During skeletal muscle stimulation, there is a summation of local events of Ca2+ release from the sarcoplasmic reticulum, known as Ca2+ sparks. Ca2+ sparks originate from groups of skeletal ryanodine receptors (RyR1) that activate and close in synchrony. This synchrony allows for the rapid and massive release of Ca2+ from the sarcoplasmic reticulum to initiate contraction and, more important, would provide a mechanism to terminate Ca2+ release under conditions where independent RyR1 are normally active. RyR1 mutations can result in abnormal intracellular Ca2+ signaling that is associated with numerous skeletal muscle disorders including malignant hyperthermia and central core disease. Therefore, investigating the mechanisms that control RyR1 function can help identify how these mutations cause deleterious Ca2+ handling. Currently, most published research on RyR1s gating utilizes single RyR1 reconstituted into planar lipid bilayers to test isolated RyR1. However, in vivo, arrays of RyR1 function in synchrony. Attempts to reconstitute RyR1s into planar lipid bilayers result in experiments that contain multiple channels, which under specific conditions may gate in synchrony, also known as coupled gating. Coupled RyR1 gating was first reported by A. Marks' laboratory and attributed to FK-506 binding protein 12 (FKBP12) associating with neighboring RyR1s the stabilization of RyR1-RyR1 interactions that promote coupled gating. Previous studies suggested that ATP is required for coupled RyR1 gating; however, the mechanism by which ATP promotes the coordinated activity of RyR1s has not been elucidated and is the focus of this thesis. Therefore, my hypothesis is that the agonist action of ATP and FKBP12 bound to RyR1 are required for coupled RyR1 gating. In addition, new pharmacological tools are required to better understand coupled RyR gating. Thus, an additional goal is to identify pharmacological agents that modulate RyR1s in an innovative manner, i.e., help to uncover novel aspects of RyR1 gating and conduction. This investigation suggests that the adenosine based nucleotides, ATP, ADP and AMP, are agonists of RyR1s and promote coupled RyR1 gating in planar lipid bilayers. However, ADP and AMP were unable to maintain coupled RyR1 gating with physiological levels of Mg2+. This suggests that coupled gating would be impaired when the levels ATP decrease, as in muscle fatigue. When ATP was compared to other nucleotides (GTP, ITP, and TTP), the results suggest that the nucleotide agonist action on RyR1s is dependent on the phosphate groups and amino group on the nucleobase. As ATP is the most efficient nucleotide for coupled gating, I also investigated the indirect action of ATP to act as a kinase substrate or alter the cytoskeletal network. The addition of kinases, phosphatases and cytoskeletal modulators did not produce a significant disruption of coupled RyR1 gating. I also tested the role of addition of exogenous FKBP12 to RyR1s that gated independently or had partial coupling, but coupled gating was never improved. Also, the addition of high doses of rapamycin to remove FKBP12 from coupled RyR1 failed to functionally uncouple the channels. Finally, I attempted to find pharmacological agents that could aid in the understanding of coupled RyR1. Some agents were found to modulate RyR1s; however, I did not find a probe that would affect kinetics/conductance of RyR1s and was suitable for comparing coupled gating in bilayers with Ca2+ sparks in cells. Overall, coupled RyR gating is dependent on the physiological modulators ATP and Mg2+. This thesis represents a step forward in identifying the requirements for coupled RyR1 gating and understanding how RyR1s function in cells. Until an understanding of how these receptors communicate in cells is obtained, how different mutations alter the Ca2+ leak will continue to be quite difficult to study.

Although F1-ATPase is extremely conserved among organisms, a putative subunit p18 was identified in Trypanosoma brucei F1-ATPase complex. To explore its function in the procylic, bloodstream and dyskinetoplastic trypanosomes, three different RNAi cell lines were created. Upon p18 silencing the F1-moiety structural integrity was impaired suggesting that p18 is indeed a bona fide subunit of this complex. Since F1-ATPase is crucial for the bloodstream form survival, its potential inhibitor from the 4-oxopiperidine-3,5-dicarboxylates class (JK-11) was examined. JK-11 inhibited growth of the bloodstream trypanosomes, decreased mitochondrial membrane potential and reduced ATPase and ATP synthase activity in mitochondrial lysates. Our results suggest that JK-11 may act on FoF1-ATP synthase/ATPase and its inhibition may contribute to the cytotoxicity of this drug.

Programa de Pós-Graduação em Biotecnologia. Núcleo de Pesquisas em Ciências Biológicas, Pró-Reitoria de Pesquisa de Pós Graduação, Universidade Federal de Ouro Preto. / Submitted by giuliana silveira (giulianagphoto@gmail.com) on 2016-03-22T17:27:01Z No. of bitstreams: 1 DISSERTAÇÃO_CorrelçaoCapacidadeEctonucleotidásica.pdf: 1915336 bytes, checksum: 791ba29f673a9a0f4566d6f2432bc73b (MD5) / Approved for entry into archive by Gracilene Carvalho (gracilene@sisbin.ufop.br) on 2016-04-05T14:05:46Z (GMT) No. of bitstreams: 1 DISSERTAÇÃO_CorrelçaoCapacidadeEctonucleotidásica.pdf: 1915336 bytes, checksum: 791ba29f673a9a0f4566d6f2432bc73b (MD5) / Made available in DSpace on 2016-04-05T14:05:46Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO_CorrelçaoCapacidadeEctonucleotidásica.pdf: 1915336 bytes, checksum: 791ba29f673a9a0f4566d6f2432bc73b (MD5) Previous issue date: 2014 / Probióticos são microrganismos vivos que, quando administrados de forma adequada, agregam benefícios à saúde do hospedeiro. Várias espécies do gênero Lactobacillus apresentam efeito probiótico comprovado e são importantes moduladoras do sistema imune intestinal. Os probióticos estimulam as células dendríticas (DCs) a produzirem citocinas que diferenciam as células T em Th1, Th2, Th17 ou Treg. As ectonucleotidases são enzimas capazes de hidrolisar nucleotídeos di e trifosfatados. Essas ectoenzimas são importantes para a modulação da resposta imune intestinal devido à hidrólise de ATP, que quando aumentada no meio extracelular é considerada uma molécula inflamatória. Por outro lado o acúmulo de adenosina, através da hidrólise de ATP em AMP, pela 5’-nucleotidase, estimula a resposta anti-inflamatória. O objetivo do trabalho foi relacionar a atividade ectonucleotidásica de diferentes linhagens de Lactobacillus com a capacidade de ativação de DCs e a produção de citocinas. Os Lactobacillus foram incubados com ATP, ADP ou AMP e os níveis de hidrólise foram avaliados através da liberação de fosfato inorgânico (Pi). Demonstrouse que os lactobacilos hidrolisam ATP diferentemente, dependendo da linhagem, sendo que Lactobacillus mali L.11 e Lactobacillus acidophilus L.36 apresentaram maior capacidade de hidrólise de ATP. Não foi observado hidrólise significativa de ADP e AMP. Ao agruparmos as linhagens com e sem atividade, foi possível verificar o aumento significativo da atividade de L. mali L.11 e L. acidophilus L.36 quando comparado às linhagens que apresentaram pouca atividade ectonucleotidasica. Além disso, as DCs foram estimuladas com os lactobacilos, por 20 horas e os resultados dessa estimulação demonstrou que as linhagens não alteraram. Além disso, as DCs foram estimuladas com Lactobacillus, por 20 horas e não demonstrou alteraração na expressão de CD40, CD86 e MHCII. Por fim observou-se a produção de citocinas após a estimulação por 20 horas. Houve um aumento de IL-6, TNF- α e IL-10 em relação às DCs não estimuladas. De maneira interessante, IL-12 teve a produção aumentada quando da estimulação de DC com L. mali L.10, que também mostrou baixa capacidade de hidrolisar ATP. Curiosamente, L. mali L.11 e L. acidophilus L.36 mostraram uma diminuição significativa da produção de IL-12, o que pode está relacionado com a alta capacidade de hidrólise dessas cepas. Conclui-se então que Lactobacillus possuem capacidade ectonucleotidásica diferenciada e que a hidrólise de ATP pode ser relacionada com a produção de citocinas por DCs e, consequentemente, com a diferenciação de linfócitos T durante a resposta imune intestinal. __________________________________________________________________________________________________________________________ / ABSTRACT : Probiotics are live microorganisms which, when administered properly, add health benefits to the host. Several species of the genus Lactobacillus have proven probiotic effect and are important modulators of the intestinal immune system. Probiotics stimulate dendritic cells (DCs) to produce cytokines that T cells differentiate into Th1, Th2, Treg and Th17. The ectonucleotidases are enzymes that hydrolyze nucleotide diand triphosphates. These ectoenzymes are important for the modulation of intestinal immune response due to ATP hydrolysis, which when raised in the extracellular medium is considered an inflammatory molecule. On the other hand, the accumulation of adenosine by hydrolysis of ATP to AMP by 5'-nucleotidase, stimulates antiinflammatory response. The objective was to relate the ectonucleotidásica activity of different strains of Lactobacillus with the ability of DCs activation and cytokine production. Lactobacilli were incubated with ATP, ADP or AMP and hydrolysis levels were assessed by the release of inorganic phosphate (Pi). It has been shown that lactobacilli hydrolyze ATP differently, depending on the strain, and Lactobacillus acidophilus and Lactobacillus mali L.11 L.36 showed greater capacity for ATP hydrolysis. No significant hydrolysis of ADP and AMP was observed. When we group the strains with and without activity, we observed a significant increase in activity of L. acidophilus and L. mali L.11 L.36 compared to strains that showed little ectonucleotidasica activity. Furthermore, DCs were stimulated with lactobacilli for 20 hours and the results of stimulation showed that the strains do not change. Furthermore, DCs were stimulated with Lactobacillus for 20 hours and showed no alteraração expression of CD40, CD86 and MHCII. Finally the observed production of cytokines after stimulation for 20 hours. There was an increase in IL-6, TNF-α and IL- 10 compared to non-stimulated DCs. Interestingly, IL-12 production was increased when the stimulation of DC with L. mali l.10, which showed poor ability to hydrolyze ATP. Interestingly, L. acidophilus and L. mali L.11 L.36 showed a significant decrease of IL-12, which can associated with high hydrolysis capability of these strains. It is concluded that Lactobacillus ectonucleotidásica have different capabilities and the hydrolysis of ATP may be related cytokine production by DCs and therefore with the differentiation of T lymphocytes in the intestinal immune response. Keywords: Probiotics,

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Tecnologia, 2013. / Submitted by Alaíde Gonçalves dos Santos (alaide@unb.br) on 2014-03-25T14:22:49Z No. of bitstreams: 1 2013_PrisciladeLimaVianna.pdf: 1762239 bytes, checksum: ed367092a4676accadf410a9a151a631 (MD5) / Approved for entry into archive by Guimaraes Jacqueline(jacqueline.guimaraes@bce.unb.br) on 2014-03-31T12:44:09Z (GMT) No. of bitstreams: 1 2013_PrisciladeLimaVianna.pdf: 1762239 bytes, checksum: ed367092a4676accadf410a9a151a631 (MD5) / Made available in DSpace on 2014-03-31T12:44:09Z (GMT). No. of bitstreams: 1 2013_PrisciladeLimaVianna.pdf: 1762239 bytes, checksum: ed367092a4676accadf410a9a151a631 (MD5) / Os barramentos das subestações são os responsáveis pela interligação de vários elementos de um sistema elétrico de potência, de modo que faltas nesses componentes ocasionam um grande número de desligamentos. Nesse contexto, uma avaliação detalhada da sua proteção torna-se eminente, a fim de garantir que faltas sejam extintas rápida e apropriadamente, preservando-se a integridade dos seus componentes e mitigando-se a ocorrência de blackouts de grandes proporções. Nesse sentido, esta dissertação de mestrado apresenta a modelagem e a simulação da proteção diferencial de baixa impedância de barramentos no software ATP. Para tanto, foi realizada uma simulação em malha fechada dos modelos dos elementos do sistema elétrico de potência residentes no software ATP (Alternative Transients Program), com os modelos dos componentes do sistema de proteção desenvolvidos no ambiente MODELS. Escolheu-se como estudo de caso um barramento com configuração de barra dupla com disjuntor simples a cinco chaves, que pode apresentar uma grande variedade de arranjos. Os resultados obtidos indicam as vantagens da utilização desse tipo de modelagem na avaliação do desempenho dos sistemas de proteção de barramentos, por meio da qual é possível verificar o comportamento do sistema de potência frente à operação da sua proteção, bem como o da proteção frente às mais diversas situações de operação que pode ser submetida. ______________________________________________________________________________________ ABSTRACT / The busbars are responsible for the interconnection of various elements of an electric power system, so that faults in these components cause the shutdown of several loads. In this context, a thorough study of their protection is of paramount importance, in order to ensure that faults are extinguished quickly and appropriately, preserving the integrity of its components and mitigating the occurrence of widespread blackouts. Therefore, this master thesis presents the modeling and simulation of the low impedance busbar differential protection principle in ATP software. In order to do so, a closed-loop simulation between the resident models of the electric power system elements in ATP and the models of protection system components developed in MODELS was realized. It was chosen as a case of study a system with double-bus single-breaker busbar topology, which can assume a wide variety of operating configurations. The obtained results reveal the advantages of using this type of modeling to thereby one can evaluate the performance of busbar protection systems, through which it is possible to evaluate the behavior of the power system against the operation of its protection, as well as the protection performance for the different power systems operational conditions.

Conceitos prévios, ou alternativos, são ideias introjetadas no universo cognitivo dos estudantes que diferem daqueles credenciados pela ciência estabelecida. São bastante estudadas as consequências da presença deste tipo de conceitos para o aprendizado. Entre outras dificuldades provocadas pelos conceitos alternativos está a impossibilidade de utilizá-los para embasar novos conhecimentos. Nestas condições, incapazes de decodificar apropriadamente as novas informações apresentadas pelas disciplinas, os alunos são inconscientemente encaminhados para a memorização. A origem dos conceitos prévios é variada. Uma das causas já detectadas é a precariedade da transposição didática, feita pelos autores dos livros textos ou por docentes. Uma das moléculas fundamentais para os estudos bioquímicos é a adenosina trifosfato (ATP), que desempenha múltiplas funções. Uma das principais é participar de processos que requerem energia. A compreensão do papel desta molécula é fundamental para o entendimento dos processos dos quais ela participa. Um dos objetivos do presente trabalho foi investigar equívocos sobre o papel termodinâmico do ATP nos processos celulares. Os testes foram realizados com alunos do ensino médio (EM), graduação (G) e pós-graduação (PG). A existência de concepções alternativas foi verificada, assim como sua estabilidade nos diferentes níveis de escolaridade: um resultado mostra que 68% EM, 92% G e 91% PG afirmaram que a energia da hidrólise de ATP é responsável por conduzir os processos celulares. Os resultados gerais mostram que os estudantes carregam equívocos em conceitos termodinâmicos básicos, tais como transferência de energia e espontaneidade de reações químicas. Duas possíveis fontes de conceitos alternativos da termodinâmica do ATP são o professor e o livro didático. Nesse trabalho foi verificado que os livros de ensino médio e graduação podem contribuir para a instalação de conceitos alternativos referentes ao ATP. Nos livros analisados, principalmente os de ensino médio, foram encontrados passagens, analogias e esquemas que podem contribuir para isso. E por fim, uma interferência didática foi feita com o intuito de corrigir o entendimento dos alunos no que diz respeito ao papel do ATP nas reações químicas. Alunos monitorados por meio de pré e pós-testes apresentaram resultados animadores em relação à atenuação de conceitos relacionados à termodinâmica do ATP. Dos alunos que participaram da intervenção, mais de 80% responderam e justificaram corretamente os testes feitos pós-intervenção / Misconceptions, or alternative concepts, are introjected ideas in the students` cognitive universe that differ from those established by science. The consequences of alternative concepts for the learning process are widely studied. Among other difficulties caused by alternative concepts is the impossibility to use them to support new knowledge. Under these conditions, unable to properly decode the new information submitted by the disciplines, students are unconsciously driven to memorization. The origin of the alternative concepts is varied and an already detected reason is the precariousness of didactic transposition by textbooks writers and teachers. One of the fundamental biochemical molecules is ATP which play multiple roles. A key role is its participation in energy requiring processes. Understand this molecule role is fundamental to understand the processes which it takes part. The present work aims at the investigation of misconceptions on the ATP\'s thermodynamic role on cellular processes. Tests were carried out with high school students (HS), undergraduate (U) and graduate students involved in PhD programs (G). Misconceptions were observed as well as its stability along different levels of education: 68% (HS), 92% (U) and 91% (G) students stated that the energy from ATP hydrolysis is responsible for driving cellular energy-demanding processes. The overall results show that students carry misconceptions on basic thermodynamic concepts such as energy transfer and chemical reactions spontaneity. Misconceptions on ATP thermodynamics have the teacher and the textbook as possible sources. In this study it was found that books from all levels may contribute to insert or preserve misconceptions on ATP. From the analyzed books, especially for the high school, there were quotes, analogies and diagrams that can contribute to it. Finally, in order to correct the students\' understanding regarding the role of ATP in chemical reactions a didactic intervention was made. Students were tested through pre- and post-tests dealing with the subject in study. Of the students who participated in the intervention, over 80% correctly answered and explained the post-intervention test

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22314号 / 医博第4555号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 山下 潤, 教授 Shohab YOUSSEFIAN, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Bacterial cell division relies on the cell division septum to form at the mid-cell position. In gram negative bacteria, this is mediated by three proteins, MinC, MinD and MinE. Together these proteins interact with each other and the membrane in a dynamic, oscillating process which prevents cell division septum formation at the cell poles. The early phase of this process involves MinD binding to the membrane, which is triggered upon binding of ATP. Subsequent interactions with MinE result in stimulation of the ATPase activity of MinD. After hydrolysis, MinD is released from the membrane and diffuses to a new binding site. Many in silico models have been constructed of the Min system in an attempt to describe its self-organizing behaviour. A limitation of these models is that, in order to prevent rapid re-binding of MinD to the membrane after hydrolysis of ATP, the exchange of bound ADP for ATP is assumed to be a slow process, on the order of 1/s . In order to provide experimental evidence of the rate of nucleotide binding, we performed a series of triple-resonance NMR experiments to complete a partial assignment of backbone atom resonances, which required the application of deuterium labelling and amino acid-specific selective unlabelling. After the introduction of ATP, it was discovered that no dimerization had been induced, in contrast with existing literature. It was proposed that MinD from N. gonorrhoeae only forms a dimer in the presence of a membrane, while literature with MinD from E. coli shows it does not have this requirement. Interestingly while dimerization had not been induced, there was a persistent population of dimeric species even in the absence of nucleotide. This was discovered to be the result of disulfide formation, likely an artifact of established purification protocols. Binding of both ADP and ATP to MinD were studied by titration using NMR, with the relative affinity of both nucleotides to MinD being indistinguishable. By analyzing peak coalescence in the half-bound condition, a maximum rate was determined for nucleotide binding, with the lifetime being on the order of 170ms. Results from this experiment support models requiring a slow nucleotide binding step, and help enhance understanding of how Min proteins sustain oscillations required for normal cell division.

京都大学 / 0048 / 新制・論文博士 / 博士(医学) / 乙第13067号 / 論医博第2122号 / 新制||医||1019(附属図書館) / 33218 / (主査)教授 長船 健二, 教授 松田 道行, 教授 岩井 一宏 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21255号 / 医博第4373号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 柳田 素子, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

These experiments were conducted to determine the membrane K+ currents and channels in human urinary bladder (HTB-9) carcinoma cells in vitro. K+ currents and channel activity were assessed by the whole-cell voltage clamp and by either inside-out or outside-out patch clamp recordings. Cell depolarization resulted in activation of a Ca2+-dependent outward K+ current, 0.57 ± 0.13 nS/pF at -70 mV holding potential and 3.10 ± 0.15 nS/pF at 30 mV holding potential. Corresponding patch clamp measurements demonstrated a Ca2+-activated, voltage-dependent K+ channel (K(Ca)) of 214 ± 3.0 pS. Scorpion venom peptides, charybdotoxin (ChTx) and iberiotoxin (IbTx), inhibited both the activated current and the K(Ca) activity. In addition, on-cell patch recordings demonstrated an inwardly rectifying K+ channel, 21 ± 1 pS at positive transmembrane potential (V(m)) and 145 ± 13 pS at negative V(m). Glibenclamide (50 μM), Ba2+ (1 mM) and quinine (100 μM) each inhibited the corresponding nonactivated, basal whole-cell current. Moreover, glibenclamide inhibited K+ channels in inside/out patches in a dose-dependent manner, and the IC50 = 46 μM. The identity of this K+ channel with an ATP-sensitive K+ channel (K(ATP)) was confirmed by its inhibition with ATP (2 mM) and by its activation with diazoxide (100 μM). We conclude that plasma membranes of HTB-9 cells contain the K(Ca) and a lower conductance K+ channel with properties consistent with a sulfonylurea receptor-linked K(ATP).