Rozprawy doktorskie na temat „GTP Homeostasis”

Obesity has grown to epidemic proportions, and in lack of efficient life-style and medical treatments, the bariatric surgeries are performed in rising numbers. The most common surgery is the Gastric Bypass (GBP) surgery, with the Biliopancreatic diversion with duodenal switch (DS) as an option for the most extreme cases with a BMI>50 kg/m2. In paper I 20 GBP-patients were examined during the first post-operative year regarding the natriuretic peptide, NT-ProBNP, which is secreted from the cardiac ventricles. Levels of NT-ProBNP quickly increased during the first post-surgery week, and later established itself on a higher level than pre-surgery. In paper II we report of 5 patient-cases after GBP-surgery with severe problems with postprandial hypoglycaemia that were successfully treated with GLP-1-analogs. The effect of treatment could be observed both symptomatically and in some cases using continuous glucose measuring systems (CGMS). In paper III three groups of subjects; 15 post-GBP patients, 15 post-DS, and 15 obese controls were examined for three days using CGMS during everyday life. The post-GBP group had high glucose variability as measured by MAGE and CONGA, whereas the post-DS group had low variability. Both post-operative groups exhibited significant time in hypoglycaemia, about 40 and 80 minutes per day <3.3mmol/l and 20 and 40 minutes < 2.8mmol/l, respectively, longer time for DS-group. Remarkably, only about 20% of these hypoglycaemic episodes were accompanied with symptoms. In Paper IV the hypoglycaemia counter regulatory system was investigated; 12 patients were examined before and after GBP-surgery with a stepped hypoglycaemic hyperinsulinemic clamp. The results show a downregulation of symptoms, counter regulatory hormones (glucagon, cortisol, epinephrine, norepinephrine, growth hormone), incretin hormones (GLP-1 and GIP), and sympathetic nervous response. In conclusion patients post bariatric surgery exhibit a downregulated counter regulatory response to hypoglycaemia, accompanied by frequent asymptomatic hypoglycaemic episodes in everyday life. Patients suffering from severe hypoglycaemic episodes can often be treated successfully with GLP-1-analogues.

L'épithélium intestinal, en constant renouvellement, assure de nombreuses fonctions vitales comme l'absorption des nutriments et le maintien d'une barrière entre le milieu extérieur et l'organisme. L'absorption intestinale des sucres est assurée par de nombreux transporteurs au niveau de l'intestin proximal. Parmi eux, GLUT2, localisé dans les entérocytes et les cellules endocrines de l'intestin, transporte le glucose, le fructose et le galactose. Les cellules L entéroendocrines produisent le GLP-1, un puissant stimulateur de la sécrétion d'insuline en réponse au glucose. L'objectif de ma thèse a été d'élucider le rôle de GLUT2 intestinal dans l'absorption des sucres et la fonction entéroendocrine grâce à l'étude d'un modèle murin spécifiquement invalidé pour ce transporteur dans les cellules épithéliales intestinales. La délétion intestinale de GLUT2 entraîne une malabsorption intestinale modérée des sucres associée à une distribution retardée du glucose aux tissus périphériques. Le retard spatial et temporel de l'absorption des sucres provoque une dysbiose intestinale au profit de bactéries ayant un rôle protecteur de l'homéostasie intestinale. De façon surprenante, l'invalidation de GLUT2 intestinal s'accompagne d'une chute de la densité de cellules L entéroendocrines, sans modification des niveaux plasmatiques de GLP-1. Cette étude met en exergue le rôle primordial de GLUT2 intestinal dans l'absorption des sucres et la fonction endocrine de l'intestin. Elle permet d'envisager le criblage de molécules capables d'inhiber l'activité de GLUT2 intestinal, pour atténuer la prise de poids et limiter les perturbations métaboliques induites par des régimes riches en sucres
The constantly renewing intestinal epithelium handles various essential functions including nutrient absorption and persistence of a barrier between our internal and external environments. Several transporters mediate sugar absorption in the proximal intestine. Among them, GLUT2, a very efficient glucose, fructose and galactose transporter and receptor, is located at the membranes of enterocytes and enteroendocrine cells. The enteroendocrine L-cells produce GLP-1, a strong activator of glucose-induced insulin secretion. This thesis aimed to further decipher the role of intestinal GLUT2 in sugar absorption and enteroendocrine cell function. To address this question, mice lacking GLUT2 specifically in intestinal epithelial cells have been generated and studied. Intestinal GLUT2 invalidation alters intestinal glucose absorption and delays glucose biodistribution to peripheral tissues. This spatial and temporal sugar absorption delay provokes intestinal dysbiosis, favoring gut microbiota having a protective impact on gut homeostasis. Surprisingly, intestinal GLUT2 deletion leads to a strong loss in enteroendocrine L cell density, with no impact on GLP-1 plasma levels. This study highlights critical roles for GLUT2 in sugar absorption and enteroendocrine cell function management. The use of specific GLUT2 inhibitors could be considered to limit body weight gain and metabolic disorders induced by sugar rich diets

A possível variabilidade na resposta fisiológica hormonal após a alimentação, relacionada à elevação do GIP pós-prandial entre indivíduos, despertou o interesse para avaliar a influência do polimorfismo no gene para o receptor do polipeptídeo insulinotrópico dependente de glicose (GIPR) na homeostase glicêmica e na função da célula beta pancreática. Foram estudados 25 adultos (12 mulheres, 13 homens) com IMC<30 e sem doenças relacionadas à síndrome metabólica. Foram coletadas amostras de sangue nos tempos basal (T0) e após refeição contendo 58g de carboidratos e 28g de lipídeos a cada 30 minutos até o T150, para dosagem de insulinemia e glicemia. Foi realizada pesquisa da variante Glu354Gln para o GIPR (SNPGIPR, rs1800437) em amostra de sangue periférico pela técnica de PCRRFLP. Foi avaliada a homeostase glicêmica de jejum e pós-prandial através dos cálculos de HOMA-IR, HOMA-B, área sob a curva (AUC) de insulina e glicemia e sua razão (AUCi/AUCg), índice insulinogênico (IGI) em cada tempo relativo ao basal e razão IGI/HOMA-IR. Na curva pós-prandial, 72% dos indivíduos apresentaram picos de glicose e 64% de insulina até o T60, com média de 107 mg/dl e 42,1 UI/mL, respectivamente. A pesquisa do SNP-GIPR revelou alelo C presente (C+, polimórfico) em 7 indivíduos, com 72% dos genótipos GG, 24% GC e 4% CC. Houve diferença significativa entre C+ e o grupo com alelo C ausente (C-) em relação ao HOMA-B (100+/-26 vs 160+/- 189%, respectivamente; p=0,04). Não houve diferença estatisticamente significativa quanto aos seguintes parâmetros: AUCg, AUCi, HOMA-IR, IGI e razões AUCi/AUCg e IGI/HOMA-IR. Porém, em todos os tempos, o IGI foi superior no grupo C+ (T30, 189%; T60, 147%; T90, 1066%; T120, 29% e T150, 93%), enquanto que uma tendência semelhante ocorreu para a razão IGI/HOMA-IR exceto no T150. O indivíduo portador do SNP-GIPR em homozigose (CC) apresentou resposta precoce de insulina. Além disso, 42,9% dos indivíduos C+ apresentaram história familiar de diabetes em comparação com 22,2% do grupo C- (p=0,29). Em conclusão, estudamos o perfil de glicemia e insulina de jejum e na curva pós-prandial incluindo 1ª e 2ª fases de secreção insulínica de uma amostra de indivíduos sadios com distribuição genotípica do SNP-GIPR semelhante à população reportada na literatura. Indivíduos euglicêmicos C+ e C- apresentaram semelhanças em diversos dos parâmetros utilizados para avaliar a função da célula beta pancreática e resistência insulínica. Por outro lado, foi encontrada variação no HOMA-B entre os genótipos estudados, sugerindo redução na função da célula beta relacionada ao polimorfismo do GIPR, sem alteração na sensibilidade à insulina. A redução do HOMA-B em indivíduos sadios, independente do histórico familiar de diabetes, sugere aprofundar o estudo deste SNP-GIPR como possível marcador precoce para o risco de falência pancreática associada ao diabetes tipo 2.
The potential variability in hormonal physiological response after feeding, related to the elevation of postprandial GIP between individuals, arouse interest to evaluate the influence of polymorphisms in the gene for glucose-dependent insulinotropic polypeptide receptor (GIPR) in glucose homeostasis and in pancreatic beta cell function. Altogether 25 adults (12 women, 13 men) with BMI <30 and without metabolic syndrome related diseases were studied. After diet containing 58g of carbohydrates and 28g of lipids, blood samples at baseline (T0) and every 30 minutes until the T150 were collected for measurement of insulin and glucose. The polymorphic GIPR variant Glu354Gln, rs1800437 (SNP-GIPR) was studied in peripheral blood sample through PCR-RFLP. Glucose homeostasis in fasting and postprandial was assessed by calculating HOMA-IR, HOMA-B, area under the curve (AUC) of insulin and glucose, AUCi/AUCg ratio, insulinogenic index (IGI) at each time relative to baseline and IGI/HOMA-IR ratio. In the postprandial curve, 72% of subjects showed glucose peaks and 64% insulin until the T60, with mean of 107 mg/dl and 42.1 IU/mL, respectively. The SNP-GIPR study revealed presence of C allele (C+) in 7 individuals, with 72% of the genotypes GG, 24% GC and 4%CC. There was a significant difference between C + or the group without C allele (C-) in HOMA-B (100 +/- 26 vs. 160 +/- 89 % respectively; p = 0.04).There was no significant difference in the following parameters: AUCg, AUCi, HOMA-IR, IGI, AUCi/AUCg and IGI/HOMA-IR. However, the IGI was higher in the C+ allele group at all time-points (T30, 189%; T60, 147%; T90, 1066%; T120, 29% and T150, 93%), while a similar trend occurred for the IGI/HOMA-R ratio, except in T150. Furthermore, family history of diabetes was positive in 42.9% of C+ subjects, compared with 22.2% from C- group (p = 0.29). In conclusion, we have studied the fasting and postprandial variation of glucose and insulin, including insulin secretion’s 1st and 2nd phases of healthy subjects; our sample presented genotype distribution similar to the reported population in the literature. C+ and C- euglycemic subjects had similar parameters of beta cell function and insulin sensitivity. In other way, we found variation in HOMA-B according to the genotype, suggesting a reduction in beta cell function related to GIPR polymorphism, without change in insulin sensitivity. The reduction in HOMA-B in healthy individuals, regardless of family history of diabetes, suggests further study of this SNP-GIPR as a possible early marker for the risk of pancreatic failure associated with type 2 diabetes.

L’obésité et le diabète de type 2 (DT2) sont des pathologies métaboliques associées à des perturbations de l’homéostasie glucidique et énergétique. Les enterohormones sont des acteurs importants de la regulation des mécanismes perturbés lors de ces pathologies. Parmi ces enterohormones, le GLP-1, sécrété par les cellules entéroendocrines de type L suite à un repas, permet d’amplifier la sécrétion d’insuline par les cellules β-pancréatiques et de diminuer la prise alimentaire. L’objectif de ma thèse a été de caractériser le rôle du récepteur nucléaire HNF-4γ dans l’homéostasie énergétique et la fonction endocrine de l’intestin.A l’aide d’un modèle murin d’invalidation totale et constitutive du facteur de transcription HNF-4γ, notre équipe a mis en évidence que l’absence de HNF-4γ induit une amélioration de la tolérance au glucose grâce à une augmentation du nombre de cellules L et de la quantité plasmatique de GLP-1 en réponse au glucose. L’ensemble de ces données démontre pour la première fois un rôle de HNF-4γ dans l’homéostasie glucidique via une modulation du lignage enteroendocrine spécifique du GLP-1 et suggère que son absence pourrait protéger les souris de l’établissement d’un DT2.Par ailleurs, la perte d’expression de HNF-4γ confère une protection vis-à-vis de la prise de poids et de l’intolérance au glucose normalement induites par six semaines d’un régime riche en lipides et en fructose grâce une perte énergétique accrue dans les fécès essentiellement due à une malabsorption des acides gras.En conclusion, cette étude met en exergue le rôle du récepteur nucléaire intestinal HNF-4γ dans la fonction enteroendocrine et la susceptibilité à l’obésité et au DT2
Obesity and type 2 diabetes (T2D) are metabolic pathologies associated with glucose and energy homeostasis perturbations. Enterohormones are important players in the regulation of the mechanisms disturbed during these pathologies. Among these enterohormones, GLP-1, secreted by enteroendocrine L cells in response to a meal, potentiates insulin secretion by pancreatic β cells and inhibits food intake. The aim of my thesis was to characterize the role of the nuclear receptor HNF-4γ in the energy homeostasis and the endocrine function of the intestine.By using a total and constitutive HNF-4γ knock-out mouse model, our team has highlighted that the loss of hnf-4γ induces an improved glucose tolerance. This effect is due to an increased GLP-1 cell number and GLP-1 plasma levels in response to glucose. All together these data demonstrate for the first time a role of HNF-4γ in glucose homeostasis through a modulation of the enteroendocrine lineage specific for GLP-1 and suggest that its absence could protect mice from the T2D establishment.The loss of HNF-4γ protects mice from body weight gain and glucose intolerance normally induced by six weeks of a high-fat/high-fructose diet demonstrating its involvement in obesity and T2D. HNF-4γ -/- mice are protected from obesity by a greater energy loss in faeces mainly due to lipid malabsorption. These results demonstrate that HNF-4γ is necessary for the intestinal fatty acids uptake.In conclusion, this study highlights the role of the intestinal nuclear receptor HNF-4γ in enteroendocrine function and susceptibility to obesity and T2D

Mestrado em Biologia Molecular e Celular
A síntese proteica é um processo essencial para que todos os organismos mantenham a homeostasia celular. Os tRNAs são elementos cruciais na síntese proteica, uma vez que codificam a informação genética presente no mRNA. A linha celular HeLa, utilizada neste estudo, foi primeiramente isolada de uma mulher com cancro do colo do útero e desde então tem sido bastante usada na investigação, sendo muito importante no estudo das bases moleculares de muitas doenças. De modo a monitorizar a agregação proteica nesta linha celular, um sistema repórter foi desenvolvido utilizando uma fusão entre HspB1 (Hsp27) e a GFP. HspB1 é um chaperone molecular com capacidade de recrutar outros chaperones e restabelecer a conformação ideal das proteínas em situações de stress. A GFP é uma proteína fluorescente que marca certas condições biológicas de interesse. Para perceber o impacto dos erros da tradução na agregação de proteínas e no surgimento das doenças, o principal objetivo deste estudo foi desenvolver uma linha celular estável (HeLa) expressando um sistema repórter HspB1-GFP, de modo a monitorizar os erros no enovelamento das proteínas em resposta ao stress proteotóxico. Ao longo deste estudo o sistema repórter expressando HspB1-GFP foi desenvolvido com sucesso, permitindo assim a sua utilização para identificar situações fisiológicas e patológicas em que a agregação de proteínas ocorre em células de mamífero.
Protein synthesis is essential for all organisms to maintain cell homeostasis. tRNAs are crucial elements in protein synthesis as they decode the genetic information organized in the mRNA codons. A HeLa cell line, used in this study, was first isolated from a woman with cervical cancer and since then was highly used in biological studies, being extremely important in the study of the molecular basis of several diseases. In order to monitor protein aggregation in this cell line, a reporter system was developed using an HspB1 (Hsp27) and a GFP fusion. HspB1 is a small heat shock protein that, in stress situations, recruits other proteins in order to restore the conformation of the proteins. GFP is a biosensor that reports several cellular conditions of interest. To understand the impact of translation errors on protein aggregation and on the disease arising, the main goal of this study was to develop a stable cell line (HeLa) expressing a reporter system HspB1-GFP to monitor the protein misfolding in response to proteotoxic stress. During this study, the reporter system expressing HspB1-GFP was developed successfully, allowing the identification of physiological and pathological situations where protein aggregation occurs in mammalian cells.

Nucleotide based second messengers are known to regulate wide variety of processes in all domains of life. Two such bacterial second messengers are (p)ppGpp (guanosine tetra- or pentaphosphate) and c-di-GMP (cyclic dimeric guanosine monophosphate). The alarmone (p)ppGpp is synthesized by bacteria to face any kind of stress; while the signalling nucleotide c-di-GMP is synthesized principally to switch from motile (planktonic) to sessile (biofilm) life style. Apart from mediating the said functions, these nucleotides also regulate transcription, translation, replication, virulence and pathogenicity of the several bacterial species. In this work, we have tried to uncover novel functions or phenotypes that are governed by the second messengers (p)ppGpp and c-di-GMP in Mycobacterium smegmatis. In M. smegmatis, (p)ppGpp and c-di-GMP are synthesized and degraded by the bifunctional proteins RelMsm and DcpA, respectively. The architecture of both the proteins is similar; the synthesis and hydrolysis domains for the second messengers occur in tandem. The knockout mutants of relMsm and dcpA genes, ∆relMsm and ∆dcpA, have been used in this study to uncover the novel functions of these second messengers in mycobacterial physiology. Chapter 1 provides is an overview of the current literature pertaining to (p)ppGpp and c-di-GMP. An historical perspective with regard to the discovery of the (p)ppGpp and c-di-GMP is given. The metabolism of these second messengers has been discussed. This has been followed by the description of various functions governed by the second messengers. Finally, the scope of the current work has been outlined. Chapter 2 investigates the effect of disrupting (p)ppGpp and c-di-GMP signalling on the antibiotic sensitivity in M. smegmatis. Using Phenotype Microarray (PM) technology, the growth of ∆relMsm and ∆dcpA knock out strains was compared to those of the wild-type and respective complemented strains in 240 different antimicrobials. It was found that the knockout mutants displayed enhanced survival in the presence of multiple antibiotics. The PM data was corroborated by the independent determination of minimum inhibitory concentrations of seven different antibiotics. Finally, the plausible reasons for the multidrug resistance of ∆relMsm and ∆dcpA strains have been discussed. Chapter 3 explores how the impairment of (p)ppGpp and c-di-GMP alters the cell wall of M. smegmatis. Thin layer chromatography analysis of cell wall fractions such as glycopeptidolipids (GPLs), mycolic acids, polar and apolar lipids was carried out. It was found that the amount of GPLs and polar lipids were reduced in the ∆relMsm and ∆dcpA knockout strains. Chapter 4 explores the effect of (p)ppGpp and c-di-GMP on the growth, cell morphology and cell division in M. smegmatis. It was found that the ∆relMsm and ∆dcpA knockout strains have slow growth compared to those of the wild type and respective complemented strain. The overproduction of (p)ppGpp and c-di-GMP, achieved through overexpression of Rel and DcpA proteins, encased the overexpression strains relOE and dcpAOE in a biofilm like matrix. The higher levels of (p)ppGpp and c-di-GMP caused M. smegmatis assume coccoid morphology. Microscopy analyses revealed that the ∆relMsm and ∆dcpA strains are elongated, multinucleate and multiseptate. Chapter 5 explores effects of (p)ppGpp and c-di-GMP on the global gene expression profile in M. smegmatis. Many genes were shown to be differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Genes regulating cell division, cell wall biosynthesis, superoxide metabolism or reactive oxygen species metabolism and genes encoding transporters were differentially expressed in the ∆relMsm and ∆dcpA knockout mutants. The microarray data were corroborated by quantitative real-time PCR. Gene expression data explained the multidrug resistance, the reduction in the level of GPLs and polar lipids, slow growth, changes in cell morphology and defective cell division exhibited by the ∆relMsm and ∆dcpA knockout mutants. Chapter 6 summarizes the entire work embodied in the thesis. Appendix 1 lists the 240 antimicrobials compounds and their mode of action for which antibiotic sensitivity of the ∆relMsm and ∆dcpA knockout mutants was tested. Appendix 2 lists the growth differences among the knockout, wild type and complemented strains in the form of area under curve values. Appendix 3 lists the genes that were differentially expressed in the ∆relMsm and ∆dcpA knockout strains. Appendix 4 is a comprehensive review on the kinetic and thermodynamic parameters governing the sigma factor competition in Escherichia coli and how (p)ppGpp and anti-sigma factors regulate this competition among sigma factors for the limited pool of core RNA polymerase in E. coli.

Mechanisms to augment the cellular function and mass of beta-cells may be effective means of treating type 2 diabetes. Important in the physiological control of beta-cell function and nutrient disposal are factors released from gut enteroendocrine cells during nutrient digestion. In enteroendocrine L-cells, post-translational processing of proglucagon gives rise to a number of proglucagon-derived peptides. One such peptide, glucagon-like peptide-1 (GLP-1), acts via its own receptor (GLP-1R) to stimulate beta-cell insulin secretion, proliferation and survival. Another, oxyntomodulin (OXM), weakly activates the GLP-1R and inhibits food intake in a GLP-1R-dependent manner in rodents, which led us to hypothesize that OXM modulates GLP-1R-dependent glucoregulation. While OXM did not mimic the inhibitory effect of GLP-1 on gastric emptying in mice, OXM stimulated insulin secretion, beta-cell survival and improved glucose tolerance in a GLP-1R-dependent manner. In a similar manner to GLP-1, glucose-dependent insulinotropic polypeptide (GIP), secreted from enteroendocrine K-cells, physiologically stimulates insulin secretion via a distinct GIP receptor (GIPR) in beta-cells. Beyond the beta-cell, GIP and GLP-1 appear to exert divergent actions for the control of glucose homeostasis. Moreover, I illustrate that physiological and pharmacological GLP-1R signalling may be comparatively more important for the preservation of beta-cell mass and glucose homeostasis in murine streptozotocin-induced diabetes. Lastly, studies in rodents and humans have showed that metformin increases circulating levels of GLP-1, leading us to hypothesize that GIP and GLP-1 may be involved in the glucoregulatory effects of metformin. Interestingly, transcripts for the Glp1r and Gipr were significantly increased within islets of metformin-treated mice, and metformin treatment enhanced the sensitivity of cultured beta-cells to GIP and GLP-1. In summary, these studies illustrate mechanisms by which enteroendocrine peptides compare and contrast with respect to beta-cell survival and function and the control of glucose homeostasis.

Our laboratory has demonstrated previously that the proglucagon gene (gcg), which encodes the incretin hormone GLP-1, is among the downstream targets of the Wnt signaling pathway; and that Pak1 mediates the stimulatory effect of insulin on Wnt target gene expression in mouse gut non- endocrine cells. Here, I asked whether Pak1 controls gut gcg expression and GLP-1 production, and whether Pak1 deletion leads to impaired metabolic homeostasis in mice. I detected the expression of Pak1 and two other group I Paks in the gut endocrine L cell line GLUTag, and co-localized Pak1 and GLP-1 in the mouse gut. Insulin was shown to stimulate Pak1 Thr423 and β-cat Ser675 phosphorylation. The stimulation of insulin on β-cat Ser675 phosphorylation, gcg promoter activity and gcg mRNA expression could be attenuated by the Pak inhibitor IPA3. Male Pak1-/- mice showed significant reduction in both gut and brain gcg expression levels, and attenuated elevation of plasma GLP-1 levels in response to oral glucose challenge. Notably, the Pak1-/- mice were intolerant to both intraperitoneal and oral glucose administration. Aged Pak1-/- mice showed a severe defect in response to intraperitoneal pyruvate challenge (IPPTT). In primary hepatocytes, however, IPA3 reduced basal glucose production, attenuated glucagon-stimulated glucose production, and inhibited the expression of Pck1 and G6pc. This implicates that the direct effect of group I Paks in hepatocytes is the stimulation of gluconeogenesis, and that the impairment in IPPTT in aged Pak1-/- mice is due to the lack of Pak1 elsewhere. The defect in IPPTT in aged Pak1-/- mice could be rescued by stimulating gcg expression with forskolin injection or by enhancing the incretin effect via sitagliptin administration. In summary, my study demonstrates that: 1) Pak1 positively regulates GLP-1 production, 2) Pak1/β-cat signaling plays a role in gut/liver axis or gut/pancreas/liver axis governing glucose homeostasis, and 3) Pak1-/- mice can be utilized as a novel model for metabolic research.

Tese de doutoramento em Ciências da Saúde, no ramo de Ciências Biomédicas, apresentada à Faculdade de Medicina da Universidade de Coimbra
O envelhecimento é um processo complexo que ocorre em todos os organismos, da levedura ao Homem. Apesar de um século de pesquisa e discussão científica, os factores subjacentes à progressão do envelhecimento permanecem por clarificar. Mas os ângulos sob os quais este processo é visto sofreram grandes mudanças com o tempo. As primeiras teorias sugeriam que o envelhecimento decorre da acumulação estocástica de danos nas macromoléculas, levando ao mal funcionamento dos organismos e, por fim, à morte dos mesmos. Esta área de investigação foi radicalmente transformada nas últimas décadas por uma série de estudos pioneiros em diferentes modelos animais que mostraram claramente que o envelhecimento pode ser alterado através da manipulação de várias vias metabólicas e genéticas. Estas descobertas sugeriram que o nível de protecção de um organismo contra danos estocásticos pode ser regulado e, consequentemente, também o período de vida durante o qual o mesmo permanece saudável. No entanto, à medida que o conhecimento acerca destes mecanismos foi maturando, tornou-se evidente que a duração de vida, a resistência a stress e a homeostase proteica, aspectos que são regulados pelas vias que regulam o envelhecimento, podem ser desacopladas sem se influenciarem mutuamente. Mais recentemente, o processo de envelhecimento revelou possuir um nível adicional de complexidade quando se mostrou que pode ser coordenado por diferentes tecidos ao nível do organismo. Neste trabalho, o nosso interesse focou-se nos princípios subjacentes à orquestração do envelhecimento ao nível do organismo, bem como na dissociação entre duração de vida, resistência a stress e homeostase proteica. De modo a abordar estes temas, usámos o nemátode Caenorhabditis elegans (C. elegans), modelo animal que oferece inúmeras vantagens no estudo do envelhecimento. Começámos por investigar os mecanismos de comunicação entre tecidos que regulam a heat shock response (HSR) a nível do organismo no modelo C. elegans, procurando, mais concretamente, esclarecer que receptores neuronais estão envolvidos neste mecanismo de sinalização e em que neurónios desempenham a sua função. Para responder a estas questões, empregámos nemátodes geneticamente modificados que apresentam hipersensibilidade a RNA de interferência (RNAi) no tecido nervoso e identificámos um presumível receptor acoplado a proteínas G (GPCR) como sendo um componente-chave deste mecanismo. Este gene, a que atribuímos o nome gtr-1, é expresso em neurónios quimiosensoriais e desempenha um papel fundamental na indução de genes que codificam proteínas de heat shock nos tecidos somáticos após exposição a temperaturas elevadas, apesar de não ser necessário à percepção de calor. Surpreendentemente, o knockdown do gtr-1 através de RNAi tem um efeito protector em nemátodes que expressam nos músculos Aβ3-42 (um péptido com tendência agregativa associado à doença de Alzheimer), mas não influencia a duração de vida, a resistência a outros stresses ou funções associadas ao desenvolvimento. Na segunda parte deste trabalho pretendemos fazer uma caracterização mais detalhada dos elementos downstream à via de sinalização da insulina/IGF-1 (IIS) que estão directamente envolvidos na regulação da toxicidade proteica em C. elegans. Com este objectivo, procurámos genes previamente citados na literatura como reguladores da homeostase proteica e identificámos o tor-2 como sendo regulado ao nível da transcrição pela via IIS. Nesta tese mostramos que a expressão do tor-2 é induzida após a supressão desta via pelos factores de transcrição DAF-16 e SKN-1. Este gene revelou-se importante na resistência a temperaturas elevadas mas não na regulação do tempo de vida do animal ou na resistência a outros tipos de stress tais como exposição a bactérias patogénicas ou a radiação UV. Curiosamente, o tor-2 parece ser importante no combate à toxicidade proteica em neurónios, onde se mostrou anteriormente que este gene é expresso, ao passo que o seu knockdown protege os nemátodes que expressam proteínas agregativas tóxicas nos músculos. Este estudo oferece novas ideias: (1) que os neurónios quimiosensoriais desempenham um papel importante nos mecanismos que regulam a HSR no nemátode; (2) que o tempo de vida e a resistência a heat shock são separáveis; (3) consolida o conceito emergente de que a habilidade para responder o calor existe em detrimento da manutenção da proteostase; e (4) sugere que a homeostase proteica pode ser diferencialmente regulada de tecido para tecido por um único gene.
Aging is a complex process that occurs in organisms ranging from yeast to humans. The factors underlying the progression of aging still elude us, despite a century of scientific inquiry and discussion. Nevertheless, the angles from which aging was perceived have greatly changed over time. Early theories suggested that aging results from the accumulation of stochastic damage to macromolecules, leading to organismal malfunction and ultimately death. The field was however revolutionized over the last decades by a series of pioneering studies carried out in model organisms that showed that aging can actually be altered by the modification of several metabolic and genetic pathways. These findings suggested that the level of protection against stochastic damage can be regulated and, hence, the length of time an organism remains healthy. However, as the knowledge on these mechanisms matured, it became evident that lifespan, stress resistance, and protein homeostasis (proteostasis), aspects that are regulated by the aging-modulating pathways, can be uncoupled without influencing one another. The aging process revealed another level of complexity when it was shown to be coordinated by different tissues in an organismal-fashion. In this work, we were interested in the principles underlying the orchestration of aging at the organismal level, as well as in the uncoupling between lifespan, stress resistance, and proteostasis. To address these questions, we employed the nematode Caenorhabditis elegans (C. elegans), which offers key advantages in the study of aging. We started by focusing on the inter-tissue communication mechanisms that regulate the heat shock response (HSR) at the organismal level in C. elegans and attempted to clarify which neuronal receptors are required for this signaling mechanism and in which neurons they function. To answer these questions, we employed worms that were engineered to exhibit RNA interference (RNAi) hypersensitivity in neurons and identified a putative G protein-coupled receptor (GPCR) as a novel key component of this mechanism. This gene, which we termed GPCR thermal receptor 1 (gtr-1), is expressed in chemosensory neurons and has no role in heat sensing but is critically required for the induction of genes that encode heat shock proteins in non-neural tissues upon exposure to heat. Surprisingly, the knockdown of gtr-1 by RNAi protected worms expressing the Alzheimer's-disease-linked aggregative peptide Aβ3-42 in their body-wall muscles from protein toxicity (proteotoxicity) but had no effect on lifespan, resistance to other stresses, or developmental functions. In the second part, we aimed at better characterizing the insulin/IGF-1 signaling (IIS)-downstream components involved in the direct regulation of protein toxicity (proteotoxicity) in the C. elegans model. For this, we searched for genes that are known regulators of proteostasis and identified tor-2 as a transcriptional target of the IIS pathway. Here we show that tor-2 is upregulated upon suppression of the IIS by both DAF-16 and SKN-1transcription factors. This gene is important for the resistance to heat shock but has no role in the determination of lifespan or in the resistance to other acute stresses such as exposure to pathogenic bacteria or to UV radiation. Interestingly, tor-2 seems to be important to counteract proteotoxicity in neurons, previously shown to be its main site of expression, whereas its knockdown protects worms that express toxic, aggregative-proteins in their body-wall muscles. In this work we provide several novel insights: (1) we show that chemosensory neurons play important roles in the nematode's HSR-regulating mechanism; (2) that lifespan and heat stress resistance are separable; (3) we strengthen the emerging notion that the ability to respond to heat comes at the expense of proteostasis; and (4) suggest that proteostasis can be differentially regulated in a tissue-specific manner by a sole gene.
FCT - SFRH/BD/70502/2010

Caloric restriction (CR), due to its negative energy balance and GLP-1, for being an incretin, constitute two weight loss protocols that remain poorly investigated, particularly its impact on male reproductive function, whose proper functioning depends on a balanced energy homeostasis. That energy homeostasis has been related to genes linked to energy metabolism control, namely the obesity-related genes (ORGs), such as fat mass and obesity associated (FTO), melanocortin-4 receptor (MC4R), glucosamine-6- phosphate deaminase 2 (GNPDA2) and transmembrane protein 18 (TMEM18). Thus, this project aimed to use an animal model to study the potential of CR and GLP-1 administration in the regulation of those ORGs expression in testes and sperm of Wistar rats and verify if there is an impact on sperm quality. Firstly, we observed that CR promoted a lower weight gain and a decrease in insulin resistance. On the other hand, GLP-1 administration, beyond the expected increase in active GLP-1 levels, did not promote any change in glucose metabolism, hormonal profile and consequently in body weight. Furthermore, CR promoted an increase in sperm head defects, while the GLP-1 administration improved sperm morphology. Regarding the ORGs we observed the presence of FTO, MC4R and TMEM18 transcripts in rat testes and identified those transcripts, for the first time, in rat sperm. Additionally, we identified, for the first time, the presence of GNPDA2 transcripts in rat testes and sperm. The corresponding proteins were also identified in rat testes, being that they all presented distinct cellular locations. CR and GLP-1 administration promoted an increase in the expression of the ORGs in testes, however in spermatozoa the expression was not altered. We also identified the presence of NFE2L2 (encodes for an important antioxidant transcription factor) transcripts in rat testes and sperm and verified that their abundance is increased, in testes, by CR and GLP-1 administration. Then we explored, individually, the role the ORGs at the testicular and sperm levels, namely in the response to CR and GLP-1 administration and we obtained some clues related to the potential involvement of each of the ORGs in the male reproductive function. Overall, we were able to perceive that CR and GLP-1 administration promoted a general association of all ORGs with an improvement in oxidative status both in testes and sperm.
A restrição calórica (CR), pelo seu balanço energético negativo e o GLP-1, por ser uma incretina, constituem dois protocolos de perda de peso, que permanecem pouco investigados, principalmente no que toca ao seu impacto na função reprodutiva masculina, cujo correto funcionamento depende de uma homeostase energética equilibrada. Essa homeostase energética tem sido relacionada a genes ligados ao controlo do metabolismo energético, nomeadamente os genes relacionados à obesidade (ORGs), como gene de obesidade e de massa de gordura associada (FTO), recetor de melanocortina-4 (MC4R), glucosamina-6-fosfato desaminase 2 (GNPDA2) e proteína transmembranar 18 (TMEM18). Assim, este projeto teve como objetivo utilizar um modelo animal para estudar o potencial da CR e da administração de GLP-1 na regulação da expressão desses ORGs nos testículos e esperma de ratos Wistar e verificar se há impacto na qualidade espermática. Primeiramente, observamos que a CR promoveu menor ganho de peso e diminuição da resistência à insulina. Por outro lado, a administração de GLP-1, além do esperado aumento nos níveis de GLP-1 ativo, não promoveu qualquer alteração no metabolismo da glucose, no perfil hormonal e consequentemente no peso corporal. Para além disso, a CR promoveu um aumento nos defeitos da cabeça dos espermatozoides, enquanto a administração de GLP-1 melhorou a morfologia espermática. Em relação aos ORGs, observamos a presença de transcritos de FTO, MC4R e TMEM18 em testículos de ratos e identificamos esses transcritos, pela primeira vez, em esperma de ratos. Além disso, identificamos, pela primeira vez, a presença de transcritos GNPDA2 em testículos e esperma de ratos. As proteínas correspondentes também foram identificadas em testículos de ratos, sendo que todas apresentavam localizações celulares distintas. A CR e a administração de GLP-1 promoveram um aumento na expressão dos ORGs nos testículos, porém no esperma a expressão não foi alterada. Também identificamos a presença de transcritos de NFE2L2 (codifica para um importante fator de transcrição antioxidante) em testículos e esperma de ratos e verificamos que sua abundância é aumentada nos testículos, pela CR e administração de GLP-1. Em seguida, exploramos, individualmente, o papel dos ORGs nos níveis testicular e espermático, nomeadamente na resposta à CR e à administração de GLP-1 e obtivemos algumas pistas relacionadas com o envolvimento potencial de cada um dos ORGs na função reprodutiva masculina. No geral, pudemos perceber que as duas intervenções promoveram uma associação geral de todos os ORGs com uma melhoria no estado oxidativo dos testículos e esperma.
Mestrado em Bioquímica

碩士
國立清華大學
生物資訊與結構生物研究所
106
ABSTRACT Tripartite split-GFP complementation assay is a new protein-protein interaction technique improved from bimolecular fluorescence complementation (BiFC). Tripartite split GFP system is composed of a larger fragment, GFP1-9 (residues 1–193), and two shorter β-strands : GFP10 (residues 194–212) and GFP11 (residues 213–233). There are some disadvantages of BiFC because BiFC is based on bulky fragments that may increase the difficulty of protein folding and interfere with protein function. To test whether tripartite split-GFP is a useful tool in planta, we first chose two proteins involved in the phosphate starvation reponse: the phosphate starvation response 1 (PHR1) and the SPX domain-containing protein 1 (SPX1) proteins. The PHR1 and SPX1 sandwitch proteins (GFP10-PHR1-GFP11 and GFP10-SPX1-GFP11) were used for transient expression in tobacco leaves and localized in the nucleus. Next, we confirmed the previous results that PHR1 interacts with SPX1 using tripartite split-GFP system. This assay showed that PHR1 and SPX1 can form homo-dimer/oligomers itself. We used the Arabidopsis nitrogen limitation adaptation (NLA/BAH1) to verify the specificity of the interaction between PHR1 and SPX1. Our results showed that NLA interacts with both PHR1 and SPX1. In addition, we chose the other PHR1 family protein PHR1-like 3 (PHL3) to test the interaction of PHL3 with PHR1 and SPX1 respectively. The signal strength between PHR1, SPX1 and PHL3 are different. From the strongest to the weakest is PHR1 and PHL3, PHR1 dimer, SPX1 and PHR1, and then the signal of SPX1 and PHL3 was the strongest. Furthermore, we generated and obtained the Arabidopsis transgenic liens overexpessing GFP10-PHR1, SPX1-GFP11 and GFP1–9. Confonal analysis of these lines showed few signals in the root hair of seedlings, indicating that the tripartite spli-GFP system used in transgenic plants of Arabidopsis needs to be improved.

Diet-induced obesity causes a central inflammatory process in the brain, more specifically in the arcuate nucleus of the hypothalamus; in addition to substantially altering cholesterol homeostasis in the brain. The brain is one of the richest organs in cholesterol and cholesterol homeostasis has proved to be very important not only to maintain healthy brain physiology, but also directly influencing the whole-body homeostasis. In addition, several metabolic disorders are correlated with different neurodegenerative diseases. Cholesterol in the brain is converted primarily to 24-hydroxycholesterol (24-OHC) by CYP46A1. Changes in oxysterol metabolism have been correlated with obesity. Previous studies from our laboratory have identified CYP46A1 as a relevant therapeutic target, not only for Machado de Joseph disease, but also for other neurodegenerative diseases. One of these studies revealed that the silencing of the expression of the Cyp46a1 gene (using AAV5-shCyp46A1), in the hypothalamus of C57BL/6J mice fed a low fat diet (Chow) (control diet) and a high fat diet (HFD), had a profound impact on the dysregulation of the entire physiological process of body homeostasis. In this sense, additional studies became necessary to demonstrate that the effect of silencing the Cyp46a1 gene in the arcuate nucleus was specific, discarding the effect of the surgical procedure or viral vectors administration. Thus, in this study, our main objective was to control the effect of Cyp46a1 silencing on the hypothalamus (arcuate nucleus). For this, through stereotaxic surgery, a control gene (GFP) was delivered by AAV vectors in the arcuate nucleus; both in animals fed with Chow ration and those fed with HFD ration. The project's main hypothesis is that the GFP protein does not interfere in the physiology of the hypothalamus and consequently in the metabolism of the whole-body, regardless of diet. Our data confirm that stereotaxic surgery and GFP expression did not alter the homeostasis of the hypothalamus and, consequently, there was no change in the whole-body metabolism of the mice.
A epidemia do excesso de peso e obesidade representa um dos mais importantes problemas de saúde pública do século XXI. A obesidade induzida por dieta causa um processo inflamatório central no cérebro, mais especificamente no núcleo arqueado do hipotálamo. O núcleo arqueado é uma região especial do cérebro responsável pela regulação da homeostasia energética corporal, possuindo uma importância crucial na manutenção do equilíbrio entre consumo e gasto energético. Na obesidade há disfunção hipotalâmica e esta disfunção pode alterar substancialmente a homeostasia do colesterol no cérebro. O cérebro é um dos órgãos mais ricos em colesterol e a sua homeostasia do colesterol é muito importante para manter a fisiologia cerebral saudável e também influencia diretamente a homeostasia do resto do organismo. Além disso, o facto de os distúrbios metabólicos estarem correlacionados com diferentes doenças neurodegenerativas, como: doença de alzheimer, doença de Parkinson e doença de Huntington. O colesterol no cérebro é convertido principalmente em 24S-hidroxicolesterol pela enzima Cyp46a1. Alterações no metabolismo do oxisterol foram correlacionadas com a obesidade. Estudos do nosso laboratório identificaram o CYP46A1 como um alvo terapêutico relevante, não apenas para a doença Machado de Joseph, mas também para outras doenças neurodegenerativas. Um desses estudos, revelou que o silenciamento da expressão do gene Cyp46a1 (utilizando-se AAV5-shCyp46A1), no hipotálamo (núcleo arqueado), de morganhos C57BL/6J alimentados com dieta com baixo teor de gordura (Chow – low fat control diet) (dieta controlo) e com uma dieta com alto teor de gordura (HFD – high fat diet), tem impacto profundo na desregulação de todo o processo fisiológico da homeostasia corporal destes ratinhos. Tem ainda grande impacto no perfil fisiológico dos órgãos metabólicos e alterações na morfologia estrutural de cada um deles, como: WAT, BAT, fígado, pâncreas, entre outros. Desta forma, os resultados deste estudo sugerem que o silenciamento do Cyp46a1 nos animais Chow AAV5-shCyp46a1 e HFD AAV-shCyp46a1 resulta em um fenótipo de obesidade, alterações nos níveis de oxisteróis cerebrais e diabetes mellitus tipo 2; além de modificações significativas no comportamento. Os resultados sugerem o papel importante do gene Cyp46a1 na manutenção do metabolismo do colesterol no cérebro e no controlo da homeostasia energética corporal. Diante destas observações, estudos adicionais tornaram-se necessários para comprovar o efeito do silenciamento do gene Cyp46a1 no núcleo arqueado, descartando o efeito do procedimento cirúrgico. Desta forma, neste estudo, o nosso principal objetivo foi controlar o efeito do silenciamento de Cyp46a1 no hipotálamo (núcleo arqueado). Para tal, por meio de cirurgia estereotáxica foi realizada a injeção de um gene controlo (GFP) mediado por vetores AAV no núcleo arqueado; tanto em morganhos alimentados com dieta Chow, como em morganhos alimentados com dieta HFD. A hipótese do projeto é que a proteína GFP não interfere na fisiologia do hipotálamo e consequentemente no metabolismo de todo o corpo, independentemente da dieta. Este estudo foi conduzido durante um período de 12 semanas; no início do estudo os morganhos (C57BL/6J) foram divididos aleatoriamente em dois grupos: Chow (controlo) e HFD. A partir deste momento, cada grupo foi alimentado com a dieta específica por 12 semanas. Na quarta semana do estudo, os dois grupos foram submetidos a injeção estereotáxica bilateral no núcleo arqueado. Os resultados demonstraram que a proteína GFP não interferiu na fisiologia do hipotálamo (núcleo arqueado) e que as injeções estereotáxicas (AAVGFP) não causaram inflamação comprometedora que tenha alterado a fisiologia e o fenótipo alcançado através exclusivamente da dieta administrada (Chow e HFD) aos grupos dos morganhos utilizados. O fenótipo obeso foi previsivelmente alcançado pela indução de dieta gordurosa apenas nos morganhos HFD, assim como, a alteração da morfologia de vários tecidos metabólicos (WAT, BAT, pâncreas e fígado) e houve uma grande acumulação de lípidos nos órgãos. Os morganhos controlo (dieta Chow) não desenvolveram obesidade, não apresentaram alterações na morfologia dos tecidos metabólicos (WAT, BAT, pâncreas e fígado) e também não apresentaram modificações no acúmulo lipídico dos órgãos. Como este estudo é um controlo importante para o projeto de silenciamento do gene Cyp46a1 realizado anteriormente, os resultados aqui apresentados confirmam que a cirurgia estereotáxica e a expressão de GFP não alteram a homeostasia do hipotálamo e consequentemente não há alteração no metabolismo corporal dos morganhos.

The mind-body problem is the problem: what is the relationship between mind and body? In this project, I claim that the relationship between the experience of pain and specific physiological mechanisms is best understood as one of type identity. Specifically, the personal experience of pain is an allostatic stress mechanism comprised of interdependent nervous, endocrine and immune operations. In Chapter One, I provide five reasons to prefer type identity theory of mind to dualistic philosophies of mind: it has greater explanatory power; it is more respectful of philosophical and folk intuitions about the causal powers of qualia; it is simpler; it is supported by the causal closure of the physical; and it is continuous with the natural sciences and not separate from them. I describe and challenge four philosophical objections to type identity theory of mind: mental states are excluded; mental states disappear; inverted qualia; and Saul Kripke‟s claim that type identity theory is false because two individuals could have the same mental state while having different physiological states. In Chapter Two, I advance a type identity theory of pain supported by a robust theoretical schema as the best description of the mind-body puzzle: what is pain? I frame a well established multilevel descriptive view of the physiological mechanisms that describe pain qualia within the context of advancing theoretical descriptions of the nervous, endocrine and immune systems and their functional interdependencies, and descriptions of allostasis, homeostasis, stress and wounds, all constrained in turn by complex adaptive systems theory. A biological individual is a complex adaptive system coping with a physical and social environment, but possessing nested subsystems. Taken together, these descriptions show how pain qualia are type identified with specific neurophysiological mechanisms; namely: (1) somatosensory qualia of pain, including submodality, intensity, duration and location, are best described as the operations of multisubsystem mechanisms in the neospinothalamic tract; (2) negative emotional pain qualia are the operations of multisubsystem mechanisms in the paleospinothalamic tract; (3) cognitive pain qualia (pain anticipation) are the operations of primary somatosensory cortex (neospinothalamic tract); (4) pain suppression (stress-induced analgesia) are the operations the dorsolateral funiculus pathway and opiate systems (paleospinothalamic tract). The simplest and most parsimonious metaphysical description of these robust relationships is that pain is mechanism. In Chapter Three, I advance a novel polyvagal-type identity theory of pain facial expression to best explain the explanatory mind-body puzzle: how can pain exist? According to some philosophers, assuming neuroscience explains with what mechanistic operations being aware of a burning arm pain is type identical, it is still impossible for any neuroscientific theory to explain how a specific pain must be correlated with a specific mechanism, as opposed to a different mechanism. Thus, there appears to be an explanatory gap. In this chapter, I will attempt to bridge the explanatory gap in two ways. First, based on the theoretical approach for a type identity theory of pain offered in Chapter Two, I offer a polyvagal-type identity theory of the mammalian pain face. I claim type identity theory of mind best explains how the gap can be bridged. Type identity theory of mind makes a realist assumption that pain is causally responsible for behaviours such as facial pain grimaces and screaming. Second, I attempt to bridge the gap by arguing that the supposed gap assumes that type identity theory must reconstruct type pain identities as formal derivations from laws of nature. Based on actual scientific practice and philosophical considerations concerning explanatory levels, I will show that this is a false assumption. Type identity statements that successfully emerge from mechanistic pain explanation are between different delineations of pain phenomena at the same explanatory level; they are intralevel. Although the explanatory gap puzzle correctly shows our incomplete understanding of how pain might be explained by mechanism, the gap merely asserts a practical limit on our present explanatory successes, and is not in principle unbridgeable on a priori grounds, as some philosophers claim. Eliminative materialists also assert that there is nothing more to pain than mechanism, but deny that pain can be type identified with neurophysiological mechanism. The reason is that pain does not really exist. Eliminativists propose that pain is part of folk psychology which consists of false generalizations („wounding is the cause of pain‟) and false theoretical claims ("pain always hurts‟). Thus, pain should be eliminated and replaced by an accurate neuroscientific successor theory. In Chapter Four, I assess philosophical arguments and data for and against eliminative materialism of pain. If eliminativism is correct, then the version of type identity theory of pain I propose in this project is strictly false. While pain folk psychology has stimulated much psychological research resulting in improved clinical outcomes for some pain patients, our own intellectual history reveals that any theory can seem successful even when it is radically false. Neuroscience already shows that many folk pain claims are false, while the truth of many others is not known. Eliminative materialism implies the disquieting consequence that the limit of what can be scientifically eliminated or reduced is much closer than we may conventionally think. Alternately, since intellectual history also offers modest evidence of theoretical co-existence between type identity theory of mind and folk psychology, radical theory change as advocated by eliminativism is just one end-point on a continuum of many possibilities. Still, accommodation such as this cannot change the sobering insight that all human knowledge is ultimately provisional. This realization encourages guarded humility about the ontological status of existing folk and scientific pain theories, including the type identity theory of pain, while it fosters an equally guarded optimism concerning our future theoretical prospects.