Introdução: após os tumores de pele não - melanoma o câncer de próstata é o tumor maligno mais comumente diagnosticado e a segunda causa de morte relacionada ao câncer na população masculina. A maioria dos pacientes que morrem por câncer de próstata possuem metástases ósseas e esta doença é pouco responsiva ao tratamento quimioterápico. O microambiente ósseo, através de uma interação bidirecional com as células cancerígenas prostáticas provoca, nessas células, alterações fenotípicas. Uma dessas alterações é a expressão das células cancerígenas metastáticas anteriormente c-KIT negativas de receptores c- KIT. Hipotetizamos, aqui, que essas alterações poderiam contribuir para a quimiorresistência desses tumores. Objetivo: avaliar a hipótese de que a expressão de c-KIT pelas células de câncer de próstata contribui para a reduzida sensibilidade dessa neoplasia ao agente quimioterápico Docetaxel. Métodos: estudo experimental, utilizando células PC3, derivadas de metástases de câncer de próstata humano andrógeno-independente e C42b derivadas de metástases ósseas de câncer de próstata de coluna lombar de ratos atímicos . Ambas linhagens celulares c-KIT negativas foram tratadas em meio celular adequado e, sempre que atingiram a confluência de 90 %, foram isoladas para experimento ou expansão celular. Com o objetivo de mimetizar a expressão de c-KIT in vitro, parte das células foram submetidas a transfecção celular estável de c-KIT. Para isso foi utilizada uma formulação para transfecção de ácidos nucleicos em células eucarióticas (LIPOFECTAMINE 2000, Invitrogen) e, após isso, foi realizada a seleção de clones transfectantes estáveis. Os grupos foram divididos em c-KIT positivo, com ou sem o tratamento de SCF e c- KIT negativo com ou sem o tratamento de SCF, submetidos a doses crescentes do quimioterápico Docetaxel. Resultados: utilizando-se o índice da metade da concentração de inibição máxima (IC50), foi observado que nos experimentos com tratamento com Docetaxel por 2 dias os IC50 dos grupos C42b c-KIT e PC3 c-KIT, na presença ou não do ligante SCF, foram bastante superiores aos IC50 dos grupos C42b e PC3 que não sofreram a transfecção estável do c-KIT, na presença ou não do ligante SCF (p<0,001 grupoxdose). Nos experimentos com tratamento de Docetaxel por 3 dias, não foi observada diferença com significância estatística dos IC50 entre os grupos das células C42b transfectadas ou não com c-KIT e PC3 transfectadas ou não com c-KIT, na presença ou não do ligante SCF, principalmente quando foram usadas doses de Docetaxel mais próximas aos IC50 encontrados nos experimentos precedentes (tratamento com Docetaxel por 2 dias, doses inferiores a 10 nM de Docetaxel). Na tentativa de entender o aumento da quimiorresistência ao tratamento do Docetaxel por 3 dias aos grupos sem a transfecção de c-KIT, realizou-se o tempo de duplicação celular (TD) nos grupos C42b e PC3, o qual também não demonstrou diferença entre os grupos (PC3 EV TD = 40 horas, PC3 c-KIT TD 32 horas, C42b EV TD = 12 horas, C42b c-KIT TD = 14 horas). Conclusões: o presente estudo não demonstrou aumento da quimiorresistência das células C42b e PC3 ao quimioterápico Docetaxel quando transfectadas com c-KIT. / Introduction: after non-melanoma skin tumors, prostate cancer is the most commonly diagnosed malignant tumor and the second greatest cancer-related cause of death among men. Most patients who die of prostate cancer have bone metastases. This disease does not respond well to chemotherapy. The bone microenvironment, through a bidirectional interaction with prostatic cancerous cells, causes phenotypic alterations in these cells. One of these alterations is the expression of previously c-KIT negative metastatic cancerous cells of c-KIT receptors. Here we hypothesize that these alterations might contribute to the chemoresistance of such tumors. Objective: evaluating the hypothesis that the c-KIT expression by prostate cancer cells contributes to the reduced sensibility of this neoplasia to chemotherapeutic agent Docetaxel. Methods: experimental study, using PC3 cells derived from metastases of androgen-independent human prostate cancer and C42b cells derived from prostate cancer bone metastases from the lumbar spine of athymic mice. Both c-KIT negative cell lines were treated in the proper cellular medium and isolated for experiment or cellular expansion whenever they reached 90% confluence. Aiming at mimicking the c-KIT expression in vitro, part of the cells was submitted to stable c-KIT cell transfection. In order to do so, we used a formulation for the transfection of nucleic acids into eukaryotic cells (LIPOFECTAMINE 2000, Invitrogen). Following, the selection of stable transfectant clones was made. The groups were divided into c-KIT positive, with or without SCF treatment, and c-KIT negative, with or without SCF treatment, receiving increasing doses of the chemotherapeutic agent Docetaxel. Results: using the half maximal inhibitory concentration index (IC50), we observed that in the experiments treated with Docetaxel for 2 days, the IC50 values of the groups C42b c-KIT and PC3 c-KIT, in the presence or absence of the ligand SCF, were very superior to the IC50 values of the groups C42b and PC3 that had not undergone the stable c-KIT transfection, in the presence or absence of the ligand SCF (p<0.001 groupxdose). In experiments treated with Docetaxel for 3 days, we did not observe any statistically significant difference of IC50 values between the groups of C42b cells transfected or not transfected with c-KIT and PC3 cells transfected or not transfected with c-KIT, in the presence or absence of the ligand SCF, especially when using doses of Docetaxel that were more similar to the IC50 values found in previous experiments (treatment with Docetaxel for 2 days, doses below 10 nM of Docetaxel). In an attempt to understand the increased chemoresistance to the treatment with Docetaxel for 3 days in the groups without c-KIT transfection, we performed the cell doubling time (DT) in the groups C42b and PC3, which also showed no difference between groups (PC3 EV DT = 40 hours, PC3 c-KIT DT = 32 hours, C42b EV DT = 12 hours, C42b c-KIT DT = 14 hours). Conclusions: the present study showed no increased chemoresistance of C42b and PC3 cells to the chemotherapeutic agent Docetaxel when they are transfected with c-KIT.

In den letzten Jahren haben regenerative Therapieansätze zur Behandlung der chronischen Herzinsuffizienz mit Einsatz adulter Stammzellen größere Aufmerksamkeit erfahren. Zahlreiche klinische Studien an Patienten mit ischämischer Kardiomyopathie zeigten erfolgversprechende Ergebnisse mit der Transplantation autologer Knochenmarkstammzellen (BMC). Es existieren Hinweise, dass BMC Connexin43 exprimieren könnten. In der hier vorgelegten Arbeit erfolgte in einem Ischämie-Reperfusions-Modell am autonom schlagenden Langendorff-Herzen nach passagerer Ligatur einer Koronarie mit Induktion einer Myokardischämie eine unselektive transkoronare Injektion autologer BMC in der frühen Reperfusionsphase. Zu überprüfen war die Hypothese, dass BMC in dieser Phase der Reperfusion sicher administriert werden können und bereits ein ischämiegetriggertes Homing im Infarktareal zeigen. Zudem sollte überprüft werden, ob c-kit+ (CD117) BMC in der Lage sind Connexin43 zu exprimieren. Additiv wurde am Beispiel von NF-κB postuliert, dass auch die Aktivierung früher Gene einen Einfluss auf das Homing von c-kit+ BMC haben. Die Applikation einer großen Zellzahl (durchschnittlich 1,8 Mio. /g Myokard) mit Infusionsraten von 0,5 - 2,6 Mio. Zellen pro Minute hatte hierbei keinen negativen Einfluss auf Hämodynamik und rhythmogene Stabilität. Akute Myokardischämien traten nicht auf. Der gewählte Applikationsweg und -zeitpunkt führten zu einer quantitativ adäquaten Zellretention. Immunhistologisch ließen sich die Tendenz zum 'Homing' von c-kit+ BMC im ischämischen linksventrikulären Myokard sowie Zeichen der ischämiegetriggerten Extravasation nachweisen. Eine signifikante Expression von Connexin43 an c-kit+ BMC konnte nicht gezeigt werden. Somit kann eine gap-junction-vermittelte Kommunikation zwischen c-kit+ BMC zu diesem Zeitpunkt nicht bestätigt werden. Im direkten Umfeld c-kit+ BMC fand sich keine vermehrte Aktivierung von NF-κB.

In der vorliegenden Arbeit wurden die Effekte einer frühzeitigen Zelltherapie im Langzeit in-vivo Infarktmodell studiert. Erstmals wurden dabei auch Veränderungen der kardialen -Adrenozeptoren untersucht und Zelltherapie mit einer reversiblen präinfarziösen Ischämie kombiniert. Initial wurden dafür bei 38 männlichen weißen Neuseeländer Kaninchen Knochenmarkspunktionen durchgeführt, MSC durch Kultur isoliert und 60 Minuten nach induziertem Infarkt und ohne Reperfusion in den Randbereich des Infarktgebietes injiziert. Zur Untersuchung möglicher Interaktionen zwischen Zelltherapie und Präinfarktgeschehen wurde bei einigen Tieren das Myokard durch eine kurzzeitige Präinfarktischämie präkonditioniert. Die Ergebnisse der vorliegenden Arbeit zeigen, dass auch die frühzeitige Zellinjektion ohne Reperfusion mit signifikanten Effekten auf die Kontraktilität und spezifischen sympathoadrenergen Veränderungen verbunden ist.:Abkürzungsverzeichnis vii Literaturübersicht 1 Myokardinfarkt und postischämische Herzinsuffizienz 1 sympathoadrenerge Veränderungen bei Myokardinfarkt und Herzinsuffizienz 1 akuter Myokardinfarkt 1 Herzinsuffizienz 2 Veränderungen der -Adrenozeptoren bei Herzinsuffizienz 3 Stammzellen und Zelltherapien 4 Zelltherapien bei Myokardinfarkt 5 Knochenmark 5 hämatopoetische Stammzellen 5 mesenchymale Stammzellen 6 endotheliale Progenitorzellen 8 klinische Studien 8 Effekte adulter Stammzellen und anderer Zelltypen 10 Angiogenese 11 Zytoprotektion, Apoptosehemmung und antiinflammatorische Effekte 12 Unterstützung endogener Reperaturmechanismen 12 Stabilisierung der extrazellulären Matrix 13 Optimierung der Stammzelltherapien 13 Injektionszeitpunkt 14 Stammzellmigration 15 Ziel der Arbeit, Thesen und Fragestellung 17 Methoden 18 Versuchsaufbau 18 Tierhaltung 19 Tiermodell und Versuchsprotokoll 19 Knochenmarksgewinnung 19 Zellaufbereitung 19 Induktion des Myokardinfarktes und Zellapplikation 20 Sakrifizierung und Probenentnahme 21 Auswertung 22 transthorakale Echokardiografie 23 Durchführung 23 Radioligandenbindungsstudien 25 Rezeptortheorie 25 -Adrenozeptoren 25 Grundlagen der Radioligandenbindungsstudien 27 Auswertung der Radioligandenbindungsstudien 28 -Adrenozeptorbestimmung durch Radioligandenbindungsstudien 30 Durchführung 30 Radioligand 31 Probenvorbereitung 31 Proteinbestimmung 32 Bindungsstudien 32 Auswertung 34 Noradrenalinbestimmung durch HPLC 36 Flüssigkeitschromatografie und HPLC 36 Prinzipien der Auftrennung 36 Aufbau eines HPLC Systems 37 chromatografische Kenngrössen 39 Probenvorbereitung 42 Durchführung 42 Katecholaminextraktion 43 Chromatografie 43 Auswertung 44 Immunhistochemie 45 c-Kit 45 Durchführung 46 Gewebeproben 46 Entparaffinierung und Gewebevorbehandlung 46 immunhistochemische Färbung 47 Auswertung der histologischen Schnitte 47 statistische Auswertung 48 Ergebnisse 49 allgemeine Daten 49 Echokardiografie 50 linksventrikuläre Funktion 50 Infarktausdehnung und Wandstärke im Infarktbereich 54 sympathoadrenerge Veränderungen 55 Radioligandenbindungsstudien 55 -Adrenozeptordichte im LV, S und RV 57 Noradrenalin Plasmakonzentration 64 c-Kit positive Zellen im Infarktbereich und infarktfernen Myokard 66 Diskussion 68 Ziel und Fragestellung 68 Tiermodell 68 Echokardiografie 70 Radioligandenbindungsstudien 73 Veränderungen der -Adrenozeptoren nach Myokardinfarkt 74 Therapiebedingte Veränderungen 76 plasmatisches Noradrenalin 79 Immunhistochemie 82 Zusammenfassung 84 Literaturverzeichnis I Anhang a Abbildungsverzeichnis a Tabellenverzeichnis b Tabellen c Materialien und Geräte f Tierversuche und Laborgeräte f Apperaturen und Geräte zur Durchführung der RLBS f HPLC System f statistische Auswertung und grafische Darstellung g Verbauchsmaterialien g allgemeine Laborchemikalien g Verbrauchsmateralien zur Durchführung der HPLC h Verbrauchsmateralien zur Durchführung der RLBS h Verbrauchsmateralien zur Durchführung der Immunhistochemie h Erklärung über die eigenständige Abfassung der Arbeit i Danksagung j Lebenslauf k Publikationsverzeichnis l

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We previously demonstrated that embryonic stem (ES) cells over-expressing tissue inhibitor of metalloproteinase-1 (TIMP-1) have increased potential to engraft and differentiate into cardiac myocytes following transplantation into the infarcted heart. However, the ability of TIMP-1 to activate endogenous stem cells and enhance their differentiation into cardiac regenerative cell types is still unknown. We postulate that TIMP-1 may additionally activate a stem cell population that enhances cardiac cell type differentiation in the infarcted myocardium. To prove this hypothesis, we isolated c-kit+ve cells from four weeks old C57BL/6 mice and cultured them in vitro in presence of ES conditioned media (ESCM), ES-TIMP-1-CM or TIMP-1. Our immunostaining data validate the existence of a novel CD63+ve/c-kit+ve cells. When treated with TIMP-1, these cells showed significantly (p < 0.05) increased proliferation and differentiation into cardiac myocytes, vascular smooth muscle cells, and endothelial cells. Western blot analysis revealed significantly (p < 0.05) increased expression of CD63, phosphorylated and total β-catenin proteins. Furthermore, our RT-PCR data showed increased cardiac gene expression (GATA-4, Mef2C, and Nkx-2.5) when compared to ESCM and control cells. Based on the in vitro findings, we investigated the effect of intramyocardial delivery of TIMP-1 on endogenous CD63+ve/c-kit+ve cells following myocardial infarction (MI). C57BL/6 and TIMP-1 KO mice underwent coronary artery ligation followed by intramyocardial delivery of 20μl of culture media (CC), ESCM, ES-TIMP-1-CM or TIMP-1. Subsequent immunohistochemistry analysis demonstrated the presence of a CD63+ve/c-kit+ve cell population within the peri-infarct area and confirmed intramyocardial delivery of ES-TIMP-1-CM or TIMP-1 significantly (p < 0.05) enhanced their proliferation. Percentage of CD63+ve/c-kit+ve cells was significantly (p < 0.05) lower in TIMP-1 KO mice compared to C57BL/6 animals. RT-PCR analysis revealed TIMP-1 KO animals expressed significantly less CD63 and TIMP-1 mRNAs compared to C57BL/6 mice. Activated CD63+ve/c-kit+ve cells were also able to differentiate into major cardiac cell types as previously shown in vitro. The differentiation potential of these cells was however higher in C57BL/6 mice compared to TIMP-1 KO mice. We also demonstrate that CD63+ve/c-kit+ve cells differentiation is regulated by CD63/β-catenin pathway in vivo. Additionally, we provide evidence that TIMP-1 protects the heart from adverse cardiac remodeling through inhibition of cardiac apoptosis and fibrosis leading to significantly (p < 0.05) improved contractile function. Collectively, our data show TIMP-1 plays a dual protective role in the MI heart. It activates a unique stem cell population, CD63+ve/c-kit+ve, which proliferates and differentiates into functional myocytes, smooth muscle cells and endothelial cells mediated through CD63/β-catenin pathway. TIMP-1 also protects the heart from adverse cardiac remodeling. Increased cardiac regeneration and inhibition of adverse cardiac remodeling consequently lead to restored cardiac function.

A incidência do melanoma cutâneo em pacientes adultos jovens tem aumentado consideravelmente nos últimos anos. Há, contudo, carência de conhecimentos clinicopatológicos e moleculares sobre os melanomas que ocorrem nessa faixa etária. O presente estudo teve por objetivo avaliar 132 casos de melanoma cutâneo primário em pacientes com idade entre 18 e 30 anos, com ênfase no estudo das características clínicas, histopatológicas e avaliação molecular das mutações nos genes BRAF, NRAS e KIT. Em relação aos achados clínicos e histopatológicos, houve predomínio de indivíduos do sexo feminino (61,4%), sendo o tronco o sítio anatômico mais comumente envolvido (44,3%) e o melanoma extensivo superficial o tipo histológico predominante (79,5%). A mutação V600E no gene BRAF (BRAFV600E) foi analisada em 93 casos, utilizando-se a técnica de RT-PCR. Essa mutação foi identificada em 38,7% (36/93) e, estatisticamente, associada à fase vertical de crescimento (p = 0,01), infiltrado inflamatório discreto (p = 0,02) e presença de mitose intradérmica (p = 0,004). Houve, ainda, forte indício de associação com a presença de ulceração (p = 0,05). Todas essas variáveis apresentaram associação com pior prognóstico do melanoma cutâneo. Observou-se predomínio da mutação BRAFV600E em regiões anatômicas relacionadas à exposição solar intermitente. Nenhum caso de melanoma com fenômeno de regressão apresentou mutação BRAFV600E (p < 0,05). Não houve associação significativa entre BRAFV600E e sexo, tipo histológico, nível de Clark, índice de Breslow, elastose solar, invasão angiolinfática e perineural, satelitose, nevo melanocítico coexistente e sobrevida. A pesquisa de mutações NRAS, pela técnica de RT-PCR, detectou frequência de 3,95% (3/76). As três mutações encontradas foram do tipo 61K e ocorreram em pacientes do sexo masculino e em região de cabeça e pescoço. As mutações BRAFV600E e NRAS, quando presentes, eram mutuamente exclusivas. A frequência de mutações KIT, analisadas por sequenciamento, foi de 11,1% (3/27). As três mutações identificadas estavam localizadas no éxon 9 (G510, G498S e 489I). Houve concomitância de casos com mutação KIT tanto com NRAS, como com BRAFV600E. Devido ao pequeno número de casos com mutação em KIT e NRAS, não foi possível estabelecer correlações clínicas e histopatológicas com esses genes. Este estudo é o primeiro a descrever as mutações G510D e G498S no gene KIT em melanomas cutâneos. No presente estudo, a mutação BRAFV600E, em melanomas cutâneos de adultos jovens, correlacionou-se com características anatomoclínicas de pior prognóstico em relação aos melanomas selvagens para BRAFV600E / The incidence of cutaneous melanoma in young adults has dramatically increased in recent years. However, there is scarce data about the clinicopathological and molecular characteristics on the melanomas occurring at this age group. The present study aimed to evaluate 132 patients aged between 18 and 30 years with primary cutaneous melanoma with emphasis on the study of clinical, histopathological characteristics and molecular evaluation of mutations in BRAF, NRAS and KIT genes. Regarding the clinical and histopathological findings, the following results were found: female predominance (61.4%), trunk was the most commonly anatomical site involved (44.3%) and superficial spreading melanoma, was the most common histological type (79.5 %). The V600E mutation in BRAF (BRAFV600E) gene was analyzed in 93 cases, using RT-PCR. It was present in 38.7% (36/93) and statistically related to the vertical growth phase (p = 0.01), mild inflammatory infiltration (p = 0.02) and the presence of intradermal mitosis (p = 0.004). There was, also, strongly evidence of an association with the presence of ulceration (p = 0.05). Worse prognosis was associated with these variables. There was a predominance of BRAFV600E mutation in anatomical regions related to intermittent sun exposure. No cases of melanoma with BRAFV600E mutation showed regression phenomenon (p < 0.05). There was no significant association between BRAFV600E and gender, histological type, Clark level, Breslow thickness, solar elastosis, angiolymphatic and perineural invasion, sattelitosis, coexisting melanocytic nevus and survival. The presence of a mutation in NRAS, by RT-PCR was seen in 3.95% (3/76) of the cases. All these three mutations were of type 61K, occurred in male patients and the head and neck region. BRAFV600E and NRAS mutations, when present, were mutually exclusive. The frequency of KIT mutations, analyzed by sequencing, was 11.1% (3/27). The three mutations identified in this gene were located in exon 9 (G510, G498S and 489I). Concomitant mutations were found between KIT and NRAS and BRAFV600E. Due to the small number of KIT and NRAS mutated cases, it was not possible to establish clinical and histopathological correlations and mutation status in these genes. This study was the first to describe the G510D and G498S mutations in KIT gene in cutaneous melanomas. In the present study, BRAFV600E mutation in cutaneous melanoma of young adults correlated with anatomic and clinical features of worse prognosis compared to wild type

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2014-09-30T18:51:57Z No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Jaqueline Silva (jtas29@gmail.com) on 2014-09-30T19:29:06Z (GMT) No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-09-30T19:29:06Z (GMT). No. of bitstreams: 2 Dissertação- Maria Helena Tavares Vilela-2013.pdf: 2276556 bytes, checksum: 772ad6ab585b17765e734a42bc50eb7e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2013-10-09 / The proper management of breast phyllodes tumors (PTs) remains challenging, due to the difficulty of the correct preoperative diagnosis. The aim of this study was to evaluate the usefulness of the Ki-67, CD10, CD34, p53, CD117 and the number of mast cells for the differential diagnosis between benign PTs and cellular fibroadenomas (CFs), and also at the grading of PTs. 51 primary PTs and 14 FCs were examined by immunohistochemistry (IH). Through the evaluation of the expression of CD117, greater epithelial expression was found at the FCs and an increased number of mast cells in benign TFs. The stromal expression of the Ki-67, CD10, CD34 and p53 showed relevance to the grading of PTs. / O manejo adequado dos tumores filóides (TFs) mamários continua desafiador, devido à dificuldade do diagnóstico pré-operatório correto. O objetivo deste estudo foi avaliar a utilidade do Ki-67, do CD34, do CD10, do CD117, da p53 e do número de mastócitos no diagnóstico diferencial entre os TFs benignos e fibroadenomas celulares (FCs), bem como na graduação dos TFs. Foram examinados 51 TFs primários e 14 FCs por imunohistoquímica (IH). Na marcação pelo CD117, houve maior expressão epitelial nos FCs e maior número de mastócitos nos TFs benignos. A expressão estromal do Ki-67, da p53, do CD10 e CD34 mostrou-se significante na graduação dosTFs.

A incidência do melanoma cutâneo em pacientes adultos jovens tem aumentado consideravelmente nos últimos anos. Há, contudo, carência de conhecimentos clinicopatológicos e moleculares sobre os melanomas que ocorrem nessa faixa etária. O presente estudo teve por objetivo avaliar 132 casos de melanoma cutâneo primário em pacientes com idade entre 18 e 30 anos, com ênfase no estudo das características clínicas, histopatológicas e avaliação molecular das mutações nos genes BRAF, NRAS e KIT. Em relação aos achados clínicos e histopatológicos, houve predomínio de indivíduos do sexo feminino (61,4%), sendo o tronco o sítio anatômico mais comumente envolvido (44,3%) e o melanoma extensivo superficial o tipo histológico predominante (79,5%). A mutação V600E no gene BRAF (BRAFV600E) foi analisada em 93 casos, utilizando-se a técnica de RT-PCR. Essa mutação foi identificada em 38,7% (36/93) e, estatisticamente, associada à fase vertical de crescimento (p = 0,01), infiltrado inflamatório discreto (p = 0,02) e presença de mitose intradérmica (p = 0,004). Houve, ainda, forte indício de associação com a presença de ulceração (p = 0,05). Todas essas variáveis apresentaram associação com pior prognóstico do melanoma cutâneo. Observou-se predomínio da mutação BRAFV600E em regiões anatômicas relacionadas à exposição solar intermitente. Nenhum caso de melanoma com fenômeno de regressão apresentou mutação BRAFV600E (p < 0,05). Não houve associação significativa entre BRAFV600E e sexo, tipo histológico, nível de Clark, índice de Breslow, elastose solar, invasão angiolinfática e perineural, satelitose, nevo melanocítico coexistente e sobrevida. A pesquisa de mutações NRAS, pela técnica de RT-PCR, detectou frequência de 3,95% (3/76). As três mutações encontradas foram do tipo 61K e ocorreram em pacientes do sexo masculino e em região de cabeça e pescoço. As mutações BRAFV600E e NRAS, quando presentes, eram mutuamente exclusivas. A frequência de mutações KIT, analisadas por sequenciamento, foi de 11,1% (3/27). As três mutações identificadas estavam localizadas no éxon 9 (G510, G498S e 489I). Houve concomitância de casos com mutação KIT tanto com NRAS, como com BRAFV600E. Devido ao pequeno número de casos com mutação em KIT e NRAS, não foi possível estabelecer correlações clínicas e histopatológicas com esses genes. Este estudo é o primeiro a descrever as mutações G510D e G498S no gene KIT em melanomas cutâneos. No presente estudo, a mutação BRAFV600E, em melanomas cutâneos de adultos jovens, correlacionou-se com características anatomoclínicas de pior prognóstico em relação aos melanomas selvagens para BRAFV600E / The incidence of cutaneous melanoma in young adults has dramatically increased in recent years. However, there is scarce data about the clinicopathological and molecular characteristics on the melanomas occurring at this age group. The present study aimed to evaluate 132 patients aged between 18 and 30 years with primary cutaneous melanoma with emphasis on the study of clinical, histopathological characteristics and molecular evaluation of mutations in BRAF, NRAS and KIT genes. Regarding the clinical and histopathological findings, the following results were found: female predominance (61.4%), trunk was the most commonly anatomical site involved (44.3%) and superficial spreading melanoma, was the most common histological type (79.5 %). The V600E mutation in BRAF (BRAFV600E) gene was analyzed in 93 cases, using RT-PCR. It was present in 38.7% (36/93) and statistically related to the vertical growth phase (p = 0.01), mild inflammatory infiltration (p = 0.02) and the presence of intradermal mitosis (p = 0.004). There was, also, strongly evidence of an association with the presence of ulceration (p = 0.05). Worse prognosis was associated with these variables. There was a predominance of BRAFV600E mutation in anatomical regions related to intermittent sun exposure. No cases of melanoma with BRAFV600E mutation showed regression phenomenon (p < 0.05). There was no significant association between BRAFV600E and gender, histological type, Clark level, Breslow thickness, solar elastosis, angiolymphatic and perineural invasion, sattelitosis, coexisting melanocytic nevus and survival. The presence of a mutation in NRAS, by RT-PCR was seen in 3.95% (3/76) of the cases. All these three mutations were of type 61K, occurred in male patients and the head and neck region. BRAFV600E and NRAS mutations, when present, were mutually exclusive. The frequency of KIT mutations, analyzed by sequencing, was 11.1% (3/27). The three mutations identified in this gene were located in exon 9 (G510, G498S and 489I). Concomitant mutations were found between KIT and NRAS and BRAFV600E. Due to the small number of KIT and NRAS mutated cases, it was not possible to establish clinical and histopathological correlations and mutation status in these genes. This study was the first to describe the G510D and G498S mutations in KIT gene in cutaneous melanomas. In the present study, BRAFV600E mutation in cutaneous melanoma of young adults correlated with anatomic and clinical features of worse prognosis compared to wild type

Pulmonary arterial hypertension (PAH) is a group of diseases characterized by avascular obstruction leading to a progressive increase of the resistances in the pulmonary blood flow. The recent progress in the understanding of mechanisms at the origin of this disease underlines the role of extrapulmonary cells, such as circulating stem cells and bone marrow-derived progenitor cells in vascular remodeling and in PAH development. In this thesis we have shown implication of the progenitor cells and chemotactic axis in the vascular remodeling in human and experimental PAH. This work could help to develop new therapies allowing more specific and more effective treatments leading to improved survival of PAH patients.

Atherosklerotische Arterien unterliegen strukturellem Umbau und chronischer Inflammation, die von einer dynamischen Entwicklung von Vasa vasorum (VV) begleitet wird. Die Beteiligung von Leukozyten und von vaskulären Vorläuferzellen an der Neovaskularisierung sowie die intimale Hyperplasie stehen im Zentrum der Atheroskleroseforschung. Damit verbundene Erkenntnisse könnten neue therapeutische Ansätze ermöglichen. Die vorliegende Arbeit befaßt sich mit der morphologischen Verteilung von Leukozyten (CD45, CD68, Mastzellen) und von Zellen mit Vorläufermarkern (CD34, CD117, VEGFR-2) in menschlichen Coronararterien mit verschiedenen atherosklerotischen Schweregraden. Mittels immunhistologischer Technik wurden Intima und Adventitia untersucht und die Ergebnisse zu den atherosklerotischen Schweregraden und der Neovaskularisierung korreliert. In Intima, Adventitia und dem perivaskulären Fettgewebe hat die Dichte der CD45+ Lymphozyten ihr Maximum im atherosklerotischen Grad 3. Dabei konnte sowohl in der Intima als auch in der Adventitia gezeigt werden, daß eine lineare Korrelation der CD45+ Lymphozyteninfiltration und VV-Dichte vorliegt. Es wurden zwei unterschiedliche Entzündungsmuster festgestellt. Beide zeigen in Grad 3 eine Zunahme der Zelldichten. In Grad 4-5 fällt die Dichte des einen Musters (CD45+, VEGFR-2+, VV) jedoch ab, während die Dichte des anderen Musters (CD34+, CD68+, Tryptase+, CD117+) in Grad 4-5 keine Veränderung aufweist. Die Ergebnisse deuten darauf hin, daß Leukozyten und vaskuläre Vorläuferzellen im Verlauf der Atherogenese wechselnde Funktionen wahrnehmen können. Sie nehmen offensichtlich VV als Eintrittspforte in die Gefäßwand.