Dissertations / Theses on the topic 'Nachisu'

Chemotherapy-Induced Peripheral Neuropathy (CIPN) is a major dose-limiting side effect of several anticancer drugs. The prevalence of CIPN ranges from one-third to two-thirds of cancer patients. CIPN can persist for months to years after completion of chemotherapy. Despite the efficacious use of paclitaxel in the treatment of tumors, it can induce many sensory symptoms, such as paresthesia, numbness, tingling and burning pain, and mechanical and cold allodynia, which typically are present in the hands and feet. Similar to other types of chronic pain, paclitaxel-induced CIPN is comorbid with depression and anxiety in cancer survivors, and paclitaxel induces changes in affect-like behavior in cancer-free animal models, suggesting that paclitaxel can cause long-lasting changes in mood, reducing the quality of life. While adjuvant therapies, such as duloxetine, tricyclic antidepressants, and gabapentin are prescribed to treat CIPN symptoms, none of these compounds can consistently reverse or prevent the development of CIPN. With no FDA-approved medication to treat CIPN, the purpose of the dissertation was to: i) characterize and develop a mouse model of paclitaxel-induced CIPN, ii) identify putative targets for CIPN treatment, and iii) test novel compounds for their ability to prevent and reverse CIPN in C57BL/6J mice. In the first Aim, we demonstrate that paclitaxel induces time- and dose-dependent hypersensitivity (mechanical and cold), which is potentiated by combination therapy with the chemotherapeutic carboplatin. In addition, paclitaxel-treated mice show changes in affect-like behaviors (anxiety-like, depression-like). In the second Aim, we used the prototypic nicotinic receptor (nAChR) agonist nicotine to reverse or prevent paclitaxel-induced mechanical hypersensitivity and degeneration of Intra-Epidermal Nerve Fibers (IENFs). Further, we discovered that nicotine’s antinociceptive effects in this mouse model of CIPN are mediated by the nicotinic receptor subtype α7. The third Aim used genetic and pharmacological approaches to dissect the role of α7 on the development and maintenance of paclitaxel-induced CIPN. Null mutant α7 mice (KO) hastens the onset, increases the magnitude, and delays the recovery of paclitaxel-induced mechanical hypersensitivity, as compared to littermate wildtype controls, whereas the selective α7 silent agonist R-47 to reverses and prevents paclitaxel-induced CIPN in C57BL/6J mice. We also examined the impact of R-47 on the paclitaxel-induced reduction of intraepidermal nerve fiber (IENF), as well as microglial morphology in the dorsal horn of the spinal cord. The data show that R-47 prevents paclitaxel-induced changes in microglial morphology and mechanical hypersensitivity behavior, without producing tolerance upon repeated administration. Finally, R-47 induces preference using the conditioned place test in paclitaxel-treated mice but vehicle-treated animals, suggesting that R-47 is a viable candidate for ongoing, spontaneous pain, with limited risk of abuse potential. Overall, these results support that the α7 nAChR subtype is an important target for the treatment and prevention of CIPN.

Consumption of alcohol and tobacco is common all around the world and these drugs are frequently consumed concomitantly. It has been estimated that 70-80 % of alcoholics are smokers and non-alcoholic drinkers are more often smokers than teetotallers. Alcohol and tobacco may affect the risk of developing neurological diseases and might influence this risk differently when combined compared to when only one of these compounds is consumed. Some in vitro-research have shown that non-toxic concentrations of ethanol and nicotine, in combination, can exert toxicity, and might do so in a synergistic way. In this work, investigations were made to see if the neuronal nicotinic acetylcholine receptors (nAChRs) and NMDA receptors are involved in this interactive behaviour between ethanol and nicotine. A human neuroblastoma SH-SY5Y cell line was treated with ethanol and nicotine at different concentrations and cell viability was measured through an MTT-assay. A significant reduction in cell viability was induced by chronic treatment with a low-dose combination of ethanol and nicotine. The cell viability reduction was completely inhibited by pretreatment with the non-specific nAChR antagonist mecamylamine. This suggests that nAChRs are involved in low-dose ethanol-nicotine interactions. The NMDA receptor antagonist memantine did not affect the ethanol-nicotine effect, which implies that NMDA receptors are not involved in low-dose ethanol-nicotine interactions in SH-SY5Y cells. However, it is unclear if the SH-SY5Y cell line expresses fully functional NMDA receptors. The expression of NMDA receptors might vary with cell passage number. Further research has to be done to uncover the contribution of specific nAChR subtypes to the ethanol-nicotine interaction. There also remains to be revealed if human neuroblastoma SH-SY5Y cells express fully functional NMDA receptors and how cell passage number affects the expression of these receptors.

Made available in DSpace on 2014-06-11T19:23:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-17Bitstream added on 2014-06-13T20:10:23Z : No. of bitstreams: 1 souza_pat_me_botib.pdf: 590527 bytes, checksum: 1859d3811e273778bf9c06965cfbdf4b (MD5)<br>Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>A insuficiência cardíaca (IC) é uma síndrome iniciada por uma redução na função cardíaca e caracterizada pela ativação de mecanismos compensatórios e atrofia. Fadiga e dispnéia são os sintomas mais comuns desta doença, e estão associados à miopatia do músculo esquelético; ocorre redução da capacidade oxidativa, mudança nas fibras do tipo I para tipo II, alteração na expressão dos fatores reguladores miogênicos (MRFs) e na JNM. O treinamento físico aeróbico é considerado uma conduta amplamente aceita para minimizar as consequências dos sintomas causados pela IC, com melhora na qualidade de vida e pode reverter anormalidades do músculo cardíaco e esquelético, além de modular a junção neuromuscular. O músculo diafragma é o mais importante músculo inspiratório dos mamíferos sendo um dos mais acometidos na IC; o treinamento físico aeróbico altera as propriedades eletrofisiológicas do diafragma durante a fadiga e favorece a resistência muscular. A hipótese do presente estudo é que as alterações fenotípicas musculares que ocorrem na IC estão associadas com alterações na distribuição e expressão dos nAChRs e que o treinamento físico aeróbico atenue estas alterações. O objetivo deste trabalho foi avaliar a distribuição e expressão dos nAChRs em ratos com IC submetidos ao treinamento aeróbico. Foram utilizados 44 ratos Wistar machos (90 a 100g), divididos em quatro grupos experimentais: controle (C), treinamento aeróbico (TR), estenose aórtica (EAo) e estenose aórtica com treinamento aeróbico (EAoTR). A estenose aórtica foi induzida pela inserção de um clip de prata de 0.6 mm de diâmetro na aorta ascendente. Após 18 semanas da cirurgia, os animais com EAo, foram avaliados por ecocardiograma para verificar a hipertrofia do VE. Os demais animais foram divididos aleatoriamente em...<br>Heart failure (HF) is a syndrome initiated by a reduction in cardiac function and characterized by the activation of compensatory mechanisms and muscle myopathy. Fatigue and dyspnea are the most common symptoms of this disease, associated with the reduction of the oxidative capacity, changes in the fibers type I to type II, alterations in the expression of myogenic regulatory factors (MRFs) and in the neuromuscular junction (NMJs). Aerobic training is considered a widely accepted practice to minimize the consequences of the symptoms caused by HF, improving the life quality and can reverse abnormalities of cardiac and skeletal muscle, and modulate the NMJ. The diaphragm is the most important inspiratory muscle in mammals and is one of the most affected in HF; aerobic training changes the electrophysiological properties of the diaphragm during fatigue and promotes muscle endurance. Thus, the hypothesis of this study is that the changes that occur in HF are associated with alterations in the NMJs, in the expression of nAChRs and aerobic training can attenuate these changes. The aim of this study was to evaluate the distribution and the expression of nAChRs subunits in rats with HF submitted to an aerobic training. We used 44 male wistar rats (90 to 100g), divided into four groups: Control (C), Aerobic Training (TR), Aortic Stenosis (AS) and Aortic Stenosis with Aerobic Training (ASTR). Aortic stenosis was induced by the insertion of a silver clip of 0.6 mm in diameter in the ascending aorta. After 18 weeks of surgery, the animals with AS were evaluated by echocardiography for evaluating LV hypertrophy. The others were randomly divided into C, TR and ASTR groups. The animals of TR and ASTR groups were submitted to aerobic training on a treadmill for 10 weeks (5 days/ week). The diaphragm muscle was used to assess the expression... (Complete abstract click electronic access below)

Orientador: Maeli Dal Pai Silva<br>Coorientador: Selma Maria Michelin Matheus<br>Banca: Elaine Minatel<br>Banca: Antonio de Castro Rodrigues<br>Resumo: A insuficiência cardíaca (IC) é uma síndrome iniciada por uma redução na função cardíaca e caracterizada pela ativação de mecanismos compensatórios e atrofia. Fadiga e dispnéia são os sintomas mais comuns desta doença, e estão associados à miopatia do músculo esquelético; ocorre redução da capacidade oxidativa, mudança nas fibras do tipo I para tipo II, alteração na expressão dos fatores reguladores miogênicos (MRFs) e na JNM. O treinamento físico aeróbico é considerado uma conduta amplamente aceita para minimizar as consequências dos sintomas causados pela IC, com melhora na qualidade de vida e pode reverter anormalidades do músculo cardíaco e esquelético, além de modular a junção neuromuscular. O músculo diafragma é o mais importante músculo inspiratório dos mamíferos sendo um dos mais acometidos na IC; o treinamento físico aeróbico altera as propriedades eletrofisiológicas do diafragma durante a fadiga e favorece a resistência muscular. A hipótese do presente estudo é que as alterações fenotípicas musculares que ocorrem na IC estão associadas com alterações na distribuição e expressão dos nAChRs e que o treinamento físico aeróbico atenue estas alterações. O objetivo deste trabalho foi avaliar a distribuição e expressão dos nAChRs em ratos com IC submetidos ao treinamento aeróbico. Foram utilizados 44 ratos Wistar machos (90 a 100g), divididos em quatro grupos experimentais: controle (C), treinamento aeróbico (TR), estenose aórtica (EAo) e estenose aórtica com treinamento aeróbico (EAoTR). A estenose aórtica foi induzida pela inserção de um clip de prata de 0.6 mm de diâmetro na aorta ascendente. Após 18 semanas da cirurgia, os animais com EAo, foram avaliados por ecocardiograma para verificar a hipertrofia do VE. Os demais animais foram divididos aleatoriamente em... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Heart failure (HF) is a syndrome initiated by a reduction in cardiac function and characterized by the activation of compensatory mechanisms and muscle myopathy. Fatigue and dyspnea are the most common symptoms of this disease, associated with the reduction of the oxidative capacity, changes in the fibers type I to type II, alterations in the expression of myogenic regulatory factors (MRFs) and in the neuromuscular junction (NMJs). Aerobic training is considered a widely accepted practice to minimize the consequences of the symptoms caused by HF, improving the life quality and can reverse abnormalities of cardiac and skeletal muscle, and modulate the NMJ. The diaphragm is the most important inspiratory muscle in mammals and is one of the most affected in HF; aerobic training changes the electrophysiological properties of the diaphragm during fatigue and promotes muscle endurance. Thus, the hypothesis of this study is that the changes that occur in HF are associated with alterations in the NMJs, in the expression of nAChRs and aerobic training can attenuate these changes. The aim of this study was to evaluate the distribution and the expression of nAChRs subunits in rats with HF submitted to an aerobic training. We used 44 male wistar rats (90 to 100g), divided into four groups: Control (C), Aerobic Training (TR), Aortic Stenosis (AS) and Aortic Stenosis with Aerobic Training (ASTR). Aortic stenosis was induced by the insertion of a silver clip of 0.6 mm in diameter in the ascending aorta. After 18 weeks of surgery, the animals with AS were evaluated by echocardiography for evaluating LV hypertrophy. The others were randomly divided into C, TR and ASTR groups. The animals of TR and ASTR groups were submitted to aerobic training on a treadmill for 10 weeks (5 days/ week). The diaphragm muscle was used to assess the expression... (Complete abstract click electronic access below)<br>Mestre

Introduction: The α4β2 subtype of the nicotinic acetylcholine receptor (nAChR) has been pursued as a drug target for treatment of psychiatric and neurodegenerative disorders and smoking cessation aids for decades. Particular halogen containing compounds have showed promising results at α4β2 subtype of nAChR. Aims: To explore and understand the structure activity relationships of halogen containing α4β2 agonists. Potentially, this could lead to rational design of superagonists. Methods: Consecutive series of halogen containing compounds were investigated by using two-electrode voltage-clamp electrophysiology. Discussion: The results of the tested series of compounds show a tendency in increasing efficacy with the increasing size of the substituent: H< F < Cl < Br; this applies for both the (α4)2(β2)3 and the (α4)3(β2)2 stoichiometries of the α4β2 nAChR subtype. Interestingly, the Br and Cl containing homopiperazines are superagonists at the (α4)3(β2)2 subtype. Activation of the cholinergic pathways elicits antinociceptive effects, which means finding a successful agonist or a superagonist could lead to a new type of analgesic dealing with chronic pain.

La découverte de l’acétylcholine binding protein (AChBP) a permis des avancées majeures dans la caractérisation des récepteurs nicotiniques de l’acétylcholine (nAChRs). Dans le cadre de ce travail, une approche combinant la mise en oeuvre de méthodes expérimentales et de calculs quantiques issus de la théorie de la fonctionnelle de la densité (DFT) a été utilisée pour (i) déterminer les conformations de basse énergie et les interactions d’une série de ligands des nAChRs (l’ACh, la nicotine, l’épibatidine, la galanthamine et la codéine) (ii) mettre à jour un motif structural de l’AChBP jouant un rôle majeur dans l’interaction avec les agonistes (iii) étudier la complexation de trois ligands avec un modèle tridimensionnel du site de fixation par une méthode mixte QM/QM’. Ainsi, malgré l’importante similitude structurale de la galanthamine et de la codéine, la confrontation des résultats obtenus au cours de ce travail révèle des différences d’interactions par liaison H significatives entre les deux composés. Par ailleurs, le motif structural mis à jour, qui comprend le Trp 143, impliqué dans la fixation des agonistes, et l’Asp 85, hautement conservé dans la famille des canaux ioniques, a été caractérisé de façon approfondie, l’influence d’un effet coopératif sur le réseau de liaison H formé étant démontrée. Enfin, l’ordre énergétique d’interaction ACh<br>The discovery of the Acetylcholine Binding Protein (AChBP) has allowed major breakthroughs in the characterization of nicotinic acetylcholine receptors (nAChRs). In this work, a combined approach based on the use of various experimental methods and of theoretical calculations from density functional theory (DFT) has allowed to (i) determine the conformations and interactions of five nAChRs ligands (ACh, nicotine, epibatidine, galanthamine and codeine) in various environments (ii) shed light on a structural motif of AChBP playing a key role in the agonist binding (iii) study the complexation of ACh, nicotine and epibatidine with a three dimensional model of the AChBP binding site through a mixed QM/QM’ approach. Thus, despite the important similarities of galanthamine and codeine, the confrontation of the results obtained from the various approaches used in this work reveals significant differences of hydrogen-bond interactions of the two compounds. Furthermore, the structural motif we have found, which includes the Trp 143, involved in the agonists binding, and the Asp 85, highly conserved in the ion channels family, has been deeply characterized, the influence of a cooperative effect on the H-bond network formed being demonstrated. Finally, the energetic ranking of interaction of the three ligands ACh

Made available in DSpace on 2014-06-11T19:30:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-08Bitstream added on 2014-06-13T19:19:46Z : No. of bitstreams: 1 cabeco_lc_dr_botib.pdf: 3413131 bytes, checksum: 235f28b6b47318c9d3eae77eee4e421c (MD5)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>A restrição protéica materna pode afetar vários parâmetros morfofuncionais da prole com conseqüências na vida adulta. Esse fenômeno, conhecido como programação fetal, pode causar mudanças na musculatura esquelética a longo prazo. Neste trabalho foram analisadas a morfologia e histoquímica dos tipos de fibras musculares, a expressão gênica e protéica dos fatores reguladores miogênicos MyoD e Miogenina, a morfologia das junções neuromusculares (JNMs) e a expressão gênica das subunidades ( , e ) que compõe os receptores nicotínicos de acetilcolina, nos músculos Soleus (SOL) e Extensor longo dos dedos (EDL) de ratos machos (30 dias e 16 semanas de idade), provenientes de mães alimentadas com dieta de baixa proteína (6%, LP, n=8) durante a gestação e alimentadas com dieta normoprotéica (17%, NP=8) após o nascimento dos filhotes. Nos períodos analisados, os músculos SOL e EDL foram submetidos à reação histoquímica mATPase para a análise da freqüência (%) e área (CSA) das fibras; a morfologia das JNMs foi analisada por microscopia eletrônica de transmissão; a expressão gênica e protéica foram determinadas pelo RT-qPCR e Western Blot, respectivamente. Nossos resultados mostraram que ao nascimento e com 30 dias de idade, o peso corporal dos filhotes do grupo LP foi estatisticamente menor comparado aos filhotes do grupo NP e com 16 semanas de idade, nenhuma diferença foi observada no peso corporal. Aos 30 dias de idade, o peso do músculo SOL foi menor no grupo LP comparado ao NP (P<0,05) e nenhuma diferença estatística foi observada no peso do músculo EDL. Com 16 semanas de idade, a razão peso muscular/peso corpóreo foi menor no músculo SOL e nenhuma diferença estatística foi observada no músculo EDL. Aumento na freqüência das fibras tipo IIB e diminuição das fibras tipo...<br>The maternal protein restriction can affect various morphophysiological parameters in offspring with consequences in adulthood. This phenomenon, known as fetal programming, can cause changes in skeletal muscle phenotype in long term. We investigated muscle fiber type changes, MyoD and Myogenin expression, neuromuscular junction (NMJs) morphology and , and nAChRs subunits expression in SOL and EDL muscles in offspring rats, with 30 and 16-weeks-old, from dams fed with low protein diet (6% protein, LP, n=8) and normoproteic diet (17% protein, NP, n=8). Muscles collected were submitted to myofibrillar adenosine triphosphatase (mATPase) histochemistry reaction for muscle fiber types frequency and the cross sectional areas (CSA) analysis. The neuromuscular junction (NMJs) morphology was analyzed using transmission electron microscope. The gene and protein expression were determined by RT-qPCR and Western Blot, respectively. Our results showed that at birth and with 30-days-old, the body weight of pups was statistically lower in LP compared to NP group; at 16-week-old, no significant difference were observed. At 30-days, the SOL muscle weight (g) was lower in LP compared to NP group (P<0.05) and no statistical difference was observed in the EDL muscle weight. At 16-week-old, SOL muscle weight/body weight ratio was lower in LP group compared to NP group, P<0.05. Increasing in IIB and decreased in I and IIA+IID fiber frequency in EDL muscle and no difference in fiber frequency in SOL muscle were observed in LP group, with 30-days-age. At 16-weeks-age, there were no differences in muscle fiber types frequency for both muscles. In LP group, muscle fiber I CSA decreased only in SOL muscle in all periods studied. MyoD and Myogenin expression no changes in response to maternal... (Complete abstract click electronic access below)

Orientador: Maeli Dal Pai Silva<br>Coorientador: Selma Michelin Matheus<br>Banca: José Antonio Rocha Gontijo<br>Banca: Maria de Lourdes Mendes Vicentini Paulino<br>Banca: Márcia Galaci<br>Banca: Débora Cristina Damasceno<br>Resumo: A restrição protéica materna pode afetar vários parâmetros morfofuncionais da prole com conseqüências na vida adulta. Esse fenômeno, conhecido como programação fetal, pode causar mudanças na musculatura esquelética a longo prazo. Neste trabalho foram analisadas a morfologia e histoquímica dos tipos de fibras musculares, a expressão gênica e protéica dos fatores reguladores miogênicos MyoD e Miogenina, a morfologia das junções neuromusculares (JNMs) e a expressão gênica das subunidades ( , e ) que compõe os receptores nicotínicos de acetilcolina, nos músculos Soleus (SOL) e Extensor longo dos dedos (EDL) de ratos machos (30 dias e 16 semanas de idade), provenientes de mães alimentadas com dieta de baixa proteína (6%, LP, n=8) durante a gestação e alimentadas com dieta normoprotéica (17%, NP=8) após o nascimento dos filhotes. Nos períodos analisados, os músculos SOL e EDL foram submetidos à reação histoquímica mATPase para a análise da freqüência (%) e área (CSA) das fibras; a morfologia das JNMs foi analisada por microscopia eletrônica de transmissão; a expressão gênica e protéica foram determinadas pelo RT-qPCR e Western Blot, respectivamente. Nossos resultados mostraram que ao nascimento e com 30 dias de idade, o peso corporal dos filhotes do grupo LP foi estatisticamente menor comparado aos filhotes do grupo NP e com 16 semanas de idade, nenhuma diferença foi observada no peso corporal. Aos 30 dias de idade, o peso do músculo SOL foi menor no grupo LP comparado ao NP (P<0,05) e nenhuma diferença estatística foi observada no peso do músculo EDL. Com 16 semanas de idade, a razão peso muscular/peso corpóreo foi menor no músculo SOL e nenhuma diferença estatística foi observada no músculo EDL. Aumento na freqüência das fibras tipo IIB e diminuição das fibras tipo... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: The maternal protein restriction can affect various morphophysiological parameters in offspring with consequences in adulthood. This phenomenon, known as fetal programming, can cause changes in skeletal muscle phenotype in long term. We investigated muscle fiber type changes, MyoD and Myogenin expression, neuromuscular junction (NMJs) morphology and , and nAChRs subunits expression in SOL and EDL muscles in offspring rats, with 30 and 16-weeks-old, from dams fed with low protein diet (6% protein, LP, n=8) and normoproteic diet (17% protein, NP, n=8). Muscles collected were submitted to myofibrillar adenosine triphosphatase (mATPase) histochemistry reaction for muscle fiber types frequency and the cross sectional areas (CSA) analysis. The neuromuscular junction (NMJs) morphology was analyzed using transmission electron microscope. The gene and protein expression were determined by RT-qPCR and Western Blot, respectively. Our results showed that at birth and with 30-days-old, the body weight of pups was statistically lower in LP compared to NP group; at 16-week-old, no significant difference were observed. At 30-days, the SOL muscle weight (g) was lower in LP compared to NP group (P<0.05) and no statistical difference was observed in the EDL muscle weight. At 16-week-old, SOL muscle weight/body weight ratio was lower in LP group compared to NP group, P<0.05. Increasing in IIB and decreased in I and IIA+IID fiber frequency in EDL muscle and no difference in fiber frequency in SOL muscle were observed in LP group, with 30-days-age. At 16-weeks-age, there were no differences in muscle fiber types frequency for both muscles. In LP group, muscle fiber I CSA decreased only in SOL muscle in all periods studied. MyoD and Myogenin expression no changes in response to maternal... (Complete abstract click electronic access below)<br>Doutor

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are necessary in memory and cognition. They are pentameric and consist of α and β subunits. They are most commonly heteromeric but, can sometimes be homomeric. nAChRs are activated by many ligands including nicotine (exogenous) and acetylcholine (endogenous).nAChRs are located on hippocampal interneurons. The interneurons, although sparse, control the synchronous firing of the pyramidal cells. However, the hippocampal interneuron structure and function is quite diverse and not fully characterized. Therefore, we sought to quantify nAChR subunit mRNA levels using real-time PCR of CA1 hippocampal interneurons.Surprisingly we found that the α3 and β2 mRNA subunits were the highest expressed and highest co-expressed subunits. Additionally, the α4 mRNA subunit was the lowest expressed of the subunits detected. The α4 subunit is one of the most pharmacologically targeted nAChR subunits and is found throughout the rest of the brain at much higher levels than the α3 mRNA subunit. Upon PCR analysis two subpopulations of the α3 and β2 subunits emerged: those that contained 3X more α3 than β2 and those that contained 3X more β2 than α3. Therefore, we hypothesized that two likely α3β2 nAChR stoichiometries are present in hippocampal interneurons. We differentiated their kinetic properties using electrophysiology.Additionally, like the α4 subunit, the α7 subunit is highly targeted in cognitive therapeutics. Since, the α7 subunit is the most characterized nAChR subunit, there are current efforts to develop allosteric modulators of the α7 subunit. The α7 subunit is found at moderate levels within hippocampal interneurons and remains a valid target. Current treatment options for Alzheimer's disease, and other dementias are limited and only mildly effective. Therefore, we sought to characterize the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) on α7.Furthermore, there are no current methods to distinguish the α7 from the α7β2 nAChRs during whole cell electrophysiological recordings. Therefore, we also characterized the PAM-2 effect on α7β2 nAChRs. Our results highlight at least 2 ways PAM-2 can be used to differentiate α7 from the α7β2 during whole-cell recordings.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc) underlies the rewarding properties of ethanol (EtOH) and nicotine (NIC). Although the dogma is that EtOH enhancement of DA neural activity contributes to enhancement of DA transmission, DA neurons are not sensitive to rewarding levels of EtOH. However, VTA GABA neurons are sensitive to low-dose EtOH. We have shown previously that EtOH modulation of DA release in the NAc is mediated by α6-containing nicotinic receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and α6*-nAChRs may be a molecular target for low-dose EtOH. Thus, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on VTA GABAergic input to CINs and DA release in the NAc. Using DIO channel rhodopsin-2 (ChR2) viral injections into the VTA of VGAT Cre mice, we found that VTA GABA neurons send an inhibitory projection to CINs, replicating what has been demonstrated by others. This study investigated the acute and chronic effects of EtOH at this synapse. We demonstrate that EtOH markedly enhances CIN firing rate and that these effects are blocked by the α6-conotoxin MII (α-Ctx MII), knockout of accumbal α6*-nAChRs with α6-shRNA, and atypical GABA receptor antagonists. This study also investigated plasticity at this synapse. We demonstrate that a low frequency stimulation (LFS; 1 Hz, 240 pulses) causes inhibitory long-term depression at this synapse (CIN-iLTD) which is also blocked by α-Ctx MII, α6-shRNA, and atypical GABA receptor antagonists. We also show that CIN-iLTD is blocked in EtOH-dependent mice. Taken together, these findings suggest that EtOH affects the VTA GABAergic projection to CINs via α6*-nAChRs and that atypical GABA receptors also play a role.

The CHRNA5/A3/B4 genomic cluster encodes for the alpha5, alpha3 and beta4 subunits of the nicotinic acetylcholine receptors (nAChRs). Human genetic studies have revealed a significant association of variants in this genomic region with nicotine dependence. However, the mechanisms through which overexpression of these three subunits may influence smoking-related behaviours is not understood. To gain insight in the possible mechanisms, we used a BAC transgenic mouse model overexpressing this cluster containing the three genes together with their transcriptional regulatory elements. We found that overexpression of the cluster: i) increases sensitivity to the pharmacological effects of nicotine; ii) modifies particular cognitive domains associated to drug addiction and hippocampal neuronal complexity and synaptic plasticity; and iii) shifts the rewarding and aversive properties of nicotine and the manifestation of nicotine-withdrawal syndrome. Our study suggests that the genomic cluster CHRNA5/A3/B4 contributes to genetic vulnerability to nicotine addiction and promotes smoking-related behaviours possibly through hippocampal plasticity changes.<br>El cluster genòmic CHRNA5/A3/B4 codifica per les subunitats alfa5, alfa3 i beta4 dels receptors d’acetilcolina (nAChRs). Estudis de genètica humana han revelat que variants en aquesta regió genòmica estan significativament associats a la dependencia a nicotina. Malauradament, els mecanismes pels quals la sobreexpressió d’aquestes tres subunitats influencia comportaments relacionats amb el consum de tabac no són del tot coneguts. Per tal d’entendre els possibles mecanismes, hem utilitzat un model de ratolí transgènic que sobreexpressa aquest cluster amb els tres gens i les seus elements de regulació transcripcional. Hem trobat que la sobreexpressió del cluster: i) incrementa la sensibilitat als efectes farmacològics de la nicotina; ii) modifica determinats dominis cognitius associats a l’addicció a droges i la complexitat neuronal i plasticitat sinàpica de l’hipocamp; a més a més iii) canvia les propietats de recompensa i aversió de la nicotina i la manifestació del síndrome d’abstinència. El nostre estudi suggereix que el cluster genòmic CHRNA5/A3/B4 contribueix a la vulnerabilitat genètica a l’adicció a la nicotina i promou comportaments relacionats amb el consum de tabac possiblement a través de canvis de plasticitiat a l’hipocamp.

Au cours de ce travail, nous avons développé des voies de synthèses convergentes et énantiosélectives en vue de préparer des analogues pyrrolidiniques des alcaloïdes de Lobelia comme nouveaux ligands des récepteurs nicotiniques à l’acétylcholine. Deux familles de ligands ont été réalisées par des méthodes d’élongation mono- ou bi-directionnelle basées respectivement sur des stratégies de désymétrisation précoce ou tardive au départ du succinaldéhyde.La première partie de ce manuscrit aborde la conception d’hybrides de lobéline, nicotine et d’agonistes naturels. Ces structures originales ont été obtenues diastéréosélectivement grâce à un intermédiaire commun issu d’une élongation monodirectionnelle du succinaldéhyde. Cette voie exploitera la chimie des iminiums masqués. La mise au point de cette synthèse s’est enrichie par la découverte et la valorisation d’une nouvelle famille de ligands chimériques.La seconde partie étudie la voie d’élongation bidirectionnelle basée sur des réactions de double aza-Michael suivies de la réduction désymétrisante tardive de pyrrolidines 2,5-phénacyl méso et pseudo-méso. Cette stratégie asymétrique s’inscrit dans une démarche d’économie d’atomes et d’étapes. La perspective majeure de ce travail est l’évaluation par électrophysiologie sur différents sous-types de récepteurs à l’acétylcholine d’une sélection de ligands hybrides.Les études de RSA menées sur ces familles de composés de haute homologie structurale permettront in fine d’améliorer les modèles prédictifs décrivant les transitions allostériques des récepteurs nicotiniques à l’acétylcholine<br>During this PhD work, convergent and diastereoselective routes for the preparation of pyrrolidine Lobelia alkaloid analogues have been developed as novel neuronal nicotinic receptor ligands. Two families of ligands have been synthesized by a strategy of mono- or bi-directional elongation of the succinaldehyde including early or late desymmetrization process respectively.The first part of this manuscript is dedicated to the preparation of hybrids of lobeline, nicotine and other natural agonists. These original structures have been diastereoselectively obtained thanks to a common intermediate resulting of the mono-elongation of succinaldehyde. This synthetic pathway uses the chemistry of masked iminium. The development of this strategy has been enriched by the discovery and the valorisation of a new chimeric ligand family.The second part studies the “bidirectional” elongation route, based on a ring-closing double aza-Michael reaction followed by the desymmetrizing reduction of meso and pseudo-meso 2,5-diphenacyl pyrrolidines. This asymmetric strategy constitutes a step- and atom-economical approach. The major perspective of this work is the biological evaluation of selected ligands by electrophysiology made on different nAChR subtypes.The SAR studies realized on these structurally homologue ligand families could allow the improvement of the predictive molecular models describing the allosteric conformations of the nAChRs

Au cours de ce travail, nous nous sommes intéressés à développer des voies de synthèse convergentes et diastéréosélectives en vue de préparer des analogues pyrrolidiniques des alcaloïdes de Lobelia comme nouveaux ligands des récepteurs nicotiniques centraux à l'acétylcholine. Ainsi, nous avons mis au point une méthode " bidirectionnelle " basée sur des réactions de double aza-Michael et donnant accès à des pyrrolidines 2,5-disubstituées. Une étude de réactivité a également été mené afin d'améliorer la chimiosélectivité des différents processus réactionnels impliquant des réactions d'aza-Michael dans des séquences de cyclisation tandem. Dans un second temps, nous avons décrit une voie dite " d'élongation monodirectionnelle " permettant d'accéder à des 2,5-trans-pyrrolidines énantiopures. Enfin, la dernière partie de ce manuscrit aborde une étude prospective de réductions désymétrisantes pour la synthèse d'homologues pyrrolidiniques de la lobéline.

碩士<br>國立臺東大學<br>兒童文學研究所<br>92<br>Japan has been called the “Kingdom of Comics”. The Japanese even think that comics have been one of the four national treasures. According to the quantity of output and sales volume, it is not exaggerating to call comics “the national treasure”. The commercial effects related to the comics may have contributed to create the miracle of Japanese economy. We can say that Japanese economy could be recovered so quickly after the Second World War in part because of comics. I rarely read the comics. While taking Nachi Misako’s works as my thesis, I started to read the comics. After taking a glance at all kinds of comics, I realized how greatness the comic world is. Japanese comics have been classified into numerous types. The comics they have not only for teenagers, but also for children and adults. The contents of the comics are very diversified, including histories, science, sports and people’s lives, even office events, that are connected to the real world. Nachi Misako first appeared in 1984 with the short story of the comic work ‘The Legend of the Sheep Valley’, and by the work ‘The Gold of Echigoya’, she has won the best award of Japanese Cartoonist Associate in 1997. The figures of Nachi’s work are pithy and brisk, and the stories are warm and touching, filled with peace and silence. This dissertation is mainly written under the assumption of the combination between comics and folk tales through Nachi Misako’s comics, and it also brings up the idea of identifying comics. Chapter One is an introduction. It describes the motive, the issues, the purpose, the process and the range of this thesis, and also the problems that may be encountered. Chapter Two generalizes the development of comic books in Japan, and it also introduces the operation of comic books in Japanese publishing circles and all kinds of Japanese comics. Chapter Three contains the introduction of the cartoonist, Nachi Misako and the works she created. Through Nachi’s pictures and the plots, I tried to analyze the styles and the characteristics of Nachi’s works. Chapter Four focuses on the themes presented in the works, including the death and the existence, the relationship between people and nature, the variations, the subversiveness of classical fairy tales, the ancient legends, etc.. Through these themes of Nachi’s comics, I have been trying to find out the origins of Japanese folk tales. Chapter Five is a conclusion. Actually, good comics would be the same as other literatures. They should be read and admired delicately. Reading comic books is like reading popular fictions. Though the language of comics is not as glorious as literature, the cartoonists who plunged themselves to the creation of comics also tried their best to devise the characters and backgrounds, making up appealing plots. In the visual world of today, written language has been replaced by multimedia. We should see comics with more spacious insight and horizons, and the elegant illustrations and innovative comics should also be encouraged and supported.

博士<br>慈濟大學<br>醫學科學研究所<br>100<br>Background and purpose: The α7-nicotinic acetylcholine receptor (α7-nAChR) on sympathetic neurons innervating basilar arteries of pigs crossed breed between Landrace and Yorkshire (LY) is known to mediate nicotine-induced, β-amyloid (Aβ)-sensitive nitrergic neurogenic vasodilation. Preliminary studies, however, demonstrated that nicotine-induced cerebral vasodilation in pigs crossbreed among Landrace, Yorkshire and Duroc (LYD) was insensitive to Aβ and α-bungarotoxin (α-BGTX). We investigated nAChR subtype on sympathetic neurons innervating LYD basilar arteries. Experimental approach: Nicotine-induced relaxation of isolated porcine basilar arteries was examined by tissue bath myography, inward currents on nAChR-expressing oocytes by two-electrode voltage recording, and mRNA and protein expression in the superior cervical ganglion (SCG), and middle cervical ganglion (MCG) by reverse transcription PCR and western blotting. Key results: Nicotine-induced basilar arterial relaxation was not affected by Aβ, α-BGTX and α-conotoxin IMI (α7-nAChR antagonists), or α-conotoxin AuIB (α3β4-nAChR antagonist), but was inhibited by tropinone and tropane (α3-containing nAChR antagonists), and α-conotoxin MII (selective α3β2-nAChR antagonist). Nicotine-induced inward currents in α3β2-nAChR-expressing oocytes were inhibited by α-conotoxin MII but not by α-BGTX, Aβ, or α-conotoxin AuIB. mRNAs of α3-, α7-, β2- and β4-subunits were expressed in both SCGs and MCGs. The mRNAs of α3-, β2- and β4-subunits were significantly higher than those of α7-subunit. Conclusions and implications: The Aβ-insensitive sympathetic α3β2-nAChR mediates nicotine-induced cerebral nitrergic neurogenic vasodilation in LYD pigs. The different finding from Aβ-sensitive α7-nAChR in basilar arteries of LY Pigs may offer a partial explanation for different sensitivities of individuals to Aβ in causing diminished cerebral nitrergic vasodilation in diseases involving Aβ.

abstract: A Japanese national identity is generally thought to have originated in the 17th century, with the advent of the Kokugaku movement. I will argue that there is earlier evidence for the existence of a Japanese national identity in the Kumano Nachi mandalas of the Kamakura and Muromachi periods. These mandalas employ the Nachi waterfall as a symbol of the strength and power of the Japanese land, counterbalancing Chinese Buddhist visual motifs. In this paper, I further assert that these mandalas are an early example of an artistic tradition of painting specific landscape features as symbols of a Japanese national identity, and that this tradition continues into the modern period.<br>Dissertation/Thesis<br>Masters Thesis Art History 2017

Nicotine, the major psychoactive ingredient of tobacco smoke, underlies numerous effects by activating neuronal nicotinic acetylcholine receptors. Both in vitro and in vivo studies suggest that nicotine is neuroprotective and improves cognitive performance. Epidemiology studies show that smoking is negatively correlated with the incidence of Parkinson's disease and Alzheimer's disease. Postmortem research and neuroimaging studies show that loss of nicotinic binding sites in the brain is the major feature of neurodegenerative diseases related to dementia and cognitive impairment. Caloric restriction, a regimen that extends the lifespan in all mammalian species studied so far including rodents and primates, is a highly regulated response to food deprivation. It is believed that the longevity effect of caloric restriction is mediated by SIRT1, a NAD-dependent deacetylase, and its related genes. Nicotine's effect on body weight could also lead to weight loss by decreasing caloric absorption consumption. The goal of this study was to find the possible correlation between nicotine's effects and the activation of SIRT1 and its related genes. Using beta2-/- mice that lack high affinity beta2 nicotinic acetylcholine receptors (nAChRs), we first demonstrated that beta2* nAChRs do not directly regulate expression of survival genes. However, we found that loss of beta2* nAChRs could result in augmented cellular stress, which indirectly increased expression of SIRT1, Nampt, and Ku70, possibly as an adaptive response to provide protection against neurodegeneration. We also found that loss of endogenous activation of beta2* nAChRs had less effect on synaptic connections but strongly impaired survival of hippocampal GABAergic neurons. To activate beta2* nAChRs in normal mice, we administered nicotine through drinking water. In a short-term exposure study, we determined the dose of nicotine to be used in young adult mice, and found that chronic nicotine treatment was anxiolytic, decreased caloric consumption, increased nAChR binding sites, and most importantly, increased expression of SIRT1 and its related genes. Finally, we compared long-term nicotine treatment with caloric restriction in middle-aged mice to examine their effects to brain aging, and our results indicated that in mice long term caloric restriction and nicotine treatment both tend to improve memory in aging mice, but appear to act through different mechanisms.

Οι α7 και α9 υπομονάδες των νικοτινικών υποδοχέων ακετυλοχολίνης (nAChRs) είναι οι μόνες, ανάμεσα σε μια μεγάλη ποικιλία υπομονάδων του ανθρώπινου nAChR, που σχηματίζουν ομοπενταμερείς υποδοχείς. Για την θεραπεία διαφόρων νευρολογικών διαταραχών αλλά και άλλων ασθενειών, όπου εμπλέκονται οι α7 και α9 nAChRs, απαιτούνται φαρμακευτικές ουσίες που θα στοχεύουν ειδικά σε έναν υπότυπο των nAChRs. Για τον σχεδιασμό τέτοιων φαρμάκων είναι ουσιώδης η διαλεύκανση σε ατομικό επίπεδο της δομής του nAChR. Εντούτοις, η κρυστάλλωση ολόκληρων των διαμεμβρανικών υποδοχέων, αλλά ακόμη και η έκφραση και απομόνωσή τους, σε βαθμό που να επιτρέπει δομικές μελέτες, έχει αποδειχθεί δύσκολος στόχος. Η δυσκολία έγκειται κυρίως στην παρουσία υδρόφοβων διαμεμβρανικών περιοχών και στην μεγάλη ενδοκυττάρια περιοχή που θεωρείται ευκίνητη και ότι δεν έχει σταθερή διαμόρφωση. Στο πλαίσιο αυτό, στοχεύσαμε στην παραγωγή τμημάτων των α7 και α9 υπομονάδων του nAChR, που να είναι κατάλληλα για αναλυτικές δομικές μελέτες, σχεδιάζοντας κατασκευές για την έκφραση των εξωκυτταρικών περιοχών των δύο υπομονάδων ή και κολοβών διαμεμβρανικών μορφών της α7 υπομονάδας. Σε αυτές έχουν απαλειφθεί είτε τμήματα της μεγάλης ενδοκυτταρικής περιοχής, είτε ολόκληρη αυτή η περιοχή και μεγάλα τμήματα της διαμεμβρανικής περιοχής. Στο παρελθόν, είχε εκφραστεί η α7-ΕΚΠ στο ετερόλογο σύστημα έκφρασης Pichia pastoris και είχε οδηγήσει σε συσσωματώματα μεγάλου μοριακού βάρους, ενώ η έκφραση ενός μεταλλάγματος της α7-ΕΚΠ έδειξε σημαντική βελτίωση της υδροφιλικότητας του μορίου και σχηματισμό ολιγομερών κυρίως πενταμερών μορίων (Zouridakis et al. 2009). Σε αυτήν την εργασία, έχουμε επιτύχει να απομονώσουμε τα σχηματιζόμενα πενταμερή μόρια αυτού του μεταλλάγματος, εκμεταλλευόμενοι την ιδιότητα τους να εκλούονται σε μεγάλο εύρος συγκέντρωσης ιμιδαζολίου, κατά την χρωματογραφία συγγένειας. Ακόμη, η ενζυμική απογλυκοζυλίωση του μεταλλάγματος αυτού, βοήθησε στην περαιτέρω μείωση της ετερογένειας των απομονωμένων πενταμερών μορίων. Αν και ο αρχικός έλεγχος συνθηκών κρυστάλλωσης των γλυκοζυλιωμένων πενταμερών μορίων οδήγησε στον σχηματισμό μικροκρυστάλλων, δεν στάθηκε δυνατή η βελτιστοποίηση της ανάπτυξής τους. Η έκφραση της αγρίου τύπου εξωκυτταρικής περιοχής της α9 υπομονάδας του ανθρώπινου nAChR (α9wt) στο ετερόλογο σύστημα έκφρασης P. pastoris, οδήγησε στην παραγωγή κυρίως μονομερών μορίων που διαχωρίζονται εύκολα από τα σχηματιζόμενα ολιγομερή. Η μονομερής μορφή της α9wt έδειξε αξιοσημείωτη διαλυτότητα, σταθερότητα και ομοιογένεια καθώς και ικανότητα πρόσδεσης της α-μπουγκαροτοξίνης, έναν ειδικό ανταγωνιστή του μυικού και των ομοπενταμερών νευρικών nAChRs. Προκειμένου να υποβοηθηθεί η συναρμολόγηση των εκφραζόμενων α7 και α9 ΕΚΠ προς τον σχηματισμό πενταμερών μορίων, σχεδιάσαμε μεταλλάξεις που στηρίχθηκαν σε τρισδιάστατα μοντέλα ομολογίας (3D homology modelling) αυτών, χρησιμοποιώντας ως πρότυπο την κρυσταλλική δομή της ομόλογης, διαλυτής πρωτεΐνης δεσμεύσεως της ακετυλοχολίνης (AChBP) από το μαλάκιο Lymnaea stagnalis. Οι μεταλλαγές αυτές έγιναν είτε σε υδρόφοβα επιφανειακά αμινοξικά κατάλοιπα, με στόχο να αυξήσουμε την υδροφιλικότητα του μορίου, είτε σε κατάλοιπα που εντοπίζονται στις διεπιφάνειες μεταξύ δύο πρωτομερών, ώστε να ενισχύσουμε τις διαμοριακές αλληλεπιδράσεις και να υποβοηθήσουμε την συναρμολόγηση τους προς πενταμερή μόρια. Τα προκύπτοντα μεταλλάγματα έχουν ταύτιση αμινοξικής αλληλουχίας 70-95% με την αντίστοιχη του αγρίου τύπου και σε ορισμένες περιπτώσεις η έκφρασή τους στην P. pastoris οδήγησε στον σχηματισμό αλλά και την απομόνωση πενταμερών μορίων. Η σάρωση των μεταλλαγμάτων απέτυχε στην ανεύρεση κάποιας συνθήκης κρυστάλλωσής τους. Ωστόσο, ο αρχικός έλεγχος συνθηκών κρυστάλλωσης των μονομερών μορίων της α9wt είχε ως αποτέλεσμα τον προσδιορισμό διαφορετικών συνθηκών όπου σχηματίζονται πολλαπλοί κρύσταλλοι, τόσο για την γλυκοζυλιωμένη, όσο και την απογλυκοζυλιωμένη μορφή της. Επιπλέον, η βελτιστοποίηση αυτών των κρυστάλλων στην περίπτωση της γλυκοπρωτεΐνης, οδήγησε στο σχηματισμό μονοκρυστάλλων, που περιθλούν ακτίνες-Χ σε ικανοποιητική ανάλυση, καθιστώντας αυτούς τους κρυστάλλους ως υποσχόμενο υλικό για την επίλυση της δομής της άγριου τύπου α9 ΕΚΠ. Τέλος, η έκφραση στην κυτταρική σειρά εντόμων Sf9 με το σύστημα των βακιλοϊών της ολόκληρης α7 υπομονάδας του nAChR και των «κολοβών» διαμεμβρανικών μορφών της οδήγησε στην ορθή στόχευση των σχηματιζόμενων υποδοχέων στην κυτταροπλασματική μεμβράνη, ενώ οι φαρμακολογικές τους ιδιότητες προσεγγίζουν αυτές του φυσικού α7 υποδοχέα. Παρά το γεγονός ότι η έκφραση των κατασκευών αυτών είχε χαμηλή απόδοση και παρά τις δυσκολίες διαλυτοποίησης και απομόνωσής τους, ανάλυση με χρωματογραφία μοριακού αποκλεισμού, για τουλάχιστον δύο από τα διαμεμβρανικά μεταλλάγματα, δείχνει ότι οι διαλυτοποιημένες πρωτεΐνες έχουν σχηματίσει έναν πληθυσμό ολιγομερών της πρωτεΐνης ο οποίος πιθανότατα αντιστοιχεί σε πενταμερή μόρια. Τα παραπάνω, σε συνδυασμό με την απουσία της εύκαμπτης ενδοκυττάριας περιοχής, καθιστούν αυτά τα α7 μεταλλάγματα, εφόσον ξεπεραστούν οι δυσκολίες της απόδοσης της έκφρασης και της απομόνωσης τους, κατάλληλα για λειτουργικές και δομικές μελέτες.<br>The neuronal α7 and α9 subunits of the nicotinic acetylcholine receptor (nAChR) are the only amongst the known human nAChR subunits to form homopentamers, with five cholinergic ligand-binding sites. Elucidation of their crystal structure is essential in order to design highly specific drugs for treatment of several neurological diseases and disorders related to them and will serve as the prototype for understanding the structure of all other members of the ligandgated ion channel superfamily. Crystallisation of the intact receptors is a difficult task to fulfil, partially due to their hydrophobic transmembrane regions. Therefore, we aim at the expression of crystallisable human α7 and α9 extracellular domains (ECDs) or truncated α7 forms lacking either only their large and probably unordered intracellular domain or large parts of its transmembrane domain. Regarding the α7 ECD, expression of the wild type form in yeast Pichia pastoris led into formation of aggregates (Avramopoulou et al. 2004). Yet, a previously described mutant of this ECD (α7m10, Zouridakis et al. 2009) succeeded in the formation of oligomers, mostly corresponding to pentamers, due to improved solubility and subunit assembly of this mutant. In this study, we managed to isolate apparently pentameric assemblies of the various expressed oligomeric states, by optimizing its first-step purification procedure (metal affinity chromatography), using a narrow stepwise increase of imidazole concentrations. In order to further improve the protein homogeneity, we proceeded to the isolation of its deglycosylated pentameric form. The relatively low polydispersity of both the glycosylated and deglycosylated α7m10 ECDs, allowed for crystallization trials, which have resulted in microcrystallic formations. Further optimization of these microcrystals failed. As to the α9 ECD, expression of the wild type form in yeast Pichia pastoris led to the formation of both monomers and a variety of oligomers. The monomeric α9 ECD showed significant monodispersity, solubility and stability and exhibited binding ability of α- bungarotoxin, a specific nAChR antagonist. In order to facilitate the pentameric assembly and enhance the solubility of these α7 and α9 ECDs, we designed several mutants based on generated 3D homology models, using as template the crystal structure of the homologous soluble molluscan acetylcholine binding protein (AChBP). Several solvent accessible hydrophobic residues were replaced with more hydrophilic ones and some interface-located residues were mutated so as to facilitate the formation of additional inter-subunit interactions. The resulting mutants shared moderate and considerably high sequence identities (70-95%) with the wild type ECDs and in some cases, formation of pentamers was accomplished. Crystallisation screening for mutant ECDs failed in producing any hit. However, the pilot crystallisation trials of monomeric wild-type α9 ECD resulted the formation of plate-like multi crystals for both its glycosylated and deglycosylated forms. Further optimisation of these crystals succeeded in producing single crystals of the glycoprotein, to produce single crystals, which diffracted X-rays to satisfactory resolution, in a home source X-ray generator. Therefore, these crystals seem to be a promising material for solving the wild type α9 ECD structure. The intact and truncated α7 nAChRs under study were expressed in the Sf9/baculovirus system and showed surface receptor expression, while presenting near-native ligand-binding affinities for characteristic nAChR agonists and antagonists. Despite the low expression yield and solubilisation and purification difficulties, gel filtration analysis for at least two truncated mutants revealed the presence of a monodispersed oligomeric population, probably corresponding to pentamers. All these, taken together with the lack of the flexible large intracellular domain, render these α7 mutants, after overcoming the expression yield and purification difficulties, a suitable material for performing both functional and structural studies.

abstract: Tobacco and alcohol are the most commonly abused drugs worldwide. Many people smoke and drink together, but the mechanisms of this nicotine (NIC) -ethanol (EtOH) dependence are not fully known. EtOH has been shown to affect some nicotinic acetylcholine receptors (nAChRs), which potentially underlies NIC-EtOH codependence. Ventral Tegmental Area (VTA) dopamine (DA) and &#947;-aminobutyric acid (GABA) neurons express different nAChR subtypes, whose net activation results in enhancement of DA release in the Prefrontal Cortex (PFC) and Nucleus Accumbens (NAc). Enhancement of DA transmission in this mesocorticolimbic system is thought to lead to rewarding properties of EtOH and NIC, clarification of which is relevant to public health and clinical diseases. The aim of this study was to elucidate pharmacological mechanisms of action employed by both NIC and EtOH through nAChRs in VTA neurons by evaluating behavioral, network, synaptic and receptor functions therein. It was hypothesized that VTA GABA neurons are controlled by &#945;7 nAChRs on presynaptic GLUergic terminals and &#945;6 nAChRs on presynaptic GABAergic terminals. NIC and EtOH, via these nAChRs, modulate VTA GABA neuronal function. This modulation may underlie NIC and EtOH reward and reinforcement, while pharmacological manipulation of these nAChRs may be a therapeutic strategy to treat NIC or EtOH dependence. This data demonstrates that in VTA GABA neurons, &#945;7 nAChRs on GLUergic terminals play a key role in the mediation of local NIC-induced firing increase. &#945;6*-nAChRs on GABA terminals enhances presynaptic GABA release, and leads to greater inhibition to VTA GABA neurons, which results in an increase VTA DA neuron firing via a disinhibition mechanism. Genetic knockout of these nAChRs significantly prevents EtOH-induced animal conditioned place preference (CPP). Furthermore, levo-tetrahydropalmadine (l-THP), a compound purified from natural Chinese herbs, blocks nAChRs, prevents NIC-induced DA neuronal firing, and eliminates NIC CPP, suggesting it as a promising candidate in a new generation of interventions for smoking cessation. Improved understanding of underlying mechanisms and development of new drugs will increase the number of successful quitters each year and dramatically improve the quality of life for millions suffering from addiction, as well as those around them.<br>Dissertation/Thesis<br>Doctoral Dissertation Neuroscience 2015

Diabetes, which is characterized by elevated plasma glucose, can have a devastating effect on peripheral nerves frequently leading to the clinical symptoms of neuropathy. Diabetic autonomic neuropathy (DAN) results from damage to autonomic nerves, and the most troubling forms of DAN often lead to cardiovascular abnormalities and premature death. Despite the prevalence of DAN and the impact to quality and life expectancy, the precise mechanisms underlying these pathologies are poorly understood. Recently, a new model for the onset of DAN was proposed where hyperglycemia-induced oxidative stress inactivates nicotinic acetylcholine receptors (nAChRs), the main receptor driving autonomic synaptic transmission at sympathetic ganglia. This inactivation leads to the depression of synaptic transmission, and consequently triggers the onset of autonomic neuropathy in diabetic mice. However, the source and pathways contributing to the elevation of reactive oxygen species (ROS) and oxidative stress remained unclear. In recent years it has been shown that the accelerated formation of advanced glycation end products (AGEs) and activation of their receptor (RAGE) in diabetes play a major role in the induction of oxidative stress in sensory nerve damage. Thus we hypothesized that the activation and up-regulation of RAGE during high glucose conditions is a major source of ROS production in sympathetic neurons leading to the inactivation of nAChRs and autonomic malfunction. In this thesis we show for the first time that RAGE is expressed in cultured sympathetic neurons and is also up-regulated during high glucose conditions. Our results further demonstrate that direct RAGE activation by its natural ligands leads to an increase in cytoplasmic ROS which in turn induces the inactivation of nAChRs in sympathetic neurons. We also report that high glucose-induced ROS generation and subsequent inactivation of nAChRs is prevented in sympathetic neurons from RAGE knock-out mice. The results of this dissertation suggest RAGE to be a pivotal source of ROS production leading to the functional deficits observed in sympathetic neurons during high glucose conditions.

Afin de mieux comprendre le régime postsoviétique russe et ses rapports avec la société civile, en particulier avec la jeunesse, ce mémoire propose d'étudier le mouvement de jeunes pro-Kremlin Nachi (« Les nôtres »), mis sur pied au printemps 2005 par l'administration présidentielle russe (le Kremlin). L'intérêt de se pencher sur Nachi réside dans le fait que ce mouvement représente un excellent exemple des politiques mises en place sous le régime de Vladimir Poutine (2000-2008), qualifié par plusieurs analystes de démocratie dirigée. En effet, les nombreuses mesures de contrôle instaurées au cours de sa présidence, visant à bloquer l'apparition d'une quelconque opposition pouvant se développer au sein de la société, ont grandement confirmé le caractère dirigé des libertés laissées à la population. Mais plus encore, le Kremlin s'est employé à élaborer des structures dépendantes de son pouvoir pour étouffer dans l'oeuf l'apparition de mouvements sociaux indépendants. Ces progénitures du régime russe, telles que Nachi, servent aussi plus subtilement à tracer une ligne de démarcation claire entre ce qui doit être vu, par la nation russe, comme la société civile légitime versus la société civile illégitime. Cette étude de Nachi sert donc à éclairer les intentions, les moyens, le contenu idéologico-politique, le degré de succès et les limites de ces politiques implantées par le haut, et par le fait même, tente de mettre en lumière les réactions des groupes sociaux visés, soit dans le cadre de ce travail, la jeunesse. Après tous les bouleversements survenus à la suite de l'effondrement de l'URSS et la disparition soudaine du Komsomol (l'aile jeunesse du Parti communiste de l'Union soviétique), les rapports entre l'État et la jeunesse ont inévitablement dû se redéfinir. Ainsi, bien que les raisons de la création de Nachi ne semblent correspondre qu'au désir du pouvoir de renforcer son contrôle sur la société, le régime justifie sa création avec des arguments étatistes, prétextant vouloir reprendre son rôle de mentor auprès de la jeunesse, un rôle qu'il avait délaissé pendant la période eltsinienne. D'une façon ou d'une autre, il faut comprendre que les effets et les conséquences des actions quotidiennes du mouvement sur les activistes et sur la société peuvent parfois ne pas correspondre à ce qu'avaient imaginé ses auteurs. La complexité structurelle et l'évolution de cette organisation doivent également être prises en compte dans l'analyse. Il importe donc de mieux cerner les motivations de ces jeunes activistes, afortiori lorsqu'une sérieuse remise en question a rendu l'avenir du mouvement bien incertain après les élections 2007-2008. Ce mémoire essaie ainsi de mieux comprendre, dans le contexte postsoviétique; (a) comment l'État russe cherche à construire ses rapports avec la société, en particulier avec la jeunesse; (b) l'idéologie sur laquelle il cherche à appuyer sa légitimité; (c) les attitudes des jeunes face à ces efforts du régime, au régime lui-même, et à son idéologie.

Nicotine addiction is a complex behavior linked to the alteration of nicotinic receptor (nAChR) expression within the brain caused by chronic nicotine. A crucial factor when elucidating an accurate picture of the underlying causes of addiction is the route of administration. Oral self-administration of nicotine is a non-invasive method of drug administration, able to mimic the episodic nature of nicotine consumption seen in human smokers and also provide a choice – the key feature of an accurate addiction model. Mice with YFP-tagged α4 nicotinic receptors (α4YFP) were pretreated with chronic nicotine via osmotic pumps in order to maximally upregulate their nAChRs before being subjected to a two bottle-choice assay of nicotine self-administration. The paradigm consists of periods of choice interspersed with periods of nicotine abstinence to affect withdrawal and perpetuate nicotine self-selection. Spectral confocal microscopy of the endogenous α4YFP was used to investigate the expression levels of nAChRs following chronic nicotine priming with osmotic pumps. Imaging confirms that mice exposed to chronic nicotine prior to entering the self-administration paradigm have upregulated α4-containing (α4*) nAChRs in the medial perforant path of the hippocampus, on GABAergic somata of the ventral tegmental area and on GABAergic and glutamatergic somata of the medial prefrontal cortex, areas implicated in mediating addictive behavior. Compared to control mice with basal levels of nicotinic receptors, nicotine-primed mice ingest a larger daily dose of nicotine and allocate a greater percentage of their daily fluid intake to their nicotine-containing bottle. They also show signs of withdrawal, observed as post-abstinence binging. The control mice show no withdrawal, but progress towards dependence by adjusting the percentage drank from their nicotine bottle in order to maintain a constant daily dose. Conversely, nicotine primed mice decrease their daily dose of nicotine, suggesting that maximal receptor upregulation caused by osmotic pumps is outside the physiologically relevant level that can be obtained by nicotine self-administration in mice. Taken together, these results show that our model is sufficient to yield addictive behavior in mice and also implicates nAChR upregulation as a key factor influencing nicotine self-administration.<br>Graduate<br>0306<br>0379<br>0317<br>tony.j.renda@gmail.com

--<br>The nicotinic acetylcholine receptors (nAChRs) are transmembrane proteins, composed of five subunits and belong to the superfamily of ligand gated ion channels The nAChRs are distinguished according to their topological and pharmacological characteristics in muscle and nervous type. Both the muscle and the nervous type are involved in the execution of many physiological functions (eg, nerve impulse transmission) but respectively in the pathogenesis of many diseases (eg Myasthenia Gravis,Parkinson's,Alzheimer's).This makes imperative the need to design drugs that target specific to each type of receptor. A prerequisite for achieving this objective is to study the structure of the extracellular regions of the receptor, as it is known that the specific areas are recognised by the cholinergic ligands and the abnormal antibodies. The α7 subunit of the human nicotinic acetylcholine receptor, can be used as a model for this study as It is expressed as a homopentamer. Wanting therefore to avoid the large and hydrophobic transmembrane regions of the receptor that would hinder the achievement of the objective, we focused on the extracellular domain (ECD) of the receptor .So, according to the above, a recombinant form of the extracellular region of the receptor was constructed and expressed previously in our laboratory (Zouridakis et al., 2009). The recombinant protein was (α7-mut10-myc-His), expressed in soluble form, in sufficient concentration and showed about three times greater affinity for I125-a-bgtx compared to the wild type (α7-ΔCDwt). Furthermore, studies of dynamic light scattering and electron microscopy confirmed the formation of homopentamer molecules. Moreover, the deglycosylated form of the protein displayed all these enhanced features, allowing the entry of crystallization experiments with both the glycosylated and the deglycosylated form. In order to further improve the specific mutant, new recombinant forms of the extracellular region of the α7 subunit of the nAChR were constructed. The recombinant forms were expressed with different expression tags in their N-or C-terminal in order to improve the folding of the molecule. The FLAG-α7-mut10-myc-His was produced in greater quantity and Ηts deglycosylated form differs significantly, indicating probably a more homogeneous protein population. Also, analysis of the molecule bygel filtration showed the predominant formation of a homopentamer molecule and the absence of high molecular weight aggregates. This protein, has enhanced features compared to the α7-mut10-myc-His and thus can proceed to crystallization trials. The second part of the study refers to the construction concateremers of the α7ECD. Σwo peptide linkers varying in their length were used. The mutant which carried the smaller linker (AGS)8, showed greater solubility compared to the more extended one (AGS)11.

Nicotinic acetylcholine receptors (nAChRs) are a complex class of ligand gated ion channels consisting of multiple subtypes with different stoichiometries. Of these, the two most commonly found in the brain are the α7 and α4β2 nAChRs. The binding site of agonists and competitive antagonists at nAChRs is well established. However, the binding site of many allosteric modulators remains unknown. Additionally, despite their importance in brain function, the role of specific subtypes and stoichiometries is largely unknown. This thesis deals with the synthesis of alkaloid derivatives that can be used to establish the binding sites of allosteric modulators and study the role of specific subtypes and stoichiometries in the brain. Such information can be used to develop better drugs and drug targets for the treatment of neurological diseases. Galanthamine and codeine are reported to be positive allosteric modulators at nAChRs. In order to establish their binding site, thiol reactive analogues of galanthamine, codeine and the structurally similar alkaloid, morphine, were synthesised for use as probes in covalent trapping experiments. The α,β-unsaturated ketone derivatives of each alkaloid; narwedine, codeinone and morphinone were synthesised along with the codeine mustard and a protected derivative of the benzyl chloride analogue of codeine. While the chlorinated derivatives were too unstable for use as probes, the α,β-unsaturated ketone derivatives were stable in aqueous solution and their reactivity towards thiols was assessed by monitoring their reaction with a cysteine derivative. All of the α,β-unsaturated ketone derivatives displayed sufficient reactivity for use as thiol reactive probes. Methyllycaconitine (MLA) is an antagonist at nAChRs that is known to bind at the α7–α7 interface of α7 nAChRs and at the α4–α4 and α4–β2 interfaces of α4β2 nAChRs. Small bicyclic ester analogues of MLA were synthesised with functional groups targeting key residues that are unique to the binding sites at the α4–α4 and α4–β2 interfaces of α4β2 nAChRs. Esters with the pyridine moiety were synthesised to target an aspartic acid residue in the α4–β2 binding site via salt bridge interactions. Esters with the acetamide moiety were synthesised to target a tryptophan residue in the α4–α4 binding site via hydrogen bonding interactions. Preliminary screening of the esters with both moieties for inhibition at α7 and α4β2 nAChRs revealed them to be inhibitors at α4β2 nAChRs, particularly (α4)3(β2)2 nAChRs which contain the α4–α4 binding site.