As pesquisas com os peptídeos hormonais de plantas se iniciaram na década de noventa com a descoberta da sistemina. Hoje existem diversos peptídeos identificados, e alguns deles já em avançado estágio de caracterização. O envolvimento desta classe de moléculas em diversas funções básicas e específicas da biologia dos vegetais despertou o interesse da comunidade científica. Dentre os peptídeos em fase de caracterização, destacam-se os representantes da família RALF. Os peptídeos RALF estão presentes em basicamente todo o reino vegetal, desde o musgo Physcomitrela pattens até as plantas superiores mono e dicotiledôneas. A conservação destes peptídeos no reino vegetal sugere um importante papel na fisiologia vegetal, e evidências recentes indicam a participação de RALF em processos básicos do desenvolvimento das plantas. O mecanismo pelo qual o peptídeo RALF atua e é percebido pela célula constitui etapa fundamental para sua caracterização funcional. Para tanto, no presente trabalho foram empregadas técnicas para identificação de proteínas de interação com RALF. Os resultados indicam que este peptídeo possivelmente tem sua atividade regulada pelo íon cálcio, através da interação com uma proteína de ligação a cálcio que, assim como o RALF, é secretada para o apoplasto. Estes dados colocam RALF em um cenário até o momento inédito no mecanismo de ação hormonal em plantas. A exemplo de animais e leveduras, observou-se também que o processamento de RALF ocorre em um sítio dibásico. A mutação de um dos aminoácidos deste sítio foi suficiente para impedir processamento do peptídeo in vivo e in vitro. A utilização de extratos protéicos da fração microsomal de Arabidopsis no ensaio in vitro indica que esta atividade é desempenhada por uma protease presente no sistema de endomembranas celular, provavelmente da classe das convertases. Os resultados publicados marcam o início dos estudos de caracterização do processamento de prohormônios em plantas. / The peptide hormone research has begun during the 90s decade with the systemin discovery. Nowadays several peptides have already been identified, and some of them are further characterized. The involvement of these molecules with a range of basic and specific biological functions has raised the scientific communitys interest. Among the peptides being studied, the RALF family is particularly intriguing. The RALF peptides can be found throughout the plant kingdom, from the moss Physcomitrella patens to the mono and dicot plant groups. The conserved occurrence of these peptides along the plant kingdom suggests an important role in the plant physiology field. Recent evidences indicate that RALF plays a role in basic mechanisms of plant development. The RALF mechanism of action and its perception by the cell are fundamental information in order to characterize this peptide function. In the present work experiments to identify RALF interacting proteins were employed. The results indicate that RALF peptides activity is possibly regulated by the calcium ion. This regulation is mediated by the interaction with a calcium binding protein. This calcium binding protein was found to be secreted to the apoplast. Presented data suggests that RALF is regulated by a mechanism never described before in the plant hormone research field. As previously described in animals and yeast the RALF propeptide processing takes place in a dibasic site. A single amino acid site specific mutation disrupted peptide processing in vivo and in vitro. The correct processing is mediated by proteases of the Arabidopsis microsomal fraction. This processing seems to occur at the endomembrane system, possibly catalized by a convertase class enzyme. The published results points the beginning of the peptide processing studies in plants.

Desde a década de 90, uma nova família de moléculas de origem protéica e com características hormonais vem sendo estudada em plantas. Esse grupo de novas moléculas, coletivamente chamado de peptídeos hormonais, está envolvido com defesa, reprodução, crescimento e desenvolvimento. RALF, que é um desses novos peptídeos, é ubíquo em plantas e está envolvido com desenvolvimento vegetal. Em Arabidopsis existem 34 genes semelhantes ao RALF (AtRALFs). Nosso grupo mostrou que AtRALF1 é processado de um precursor maior por uma subtilase. Arabidopsis possui 56 subtilases, o objetivo deste trabalho é identificar a subtilase responsável pelo processamento do AtRALF1. Predição da localização subcelular e análise da expressão in silico, ambas confirmadas por análise da expressão por RT-PCR e fusões proteicas com a proteína verde fluorescente, permitiram reduzir para sete o número de subtilases candidatas. Cruzamentos entre mutantes nocaute ou plantas de baixa expressão por RNAi dessas sete subtilases com plantas superexpressoras do AtRALF1 identificaram as subtilases AtSBT6.1 (At5g19660) e AtSBT5.3 (At2g04160) como prováveis envolvidas no processamento do prepropeptídeo AtRALF1. / Since the 90s, a new family of molecules of protein origin and with hormone characteristics has been studied in plants. This group of new molecules, collectively named peptide hormones, is involved in defense, reproduction, growth and development. RALF, one of these peptides, is ubiquitous in plants and is involved in plant development. In Arabidopsis there are 34 RALF-like genes (AtRALFs). Our group has shown that AtRALF1 is processed from a larger precursor by a subtilase. Arabidopsis has 56 subtilases, our goal is the identification of the specific subtilase that is responsible for the AtRALF1 processing. Prediction of subcelular localization and in silico gene expression analysis, both confirmed by RT-PCR expression analysis and chimeric proteins with green-fluorescent protein, allowed the reduction of the initial 56 candidates to only 7 subtilases. Crosses between knockout mutants or RNAi plants expressing low levels of subtilases with overexpressors of AtRALF1 identified the subtilases AtSBT6.1 (At5g19660) and AtSBT5.3 (At2g04160) as potentialy involved in the prepropeptide AtRALF1 processing.

RALFs são peptídeos hormonais de aproximadamente 5kD que regulam negativamente o alongamento celular. Dentre suas atividades biológicas, a alcalinização do meio extracelular e a inibição do alongamento celular são tidas como associadas, pois a acidificação do meio extracelular é necessária para a expansão celular. A atividade de alcalinização e a de inibição do crescimento são medidas em dois ensaios distintos: o ensaio de alcalinização do meio extracelular de células em suspensão e o ensaio do crescimento de raiz primária de plantas jovens. Buscando-se fazer ambas as avaliações da inibição da expansão celular e da alcalinização em um único modelo de estudo, tomou-se como medida da expansão celular o volume das células decantadas (VCD). Quando o tampão MES ou o pré-tratamento com \"Fusicoccin\" foi utilizado para se atenuar o efeito de alcalinização do AtRALF1, verificou-se que o efeito de inibição do alongamento celular não sofreu alteração. Em arabidopsis são encontrados nove peptídeos RALF, que apresentam alta similaridade com o RALF original de tabaco. Com exceção do AtRALF4, todos são capazes de alcalinizar o meio extracelular e inibir raízes. A comparação da estrutura primária do AtRALF4 com os demais RALFs mostra que poucos resíduos são distintos entre eles, sugerindo que estes possam ser os determinantes das atividades de inibição e alcalinização. A alteração de um destes resíduos, AtRALF4(N92A), é capaz de restaurar a capacidade do AtRALF4 de inibir as raízes sem, no entanto, recuperar a atividade de alcalinização. Quando três outros resíduos exclusivos do AtRALF4 foram substituídos pelos seus correspondentes do AtRALF1, observa-se uma restauração parcial da alcalinização, desta vez, sem alterar a capacidade de inibir as raízes. Recentemente, foi mostrado pelo nosso grupo que o peptídeo AtRALF1 induz a expressão de genes relacionados ao rearranjo da parede celular. Quando verificado por PCR quantitativa, contatou-se que somente os peptídeos mutantes que apresentam atividade de inibição do crescimento são capazes de promover uma indução semelhante. Ainda, utilizando gel indicador de pH, verificou-se que plantas transgênicas super-expressando AtRALF1 (35S:AtRALF1) acidificam o meio durante seu crescimento de maneira semelhante a plantas selvagens. O peptídeo de defesa AtPEP1, a exemplo dos peptídeos RALF, também promove a alcalinização do meio extracelular. A utilização de drogas para reproduzir ou atenuar o efeito de alcalinização promovido por este peptídeo sugere que a expressão dos genes responsivos a AtPEP1 também não está relacionada à alteração na atividade das próton-ATPases. Finalmente, quando mutantes para ambos os receptores de AtPEPs foram utilizados (atpepr1/r2) em gel indicador de pH, verificou-se que estes alcalinizam o pH da rizosfera na presença do peptídeo. Nossos dados somados sugerem uma dissociação das atividades biológicas de alcalinização do meio extracelular e de inibição da expansão celular. A alteração na atividade das próton-ATPases pode não ser apenas uma mensagem secundária do efeito biológico, mas sim outra fonte de informação independente e ainda pouco explorada como tal. / RALFs are 5kD peptide hormones that negatively regulates cell expansion. Among the biological activities of the RALF peptide, the extracellular alkalinization and cellular expansion inhibition were previously suggested to be associated, once the extracellular acidification is required to cell expansion. Usually, the alkalinization and cell expansion inhibition activities are evaluated in two different assays, the cell suspension medium alkalinization and the plantlet root growth inhibition. We manage to set an assay in which both cell expansion inhibition and extracellular alkalinization activities could be evaluated. Using the package suspension cell volume through decantation as a value of cell expansion, we evaluated the relation between alkalinization and cell expansion inhibition in different conditions. When the MES buffer or the pre-treatment with Fusicoccin was used to arrest the AtRALF1 extracellular alkalinization, the package cell volume inhibition activity was not affected. There are 9 RALF peptides encoded in arabidopsis plants that are closely related with the first isoform isolated from tobacco. With exception of the AtRALF4, all those isoforms are able to alkalinize the extracellular medium and arrest root growth. Comparing the AtRALF4 with other eight isoforms, we verified that are few different amino acids between them, suggesting that those amino acids could be essential for the biological activities. The rescue of one of those amino acids, AtRALF4(N92A), was able to regain the root growth inhibition activity of the AtRALF4 peptide, although the extracellular alkalinization activity was not restored. When three other AtRALF4 amino acids were substituted by their AtRALF1 correspondent, there is a partial rescue of the extracellular alkalinization activity, but no alterations in the root growth inhibition. We had recently shown that the AtRALF1 peptide induces the expression of genes related to cell wall rearrangement. The quantitative PCR analyses demonstrates that only the mutant peptides that are able to arrest root growth are also able to induce the gene expression. When submitted to a pH indicator, it was verified that AtRALF1 overexpressing plants acidifies it during its growth, as much as wild type plants.The defense peptide AtPEP1, similarly of AtRALF1, also triggers extracellular alkalinization. The use of proton-pump chemical modulators to simulate or arrests AtPEP1 extracellular alkalinization activity suggests that the gene expression of the AtPEP1-responsive genes is not related to changes in plasma membrane proton pump activity. Finally, when double mutants for both the AtPEP1 receptors (atpepr1/r2) were submitted to a pH gel indicator, it was seen that they alkalinize the rhizosphere pH in the presence of the AtPEP1 peptide. Our data suggests that the extracellular alkalinization and arrest of cell expansion activities are dissociated. The proton pump modulation activity may not be only a secondary messenger, but another source of information independent and yet to be explored.

囊性纖維化（CF）是由囊性纖維化跨膜電導調節器（CFTR）的突變引起的一種隱性遺傳病。CF病人的肺、肝、胰腺、腸道與生殖道受到嚴重影響，其中有50%的成年病人患有糖尿病。由CF引起的糖尿病被稱為CF相關糖尿病（CFRD）, 关于它的病因至今仍然存有爭議。2007年，人們發現CFTR在分泌胰島素的胰島β細胞上有表達。儘管如此，β細胞上的CFTR与糖尿病发病的关系却一直被忽略。我們的研究目標是闡述β細胞上的CFTR在胰島素分泌中的作用。 / 在β細胞上，葡萄糖刺激的胰島素分泌伴隨著複雜的電活動，這種電活動被描述為細胞膜電位去极化疊加的動作電位的爆發。葡萄糖引起的ATP敏感的鉀離子通道（K[subscript Asubscript Tsubscript P]）的關閉被普遍認為是β細胞去極化的初始事件，初始的去極化啟動了電壓依賴的鈣離子通道，由此產生的鈣離子內流成為構成動作電位的去極化電流，引起了細胞內鈣離子的震盪，從而引起胰島素的釋放。雖然氯離子電流被認為參與了β細胞去極化電流，但是，人們仍然不能確定是哪一種氯離子通道介導了這個去極化電流。在我們研究的第一部分，CFTR被證明功能性的表達在β細胞上，並且可以被葡萄糖激活。CFTR可以被葡萄糖激活这一性质，在CFTR超表達的CHO 细胞上被進一步驗證。在原代培養的β細胞與β細胞株RIN-5F细胞中的葡萄糖引起的全細胞電流、膜電位的去極化、動作電位的幅度與頻率、鈣震盪和胰島素的分泌可以被CFTR的抑制劑或缺陷所降低。與野生型小鼠相比，CFTR基因敲除的小鼠，禁食之後，具有更高的血糖濃度，然而其胰島素的濃度低。 / 我們研究中的第二部分，利用了數學模型去闡明CFTR 在胰島素分泌的電活動中的角色。結果顯示， CFTR電導的減低可以使細胞的細胞膜去極化，從而導致需要更高的電刺激去引發動作電位，这些結果證明了CFTR對於维持細胞膜電位的貢獻。同時增加細胞內氯離子濃度和CFTR的電導可以引起更大頻率的膜電位的震盪，這一點證明了氯離子對於細胞膜電位震盪有著重要的作用。在数学模型中，CFTR電導的降低可以消除通過改變ATP/ADP值所引起的電火花， 這與我們在試驗中發現的CFTR參與了葡萄糖引起的動作電位是一致的。總而言之，我們的数学模型證明了CFTR對於胰島素的分泌是非常重要的，它通過介導氯離子外流對細胞膜電位的產生貢獻並且參與了電火花的產生，所有這些都進一步驗證了我們在實驗部分的發現。 / 综上所述，現有的研究揭示了CFTR，通過對β細胞膜電位作用與参与了動作電位的產生，在葡萄糖刺激胰島素分泌过程中的鮮為人知的重要角色。這個發現為揭示CFRD的病理機制提供了全新的視角，並且可能為開發治療CFRD的方法帶来了曙光。 / Cystic fibrosis (CF) is a recessive autosomal genetic disease resulted from mutations of cystic fibrosis transmembrane conductance regulator (CFTR). CF affects critically the lung, liver, pancreas, intestine and reproductive tract. CF patients also exhibit a high percentage of diabetes, which almost reach 50% in adult. The pathological cause of diabetes in CF patients, also called CF related diabetes (CFRD), is still controversial. It has been reported that CFTR expressed in the islet β cells, which is responsible for insulin secretion. However, the exact role of CFTR in islet β-cell and its relation to diabetes have been ignored. The present study aims to elucidate the role of CFTR in the process of insulin secretion by pancreatic islet β cells. / Glucose-stimulated insulin secretion is associated with a complex electrical activity in the pancreatic islet β-cell, which is characterized by a slow membrane depolarization superimposed with bursts of action potentials. Closing ATP-sensitive K⁺ channels (K[subscript Asubscript Tsubscript P]) in response to glucose increase is generally considered the initial event that depolarizes the β-cell membrane and activates the voltage-dependent Ca²⁺ channels, which constitutes the major depolarizing component of the bursting action potentials giving rise to the cytosolic calcium oscillations that trigger insulin release. While Cl⁻ has been implicated in an unknown depolarization current of the β-cell, the responsible Cl⁻ channel remains unidentified. In the first part of our study, we show functional expression of CFTR and its activation by glucose in the β-cell. Activation of CFTR by glucose was also demonstrated in CHO cell over-expression system. The glucose-elicited whole-cell currents, membrane depolarization, electrical bursts (both magnitude and frequency), Ca²⁺ oscillations and insulin secretion could be abolished or reduced by inhibitors/knockdown of CFTR in primary mouse β-cells or RIN-5F β-cell line, or significantly attenuated in isolated mouse islet β-cells from CFTR mutant mice compared to that of wildtype. Significantly increased blood glucose level accompanied with reduced level of insulin is found in CFTR mutant mice compared to the wildtype. The results strongly indicate a role of CFTR in the process of insulin secretion. / In the second part of our study, mathematical model is built up to clarify the role of CFTR in the electrical activity during insulin secretion. It is shown that reduction of CFTR conductance hyperpolarizes the membrane of the β-cell, for which it requires a larger electrical stimulus to evoke an action potential, indicating the contribution of CFTR to the membrane potential as demonstrated by our experimental results. Increase in intracellular Cl⁻ concentration and the conductance of CFTR result in higher frequency of membrane potential oscillations, demonstrating that Cl⁻ is crucial for the membrane potential oscillations. The electrical spikes induced by increase of ATP/ADP in the model are abolished by decreasing CFTR conductance, which is consistent with our findings that CFTR is involved in the generation of action potentials induced by glucose. In other word, our model demonstrates that CFTR is crucial for insulin secretion by its contribution to membrane potential and participating in the generation of electrical spikes via conducting Cl⁻ efflux, which confirms our findings in the experimental study. / Taken together, the present study reveals a previously unrecognized important role of CFTR in glucose-stimulated insulin secretion via contributing to the membrane potential and the participating in the generation of action potential in islet β cells. This finding sheds new light into the understanding of the pathogenesis of CFRD and may provide grounds for the development of new therapeutic approaches for CFRD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Guo, Jinghui. / "December 2012." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 156-164). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / 摘要： --- p.iii / Acknowledgement: --- p.v / LIST OF PUBLICATIONS --- p.vi / Declaration --- p.viii / ABBREVIATIONS --- p.xi / LIST OF FIGURES --- p.xiii / Chapter Chapter 1: --- General introduction --- p.1 / Chapter 1.1 --- The function of islet β cells and diabetes --- p.1 / Chapter 1.1.1 --- The introduction of the pancreas --- p.1 / Chapter 1.1.2. --- Glucose metabolism and blood glucose regulation --- p.6 / Chapter 1.1.2.2 --- Blood glucose regulation --- p.7 / Chapter 1.1.3 --- Insulin secretion by the islet β cell --- p.10 / Chapter 1.1.4 --- Diabetes --- p.14 / Chapter 1.2 --- Cystic fibrosis-related diabetes --- p.17 / Chapter 1.2.1 --- Cystic fibrosis --- p.17 / Chapter 1.2.2 --- CFTR --- p.19 / Chapter 1.3 --- Mathematical model for insulin secretion --- p.25 / Chapter 1.4 --- Aim and hypothesis --- p.27 / Chapter 1.4.1 --- CFTR may be activated by glucose --- p.27 / Chapter 1.4.2 --- Activation of CFTR may depolarize the membrane potential --- p.28 / Chapter 1.4.3 --- CFTR-mediating Cl-efflux may be involved in the generation of electrical spikes --- p.28 / Chapter 1.4.4 --- Calcium oscillation depends on CFTR --- p.28 / Chapter 1.4.5 --- Insulin secretion --- p.29 / Chapter 1.5 --- Approaches to test the hypothesis --- p.29 / Chapter Chapter 2: --- Materials and Methods --- p.31 / Chapter 2.1 --- Cell culture --- p.31 / Chapter 2.1.1 --- RIN-5F cell --- p.31 / Chapter 2.1.2 --- CHO cell --- p.31 / Chapter 2.2 --- Islet isolation and culture --- p.32 / Chapter 2.3 --- CFTR knockdown --- p.33 / Chapter 2.4 --- Western blot --- p.35 / Chapter 2.5 --- Immunofluorescence --- p.37 / Chapter 2.6 --- Membrane potential (Vm) measurement --- p.38 / Chapter 2.7 --- Intracellular chloride imaging --- p.39 / Chapter 2.8 --- Intracellular calcium imaging --- p.40 / Chapter 2.9 --- Patch-clamp --- p.40 / Chapter 2.10 --- Blood glucose measurement --- p.42 / Chapter 2.11 --- Insulin ELISA --- p.42 / Chapter 2.12 --- Statistics --- p.42 / Chapter Chapter 3: --- Contribution of CFTR on the eletrophysiological properties in insulin secretion --- p.43 / Chapter 3.1 --- Introduction --- p.43 / Chapter 3.2 --- Results --- p.45 / Chapter 3.2.1 --- Functional expression of CFTR in mouse islet β cells --- p.45 / Chapter 3.2.2 --- CFTR activation by glucose --- p.46 / Chapter 3.2.3 --- Involvement of CFTR in the maintenance of membrane potential of islet β cells --- p.47 / Chapter 3.2.4 --- CFTR is involved in the generation of spikes induced by glucose --- p.50 / Chapter 3.2.5 --- Generation of spikes burst in the β cell depends on intracellular chloride. --- p.52 / Chapter 3.2.6 --- Inhibition/mutation of CFTR attenuates calcium oscillation induced by glucose --- p.53 / Chapter 3.2.7 --- Inhibition/mutation of CFTR impairs insulin secretion --- p.53 / Chapter 3.3 --- Discussion --- p.71 / Chapter Chapter 4: --- Mathematical model for the role of CFTR in the process of insulin secretion in islet β cell --- p.74 / Chapter 4.1 --- Introduction to the mathematical modeling in the process of insulin secretion --- p.74 / Chapter 4.2 --- Methods --- p.77 / Chapter 4.2.1 --- Components in the model --- p.77 / Chapter 4.2.2 --- Assumptions and approaches in modeling --- p.78 / Chapter 4.2.3 --- Modeling ion channels and transporters --- p.79 / Chapter 4.2.3.1 --- KATP channel --- p.79 / Chapter 4.2.3.2 --- Sodium channel --- p.82 / Chapter 4.2.3.3 --- Voltage Dependent calcium channel --- p.83 / Chapter 4.2.3.4 --- NCX --- p.84 / Chapter 4.2.3.5 --- Na-K pump --- p.85 / Chapter 4.2.3.6 --- Kv channel --- p.87 / Chapter 4.2.3.7 --- Ca pump --- p.88 / Chapter 4.2.3.9 --- CFTR --- p.90 / Chapter 4.2.3.10 --- NKCC --- p.91 / Chapter 4.3 --- Results --- p.93 / Chapter 4.3.1 --- Role CFTR in regulation of the basal membrane potential in β cells --- p.93 / Chapter 4.3.2 --- Role of intracellular chloride concentration in the burst spikes induced by glucose --- p.95 / Chapter 4.3.3 --- Role of CFTR in the burst spikes induced by glucose --- p.96 / Chapter 4.4 --- Discussion --- p.105 / Chapter Chapter 5: --- General discussion and conclusion --- p.109 / Chapter 5.1 --- General discussion --- p.109 / Chapter 5.1.1 --- Role of CFTR in endocrine pancreas and diabetes --- p.109 / Chapter 5.1.2 --- Role of CFTR as a cell metabolic sensor --- p.111 / Chapter 5.1.3 --- Role of CFTR in excitable cells --- p.113 / Chapter 5.2 --- Conclusion --- p.114 / Appendix A --- p.115 / Appendix B --- p.118 / Reference: --- p.156

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Abstract The atherosclerotic plaques develop with the adhesion of inflammatory cells and lipids onto the innermost layer of the vessel. They may eventually occlude the vessel impairing blood flow. A severe complication is the rupture of a plaque resulting in the formation of a thrombus that can cause myocardial infarction or stroke. Though a large number of risk factors for atherosclerosis have been identified, the pathogenesis of atherosclerosis is far from unravelled. The aim of the present work was to study both traditional as well as potential novel risk factors of atherosclerosis. The first study examined the relationship between IGF-I concentrations and carotid artery atherosclerosis and its metabolic risk factors. Low IGF-I concentrations were associated with several cardiovascular risk factors. A positive association was observed between IGF-I concentrations and carotid artery intima-media thickness in women. The results suggest that IGF-I may be involved in the pathogenesis of atherosclerosis. Interestingly, the effect may manifest differentially in men and women. The second study focused upon the effects of obesity and weight loss on liver gene expression. A global decrease in gene expression was observed. The down-regulated genes included genes involved in the ubiquitin cycle, which may point to a reduction in oxidative stress due to the hypocaloric diet. The down-regulation of peroxisome proliferator-activated receptor gamma cofactor 1 alpha (PGC-1α) may be related to improved insulin sensitivity. Several novel genes not previously linked to obesity and weight loss were also discovered. In the third and fourth studies, the developmental origins of atherosclerosis hypothesis was studied in a rat model of fetal undernutrition. Unfavourable changes in the obesity-related peptide hormones adiponectin and resistin were observed which could predispose to insulin resistance in later life. In addition, total cholesterol levels were elevated in the undernourished offspring. The gene expression changes in the rat pups suggest that the development of pancreas was affected, which might further contribute to disturbances in insulin and glucose metabolism. / Tiivistelmä Ateroskleroosi eli valtimonkovettumatauti on sairaus, joka saa alkunsa verisuonen sisäseinämään kiinnittyvistä tulehdussoluista ja veren rasvapartikkeleista, joista muodostuu pitkän ajan kuluessa ateroskleroottisia plakkeja. Plakit voivat kasvaessaan heikentää veren virtausta valtimoissa ja pahimmillaan jopa tukkia suonen kokonaan. Mikäli plakki repeää, voi muodostua verihyytymä joka sydämessä aiheuttaa sydäninfarktin ja aivoissa aivoinfarktin. Vaikka useita ateroskleroosille altistavia tekijöitä tunnetaan, taudin syntymekanismit ovat vielä suurelta osin selvittämättä. Tämän väitöskirjatyön tarkoituksena oli tutkia sekä ateroskleroosin perinteisiä että mahdollisia uusia riskitekijöitä. Ensimmäisessä osatyössä tutkittiin insuliininkaltaisen kasvutekijä I:n (IGF-I) yhteyttä kaulavaltimon ateroskleroosiin sekä perinteisiin ateroskleroosin riskitekijöihin. Matalat IGF-I pitoisuudet liittyivät moniin ateroskleroosin riskitekijöihin. Naisissa korkeammat IGF-I pitoisuudet kuitenkin yhdistyivät paksumpaan kaulavaltimoon, mikä viittaa ateroskleroosiin. Tulosten perusteella IGF-I saattaa liittyä ateroskleroosin kehitykseen ja mahdollisesti sen vaikutukset ilmenevät naisissa ja miehissa eri tavoin. Toisessa osatyössä tutkittiin maksan geenien ilmentymistä lihavuudessa ja laihdutusjakson jälkeen. Laihduttaneessa ryhmässä 142:n geenin ilmentyminen oli vähentynyt ja vain yhden lisääntynyt suhteessa kontrolliryhmään. Ubikitiini-syklin geenien ilmentymisen väheneminen voi viitata vähentyneeseen oksidatiiviseen stressiin elimistössä dieetin seurauksena. Muun muassa diabetekseen liittyvän geenin, peroxisome proliferator-activated receptor gamma cofactor 1 alpha, väheneminen puolestaan voi liittyä parantuneeseen insuliiniherkkyyteen laihduttaneissa. Lisäksi tässä työssä tuli esiin monia uusia, mielenkiintoisia geenejä, joita ei aiemmin ole yhdistetty lihavuuteen tai ateroskleroosiin. Kolmannessa ja neljännessä osatyössä selvitettiin ns. Barkerin hypoteesia, eli sitä, voisiko sairastumisalttius määräytyä jo sikiökauden ja varhaiskehityksen aikana. Rottakokeemme osoittivat, että sikiöaikaisen aliravitsemuksen seurauksena kolesteroliarvot olivat korkeammat ja että lihavuuteen liittyvien peptidihormonien, adiponektiinin ja resistiinin, pitoisuuksissa oli tapahtunut epäsuotuisia muutoksia, jotka voivat altistaa insuliiniresistenssille Tulokset viittasivat myös siihen, että aliravitsemus oli mahdollisesti vaikuttanut haiman kehitykseen, mikä voi myös osaltaan vaikuttaa mm. insuliini- ja sokeriaineenvaihduntaan. Tämänkaltaiset muutokset saattavat altistaa ateroskleroosille myöhemmällä iällä.

Indiana University-Purdue University Indianapolis (IUPUI) / Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of incretin based therapeutics which aid in blood glucose management in Type II diabetes mellitus (T2DM). Recent studies have demonstrated direct cardiovascular benefits conferred by these agents including protection in ischemia and heart failure. Despite these observations, human clinical trials fail to support improvements in cardiovascular outcomes independent of glucose lowering effects in the T2DM populations. Prior data from our lab demonstrate that obesity impairs GLP-1 associated increases in myocardial glucose uptake. However, the reasons for this impairment/resistance to cardiac effects of GLP-1 in the setting of obesity remain ill defined. This investigation tested the hypothesis that underlying differences in the cardiac proteome and microRNA (miR) transcriptome could contribute to distinct cardiac responses to ischemia and activation of GLP-1 signaling in the setting of obesity. To identify whether obesity modulated cardiac functional responses to GLP 1 related drugs, we first examined the effects of obesity on cardiac function, miR transcriptome, and proteome in response to short duration ischemia-reperfusion (I/R). We observed divergent physiologic responses (e.g. increased diastolic volume and systolic pressure in lean, decreased diastolic volumes in obese) to regional I/R in obese vs lean hearts that were associated with significant molecular changes as detected by protein mass spectrometry and miR microarray. Molecular changes were related to myocardial calcium handling (SERCA2a, histidine-rich Ca2+ binding protein), myocardial structure and function (titin), and miRs relating to cardiac metabolism, hypertrophy, and cell death, including miR-15, miR-30, miR-199a, miR-214. Importantly, these effects were modified differently by GLP-1 agonism in lean vs obese swine. Additional studies investigated the functional effects of 30 days of treatment with the GLP-1 analogue liraglutide on a model of slowly-developing, unrelieved coronary ischemia. Liraglutide failed to reduce infarct size or collagen deposition. However, analysis of left ventricular pressure-volume relationships support that liraglutide improved diastolic relaxation/filling, load-dependent indices of cardiac function, and cardiac efficiency in response to sympathetic stimulation in obese swine. Taken together, these findings support that miR and proteomic differences underlie distinct changes in functional cardiac responses to I/R and pharmacologic activation of GLP-1 signaling in the setting of obesity.

O hormônio adrenocorticotrópico, ACTH, regula função (esteroidogênese) e proliferação das células da córtex das glândulas adrenais através de um único receptor específico, ACTHR, que pertence à superfamília GPCR (G-protein coupled receptors). Embora o ACTHR tenha sido clonado há 8 anos, os mecanismos moleculares das ações mitogênica e anti-mitogênica de ACTH permanecem obscuros, cuja elucidação é objeto de estudo deste trabalho. A abordagem experimental consistiu na clonagem do ACTHR de células adrenocorticais Y-1 de camundongo e expressão funcional em fibroblastos 3T3 e células AR-1. Clones transfectantes, expressando estavelmente ACTHR, mostraram-se responsivos a ACTH através de: a) ativação de adenilato ciclase e b) indução de genes das famílias fos e jun. Por outro lado, medidas de síntese de DNA e proliferação celular indicam que ACTH não tem nenhum efeito mitogênico ou anti-mitogênico nos transfectantes ACTHR. O gene c-fos foi usado como alvo para testar vias de transdução de sinal ativadas por ACTH nos transfectantes 3T3 ACTHR. Estes testes mostraram que PKA, PKC e MAPK tem pouca ou nenhuma participação na indução de c-fos por ACTH nos clones 3T3 ACTHR, sugerindo que ACTHR pode ativar vias ainda não identificadas e motivando a busca de novas vias ativadas por ACTH nas células Y-1. Verificou-se que células Y-1 apresentam níveis constitutivamente elevados de AKT/PKB ativada (fosforilada), dependentes de PI3K e SRC, e que ACTH promove rápida desfosforilação de AKT via Gs/Adenilato Ciclase/cAMP/PKA. Ao promover a inativação de AKT, ACTH promove simultaneamente a indução da proteína p27'IND.Kip1', um mecanismo que contribui para a atividade anti-mitogênica de ACTH. / The adrenocorticotropic hormone, ACTH, regulates both function and proliferation of adrenocortical cells binding to a specific receptor, ACTHR, which belongs to superfamily of GPCR (G-protein coupled receptors). ACTHR was cloned a few years ago, but the molecular mechanisms underlying the mitogenic and anti-mitogenic actions of ACTH remain unknown, whose elucidation is aim of this project. The experimental approach was to clone the ACTHR from mouse Y-1 adrenocortical cells and to express the functional receptor in Balb 3T3 fibroblasts and AR-1 cells. Transfectant clones stably expressing the ACTHR respond to ACTH treatments by: a) adenylate cyclase activation and b) induction of fos and jun genes. On the other hand, experiments of DNA synthesis and cellular proliferation showed that ACTH has not mitogenic or anti-mitogenic effects in ACTHR transfectants. c-fos gene was used as a target to test signal transduction pathways activated by ACTH into 3T3 ACTHR transfectants. The results showed that PKA, PKC and MAPK have no relevant contribution on the ACTH c-fos induction in 3T3 ACTHR transfectants suggesting that ACTHR can activate signal transduction pathways still not identified. This conclusion prompted us to search for another signal transduction pathways triggered by ACTHR in Y-1 adrenocortical cells. This search led to the detection of high constitutive levels of activated AKT/PKB in Y-1 adrenocortical cells, which are dependent on PI3K and SRC. ACTH causes a strong and rapid downregulation of activated AKT in a Gs/Adenylate Cyclase/cAMP/PKA dependent manner. Apparently, dephosphorylation of AKT promoted by ACTH releases transcription factors that induce p27'IND.Kip1', a likely mechanism underlying ACTH anti-mitogenic effects in adrenocortical cells.

O hormônio adrenocorticotrópico, ACTH, regula função (esteroidogênese) e proliferação das células da córtex das glândulas adrenais através de um único receptor específico, ACTHR, que pertence à superfamília GPCR (G-protein coupled receptors). Embora o ACTHR tenha sido clonado há 8 anos, os mecanismos moleculares das ações mitogênica e anti-mitogênica de ACTH permanecem obscuros, cuja elucidação é objeto de estudo deste trabalho. A abordagem experimental consistiu na clonagem do ACTHR de células adrenocorticais Y-1 de camundongo e expressão funcional em fibroblastos 3T3 e células AR-1. Clones transfectantes, expressando estavelmente ACTHR, mostraram-se responsivos a ACTH através de: a) ativação de adenilato ciclase e b) indução de genes das famílias fos e jun. Por outro lado, medidas de síntese de DNA e proliferação celular indicam que ACTH não tem nenhum efeito mitogênico ou anti-mitogênico nos transfectantes ACTHR. O gene c-fos foi usado como alvo para testar vias de transdução de sinal ativadas por ACTH nos transfectantes 3T3 ACTHR. Estes testes mostraram que PKA, PKC e MAPK tem pouca ou nenhuma participação na indução de c-fos por ACTH nos clones 3T3 ACTHR, sugerindo que ACTHR pode ativar vias ainda não identificadas e motivando a busca de novas vias ativadas por ACTH nas células Y-1. Verificou-se que células Y-1 apresentam níveis constitutivamente elevados de AKT/PKB ativada (fosforilada), dependentes de PI3K e SRC, e que ACTH promove rápida desfosforilação de AKT via Gs/Adenilato Ciclase/cAMP/PKA. Ao promover a inativação de AKT, ACTH promove simultaneamente a indução da proteína p27'IND.Kip1', um mecanismo que contribui para a atividade anti-mitogênica de ACTH. / The adrenocorticotropic hormone, ACTH, regulates both function and proliferation of adrenocortical cells binding to a specific receptor, ACTHR, which belongs to superfamily of GPCR (G-protein coupled receptors). ACTHR was cloned a few years ago, but the molecular mechanisms underlying the mitogenic and anti-mitogenic actions of ACTH remain unknown, whose elucidation is aim of this project. The experimental approach was to clone the ACTHR from mouse Y-1 adrenocortical cells and to express the functional receptor in Balb 3T3 fibroblasts and AR-1 cells. Transfectant clones stably expressing the ACTHR respond to ACTH treatments by: a) adenylate cyclase activation and b) induction of fos and jun genes. On the other hand, experiments of DNA synthesis and cellular proliferation showed that ACTH has not mitogenic or anti-mitogenic effects in ACTHR transfectants. c-fos gene was used as a target to test signal transduction pathways activated by ACTH into 3T3 ACTHR transfectants. The results showed that PKA, PKC and MAPK have no relevant contribution on the ACTH c-fos induction in 3T3 ACTHR transfectants suggesting that ACTHR can activate signal transduction pathways still not identified. This conclusion prompted us to search for another signal transduction pathways triggered by ACTHR in Y-1 adrenocortical cells. This search led to the detection of high constitutive levels of activated AKT/PKB in Y-1 adrenocortical cells, which are dependent on PI3K and SRC. ACTH causes a strong and rapid downregulation of activated AKT in a Gs/Adenylate Cyclase/cAMP/PKA dependent manner. Apparently, dephosphorylation of AKT promoted by ACTH releases transcription factors that induce p27'IND.Kip1', a likely mechanism underlying ACTH anti-mitogenic effects in adrenocortical cells.