumption, baclofen has been shown to both increase (Petry, 1997; Smith et al., 1999; Czachowski et al., 2006; Moore et al., 2007) and decrease (Colombo et al., 2000; 2002; 2005; Stromberg, 2004; Moore et al., 2009) drinking. A critical issue in determining pharmacological effects of a drug is using the appropriate animal model. The drinking-in-the-dark (DID) model, developed by Rhodes et al. (2005, 2007), produces high levels of drinking in a binge-like paradigm and has been used to assess many pharmacological targets (e.g. Kamdar et al., 2007; Gupta et al., 2008; Moore et al., 2007; 2009). While DID produces high-levels of binge drinking, it is unclear what areas of the brain are involved in this behavior. A direct way to target areas that are believed to be involved in the circuitry of particular behaviors is through microinjection of drugs (Kiianmaa et al., 2003). Of particular recent interest involving motivated behaviors and addiction is the nucleus accumbens (Acb) (Everitt & Robbins, 2005); specifically the accumbens shell (AcbSh) (e.g. Rewal et al., 2009, 2012; Nie et al., 2011; Leriche et al., 2008). The current study aimed to investigate the role of GABAB receptors in the AcbSh by examining the ability of two different enantiomers of baclofen to alter ethanol and saccharin intake in male C57BL/6J (B6) mice. B6 mice underwent bilateral cannulation surgery targeting the AcbSh. After 48 hours of recovery time, animals began a five day Drinking-in-the-Dark (DID) procedure where they received 20% ethanol or 0.2% saccharin for two hours, three hours into the dark cycle, each day. Throughout the five drinking sessions, animals were kept in home-cage locomotor activity chambers to monitor activity throughout the drinking cycle. Day 4 drinking was immediately preceded by a mock microinjection, whereas Day 5 drinking was immediately preceded by a drug microinjection. Microinjection of one of five doses of baclofen was given in ng/side dissolved in 200 µl of aCSF (aCSF alone, 0.02 R(+)-, 0.04 R(+)-, 0.08 S(-)-, or 0,16 S(-)-). Intake was recorded every twenty minutes on Days 4 and 5. Retro-orbital sinus blood samples were taken from ethanol animals immediately following the Day 5 drinking period to determine blood ethanol concentrations (BECs). A one-way ANOVA on total Day 4 ethanol consumption revealed no baseline differences between dose groups. A one-way ANOVA on total Day 5 ethanol consumption revealed that the 0.04 R(+)- baclofen dose reduced total drinking, but the 0.16 S(-)- baclofen dose increased total drinking (p’s<0.05). This pattern was reflected in the BECs; 0.04 R(+)- baclofen reduced BECs, whereas 0.16 S(-)- baclofen increased BECs (p’s<0.05). These results were also time-dependent, with R(+)-baclofen reducing drinking in the first 20 minutes of the session and S(-)- increasing drinking in the last 40 minutes of the session. There were no effects on saccharin intake. An issue with the locomotor activity boxes led to unreliable locomotor activity counts. However, because there were no drug effects on saccharin consumption, it was concluded that locomotor effects did not contribute to the decreases or increases in ethanol consumption. These results further implicate the role of GABAB receptors in modulating ethanol intake. The bidirectional effects shown highlight the importance of considering enantioselective drug effects when interpreting data. Finally, these results also support previous conclusions that the AcbSh plays an important role in modulating use of drugs of abuse, but not other reinforcers.

Thesis (MScMedSc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Introduction: A westernized diet rich in saturated fats and sugars, together with a sedentary lifestyle, has contributed to the dramatic increase in obesity during the last decade (Zimmett et al, 2001; Wild et al, 2004). Obesity is associated with dyslipidemia and insulin resistance which are major risk factors for the development of type 2 diabetes (T2D) (Zimmet et al, 2001, Kahn et al, 2006; Schröder et al, 2007). High-fat feeding in rodents induces symptoms similar to the human metabolic syndrome without progression to T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). The addition of fructose to a high-fat diet exacerbates the insulin resistance and leads to impaired pancreatic function of insulin secretion and glucose intolerance (Basciano et al, 2005; Stanhope et al, 2009). Aims: The aim of this study was to establish the effect of a high-fat and sucrose/fructose diet on glucose metabolism, the development of insulin resistance and β-cell dynamics. Methods: Weanling Wistar rats were randomized into two study groups; study one over an experimental period for three months and study two for twelve months. Each study consisted of a control group that received standard rat chow and water; and two experimental groups receiving either a high-fat diet and water (HFD) or a café diet consisting of HFD with the addition of 15% sucrose/fructose (CFD). Fasting glucose and insulin concentrations, intravenous glucose tolerance test (IVGTT), glucose stimulated insulin secretion rates and 2-deoxy-[3H]-D-glucose uptake in muscle, liver and fat were measured. The pancreata were harvested for immunohistochemical labeling of β-cells (insulin), α-cells (glucagon), GLUT2 (glucose transport) and MIB5 (proliferation). Samples of the pancreata were also collected for electron microscopy. Results and discussion: Feeding Wistar rats a CFD induced obesity, insulin resistance and glucose intolerance. By twelve months the rats had an impaired glucose response with increased IVGTT peak values, area under the curve (AUC) values and glucose clearance rates. Concomitantly, the glucose stimulated insulin secretion rate (GS-ISR) was attenuated. Stimulated glucose disposal as measured by 2-deoxy-[3H]-D-glucose uptake was reduced in muscle and adipose tissue at three months. By twelve months, due to the age of the rats, stimulated glucose uptake declined compared to three months with no difference between groups. After three months the diets had no observable effect on the islets using light microscopy. However, by twelve months morphological changes were observed in both the HFD and CFD groups. In the HFD group large hypertrophied irregular islets with fibrous changes were observed. In the CFD group these morphological changes were more prominent with fibrous segregation and disruption of the normal endocrine arrangement. In addition, the presence of inflammatory cells within the affected islets is consistent with T2D. Conclusion: High-fat diet fed to Wistar rats induced obesity, abdominal adiposity and insulin resistance. The addition of sucrose/fructose to a high-fat diet exacerbated the insulin resistance and resulted in glucose intolerance and mild hyperglycemia. Morphological changes in the large islets were observed which are consistent with the development of T2D. / AFRIKAANSE OPSOMMING: Inleiding: ‘n Verwesterde dieët, ryk aan versadigde vette en suikers tesame met 'n passiewe lewenstyl, het bygedra tot die dramatiese verhoging in vetsug gedurende die laaste dekade (Zimmett et al, 2001; Wild et al, 2004). Vetsug word met dislipidemie en insulienweerstandigheid geassosieer wat hoof risikofaktore is vir die ontwikkeling van tipe 2 diabetes (T2D) (Zimmet et al, 2001; Kahn et al, 2006; Schröder et al, 2007). Hoë-vet voeding in knaagdiere induseer simptome soortgelyk aan menslike metaboliese sindroom sonder die ontwikkeling van T2D (Woods et al, 2002; Weir and Bonner-Weir, 2007). Die byvoeging van fruktose tot 'n hoë-vet dieët vererger insulienweerstandigheid en lei tot verswakte pankreas funksie, insuliensekresie en glukoseintoleransie (Basciano et al, 2005; Stanhope et al, 2009). Doelwitte: Die doelwitte van die studie was om die effek van hoë-vet en sukrose/fruktose voeding op glukosemetabolisme, die ontwikkeling van insulienweerstandigheid en β-sel dinamika te bepaal. Metodes: Gespeende Wistar rotte was in twee groepe gerandomiseer; studie een oor ʼn tydperk van drie maande en studie twee oor ʼn tydperk van twaalf maande onderskeidelik. Elke studie het 'n kontrole groep met standaard rot kos en water (control); en twee experimentele diëte wat of ʼn hoë-vet dieët en water (HFD) of 'n kafeedieët groep wat die HFD met die byvoeging van 15% sukrose/fruktose in hul drink water (CFD) ontvang. Fastende glukose en insulien, binneaarse glukose toleransie toets (IVGTT), glukose gestimuleerde insulien sekresie tempo en 2-deoxi-[3H]-D-glukose opname in spier, lewer en vet is gebruik om die effek van die dieët op glukosemetabolisme te bepaal. Die pankreata is uitgehaal vir immunohistochemiese identifisering van β-selle (insulien), α-selle (glukagoon), GLUT2 (glukose transport) en MIB5 (proliferasie). Monsters van die pankreata was ook vir elektronmikroskopie versamel. Resultate en bespreking: Voeding van ʼn CFD aan Wistar rotte induseer vetsug, insulienweerstandigheid en glukose-intoleransie Teen twaalf maande toon die rotte 'n verswakte respons tot glukose met verhoogde IVGTT piekwaardes, AUC waardes en glukose opruimingswaardes. Terselfdetyd is die glukose gestimuleerde insuliensekresie tempo (GS-ISR) ook verswak. Gestimuleerde glukose opruiming, soos deur 2-deoxi-[3H]-D-glukose opname bepaal, was verlaag in spier en vetweefsel teen drie maande. Teen twaalf maande, weens die ouderdom van die rotte, is die gestimuleerde glukose opname verlaag in vergelyking met drie maande sonder 'n verskil tussen groepe. Na drie maande kon geen sigbare morfologiese verskille met ligmikroskopie tussen die diëte waargeneem word nie. Teen twaalf maande is morfologiese verskille waargeneem in beide die HFD en die CFD groepe. In die HFD groep is groot hipertrofiese onreëlmatige eilande met fibrotiese verandering waargeneem. In die CFD groep was die morfologiese verandering meer gevorder met fibrotiese onderverdeling en ontwrigting van die normale endokriene rangskikking. Die teenwoordigheid van inflammatoriese selle in die geaffekteerde eilande is verenigbaar met T2D. Afleiding: Die voer van 'n hoë-vet dieët aan Wistar rotte veroorsaak vetsug, abdominale adipositeit en insulienweerstandigheid. Die byvoeging van sukrose/ fruktose tot die hoë-vet dieët vererger die insulienweerstandigheid en veroorsaak glukoseintoleransie en matige hiperglukemie. Morfologiese veranderings in die groter eilande was verenigbaar met T2D.

La douleur articulaire associée à l’arthrose est un problème clinique majeur, spécialement chez les personnes âgées. L’intensité de la douleur est souvent amplifiée lors de mouvement de l’articulation et principalement lors du soutien de la charge corporelle sur le membre lésé. Malheureusement, les traitements pharmacologiques proposés sont trop souvent associés à des effets secondaires néfastes et à une inefficacité pour le soulagement de la douleur à long terme. Divers modèles murins sont utilisés en laboratoire de recherche pour des études précliniques de molécules aux propriétés analgésiques. Une évaluation comparative de la réponse comportementale douloureuse des animaux d’un modèle d’instabilité articulaire induit par le sectionnement du ligament croisé antérieur accompagné d’une méniscectomie partielle (le modèle ACLT+pMMx) et d’un modèle de dégénérescence articulaire induite par le monoiodoacetate (le modèle MIA) a permis de sélectionner un modèle approprié pour la continuité du projet. Les deux modèles ont démontré des lésions tissulaires, mais le modèle MIA a démontré une réponse douloureuse plus prononcée que le modèle ACLT+pMMx. Par l’analyse de la démarche, le modèle MIA a démontré une boiterie claire dans le patron de la démarche des animaux qui est associée à une lésion unilatérale. Le modèle MIA a donc été choisi pour la suite du projet. La problématique principale dans la recherche sur la douleur associée à l’arthrose est une compréhension incomplète des mécanismes de douleur responsables de l’induction et du maintien de l’état de douleur. Il devient donc nécessaire d’améliorer nos connaissances de ces mécanismes en effectuant une caractérisation plus approfondie des modèles animaux employés pour l’évaluation de stratégies pharmacologiques analgésiantes. Afin de bien comprendre le modèle MIA, une caractérisation des événements moléculaires centraux lors de la progression du processus dégénératif des structures articulaires de ce modèle s’est effectuée aux jours 3, 7, 14, 21 et 28 post injection. Des mécanismes hétérogènes qui modulent l’information nociceptive en fonction de la progression temporelle de la pathologie ont été observés. Les changements du contenu i spinal des neuropeptides sélectionnés (substance P, CGRP, dynorphine A et Big dynorphine) ont débuté sept jours suivant l’injection de MIA. L’observation histologique a démontré que les dommages structuraux les plus importants surviennent entre les jours 14 et 21. C’est entre les jours 7 et 21 que les lésions démontrent le plus de similarités à la pathologie humaine. Cela suggère que lors d’une évaluation préclinique d’un traitement pharmacologique pour pallier la douleur articulaire utilisant le modèle MIA, l’étude doit tenir compte de ces événements afin de maximiser l’évaluation de son efficacité. Puisque les traitements pharmacologiques conventionnels proposés pour le soulagement de la douleur ne font pas l’unanimité en terme d’efficacité, d’effets non désirés et de coûts monétaires parfois onéreux, les molécules de dérivés de plante deviennent une alternative intéressante. L’eugénol, le principal constituant de l’huile de clou de girofle, a été administré oralement pour une période de 28 jours chez des rats ayant reçu l’injection intra-articulaire de MIA afin d’évaluer son efficacité pour le traitement de la douleur articulaire. L’eugénol à une dose de 40 mg/kg s’est révélé efficace pour l’amélioration du patron de la démarche des animaux ainsi que pour la diminution de l’allodynie mécanique secondaire. De plus, les concentrations spinales de neuropeptides pronocicepteurs ont diminué chez les animaux traités. Par une évaluation histopathologique, l’eugénol n’a démontré aucune évidence d’effets toxiques suite à une administration per os quotidienne pour une période prolongée. Ces résultats suggèrent le potentiel thérapeutique complémentaire de la molécule d’eugénol pour le traitement de la douleur articulaire. / Pain is the most predominant clinical symptom associated with osteoarthritis (OA), mostly among older people. Joint movement and weight bearing often increase the pain intensity. Unfortunately, the proposed pharmacological treatments are frequently associated with side effects and ineffective for pain alleviation for long time periods. Many murine models are used in laboratories for preclinical studies evaluating analgesic compounds. A comparative evaluation of the behavioral pain responses of animals with a joint instability model induced by the transection of the anterior cruciate ligament followed by a partial menisectomy (the ACLT+pMMx model) and of an articular degenerative model induced by an intra-articular injection of monoiodoacetate (the MIA model) was conducted to select an appropriate model for the continuation of the project. Both models demonstrated articular lésions, however the MIA model demonstrated a clearer behavioral pain response over the ACLT+pMMx model. The gait pattern of the MIA model revealed a clear limping gait similar to that observed with unilateral OA in humans. The MIA model was chosen for the subsequent studies. An unresolved issue in pain OA research is the lack of understanding of the pain mechanisms responsible for the induction and maintenance of the pain. Therefore, there is an urgent clinical need to improve the characterization of animal models to effectively discover novel pain relief pharmacological treatment stratégies for OA patients. A characterization of the spinal pain molecular events during the progression of the joint degenerative process in the MIA model was performed on days 3, 7, 14, 21 and 28 post injection. Heterogeneous nociceptive central molecular events were observed in respect to the time course of the pathology’s progression. Changes in selected spinal neuropeptide content (substance P, CGRP, dynorphin A, Big dynorphin) began 7 days following the MIA injection. Most severe joint structural damage on histology occured between days 14 and 21 post injection. These results suggest that preclinical drug evaluation employing this model should be conducted between 7 and 21 days post injection when the lesions resemble most those of human OA. iii As current pharmacological therapy for the alleviation of joint pain does not achieve the unanimity in respect to efficacy, side effects and cost, plant derivate compounds are now interesting alternatives to improve the situation. Eugenol, the main constituent of clove oil, was evaluated for its efficacy for alleviation of joint pain in rats who previously received an intra-articular injection of mono-iodoacetate to induce the MIA model. Eugenol, administered orally for 28 consecutive days at a dose of 40 mg/kg, improved gait pattern and reduced secondary mechanical allodynia. Furthermore, spinal concentrations of pronociceptive neuropeptides were also decreased in the treated animals. No toxic effects of the compoud were identified on histopathological assessment of the various tissues. These results suggest that eugenol could be a potential therapeutic option for alleviating OA joint pain.

Le trouble bipolaire est une maladie invalidante caractérisée par une alternance d’épisodes maniaques et dépressifs. Malgré des efforts de recherche notables, la physiopathologie et les mécanismes d’action des traitements du trouble bipolaire demeurent peu connus. La protéine kinase C (PKC) est récemment apparue comme une cible moléculaire potentielle pour le traitement du trouble bipolaire. Dans ce travail de thèse, nous avons cherché à étudier le rôle de la PKC dans les phases maniaque et dépressive du trouble bipolaire. Nous avons montré que l’inhibition de la PKC a un effet antimaniaque non seulement chez le rat naïf, mais aussi dans un modèle de manie basé sur une privation de sommeil, que nous avons validé au cours de notre étude. De plus, les inhibiteurs de la PKC sont capables de rétablir les déficits de prolifération cellulaire hippocampique que présentent les rats privés de sommeil. Ces effets prolifératifs et antimaniaques seraient indépendants, puisque le blocage de la prolifération cellulaire n’abolit pas l’efficacité antimaniaque des inhibiteurs de la PKC dans le modèle de privation de sommeil. En parallèle, nous avons montré que l’activation de la PKC a un effet antidépresseur chez le rat naïf, alors que son inhibition provoque un phénotype pseudodépressif qui s’accompagne d’une diminution de la prolifération cellulaire hippocampique. L’ensemble de ces données révèle une implication de la PKC dans les deux phases du trouble bipolaire, et soutient l’hypothèse qu’une suractivation du système PKC serait à l’origine des perturbations de neuroplasticité associées à la manie. / Bipolar disorder is a devastating long-term disease characterized by alternate episodes of mania and depression. Despite extensive research, the molecular and cellular underpinnings of bipolar disorder remain to be fully elucidated. Protein kinase C (PKC) has emerged as a potential molecular target for the treatment of bipolar disorder. The present study investigated the role of PKC in manic- and depressive-like behaviors. Our results showed that PKC inhibition produced an antimanic-like effect not only in naive rats, but also in an animal model of mania based on sleep deprivation, that we have validated in our study. Interestingly, PKC inhibitors rescued the hippocampal cell proliferation deficits displayed by sleep-deprived animals. These proliferative and antimanic effects were independent, since blockade of cell proliferation did not abolish the antimanic efficacy of PKC inhibitors in the sleep deprivation model. At the same time, we showed that PKC activation had an antidepressant-like effect in naive rats, whereas its inhibition caused a depressive-like phenotype accompanied by a decrease in hippocampal cell proliferation. Taken together, our results demonstrate the involvement of the PKC system in regulating opposite facets of bipolar disorder, and support the hypothesis that an overactivation of the PKC signaling system may be crucial for the deficits of neuroplasticity associated with mania.

Na epilepsia do lobo temporal (ELT), alterações morfofuncionais em estruturas límbicas são classicamente acompanhadas de déficits cognitivos. Estudos anteriores revelaram que disfunções eletrofisiológicas em circuitos hipocampo-corticais são observadas durante o sono NREM (non rapid eye movement), onde eventos patológicos como fast ripples e IEDs (interictal epileptiform discharges) substituem gradativamente eventos fisiológicos, como as sharp-wave ripples (SWR). Tal substituição pode estar por trás dos prejuízos cognitivos observados tanto nos modelos animais como em pacientes, já que as SWRs são fundamentais para a transferência de informação do hipocampo ao córtex durante a consolidação de memórias. De maneira complementar, o sono REM também parece exercer um papel fundamental em processos mnemônicos, facilitando eventos de plasticidade sináptica e coordenando regiões encefálicas distantes por meio de acoplamento entre diferentes frequências oscilatórias, tais como teta e gama. Entretanto, as alterações no sono REM durante os processos de epileptogênese ainda foram pouco exploradas. Neste trabalho testamos a hipótese de que disfunções na coordenação rítmica durante o sono REM estariam associadas a prejuízos de memória que se manifestam na epileptogênese. Para isso, submetemos ratos Wistar adultos machos a protocolo de abrasamento rápido da amígdala basolateral, possibilitando a avaliação de alterações eletrofisiológicas gradativas durante o processo de epileptogênese. Foram realizados implantes crônicos de eletrodos para registro do potencial local de campo (LFP, Local Field Potential) nas regiões de CA1 e do córtex pré-frontal medial (mPFC, medial prefrontal cortex), além de eletrodos bipolares para estímulo na amígdala basolateral. Os protocolos de abrasamento foram realizados durante um período de 3 dias, aplicando-se diariamente 10 trens de estímulos a 50 Hz com duração de 10 segundos. Para avaliarmos os prejuízos cognitivos, os animais foram submetidos a testes de reconhecimento de objetos antes do início a após o término dos protocolos de abrasamento. O sonosubsequente às sessões de reconhecimento de objetos e aos protocolos de estimulações foram registrados diariamente. Além do prejuízo no reconhecimento de objetos e alterações eletrofisiológicas durante o sono NREM, como a substituição gradativa de ripples por IEDs, os animais submetidos ao abrasamento elétrico apresentaram um aumento na comodulação fase-amplitude entre oscilações teta e gama durante o sono REM após as estimulações, exibindo também uma correlação negativa entre a comodulação e a duração das pós-descargas induzidas pelos estímulos elétricos do abrasamento durante o sono subsequente a aplicação dos protocolos. Nossos achados ampliam a compreensão vigente sobre como alterações de oscilações cerebrais durante o sono, especialmente da fase REM, poderiam estar subjacentes a prejuízos de memória que ocorrem na ELT. / Morphofunctional changes in limbic structures are classically followed by cognitive deficits in Temporal Lobe Epilepsy (TLE) patients. Previous studies revealed that electrophysiological dysfunctions in hippocampal-cortical circuits are observed during NREM (non-rapid eye movement) sleep, where pathological events such as fast ripples and IEDs (interictal epileptiform discharges) gradually replace physiological events, such as Sharpwave Ripples (SWR). This replacement seems to describe the cognitive impairments observed in animal models and TLE patients since SWRs are fundamental for information transfer from the hippocampus to cortex during memory consolidation. Complementary, REM sleep also plays a significant role in mnemonic processes, facilitating synaptic plasticity events and coordinating distant brain regions by coupling different frequencies, such as theta and gamma. However, alterations in REM sleep during the epileptogenesis processes are poorly investigated. In this study, we tested the hypothesis that dysfunctions on rhythmic coordination during REM sleep would be associated with memory deficits showed during epileptogenesis. For this, we submitted adult Wistar rats to a rapid kindling protocol on basolateral amygdala (BLA), allowing the evaluation of progressive electrophysiological changes during the epileptogenic process. Chronic electrodes were implanted for the local field potentials (LFP) recording in the CA1 and medial prefrontal cortex (mPFC), as well as bipolar electrodes for BLA stimulation. The kindling protocols were performed during three days, applying ten trains of 50 Hz stimulations with ten seconds duration. Object recognition tasks were performed before and after the kindling protocol to evaluate cognitive impairment. Sleep recordings were performed daily after the object recognition or kindling application. Along with object recognition impairment and electrophysiological changes during NREM sleep, such as progressive SWR substitution by IEDs, kindled rats presented an increase in phase-amplitude comodulation between theta and gamma oscillations during REM sleep after stimulation sessions, which also correlates negatively with after-discharges (AD) duration induced by the kindling stimulation. Our findingsexpand the comprehension about how changes in brain oscillations during REM sleep underlies observed memory deficits in TLE.

Dynamic molecular interactions between the microbiota and the intestinal mucosa play an important role in the establishment and maintenance of mucosal homeostasis. Aberrant host- microbiota interaction could lead to many diseases such as inflammatory bowel disease. The aim of our study was to evaluate the commensal and probiotic bacteria activities and their ability to induce pathological or exert beneficial effects. The most important trigger for immune system development is an exposure to microbial components. Here, we show that there is a time window at about three weeks of age, which enables the artificial colonization of germ free mice by a single oral dose of cecal content. The delayed colonization by either inoculation or co-housing causes permanent changes in immune system reactivity, which may downgrade the results of experiments performed on first generation of colonized animals. In this thesis we report that even non-living commensal bacteria such as Parabacteroides distasonis (mPd) or well known probiotics such as L. casei DN-114 001 (Lc) possess anti-inflammatory effects in experimental model of colitis. The mechanisms that this effect is achieved by the lysate of L. casei DN-114 001 comprise: a) improvement in the gut barrier function, b) correction of the dysbiosis, and c) modulation of the...

Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique. / Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

Prévios estudos têm demonstrado alterações nas taxas normais de razão sexual. No entanto, uma associação direta entre níveis ambientais de poluição e razão sexual, não tem sido ainda claramente demonstrada. A fim de investigarmos esta associação, desenvolvemos duas abordagens: uma epidemiológica e outra experimental. O efeito da poluição sobre a população da cidade de São Paulo foi verificado através da associação entre a razão sexual e gradientes de concentração de material particulado (PM10). A fim de conferir plausibilidade biológica aos achados epidemiológicos, camundongos machos com 10 dias de vida, foram expostos em câmaras do tipo Topo Aberto, sob condições de exposição mais controladas. Uma associação negativa e significante foi observada entre razão sexual e PM10, dentro de uma faixa estreita de concentração de PM10 (31 a 61 ?g/m3). Na região menos poluída, a razão sexual foi de 51,7% (106,8) para 34.795 nascimentos registrados, enquanto que para a mais poluída, a proporção decresceu para 50,7% (102,9) para 48.023 nascimentos registrados, com uma redução de 1% no número total de nascimentos masculinos. O grupo de camundongos da linhagem Swiss, acasalados com fêmeas virgens, maduras e não expostas, produziu uma prole com alterações significativas (p<0,041) na razão sexual (0,86) quando comparado com o grupo controle (1,34). Análises morfométricas dos testículos demonstraram alterações significativas no perfil da linhagem germinativa, com reduções no número das espermátides alongadas (p<0,020) e no número total de células (p<0,032). Um decréscimo na concentração dos espermatozóides (46,95x106 /ml) também foi observado nos animais expostos à poluição quando comparado ao grupo controle (54,60x106 /ml). A partir dos nossos resultados podemos concluir que a poluição pode interferir com a distribuição do sexo nas populações expostas aos níveis ambientais de poluição. / Some studies have observed abnormal sex ratios in industrial polluted areas but a direct association between urban levels of air pollutant and sex ratio at birth has not hitherto reported. To examine whether this relationship held true, we evaluated how ambient air pollution interferes on female/male ratio in human and mice models. The effect of air pollution was addressed by determining SSR (secondary sex ratio) across pollution gradients in the city of São Paulo, using inhalable particles (PM10) as a proxy variable for overall pollution levels. To provide biological plausibility to the foregoing epidemiological observation, we designed an experimental study exposing mice in controlled chamber conditions. Results disclosed a significant negative between SSR and PM10, within a relatively narrow range of PM10 levels (31 to 61 ?g/m3). In the least polluted area the sex ratio was 51.7% (106.8) for 34,795 births recorded, and for the most polluted area the proportion decreased to 50.7 % (102.9) for 48,023 births recorded, indicating a difference of 1% in total male births. This result corresponds to a 1,180 unborn male offspring in the highest polluted areas. The group of male Swiss mice housed 10 days after birth in open top chambers exposed to air pollution, where they mate mature and non-exposed virgin female mice, produced an offspring with a 0.86 male/female ratio. The offspring of the group of mice concurrently raised in similar but filtered open top chamber was significantly (p<0.042) higher (1.34). A remarkable result was the testicular histological morphometry with a significant (p<0,020) reduction in the number of elongated spermatids and total cells (p<0,032) of exposed males. In association, a decrease in sperm concentration in the caudal portion of the epidydimus was also found in the exposed mice (46,95x106 /ml) compared to the non-exposed (54,60x106 /ml). In conclusion the findings in our study support the concept that ambient air pollution may interfere with sex distribution of exposed populations

Le développement récent de l’endoscopie interventionnelle digestive nous a conduit à prendre en charge deux types de pathologies préoccupantes. Il s’agit d’une part des fistules digestives souvent responsables d’une morbi-mortalité élevée et d’autre part des sténoses œsophagiennes après résection tumorale endoscopique étendue. Dans ces deux situations, des phénomènes inflammatoires chroniques conduisent soit à l’absence de fermeture de la fistule soit à une fibrose importante responsable de sténose de l’œsophage. La thérapie cellulaire a déjà été utilisée pour diminuer ces phénomènes inflammatoires et entrainer une cicatrisation. La thérapie tissulaire par cellules souches organisées en construction 3D représente un avantage important en permettant de cibler le site d’action par dépôt direct du feuillet cellularisé. Notre objectif était d’évaluer l’effet thérapeutique de ces nouveaux outils pour fermer les fistules digestives et pour prévenir la survenue des sténoses œsophagiennes. La première étape a consisté a évaluer l’efficacité du traitement par des cellules souches mésenchymateuses provenant de moelle osseuse humaine, marquées puis organisées en doubles feuillets, dans un modèle de fistule entéro-cutanée post-chirurgicale chez la souris nude. L’évaluation clinique et en imagerie (IRM et microscopie confocale) a montré une meilleure cicatrisation avec une augmentation de la microvascularisation et une accélération de la fermeture de la fistule chez les souris greffées. Les effets observés semblent liés à une augmentation précoce de la synthèse des facteurs de réparation (EGF et le VEGF) et des cytokines anti-inflammatoires (TGF-ß2 et IL-10). Après avoir développé un modèle inédit de fistule oeso-cutanée chez le porc grâce à la mise en place par voie endoscopique et chirurgicale de prothèses plastiques entre la lumière œsophagienne et la peau, nous avons évalué l’efficacité thérapeutique d’un gel contenant des vésicules extracellulaires issues de cellules souches isolées du tissu adipeux de porc. Ce gel injecté dans la fistule par voie endoscopique a permis la fermeture des fistules. Enfin, la troisième partie de notre travail a consisté à évaluer l’efficacité de la greffe allogénique de doubles feuillets de cellules souches mésenchymateuses pour prévenir la survenue des sténoses œsophagiennes dans un modèle porcin après dissection sous muqueuse étendue. Il existait une réduction significative du taux de sténose œsophagienne cicatricielle dans le groupe greffé avec une fibrose moins importante. En conclusion, l’effet paracrine antifibrosant des cellules souches mésenchymateuses organisées en feuillets est efficace à la fois pour fermer les fistules entéro-cutanées chez la souris et pour prévenir les sténoses œsophagiennes chez le porc. Un gel avec des vésicules extracellulaires issues des cellules souches a de la même façon un effet cicatrisant anti-inflammatoire permettant la fermeture des fistules œsophagiennes chez le porc. Ces résultats sont très encourageants et posent la question d’une évaluation future chez l’homme. / Recent developments in digestive interventional endoscopy lead us to manage two types of digestive disease. First, it is digestive fistulas associated in many cases with high morbi-mortality; and second is oesophageal stenosis after extended superficial endoscopic resection. In both situations, chronic inflammatory process resulted in delayed or no fistula healing for the first case or oesophageal stenosis due to fibrosis. Cellular therapy has proved to be successful in reducing the inflammatory process and to promote tissue healing. Tissue therapy with 3D construct stem cells represents a major advantage by allowing a direct adaptation on the right place. Our objective was to evaluate the therapeutic effect of new strategy to close the digestive fistula and to prevent oesophageal stenosis. First step was to evaluate the effect of labelled human bone marrow derived mesenchymal stem cells engraftment in the form of double cellsheet in a post-surgical fistula model in nude mice. Clinical and radiological (MRI and probe based confocal microscopy) evaluation showed a better fistula healing with higher microvascularization and a faster fistula closing in grafted mice. These effects appear to be related to an increase production of factors involved in tissue repair (EGF et le VEGF) and anti-inflammatory cytokines (TGF-ß2 et IL-10). We developed an unpublished eso-cutaneous fistula in a porcine model after plastic catheters placement by surgical and endoscopic way between the oesophageal lumen and the skin. We evaluated the therapeutic effect of a hydrogel with extracellular vesicles extracted from porcine adipose derived stem cells. The hydrogel with vesicles was injected into the fistula by endoscopy. Radiological and histological evaluation 15 days after injection showed a fistula tract closure in treated group.The third part of this work was to evaluate the effect of allograft of adipose derived stem cells 3D construct to prevent the stenosis after extended endoscopic submucosal dissection in a porcine model. There was a significant reduction of number and degree of stenosis with decrease fibrosis infiltration in the grafted group.In summary, thanks to their paracrine and antifibrotic effect, the mesenchymal stem cells organised as 3D construct allowed fistula closure in an entero-cutaneous model in mice and prevention of stenosis after extended oesophageal submucosal dissection in a porcine model. Moreover, endoscopic hydrogel and extracellular vesicles injection allowed oesophageal fistula healing in a porcine model. These promising results pose the challenge of future clinical studies.