A fibrilação atrial (FA) é a arritmia cardíaca mais comum na prática clínica e que apresenta a maior morbidade, principalmente com o avançar da idade. O sistema nervoso autonômico, particularmente o balanço adrenérgico/colinérgico, tem profunda influência na ocorrência de fibrilação atrial. A FA pode ser gerada e mantida por uma variedade de mecanismos eletrofisiológicos e uma mudança na atividade autonômica poderá afetar cada um deles de forma diferente. Além do sistema nervoso autônomo, simpático e parassimpático, envolvidos na gênese e manutenção da FA, já é sabido que existem vários outros fatores envolvidos e, dentre eles, as células intersticiais tipo-Cajal (CITC), semelhantes às células intersticiais que contribuem para a atividade motora peristáltica do trato gastrointestinal. Essas células foram encontradas no miocárdio atrial e ventricular, e poderiam ser a origem da atividade deflagradora de focos elétricos ectópicos geradores de FA. O presente estudo teve como objetivos analisar possíveis alterações na expressão miocárdica dos receptores beta-adrenérgicos e quantificar as células intersticiais tipo-Cajal nos átrios de corações humanos, em particular, no esquerdo, e sua relação com a fibrilação atrial permanente (FAP). Para o primeiro objetivo, foram estudados 19 casos de corações de autópsias de portadores de FAP e cardiopatia crônica definida (grupo I), e 19 corações pareados com as mesmas cardiopatias, porém sem evidências de qualquer arritmia supraventricular (grupo II). Foram ressecadas uma amostra no teto do átrio direito, duas no átrio esquerdo, e uma em terminação nervosa envolvida em tecido gorduroso no epicárdio do átrio esquerdo (fat-pad). A expressão miocárdica dos receptores beta-adrenérgicos 1 a 3 e da quinase-5 do receptor adrenérgico acoplado à proteína G (GRK5) foi avaliada pela proporção positiva no miocárdio nos cortes citados. Não houve diferença estatisticamente significante entre os dois grupos quando analisamos a expressão dos receptores adrenérgicos (beta-1, beta-2, beta-3 e GRK5), independentemente do uso ou não de beta-bloqueador. Para o segundo objetivo, foram estudados 6 casos de corações de autópsias de portadores de FAP e cardiopatia crônica definida (grupo I), e 6 corações pareados com as mesmas cardiopatias, porém sem evidências de qualquer arritmia supraventricular (grupo II). As CITC foram avaliadas na região média da parede diafragmática do átrio esquerdo. Não houve alterações estatisticamente significantes entre os grupos estudados, quando avaliamos o número de células positivas no miocárdio pela área do miocárdio em mm2, o número de células positivas no corte inteiro pela área do miocárdio em mm2 ou o número de células positivas no corte inteiro/área do corte inteiro em mm2, seja em relação a cada corte individualmente, ao átrio esquerdo isoladamente e a todos os cortes juntos. Em conclusão, nem alterações na expressão de receptores beta-adrenérgicos nem a presença de células tipo-Cajal parecem ter maior papel na patogênese da fibrilação atrial permanente / Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice, presenting the highest morbidity, especially with advancing age. The autonomic nervous system, particularly the adrenergic/cholinergic balance, has a profound influence on the occurrence of AF. AF can be generated and maintained through a variety of electrophysiological mechanisms, and a change in autonomic activity may affect each of mechanism differently. In addition to the autonomous, sympathetic, and parasympathetic nervous systems involved in the genesis and maintenance of AF, there are several other factors known to be involved, including the interstitial cells of Cajal (ICCs), similar to the interstitial cells that contribute to the peristaltic motor activity of the gastrointestinal tract. These cells were found in the atrial and ventricular myocardium, and could be the source of the triggering activity of ectopic electrical foci that generate AF. In the present study, we aimed to analyze the possible changes in the myocardial expression of beta-adrenergic receptors and to quantify ICCs in the atria of human hearts, in particular in the left atrium, and its relation with permanent AF (PAF). For the first objective, we studied 19 hearts from autopsies of patients with PAF and defined chronic cardiomyopathy (group I), and 19 paired hearts with the same cardiomyopathy but without evidence of any supraventricular arrhythmia (group II). A tissue sample from the ceiling of the right atrium, two from the left atrium, and one from the nerve ending involved in the adipose tissue in the epicardium of the left atrium (fat pad) were resected. The myocardial expression of beta-adrenergic receptors 1 and 3, and of the G protein-coupled receptor kinase 5 (GRK5) was assessed according to the positive proportion in the myocardium in the mentioned sections. There was no statistically significant difference between the two groups in the expression of adrenergic receptors (beta-1, beta-2, beta-3, and GRK5), regardless of the use or nonuse of beta-blockers. For the second objective, six hearts from autopsied patients with PAF and defined chronic cardiopathy (group I) were studied, along with six paired hearts with the same cardiopathies but without evidence of any supraventricular arrhythmia (group II). The ICCs were evaluated in the middle region of the diaphragmatic wall of the left atrium. There were no statistically significant changes between the groups when we evaluated the number of positive cells in the myocardium by area of the myocardium in mm2, the number of positive cells in the full section by area of the myocardium in mm2, or the number of positive cells in the full section/area of the full section in mm2, be it in relation to each section individually, the left atrium alone, or all sections together. In conclusion, neither changes in the expression of beta-adrenergic receptors nor the presence of ICCs seem to have a large role in the pathogenesis of permanent AF

As células intersticiais de Cajal (CIC) têm sido estudadas como participante do peristaltismo em vários sistemas. Sua presença no trato geniturinário pode sustentar a importância dessas células na fisiopatologia da estenose da junção ureteropielica (JUP). O Objetivo desse estudo foi avaliar a densidade das CIC em pacientes adultos e no final da adolescência, portadores de estenose da JUP, submetidos à pieloplastia e verificar se há associação entre a densidade das CIC com os achados clínicos e de imagem pré e pós-operatórios, notadamente ultrassonografia e cintilografia renal. Foram estudados 23 pacientes com estenose da JUP, submetidos à pieloplastia desmembrada pela técnica videolaparoscópica na Divisão de Clínica Urológica do Departamento de Cirurgia do HCFMUSP, de forma consecutiva, pelo mesmo grupo de cirurgiões, no período entre fevereiro de 2011 a janeiro de 2012. Foi realizada análise imunohistoquímica para expressão do receptor de tirosina quinase (c-KIT) em todas as amostras das JUP e quantificada a densidade das CIC. Os pacientes foram acompanhados periodicamente para avaliação da resposta clínica e dos exames de imagem. Foi encontrado que a média de idade da amostra foi de 34,83 anos. Houve predomínio do gênero masculino (56,5%). O rim direito foi o mais acometido (56,5%). A hidronefrose grave foi identificada na maioria dos pacientes (52,2%). A média da função renal do rim acometido estimada pela cintilografia, pré e pós-operatória foi de respectivamente, 33,7 e 33,4%. Dos 23 pacientes, 20 apresentaram melhora do padrão cintilográfico de drenagem ureteral. Houve predomínio de pacientes que apresentavam alta densidade das CIC (52,2%). Houve significância estatística quando associado a densidade das CIC e a melhora do padrão ultrassonográfico (p= 0,032). Contudo, não houve associação entre a densidade das CIC e as outras variáveis clínicas ou de imagem. Pode-se concluir que a densidade das CIC pode ser um bom preditor da resposta ultrassonográfica pósoperatória em pacientes adultos com estenose da JUP submetidos à pieloplastia / The interstitial cells of Cajal (ICC) have been studied as peristalsis participating in various systems. Its presence in the genitourinary tract can sustain the importance of these cells in the pathophysiology of ureteropelvic junction obstruction (UPJO). The aim of this study was to evaluate the density of ICC in adults and in the late adolescence patients with UPJO, undergoing pyeloplasty and to check if there is association of changes in the ICC density with clinical findings, as well as pre and postoperative images, especially ultrasound and diuretic radioisotope renography. We selected 23 patients with UPJO, undergoing laparocopic dismembered pyeloplasty in the Urology Division of the HC-FMUSP Department of Surgery, consecutively, by the same group of surgeons in the period between February 2011 and January 2012. It was performed immunohistochemical analysis for tyrosine kinase receptor expression (c-KIT) in all samples of UPJO quantified the ICC density. The patients were followed up periodically to evaluate the clinical response and imaging. The average age of the sample was 34.83 years. There was a predominance of males (56.5%). The right kidney was the most affected (56.5%). Severe hydronephrosis was identified in most patients (52.2%). The average renal function affected estimated by diuretic radioisotope renography, pre and post-operative was respectively 33.7 and 33.4%. Of the 23 patients, 20 had an improvement on diuretic radioisotope renography pattern of ureteral drainage. There was a predominance of patients with high ICC density (52.2%). There was statistical significance when associated with ICC density and the improvement of ultrasonographic pattern (p = 0.032). However, there was no association between the ICC density and other clinical or imaging variables. It can be concluded that the density of the ICC maybe a good predictor of post-operative ultrasound response in adult patients with UPJO undergoing pyeloplasty

The present thesis project focused on the preclinical study of Neurotensin receptor 1 (Ntsr1), Endoglin/CD105 (Eng), Glypican 6 (Gpc6) and Sprouty homolog 4 (Spry4) as potential markers or molecular targets for future therapeutic interventions of gastrointestinal stromal tumors (GIST). Ntsr1 expression was characterized was reported in a paper that I co-authored entitled “Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial cells of Cajal.” by Gromova et al. PLOS ONE, 2009. As radio-labeled NTSR1 ligand analogues have already been reported for whole-body imaging and therapeutic interventions, prompting us to investigate NTSR1 as a target for in vivo imaging of GIST.Next, expression of Eng was characterized in the KitK641E murine GIST model, human GIST, GIST882 and BaF3 cells. This study has been reported in “Endoglin/CD105 is expressed in KIT positive cells in the gut and gastrointestinal stromal tumors” by Gromova et al. JCMM, 2011, a paper I co-authored. As result, an American start-up approached us to assess their proprietary compound targeting ENG on GIST882 cells. However, concerns were raised about possible non-selective action and the project was stalled by the company.Subsequently, GIST tissue microarrays were examined by immunohistochemistry using the sole commercially available GPC6 antibody. No statistical correlation could be found between GPC6-ir and GIST clinic-pathological features and concerns were raised about the reliability of the GPC6 antibody used. Ultimately, Spry4 was investigated in the last part of my thesis. In vitro, we have demonstrated that Spry4 is specifically upregulated by the ERK pathway in GIST882 cells. In vivo, Spry4 deficient mice showed an ICC hyperplasia in antrum and colon, using a new ICC quantification method developed in the lab, which was reminiscent of the oncogenic GIST murine model KitK641E. Similarities between Spry4 KO and KitK641E heterozygous animals were even further emphasized by functional studies, as both genotypes showed a delay in transit time. This study lead to the publication “Hyperplasia of interstitial cells of Cajal in Sprouty homolog 4 deficient mice” by Thys et al. 2015, PLOS ONE. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

1) Aims To explore the ultrastructure of interstitial cells in the upper lamina propria of the human bladder, to describe the spatial relationships and to investigate cell-cell contacts. 2) Methods Focused ion beam scanning electron microscopy (FIB-SEM), 3-View SEM and confocal laser scanning microscopy were used to analyze the 3D ultrastructure of the upper lamina propria in male and female human bladders. 3) Results 3View-SEM image stacks as large as 59µm x 59µm x 17µm (xyz) at a resolution of 16nm x 16nm x 50 nm and high resolution (5nm x 5nm x 10nm) FIB-SEM stacks could be analyzed. Interstitial cells with myoid differentiation (mIC) and fibroblast like interstitial cells (fIC) were the major cell types in the upper lamina propria. The flat, sheet-like ICs were oriented strictly parallel to the urothelium sheet-like morphology. No spindle shaped cells were present. We furthermore identified one branched cell (bIC) with several processes contacting urothelial cells by penetrating the basal membrane. This cell did not make any contacts to other ICs within the upper lamina propria. We found no evidence for the occurrence of telocytes in the upper lamina propria. 4) Conclusions Comprehensive 3D-ultrastructural analysis of the human bladder confirmed distinct subtypes of interstitial cells. We provide evidence for a foremost unknown direct connection between a branched interstitial cell and urothelial cells of which the functional role has still to be elucidated. 3D-ultrastructure analyses at high resolution are needed to further define the subpopulations of lamina propria cells and cell-cell interactions.

Les contractions rythmiques résultent de l’activité stimulatrice du nœud sinusal dans le cœur et des cellules interstitielles de Cajal (CICs) dans les intestins. Nous avons découvert un nouveau syndrome résultant d’une combinaison de la maladie du nœud sinusal (MNS) et de la pseudo-obstruction intestinale chronique (POIC). Ce syndrome, que nous avons nommé Chronic Atrial and Intestinal Dysrhythmia (CAID), résulte d’une mutation récessive du gène SGO1 (K23E). Cependant, les rôles connus de SGO1 n'expliquent pas l'apparition postnatale du syndrome ni la pathologie spécifique, suggérant que des rôles non canoniques de SGO1 conduisent aux manifestations cliniques observées. Cette hypothèse est supportée par la comparaison de CAID avec les autres cohésinopathies qui présentent principalement des phénotypes développementaux sans ou avec des défauts légers du cycle cellulaire. Ce projet visait à une découverte non biaisée des mécanismes non canoniques expliquant le syndrome CAID en utilisant le dogme de la biologie moléculaire (ADN→ARNm→protéine) comme ligne directrice. Pour ce faire, nous avons effectué des criblages multi-omiques sur des fibroblastes de peau de patients CAID et de contrôles sains. Les résultats des criblages ont été validés par électrophysiologie, étude des voies de signalisation pertinentes, immunohistochimie, pyroséquençage des rétrotransposons LINE-1 et quantification des marques d’histones. Nos études multi-omiques ont confirmé des changements dans la régulation du cycle cellulaire, mais aussi dans la conduction cardiaque et la fonction des muscles lisses. Plus spécifiquement, plusieurs canaux potassiques étaient sous-régulés. L’électrophysiologie a confirmé une diminution du courant potassique rectifiant entrant (IK1). L'immunohistochimie des coupes intestinales de patients CAID a confirmé l’augmentation de l’expression de SGO1 et BUB1, un régulateur de la voie de signalisation TGF-β. De plus, la voie canonique de TGF-β est augmentée et est découplée de la voie non canonique. Au niveau épigénétique, une signature unique d’hyperméthylation et de fermeture de la chromatine a été observée. Ce qui est soutenu par l’augmentation de la méthylation de H3K9me3 et de H3K27me3. En conclusion, le syndrome CAID est associé à plusieurs changements ayant possiblement un effet cumulatif plutôt que d’une seule voie de signalisation dérégulée. Nos résultats désignent la perturbation du courant IK1, la dérégulation de la signalisation TGF-β, l’hyperméthylation de l’ADN et la compaction de la chromatine comme éléments conducteurs potentiels des manifestations cliniques observées. La voie TGF-β et les changements épigénétiques peuvent être ciblées par des médicaments existants, constituant ainsi des cibles thérapeutiques prometteuses pour le traitement du syndrome CAID. / Rhythmic contractions are driven by the pacemaker activity of the cardiac sinus node and the intestinal interstitial cells of Cajal (ICC). We have discovered a new syndrome resulting from a combination of sick sinus syndrome (SSS) and chronic intestinal pseudo-obstruction (CIPO). This syndrome, which we have named Chronic Atrial and Intestinal Dysrhythmia (CAID), results from a recessive mutation in the SGO1 gene (K23E). However, the known roles of SGO1 do not explain the postnatal onset of the syndrome nor the specific pathology, suggesting that non-canonical roles of SGO1 lead to the clinical manifestations observed. This hypothesis is supported by the comparison of CAID with other cohesinopathies which mainly exhibit developmental phenotypes without or with mild cell cycle defects. This project aimed towards an unbiased discovery of noncanonical mechanisms explaining CAID using the molecular biology dogma (DNA→mRNA→protein) as a guideline. We performed multi-omic screens on skin fibroblasts from CAID patients and healthy controls. Screening results were validated by electrophysiology, study of relevant signaling pathways, immunohistochemistry, LINE-1 retrotransposon pyrosequencing, and histone marks quantification. Our multiomics analyses confirmed changes in cell cycle regulation, but also in cardiac conduction and smooth muscle function. More specifically, several potassium channels were downregulated. Electrophysiology studies confirmed a decrease in the inward rectifier potassium current (IK1). Immunohistochemistry in CAID patient’s intestinal sections confirmed overexpression of SGO1 and BUB1, a regulator of TGF-β signaling pathway. Additionally, the canonical TGF-β signaling was increased and decoupled from noncanonical signaling. At the epigenetic level, CAID patient fibroblasts have a unique signature of hypermethylation and chromatin closure. This is supported by the increased methylation of H3K9me3 and H3K27me3. In conclusion, CAID syndrome is associated with several changes that, may have a cumulative effect rather than a single deregulated signaling pathway. Our results reveal the disturbance of the IK1 current, the deregulation of TGF-β signaling, DNA hypermethylation and chromatin accessibility changes as potential conductors of intestinal and cardiac manifestations of CAID syndrome. In particular, the TGF-β pathway and epigenetic changes, may be targeted by existing drugs, thus constituting promising therapeutic targets for the treatment of CAID syndrome.