The study was conducted to identify characteristics that are associated with successful transfer to institutions of higher learning from 3 Phi Theta Kappa chapters at a large multi-campus Mississippi community college. The study examined participation rates and transfer rates of Phi Theta Kappa students. The current study also identified student characteristics that predict the successful transfer of Phi Theta Kappa members. A correlational methodological approach was employed. The Phi Theta Kappa chapters in this study have significant differences in membership, yet the factors that predict transfer to a 4-year university or college are few. The factors include age at transfer, cumulative grade point average, type of major, and associate degree completion. The analysis resulted in a model that may be used to predict the probability that a Phi Theta Kappa student will be a successful transfer. The model was able to predict the transfer of these students with significantly higher probabilities than the baseline model.

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Kappa opioid receptor (KOPR) is involved in many physiological functions and pharmacological responses such as analgesia, anti-pruritic effect, sedation, motor incoordination and aversion (Simonin et al., 1998; Liu-Chen, 2004). The cellular mechanisms following activation of KOPR involve in part Gi/o protein-dependent pathways (Law et al., 2000). Following KOPR activation, the receptor is phosphorylated and arrestins are recruited. Arrestins mediate agonist-dependent KOPR desensitization, internalization and down-regulation (Liu-Chen, 2004). In recent years, arrestins were found to initiate arrestin-dependent downstream signaling. Thus, agonist-promoted KOPR phosphorylation plays a pivotal role in KOPR regulation and signaling. Previous studies from our lab showed that in Chinese hamster ovary (CHO) cells stably transfected with the human KOPR (hKOPR), U50,488H induced phosphorylation (Li et al., 2002a); however, sites of phosphorylation were not determined. Using LC-MS/MS, our lab recently identified four residues (S356, T357, T363 and S369) to be the sites of U50,488H-promoted phosphorylation in the mouse KOPR (mKOPR) stably expressed in N2A cells (Chen et al., 2016). Antibodies were generated against phosphopeptides and purified and three antibodies were found to have high specificity for the mKOPR phosphorylated at S356/T357, T363 and S369, respectively (Chen et al., 2016). Our lab previously showed that while U50,488H promoted robust hKOPR phosphorylation and internalization, etorphine induced little phosphorylation and internalization, although both were potent full agonists in enhancing [35S]GTPγS (Li et al., 2002a; Zhang et al., 2002; Li et al., 2003). Etorphine caused lower levels of KOPR phosphorylation at all the four residues than U50,488H by immunoblotting with the phospho-specific antibodies (Chen et al., 2016). Using the SILAC (stable isotope labeling by amino acids in cell culture) approach, we have found that compared to etorphine, U50,488H promoted higher levels of single phosphorylation at T363 and S369 and double phosphorylation at T363+S369 and T357+S369 as well as triple phosphorylation at S356+T357+S369 (Chen et al., 2016). These results indicate that an above-threshold phosphorylation is required for KOPR internalization. It has been reported that KOPR is involved in neuronal differentiation and neurogenesis. In the first chapter, I focused on whether there are differences in the mechanisms underlying neurite outgrowth induced by U50,488H and etorphine. In the chapter 2, mechanisms of KOPR phosphorylation were characterized in detail using phospho-specific KOPR antibodies. Protein kinase C was found, for the first time, to be involved in agonist-promoted KOPR phosphorylation. The roles of PKC in behavioral effects induced by KOPR agonists in mice were examined. For the chapter 1, in Neuro2a mouse neuroblastoma cells stably transfected with the hKOPR (N2A-3HA-hKOPR), U50,488H robustly induced neurite outgrowth, but etorphine caused outgrowth to a much lower extent. G protein-dependent pathway was found to be involved in the actions of both agonists, but β-arrestin-dependent pathway was not. Inhibition of ERK1/2 phosphorylation decreased neurite outgrowth promoted by both agonists, indicating the roles of MAP kinase cascades in KOPR agonist-induced neuritogenesis. In contrast, β-arrestin2, 14-3-3ζ, GEC1 and Rap1 are not involved in U50,488H- or etorphine-promoted neurite outgrowth. Thus, the two agonists appear to share the same signaling pathways and the difference between two agonists is likely due to the lower efficacy of etorphine. For the chapter 2, U50,488H caused phosphorylation of the mKOPR at S356, T357, T363 and S369 in N2A cells stably transfected with FmK6H (FmK6H-N2A cells). NorBNI abolished U50,488H-induced KOPR phosphorylation at all four residues. GRKs (GRKs2, 3, 5 and 6) and PKCs were involved in U50,488H-mediated KOPR phosphorylation. In addition, PKC also participated in agonist-independent KOPR phosphorylation. This is the first time that PKC was shown to be involved in agonist-induced KOPR phosphorylation. We found that U50,488H caused KOPR phosphorylation at T363 and S369 in the mouse brain and PKC participated in phosphorylation of S369, but not T363, by using the PKC inhibitor chelerythrine (CHL). Thus, we further characterized effects of PKC inhibition on KOPR-mediated behaviors in CD1 mice. PKC was involved in KOPR-mediated sedation, motor incoordination and conditioned place aversion, but not analgesia and anti-scratching effect in mice. Studies in this thesis revealed the mechanisms of KOPR-mediated neurite outgrowth and KOPR-mediated phosphorylation and the involvement of PKC in KOPR-mediated pharmacological effects in vivo. These studies push the frontier of molecular pharmacology of the KOPR, which may be useful for development of KOPR agonists for therapeutic use. / Pharmacology

Opioid receptor modulation of pro-inflammatory cytokine production is vital for host defense and the inflammatory response. Previous results have shown the mu-opioid receptor (MOR) selective agonist, DAMGO, has the capacity to increase the expression of the pro-inflammatory chemokines, CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 in peripheral blood mononuclear cells (PBMCs). We have shown that MOR activation is able to induce the expression of TGF-β, and TGF-β appears to be required for induction of CCL5 following MOR activation. This work suggests a novel role for TGF-β in the inflammatory response. NF-κB is a transcription factor that plays a pivotal role in inflammation and the immune response. We have found that NF-kB inhibitors can prevent the MOR-induced activation of CCL2 and CCL5, and that the NF-kB subunit, p65, is phosphorylated at serine residues 311 and 536 in response to μ-opioid receptor activation. In vivo, DAMGO administration can induce binding of p. 65 to the enhancer region of the CCL2 promoter. Furthermore, we demonstrate that PKCζ is phosphorylated following DAMGO-induced MOR activation and, is essential for NF-kB activity as well as CCL2 expression and transcriptional activity. In conclusion, these data suggest a pro-inflammatory role for MOR which involves NF-κB activation and PKCζ as well as a novel role for TGF-β as a regulator of pro-inflammatory chemokines. / Molecular Biology and Genetics

Activation of the dynorphin-kappa opioid receptor (KOR) system produces a negative emotional state during drug withdrawal, thereby motivating continued cocaine-seeking behaviors. However, it is not clear whether dynorphin plays a functional role in the onset of compulsive cocaine-taking. Here, escalation of cocaine self-administration was significantly attenuated by pretreatment of a long-acting KOR antagonist, norbinaltorphimine (NBI), in long access (LgA; 6-hours) male rats, whereas there was no effect of NBI on short access (ShA; 1-hour) rats on a fixed or progressive ratio schedule of reinforcement. Additionally, optical density of prodynorphin was increased in the nucleus accumbens (NAc) core and shell, bed nucleus of the stria terminalis (BNST), central amygdala (CeA), and basolateral amygdala (BLA) of LgA rats compared to both ShA and drug-naïve rats. These results suggest dynorphin in the stress-sensitive extended amygdala (NAc shell, BNST, CeA), and BLA-NAc core circuitry mediating cue-controlled cocaine-taking may be associated with the onset of compulsive drug-taking.

Titre de l'écran-titre (visionné le 23 mai 2023) / Contexte : La génétique a une implication majeure dans la pathogenèse de la parodontite. Elle fait d'ailleurs l'objet d'un nombre grandissant de recherches qui tentent d'identifier quels facteurs prédisposent au développement de la maladie, mais également à une réponse réduite à la thérapie. Le facteur nucléaire kappa-B (NF-κB) a été identifié dans plusieurs maladies inflammatoires comme étant la principale voie de signalisation permettant l'activation de l'inflammation. Par ailleurs, toute altération génétique (mutation, polymorphisme ou encore modification épigénétique) de cette voie de signalisation est étroitement liée à certaines maladies inflammatoires. Objectif : L'objectif du projet est (1) d'étudier si le polymorphisme génétique de NF-κB est associé au développement de la parodontite dans la population québécoise; et (2) de vérifier s'il est associé à une réponse altérée à la thérapie parodontale non chirurgicale. Matériel et méthodes : 200 échantillons salivaires ont été obtenus auprès de patients québécois de la clinique de parodontite de la Faculté de médecine dentaire de l'Université Laval et l'ADN de chaque échantillon a été isolé. Quatre SNP de NF-κB ont été choisis pour génotypage : rs4648127, rs4648099, rs4648072, rs4648065. L'ADN a été extrait avec la trousse de Qiagen et quantifié avec NanoDrop. Le génotypage a été réalisé par une réaction en chaîne par polymérase (PCR) grâce à la technique de génotypage TaqMan utilisée pour amplifier et détecter des allèles spécifiques de chaque polymorphisme sélectionné de NF-κB dans l'ADN génomique (ADNg). Résultats : 146 échantillons ont pu être analysés. Le groupe test (parodontite) était formé de 58 participants tandis que le groupe contrôle en comptait 88. Le seul polymorphisme ayant montré des variations entre les sujets est rs4648127. Le génotype CT était associé au développement de la parodontite (OR = 1,04, p > 0,05), mais le résultat n'était pas statistiquement significatif. Des analyses de sous-groupe ont été réalisées pour vérifier les différences entre les hommes, les femmes et différentes tranches d'âge. Aucune valeur statistiquement significative n'a été trouvée. Les concentrations salivaires de TNF-α à la base de référence n'étaient pas les mêmes entre les groupes test et contrôle (test : 42,43 pg/mL, contrôle : 25,34 pg/mL, p = 0,000952). Concernant la réponse à la thérapie, les concentrations salivaires de TNF-α ont diminué de manière significative entre le début et le contrôle postopératoire (base de référence : 42,43 pg/mL, post-op : 28,19 pg/mL, p = 0,00435). L'écart dans la réponse à la thérapie chez les patients présentant un génotype CC ou CT n'ont pas montré de différences statistiques. Conclusion : Les polymorphismes de NF-κB (rs4648127, rs4648099, rs4648072, rs4648065) ne semblent pas associés ni au développement de la parodontite, ni à une réponse altérée à la thérapie parodontale chez les adultes québécois. Cependant, d'autres études avec de plus larges échantillons sont nécessaires pour tirer de plus franches conclusions sur ces résultats. / Introduction: The nuclear factor kappa-B (NF-κB) has been identified in several inflammatory diseases as the main signaling pathway responsible for the activation of inflammation. Furthermore, this factor's genetic variations (polymorphisms) have been associated with inflammatory diseases. Therefore, this study aimed to (1) search for the implication that polymorphisms of NF-κB have in the development of periodontitis in adults from the province of Quebec, Canada and (2) verify if different allele frequencies can affect the outcomes of the non-surgical periodontal therapy. Methods: 200 saliva samples were obtained and studied using a genotyping assay to verify the association between periodontitis and four single nucleotide polymorphisms (SNPs): rs4648127, rs4648099, rs4648072, rs4648065. DNA was extracted using a Qiagen kit and quantified by NanoDrop One. Genotyping was realized by TaqMan Real-Time PCR Assays. The reduction in inflammation following therapy was measured by comparing saliva concentration of tumor necrosis factor-α (TNF-α) at the baseline and four to eight weeks after the treatment. Results: 146 samples were subject to analysis. As 58 and 88 patients were forming the test (periodontitis) and control groups, respectively. Only the rs4648127 SNP varied between subjects. CT genotype was associated with the development of periodontitis (OR = 1.04, p > 0.05) but was not statistically significant. Subgroup analysis were done to search for associations in male, female, and different age groups but were not statistically significant. Baseline TNF-α concentration in saliva showed variations between test and control groups (test: 42.43 pg/mL, control: 25.34 pg/mL p = 0.000952). The level of inflammation reduced after therapy. The concentration of TNF-α was 42.43 pg/mL at baseline and 28.19 pg/mL after treatment (p = 0.00435). No significant results were found between subjects with CT genotype or CC genotype. Conclusion: Either the development of periodontitis or the reduction in inflammation after therapy is associated with NF-κB polymorphisms in adults from Quebec. However, other studies with larger samples are needed to confirm those results.

O perfil de ativação do NF-B foi avaliado em biópsias de 47 pacientes com diagnóstico clínico e laboratorial de hanseníase, seguidos no Ambulatório de Hanseníase do Centro de Referência Nacional em Dermatologia Tropical com Ênfase em Hanseníase do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto Universidade de São Paulo. O índice de ativação NF-B foi calculado de acordo com a porcentagem de positividade na histoquímica Southwestern. Índice de ativação NF-B >1 foi considerado representativo de ativação. Trinta e seis por cento dos pacientes apresentaram NF-B ativado na biópsia, o que foi mais frequente em multibacilares (54,6%) que em paucibacilares (20%), p=0,018. Hanseníase tuberculóide esteve associada com ausência de NF-B ativado (p=0,039). Forma dimorfa e neural pura estiveram associadas com ativação de NF-B, com odds ratio de 44 (p=0,014) e 30 (p=0,029), respectivamente. A ativação correlacionou-se com detecção in situ de fator de necrose tumoral por imuno-histoquímica (p=0,0064). Observou-se grande variação da ativação do NF-B nas formas clínicas de hanseníase. Ativação foi nula em granulomas de hanseníase tuberculóide, que representa a reação inflamatória mais efetiva contra o M. leprae. A ativação do NF-B ocorreu predominantemente em formas clínicas com maior suscetibilidade (multibacilares) e instabilidade imunológica (dimorfa), indicando condição favorável à infecção pela ativação do NF-B, por seus efeitos antiapoptóticos, visto que o bacilo depende das funções celulares para sobreviver. / NF-B activation profile was evaluated in cutaneous biopsies from 47 patients with clinical and laboratorial diagnosis of leprosy followed at a referral center for treatment of leprosy, the leprosy outpatient clinic of the Hospital of Clinics of Faculty of Medicine of Ribeirão Preto University of São Paulo. NF-B activation index (ranging from 0 to 4) was calculated according to the percentage of positivity in Southwestern histochemistry. Activation index >1 was considered representative of activation. Thirty-six percent of patients presented activated NF-B, which was more frequent in multibacillary (54,6%) than in paucibacillary (20%), p=.018. Tuberculoid leprosy was associated with absence of activated NF-B (p=.039). Borderline and pure neural leprosy clinical forms were associated with NF-B activation, with odds ratio of 44 (p=.014) and 30 (p=.029), respectively. NF-B activation was correlated with in situ detection of tumor necrosis factor- by immunohistochemistry (p=.0064). Great variation of NF-B activation was found in clinical forms of leprosy. Activation was absent in tuberculoid leprosys granulomas, which represent effective inflammatory reaction pattern against M. leprae. NF-B activation was present in clinical forms with increased susceptibility (multibacillary) and immunological instability (borderline), which suggests favorable conditions towards infection, probably due to the anti-apoptotic effects of NF-B, since bacillary survival is dependent of cellular functions.

O excesso de adenina na dieta (ADE) promove precipitação intratubular de cristais, levando à instalação de uma nefrite intersticial progressiva e perda da função renal. Observações recentes indicam que a sobrecarga de ADE em camundongos em que os genes para TLR-2, -4, MyD88, ASC ou caspase-1 foram inativados provoca menos dano renal do que quando administrado a camundongos selvagens, sugerindo a existência de um papel patogênico para a ativação de TLRs e a montagem de inflamassomas nesse modelo. O presente estudo foi concebido para investigar se outro importante componente da imunidade inata, o sistema NF-B, também exerce papel patogênico na nefropatia associada ao excesso de ADE. Ratos Munich-Wistar machos e adultos foram divididos em 3 grupos: C (N=17), ração padrão; ADE (N=17), ADE na ração, 0,7% durante 1 semana e 0,5% durante 2 semanas; ADE+PDTC (N=14), ADE administrada como descrito anteriormente, associada ao inibidor do NF-B, pirrolidina ditiocarbamato (PDTC), 120 mg/kg/dia na água do bebedouro. Após 3 semanas, observou-se deposição de numerosos cristais no tecido renal, em sua maioria no interior de granulomas de corpo estranho, acompanhada de intensa atividade proliferativa tubulointersticial. Uma parte dos cristais apareceu envolvida por uma camada de células aparentemente derivadas do epitélio tubular, que pareciam segregar os precipitados e, em alguns casos, expulsá-los ao interstício. Essas alterações associaram-se a uma grande expansão da área intersticial, com deposição de colágeno, além de hipotrofia glomerular. Foi possível ainda demonstrar um aumento da expressão da interleucina-6, do interferon-, da proteína específica de fibroblastos (FSP-1) e da proteína quimiotática para macrófagos (MCP-1). A abundância do IKK-, uma quinase ativadora do sistema NK-B, mostrou-se também acentuadamente elevada nesses ratos. O tratamento com PDTC normalizou a expressão do IKK-, diminuindo o número de granulomas e a proliferação celular, além de atenuar fortemente a fibrose e o declínio da função renal. Esses achados, que contribuem para elucidar os mecanismos de formação de granulomas no modelo de sobrecarga de adenina, são consistentes com o conceito de que o sistema NF-B é ativado pela precipitação intratubular de cristais e contribui, juntamente com outros mecanismos ligados à imunidade inata, para iniciar a intensa resposta inflamatória associada a esse modelo. Descritores: 1.Insuficiência renal crônica 2.Adenina 3.Imunidade inata 4.NF-kappa B 5.GranulomaO excesso de adenina na dieta (ADE) promove precipitação intratubular de cristais, levando à instalação de uma nefrite intersticial progressiva e perda da função renal. Observações recentes indicam que a sobrecarga de ADE em camundongos em que os genes para TLR-2, -4, MyD88, ASC ou caspase-1 foram inativados provoca menos dano renal do que quando administrado a camundongos selvagens, sugerindo a existência de um papel patogênico para a ativação de TLRs e a montagem de inflamassomas nesse modelo. O presente estudo foi concebido para investigar se outro importante componente da imunidade inata, o sistema NF-B, também exerce papel patogênico na nefropatia associada ao excesso de ADE. Ratos Munich-Wistar machos e adultos foram divididos em 3 grupos: C (N=17), ração padrão; ADE (N=17), ADE na ração, 0,7% durante 1 semana e 0,5% durante 2 semanas; ADE+PDTC (N=14), ADE administrada como descrito anteriormente, associada ao inibidor do NF-B, pirrolidina ditiocarbamato (PDTC), 120 mg/kg/dia na água do bebedouro. Após 3 semanas, observou-se deposição de numerosos cristais no tecido renal, em sua maioria no interior de granulomas de corpo estranho, acompanhada de intensa atividade proliferativa tubulointersticial. Uma parte dos cristais apareceu envolvida por uma camada de células aparentemente derivadas do epitélio tubular, que pareciam segregar os precipitados e, em alguns casos, expulsá-los ao interstício. Essas alterações associaram-se a uma grande expansão da área intersticial, com deposição de colágeno, além de hipotrofia glomerular. Foi possível ainda demonstrar um aumento da expressão da interleucina-6, do interferon-, da proteína específica de fibroblastos (FSP-1) e da proteína quimiotática para macrófagos (MCP-1). A abundância do IKK-, uma quinase ativadora do sistema NK-B, mostrou-se também acentuadamente elevada nesses ratos. O tratamento com PDTC normalizou a expressão do IKK-, diminuindo o número de granulomas e a proliferação celular, além de atenuar fortemente a fibrose e o declínio da função renal. Esses achados, que contribuem para elucidar os mecanismos de formação de granulomas no modelo de sobrecarga de adenina, são consistentes com o conceito de que o sistema NF-B é ativado pela precipitação intratubular de cristais e contribui, juntamente com outros mecanismos ligados à imunidade inata, para iniciar a intensa resposta inflamatória associada a esse modelo / Excessive dietary adenine (ADE) promotes intratubular precipitation of crystals, leading to the installation of progressive interstitial nephritis and renal function loss. Recent observations indicate that ADE overload in mice in which genes for TLR-2, -4, MyD88, ASC or caspase-1 were inactivated causes less renal damage than when administered to wild-type mice, suggesting the existence of a pathogenic role for the activation of TLRs and the assembly of inflamassomes in this model. The present study was designed to investigate whether another important component of innate immunity, the NF-B system, also plays a role in the pathogenesis of the nephropathy associated with excess ADE. Adult Male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard diet; ADE (N = 17), given ADE in the diet at 0.7% for 1 week and 0.5% for 2 weeks; and ADE + PDTC (N = 14), receiving ADE as described above, associated with the NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), 120 mg/kg/ day in drinking water. After three weeks, there was widespread deposition of crystals in the renal tissue, mostly within granulomas, accompanied by florid tubulointerstitial proliferative activity. Part of these crystals was enclosed in a layer of cells apparently derived from the tubular epithelium, which appeared to segregate the precipitates, and in some cases, to extrude them to the interstitium. These changes were associated with marked interstitial expansion, collagen deposition, and glomerular hypotrophy. Increased expression of interleukin-6, interferon-, fibroblast specific protein (FSP-1) and macrophage chemoattractant protein (MCP-1) were also shown. The abundance of IKK-, a kinase that activates the NF-B system, was also markedly elevated in these mice. Treatment with PDTC normalized the expression of IKK-, reducing the number of granulomas and cell proliferation, and strongly attenuated interstitial fibrosis and the decline of renal function. These findings, which contribute to illuminate some mechanisms of granuloma formation in the ADE overload model, are consistent with the concept that the NF-B system is activated by intratubular precipitation of crystals and contributes, along with other mechanisms related to innate immunity, to initiate the intense inflammatory response associated with this model