Introdução: A alteração dos níveis normais de marcadores séricos e urinários ocorre na presença de dano renal associado à uropatia obstrutiva. Valores séricos e e urinários de TGF beta1 e CA 19-9 ainda não foram avaliados em modelo experimental de uropatia obstrutiva. Material e Métodos: Ratos foram divididos em sete grupos: referência, sham operation, nefrectomia unilateral, ligadura completa de ureter unilateral, obstrução parcial de ureter unilateral, obstrução parcial de ambos ureteres, nefrectomia unilateral associada à obstrução parcial do ureter contralateral. Morfometria renal e ureteral, concentrações séricas e urinárias de TGF beta1 e CA 19-9 e expressão tecidual renal de CA 19-9 foram analisadas. A correlação destes marcadores com os grupos submetidos a obstrução completa, obstrução parcial ou sem obstrução foi realizada. Resultados: Achados anatomopatológicos correlacionaram-se positivamente à intensidade da obstrução ureteral e negativamente aos níveis urinários de CA 19-9. Subexpressão acentuada do CA 19-9 foi observada em unidades renais com obstrução completa. Não foram encontradas diferenças estatisticamente significativas para os marcadores TGF beta1 urinário, TGF beta1 sérico e para o CA 19-9 sérico Conclusões: O CA 19-9 urinário correlacionou-se negativamente com o grau de obstrução ureteral. A análise imuno-histoquímica demonstrou a expressão do CA 19-9 no citoplasma das células epiteliais tubulares, sugerindo produção renal do marcador. O TGF beta1 sérico e urinário não apresentaram modificações de acordo com o grau de severidade e tempo de obstrução, o que pode estar relacionado a remodelamento renal menos intenso em resposta à uropatia obstrutiva nestes ratos / Introduction: Abnormal levels of serum and urinary markers occur in the presence of renal damage associated to obstructive uropathy. Urinary and serum TGFbeta1 and CA 19- 9 have not yet been evaluated in an experimental model of obstructive uropathy. Material and Methods: Rats were divided into seven groups: reference, sham operation, unilateral nephrectomy, complete unilateral ureteral obstruction, partial unilateral ureteral obstruction, partial bilateral ureteral obstruction, and unilateral nephrectomy with contralateral partial ureteral obstruction. Kidney and ureter morphometry, TGFbeta1 and CA 19-9 serum and urinary concentrations and CA 19-9 renal tissue expression were analysed. Correlation of these markers to complete, partial obstruction or unobstructed groups was performed. Results: Pathological findings correlated positively with the degree of ureteral obstruction, but negatively with urinary CA 19-9 levels. Marked underexpression of CA 19-9 was observed in kidneys with complete ureteral obstruction. No statistically significant differences were found for urinary and serum TGFbeta1 and also for serum CA 19-9. Conclusions: Urinary CA 19-9 correlated negatively with ureteral obstruction grade. Immunohistochemistry depicted CA 19-9 expression on epithelial tubular cells cytoplasm, suggesting renal origin. Serum and urinary TGFbeta1 did not show alterations in response to severity and length of urinary obstruction, which might be associated with less intense renal remodeling

Orientador: Maria Julia Marques / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-20T07:19:08Z (GMT). No. of bitstreams: 1 Moreira_DrielendeOliveira_M.pdf: 2075338 bytes, checksum: 00bb5917e4f176b73c58453340cb3a5e (MD5) Previous issue date: 2012 / Resumo: A Distrofia muscular de Duchenne (DMD) é uma doença caracterizada pela fraqueza muscular progressiva que leva à insuficiência respiratória e cardíaca, resultando em morte por volta dos 30 anos de idade. No camundongo mdx, modelo experimental da DMD, os músculos diafragma e cardíaco são severamente afetados apresentando fibrose semelhante à observada na patologia humana. O objetivo deste trabalho foi investigar os efeitos do tratamento a longo prazo com suramina, uma droga anti-fibrótica, nos músculos diafragma e cardíaco de camundongos mdx idosos. Camundongos mdx (n=20; 8 meses de idade) receberam injeções intraperitoneais de suramina (60 mg/kg), durante 3 meses. Controles mdx (n=20; 8 meses) e C57BL/10 (n=18; 8 meses) foram injetados com solução salina. Os camundongos da linhagem C57BL/10 expressam distrofina e são utilizados como controle da linhagem mdx. A suramina diminuiu os níveis de CK e atenuou a perda da força muscular. No músculo diafragma, a suramina reduziu a área de fibrose e a mionecrose. No músculo cardíaco, houve redução da fibrose, da inflamação e melhora significativa de parâmetros funcionais cardíacos (amplitude das ondas P, Q, R e S do eletrocardiograma). Sugere-se que a suramina possa ser potencialmente útil nas distrofinopatias, atenuando a miopatia nos músculos mais afetados, o coração e o diafragma, nos estágios tardios da doença / Abstract: Duchenne muscular dystrophy (DMD) is a disease characterized by progressive muscle weakness leading to respiratory and cardiac failure, resulting in death around 30 years of age. In the mdx mice model of DMD, diaphragm and cardiac muscles are severely affected in the later stages of the disease, showing intense fibrosis similar to that observed in human pathology. The aim of the present study was to investigate the effects of long-term treatment with suramin, an anti-fibrotic agent, in the diaphragm and cardiac muscles of the mdx mice. Mdx mice (n=20; 8 months of age) received intraperitoneal injections of suramin (60 mg/kg) for 3 months. Mdx controls (n=20; 8 months) and C57BL/10 (n=18; 8 months old) were injected with saline. C57BL/10 mice express dystrophin and are the control strain for the mdx mice. Suramin decreased CK levels and reduced the loss of muscle strength. Suramin reduced fibrosis and myonecrosis in diaphragm. In the cardiac muscle, suramin decreased fibrosis, inflammation and improved cardiac functional parameters (P, Q, R and S waves of the electrocardiogram). It is suggested that suramin may be a potential therapy for distrophinopaties, attenuating the dystrophic phenotype of the most affected cardiac and diaphragm muscles of the mdx mice, during later stages of the disease / Mestrado / Anatomia / Mestre em Biologia Celular e Estrutural

La maladie d'Alzheimer représente un défi mondial pour la société. Il n'y a pas à ce jour de traitement efficace pour traiter ou ralentir les symptômes de la maladie d'Alzheimer. Cette maladie est caractérisée par une perte de synapses, une augmentation du nombre de plaques extracellulaires d'Abêta et une augmentation de Tau hyperphosphorylée agrégée intracellulaire (neurodégénérescence fibrillaire). Il est communément admis que la maladie d'Alzheimer est principalement liée à l’oligomérisation et à la fibrillation de peptides amyloïdes bêta, et que les oligomères Abêta solubles et les fibres sont des espèces neurotoxiques. Une stratégie pour réduire la présence de ces espèces toxiques de Abêta est l'utilisation de peptides ou de peptidomimétiques pour inhiber l'agrégation de Abêta, soit par interaction avec les oligomères d'Abêta pour empêcher davantage son agrégation en fibres ou en stabilisant les conformations hélicoïdales transitoires de Abêta pour empêcher sa transition vers des structures en feuillets bêta. Dans un premier temps, sur la base des résultats encourageants de glycopeptides contenant un élément polaire casseur de feuillets bêta pour moduler l'agrégation du peptide Abêta, et des propriétés uniques du groupe trifluoromethylhydroxyle, nous avons conçu et synthétisé des mimes de pentapeptides contenant la séquence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF3 Thr et (2S, 3S)-CF3 Thr), pour interagir avec le site de nucléation du peptide Abêta dans sa forme monomérique ou oligomérique de manière à perturber l'auto-assemblage en oligomères toxiques. Des études conformationnelles par RMN 2D et par modélisation moléculaire ont indiqué que ces peptides adoptent des conformations étendues dans des solvants polaires (eau et méthanol). Nous avons constaté expérimentalement et théoriquement que les pentapeptides contenant l’acide aminé non naturel (2S, 3S)-CF3-thréonine sont plus étendus que les pentapeptides contenant les acides aminés naturels L-sérine et L-thréonine. Nous avons également observé que les pentapeptides (2S, 3S)-CF3-Thr ont une propension à s’auto-associer en formant des brins bêta intermoléculaires. La capacité de ces pentapeptides à inhiber la formation de fibres amyloïdes a été évaluée sur les peptides Abêta 1-42 et IAPP (impliqué dans le diabète de type II) par des essais de fluorescence à la thioflavine T (ThT). Il a été constaté qu'aucun de ces pentapeptides ont un effet d'inhibition de l'agrégation des peptides Abêta 1-42 et IAPP. Au contraire, certains composés ont montré un effet d'accélération de l'agrégation de IAPP. Accélérer l'agrégation est moins intuitif mais cette stratégie a plus récemment suscité un intérêt. Il pourrait être intéressant d'étudier plus en détail l’intérêt de cette accélération de l'agrégation de IAPP par des techniques complémentaires.Dans une deuxième partie de cette thèse, nous avons assemblé une unité amino acide basée sur un motif CF3-1,4 disubstituted-1,2,3-triazole en homo-oligomères (trimères et tétramères) et en peptidomimétiques. Des études conformationnelles de ces oligomères fluorés ont été menées par RMN 2D et par des simulations de modélisation moléculaire. Nos études préliminaires de modélisation moléculaire prévoient des structures hélicoïdales avec des trimères et des tétramères d'acides aminés à base de ces CF3-triazoles. L'analyse par RMN 2D du tétramère affiche des corrélations NOE très intéressantes, ce qui indique une structure repliée. La capacité de ces oligomères fluorés à moduler la formation de fibres amyloïdes des peptides Abêta 1-42 et IAPP a été évaluée par test de fluorescence à la ThT. Nous avons constaté que le trimère est un faible inhibiteur de l'agrégation de Abêta 1-42, mais aussi un promoteur d'agrégation de IAPP. Le tétramère a été trouvé capable de moduler l'agrégation de Abêta 1-42 et de IAPP, mais non d'une manière classique d'inhibition ou de promotion. / It has been widely recognized that Alzheimer’s disease (AD) represents an unsettling, worldwide challenge for society. By far, there has been no effective cure for AD. Pathologically, AD is characterized by a loss of synapses, an increase in the number of extracellular Abeta plaques and an increase in intracellular aggregated hyperphosphorylated Tau (neurofibrillary tangles). It is commonly believed that AD is primarily linked to oligomerization and fibrillization of amyloid beta peptides, and the soluble Abeta oligomers and fibrils are neurotoxic species.One strategy to reduce the Abeta fibrils is the use of peptides or peptidomimetics to inhibit the Abeta aggregation either by interaction with the Abeta oligomers to prevent its further aggregation into fibrils, or by stabilizing the transient Abeta α-helical conformations to prevent its transition to beta-sheet structures.In the first direction, based on the encouraging results of the glycopeptides containing the polar sugar beta-sheet breaker element to modulate Abeta peptide aggregation and the unique properties of trifluoromethylhydroxyl group, we designed and synthesized pentapeptide mimics with the sequence (Boc) Ala-Val-X-Val-Leu-OMe (X = Ser, Thr, (2S, 3R)-CF3-Thr and (2S, 3S)-CF3-Thr) to interact with the nucleation site of Abeta peptide in its monomeric or oligomeric form so as to disrupt the self-assembly into toxic oligomeric form. Both 2D NMR and molecular modelling studies indicated that these peptides in polar solvent (water and methanol) adopt mainly extended backbone conformations. It is found both experimentally and theoretically that, the (2S, 3S)-CF3-threonine-containing pentapeptides are more extended than the L-serine- and L- threonine-containing pentapeptides. It is also observed that the (2S, 3S)-CF3-Thr pentapeptides have a propensity to self-associate of by forming intermolecular beta-strand contacts. The ability of these pentapeptides to inhibit amyloid fibril formation was evaluated on Abeta1-42 and IAPP peptides by ThT fluorescence assay. It was found that none of these pentapeptides have any inhibition effect in Abeta1-42 peptide and IAPP aggregation. On the contrary, some compounds showed an acceleration effect in IAPP aggregation. Accelerating the aggregation pathways is less intuitive but this strategy has more recently aroused interest. It could be interesting to further study the effect of accelerating IAPP aggregation by complementary techniques.In the other direction, we have assembled novel a CF3-1,4-triazole-based amino acid mimic into homo-oligomers (trimer and tetramer) and peptidomimetics. The fluorinated oligomers were investigated both by NMR conformational studies and molecular modelling simulations. Our preliminary modelling studies predict helical structures with trimer and tetramers of CF3-1,4-disubstituted- 1,2,3- triazole-based amino acid. NMR analysis of tetramer displayed very interesting NOE correlations, indicating a folded structure. The ability of these fluorinated oligomers to modulate the amyloid fibril formation of Abeta1-42 and IAPP peptides were evaluated by ThT fluorescence assay. It was found that trimer was a weak inhibitor of Abeta1-42 aggregation but also a promoter of IAPP aggregation. The tetramer was found able to modulate the aggregation of Abeta1-42 and IAPP but not in a classical inhibition or promotion manner.

Перинатальный артериальный ишемический инсульт - это цереброваскулярное заболевание, возникающее между 20-й неделей беременности и 28-м послеродовым днем, вызывающее двигательный и немоторный дефицит, причем церебральный паралич является частым исходом. Молодой мозг реагирует, реорганизуя свои поврежденные сети в ипсилезионное и/или контральезионное полушария, причем последнее больше связано с двигательными нарушениями. Префронтальная кора считается одной из наиболее уязвимых областей с когнитивным дефицитом, возникающим с задержкой, из-за ее длительного развития, достигающего своего пика синаптогенеза после первого послеродового года, в то время как другие области, такие как первичная кора, обычно проходят свою основную фазу синаптогенеза в течение первого послеродового семестра. Таким образом, раннее обнаружение низкого синаптогенеза может быть ранним признаком настоящего или предстоящего дефицита и привести к раннему вмешательству. Бета-диапазон недавно был предложен в качестве возможного биомаркера синаптогенеза, причем активность ГАМК связана с нейропластичностью и синаптогенезом. Основной целью настоящего исследования является установление роли абсолютной бета-1 мощности в синаптогенезе и исследование уязвимости префронтальной коры головного мозга. Были набраны сорок типичных детей и 10 детей с перинатальным артериальным ишемическим инсультом в их подкорковой средней мозговой артерии и были созданы 3 возрастные подгруппы: 5-месячная, 10-месячная и 24-месячная подгруппы. Запись ЭЭГ и тест Бейли-III использовались для измерения их фоновой активности и уровня развития. Хотя статистический анализ с помощью непараметрических инструментов (U-тест Манна-Уитни, тест Крускалла Уоллиса) не показал решающих результатов, потенциальная связь бета-диапазона с синаптогенезом может быть обнаружена при наблюдении низкой мощности бета-1 в моторных и когнитивных областях мозга и низкой моторной и когнитивной производительности, а также при обнаружении заднего или переднего созревания. Кроме того, ранняя уязвимость префронтальной коры может быть обнаружена в снижении двусторонней бета-1 мощности у 24-месячных детей с перинатальным инсультом, по сравнению с типичными детьми и более ранними односторонними различиями, наряду с некоторыми когнитивными дефицитами, которые начинают проявляться в той же группе. / Perinatal Arterial Ischemic Stroke is a cerebrovascular disease occurring between the 20th gestational week and the 28th postnatal day, causing motor and non-motor deficits with cerebral palsy being a frequent outcome. The young brain reacts by reorganizing its injured networks to ipsilesional and/or contralesional hemisphere with the latter relating more to motor impairment. The Prefrontal Cortex is considered one of the most vulnerable areas with cognitive deficits emerging with a delay, due to its lengthy development reaching its synaptogenesis peak after the first postnatal year, while other areas, such as the Primary Cortices undergo generally their major synaptogenesis phase during the first postnatal semester. So early detection of low synaptogenesis could be an early mark of present or upcoming deficits and lead to an early intervention. Beta band has been recently suggested as a possible biomarker of synaptogenesis with GABA’s activity being connected with neuroplasticity and synaptogenesis. The main goal of the current study is to establish the role of of the absolute beta-1 power to synaptogenesis and the investigate the vulnerability of the Prefrontal Cortex. Fourty typical children and 10 children with Perinatal Arterial Ischemic Stroke in their subcortical Middle Cerebral Artery were recruited and were created 3 age subgroups; 5month, 10month and 24month subgroup. EEG recording and Bayley-III test were used to measure their background activity and developmental level. Although the statistical analysis via non-parametric tools (Mann-Whitney U-test, Kruskall Wallis test) didn’t show decisive results, a potential connection of beta-band with synaptogenesis could be detected when observing low beta-1 power in motor and cognitive brain areas and low motor and cognitive performance and also by detecting a posterior to anterior maturation. Moreover the early vulnerability of Prefrontal Cortex may be found in the decreased bilateral beta-1 power in the 24month children with perinatal stroke, when compared with the typical children and the earlier unilateral differences, along with some cognitive deficits which begin to emerge in the same group.

The tumor microenvironment is a key modulator in cancer progression and has become a novel target in cancer therapy. An increase in hyaluronan (HA) accumulation and metabolism can be found in advancing tumor progression and are often associated with aggressive malignancy, drug resistance and poor prognosis. Wound-healing related myofibroblasts or activated cancer-associated fibroblasts (CAF) are assumed to be the major sources of HA. Both cell types are capable to synthesize new matrix components as well as reorganize the extracellular matrix. However, to which extent myofibroblasts and CAF perform these actions are still unclear. In this work, we investigated the matrix remodeling and HA production potential in normal human dermal fibroblasts (NHFB) and CAF in the absence and presence of transforming growth factor beta -1 (TGF-β1), with TGF-β1 being a major factor of regulating fibroblast differentiation. Three-dimensional (3D) collagen matrix was utilized to mimic the extracellular matrix of the tumor microenvironment. We found that CAF appeared to response insensitively towards TGF-β1 in terms of cell proliferation and matrix remodeling when compared to NHFB. In regards of HA production, we found that both cell types were capable to produce matrix bound HA, rather than a soluble counterpart, in response to TGF-β1. However, activated CAF demonstrated higher HA production when compared to myofibroblasts. The average molecular weight of produced HA was found in the range of 480 kDa for both cells. By analyzing gene expression of HA metabolizing enzymes, namely hyaluronan synthase (HAS1-3) and hyaluronidase (HYAL1-3) isoforms, we found expression of specific isoforms in dependence of TGF-β1 present in both cells. In addition, HAS2 and HYAL1 are highly expressed in CAF, which might contribute to a higher production and degradation of HA in CAF matrix. Overall, our results suggested a distinct behavior of NHFB and CAF in 3D collagen matrices in the presence of TGF-β1 in terms of matrix remodeling and HA production pointing to a specific impact on tumor modulation.

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Na maioria dos países, a incidência e prevalência da doença renal crônica (DRC) vem aumentando ao longo dos anos. Embora tenha havido uma melhora significativa no manejo da DRC com os inibidores do sistema renina-angiotensina-aldosterona, a doença ainda é progressiva, levando a necessidade do surgimento de novas estratégias protetoras. A fibrose renal progressiva está presente na DRC e envolve a participação de várias citocinas, com destaque para o fator Transforming growth factor- beta1 (TGF-beta1). Tem sido demonstrado que a mortalidade de pacientes com DRC está diretamente relacionada à função renal e está associada a riscos tradicionais como cardiovasculares e infecções. Entretanto, esses riscos tradicionais explicam apenas metade das causas de mortalidade nesses pacientes. Evidências crescentes mostram que o status de vitamina D pode ser um fator de risco não tradicional para a evolução da DRC. Tendo em vista o importante papel da vitamina D na manutenção das funções fisiológicas essenciais e a observação da queda dos níveis deste hormônio na DRC, torna-se relevante o estudo da deficiência de vitamina D nos eventos envolvidos na evolução crônica da lesão renal aguda em modelo experimental de isquemia/reperfusão renal. Ratos Wistar foram divididos em quatro grupos: controle, animais que receberam dieta padrão; dVD, animais que receberam dieta depletada em vitamina D; Isq, animais que receberam dieta padrão e foram submetidos ao insulto de isquemia/reperfusão renal bilateral no 28º dia; Isq+dVD, animais que receberam dieta depletada em vitamina D e foram submetidos ao insulto de isquemia/reperfusão bilateral no 28º dia. Ao final dos 90 dias do protocolo, os animais foram submetidos à eutanásia, amostras de sangue, urina e o tecido renal foram coletados para a análise dos mecanismos de lesão renal. Os animais submetidos ao insulto de isquemia/reperfusão renal apresentaram hipertrofia renal, aumento dos níveis de pressão arterial média, colesterol e de PTH plasmático. Além disso, foi observada expansão da área intersticial, aumento do infiltrado de macrófagos/monócitos, da expressão de colágeno IV, fibronectina, vimentina e alfa-actina e redução da expressão da proteína Klotho. A deficiência de vitamina D contribuiu para a elevação dos níveis plasmáticos de PTH e aumento da proteinúria assim como para as alterações túbulo-intersticiais crônicas importantes (fibrose e infiltrado inflamatório do interstício, dilatação e atrofia tubular), aumento da expressão da citocina TGF-beta1 expressão do receptor de vitamina D (VDR) e da proteína Klotho, observados nos animais deficientes em vitamina D submetidos ao insulto de isquemia/reperfusão renal. Portanto, através de vias inflamatórias e com participação do fator de crescimento TGF-beta1 ê um fator agravante para o dano túbulo-intersticial e formação de fibrose intersticial nesse modelo experimental de isquemia/reperfusão renal / In most countries, the incidence and prevalence of chronic kidney disease (CKD) has been increasing over the years. Although there was a significant improvement in the management of CKD with renin-angiotensin-system inhibitors, the disease is still progressive, leading to the need of emergence of new protective strategies. The progressive renal fibrosis is present in CKD and involves the participation of several cytokines, especially the Transforming growth factor-beta1 (TGF-beta1). It has been shown that the mortality of patients with CKD is directly related to renal function, which is associated to traditional risk factors such as cardiovascular diseases and infections. However, these traditional risk factors explain only half of the causes of mortality in these patients. Growing evidence shows that vitamin D status may be a non-traditional risk factor for the progression of CKD. Considering the important role of vitamin D in the maintenance of essential physiological functions and the observation of low levels of this hormone in CKD, the study of vitamin D deficiency in the events involved in chronic evolution of acute kidney injury in an experimental model of ischemia/reperfusion becomes relevant. Wistar rats were divided into four groups : control, animals received a standard diet ; dVD, animals received a vitamin D-depleted diet ; Isq, animals received a standard diet and were subjected to bilateral renal ischemia/reperfusion injury on day 28; Isq +dVD, animals received a vitamin D-depleted diet and were subjected to bilateral renal ischemia/reperfusion injury on day 28 . At the end of the 90 days of the protocol, the animals were euthanized and samples of blood, urine and kidney tissue were collected for analysis of the mechanisms of renal injury. The animals subjected to the insult of ischemia/ reperfusion showed renal hypertrophy, increased levels of mean blood pressure, cholesterol and plasma PTH. Furthermore, expansion of the interstitial area, increased infiltration of macrophages/monocytes, increased expression of collagen IV, fibronectin, vimentin and alpha-actin, and reduced expression of Klotho protein were observed. The vitamin D deficiency contributed to the elevation of plasma PTH levels and increased proteinuria as well as for important chronic tubulo-interstitial changes (fibrosis and inflammatory infiltration of the interstitium, tubular dilation and atrophy), increased expression of cytokine TGF-beta1 vitamin D receptor (VDR) and Klotho protein observed in vitamin D-deficient animals subjected to the insult of renal ischemia/reperfusion. Therefore, through inflammatory pathways and involvement of TGF-beta1 w y aggravating factor in tubulointerstitial damage and formation of interstitial fibrosis in this experimental model of renal ischemia/reperfusion