FundaÃÃo Cearense de Apoio ao Desenvolvimento Cientifico e TecnolÃgico / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Tumores sÃo populaÃÃes celulares heterogÃneas hierarquicamente organizadas, cujas cÃlulas-tronco possuem importÃncia relevante desde que sÃo cÃlulas com a capacidade de se renovarem e de gerarem linhagens em fases diferentes. Dada a sua importÃncia, a identificaÃÃo de componentes de cÃlulas-tronco à essencial para o entendimento da tumorigÃnese. Apesar de marcadores de linhagem neural terem sido identificados, a associaÃÃo destes marcadores com os tumores cerebrais ainda à escassa e nos astrocitomas sÃo relacionados principalmente aos glioblastomas. Entre esses marcadores de cÃlulas-tronco,CD133, CXCR4 e CD44 sÃo relacionados à formaÃÃo do glioma, migraÃÃo e crescimento; por outro lado, OLIG2 à envolvido no destino celular. NÃo existem estudos, atà essa data, que avaliam todos esses marcadores juntos e sua relaÃÃo com grau tumoral. Adicionalmente, alteraÃÃes epigenÃticas especÃficas, especialmente a metilaÃÃo em promotor, tem sido identificadas nestes tumores, levando a inativaÃÃo de genes, com destaque o CDKN2A (proteÃna p16INK4A), um supressor tumoral. Apesar de esse mecanismo ser apontado como o principal inativador desse gene, em astrocitomas ainda existem questÃes controversas. Para avaliar essas questÃes, este estudo objetivou determinar a expressÃo e padrÃo de metilaÃÃo em promotor de CDKN2A e sua associaÃÃo com parÃmetros clinico-patolÃgicos e se a presenÃa de cÃlulas-tronco/progenitoras, considerando a expressÃo de CD133, CXCR4, CD44 e OLIG2 poderia definir subpopulaÃÃes de cÃlulas que podem ser usadas como marcadores prognÃsticos. Para isso, em uma sÃrie de 93 astrocitomas de diferentes graus de malignidade, foram estudadas a expressÃo dos marcadores CD133, CXCR4, CD44, OLIG2 e p16INK4A, detectada pela tÃcnica de imunohistoquÃmica, e o padrÃo de metilaÃÃo em promotor de CDKN2A, por PCR especÃfico para metilaÃÃo (PCR-MS). Os dados foram entÃo associados com grau tumoral, localizaÃÃo e outros parÃmetros clinico-patolÃgicos. As anÃlises estatÃsticas foram realizadas usando o teste do X2, teste exato de Fisher, correlaÃÃo de Spearman, agrupamento de k-means e anÃlise de componentes principais, com diferenÃas consideradas significantes com p<0.05. A imunomarcaÃÃo de OLIG2 mostrou a frequÃncia maior de positividade (73,1%), seguido por CXCR4 (60,2%), CD44 (55,9%) e CD133 (45,2%). AnÃlises de correlaÃÃo e agrupamento definiram dois subtipos de populaÃÃo de acordo com os marcadores estudados, um subtipo CXCR4(+)CD133(+)CD44(+) e outro OLIG2(+). Tumores CD133, CXCR4 e CD44 positivos aumentaram de acordo com malignidade. No grau IV, este subtipo de tumores [CD133(+)CXCR4(+)CD44(+)] foi significantemente mais frequente (p=0,008) e tambÃm nos tumores difusos. Adicionalmente, tumores com CXCR4(+) e CD133(+) foram preferencialmente localizados nos hemisfÃrios cerebrais e nos ventrÃculos, e a maioria nos pacientes com idade &#8805; 30 anos. Por outro lado, tumores OLIG2(+) foram associados com o cerebelo, que à a localizaÃÃo preferencial do astrocitoma pilocÃtico. Uma forte correlaÃÃo negativa entre imunomarcaÃÃo nuclear e citoplasmÃtica e metilaÃÃo em promotor de CDKN2A foi encontrada. AlÃm do mais, uma correlaÃÃo negativa significante entre metilaÃÃo em promotor de CDKN2A e idade foi observada e pacientes do sexo feminino tiveram uma maior frequÃncia significante de CDKN2A metilado em promotor que o sexo masculino. Em conclusÃo, a presenÃa de subpopulaÃÃes de cÃlulas-tronco em astrocitomas à indicativa de progressÃo tumoral, cujos marcadores CXCR4, CD133 e CD44 podem ser potencialmente usados em conjunto como marcadores prognÃsticos. A associaÃÃo com localizaÃÃo do tumor e idade tambÃm corroboram esses achados. Adicionalmente, a inativaÃÃo de CDKN2A por metilaÃÃo em promotor à um evento frequente em astrocitomas e à relacionada à idade e sexo dos pacientes. / Currently, the concept that tumors are cell populations organized in a hierarchically heterogenous way in which stem-cells are relevantly important as these cells have the capacity of self-renew and of generating cell lineages in different phases of differentiation. So that, the identification of stem-cell components is essential to tumorigenesis understanding. Althought neural cell lineage markers have been identified, the association among these markers and neurological tumors is still scarce, and taking in consideration the astrocytomas, the association assessements are verified mainly regarding the glioblastomas. Among these stem cell markers, CD133, CXCR4 and CD44 are related to the glioma formation, migration and growth; on the other hand, OLIG2 is involved in cell destination. So far there are no studies evaluating all these markers together and their relationship to tumor grades. Additionally, specific epigenetic alterations, specially promoter methylation, have been widelly identified in these tumors, leading to gene inativation, mostly involving CDKN2A (p16INK4A protein), a tumor suppressor. Althought this mechanism is pointed as this gene main inactivator, there are still controvertial questions regarding the astrocytomas. In order to evaluate these questions, the present study aimed to determine CDKN2A pattern of methylation and expression and their association to clinicalpathological parameters, and if the presence of progenitor/stem-cells, taking CD133, CXCR4, CD44 and OLIG2 expression in consideration, could define subpopulations of cells which might be used as prognostic markers. So, in a series of 93 astrocytomas of different malignity grades, the expression of CD133, CXCR4, CD44, OLIG2 and p16INK4A was analysed by the imunohistochemistry technique, and the CDKN2A methylation status was assessed by methylation specific PCR (MS-PCR). The data was then associated to tumor grades, localization and other clinicalpathological parameters. The statistic analyses were made using X 2 test, Fisher's exact test, Spearman's correlation, kmeans groupment and principal component analyses, using p<0.05 as statistically significance. The imunopositivity of OLIG2 was predominant (73.1%), followed by CXCR4 (60.2%), CD44 (55.9%) and CD133 (45.2%). The correlation and groupment analyses defined two different population subtypes, a CXCR4(+)CD133(+)CD44(+) subtype and a OLIG2(+) subtype. CXCR4(+)CD133(+)CD44(+) tumors became more frequent as malignity grew. In grade IV, this subtype was significantly more frequent (p=0.008), being also in diffuse tumors. Additionally, CXCR4(+) and CD133(+) tumors were preferentially located in brain hemisferes and in the ventricles, and mostly in aged >30 patients. On the other side, OLIG2(+) tumors were associated to the cerebellum, which is the pylocitic tumor preferential localization. A strong negative correlation between nuclear and cytoplasmatic imunopositivity and promoter methylation in CDKN2A was observed. Also, a negative significant correlation between methylated CDKN2A and patient's age was found; moreover, feminine patients presented a higher frequency of methylated CDKN2A. In conclusion, the presence of stemcell subpopulations in astrocytomas indicates tumoral progression, in which CXCR4, CD133 and CD44 may be potentially used together as prognostic markers. The association between tumor localization and patient's age also corroborates these findings. Additionally, the CDKN2A inactivation by promoter methylation is a frequent event in astrocytomas and it is associated to patient's age and gender

Sabemos hoje que os tumores sólidos apresentam uma composição celular heterogênia e que apenas uma pequena parcela dessas células apresenta capacidade de se proliferar e gerar novos tumores. Estudos prévios sobre a formação do câncer de mama têm sido realizados com base na combinação dos marcadores de superfície celular CD44 e CD24. Já foi demonstrado que uma subpopulação de células do câncer de mama com alta expressão de CD44 e baixa expressão de CD24 (CD44+/CD24-) tem maior capacidade de gerar tumores, quando comparadas com a subpopulação de células CD44-/CD24+. O objetivo do estudo foi identificar a taxa de células com fenótipo CD44+/CD24- presentes nos tumores mamários e relaciona-la com a taxa de comprometimento dos linfonodos axilares ipsilaterais por neoplasia, além de avaliar também sua relação com outros fatores sabidamente relacionados com mal prognóstico da paciente. Pacientes e métodos: avaliamos prospectivamente 53 amostras cirúrgicas provenientes de 42 pacientes com diagnóstico histopatológico de carcinoma de mama, quantificando as células CD44+/CD24- por citometria de fluxo. Relacionamos a porcentagem destas células encontrada em cada amostra com o comprometimento axilar, os receptores hormonais e Her-2, a idade da paciente, o grau histológico do tumor, o diâmetro patológico do tumor e o tipo histológico. Resultados: verificamos um significante aumento da população de células CD44+/CD24- no grupo de carcinomas ductais invasivos em pacientes que apresentavam metástase axilar [mediana 8,53% (3,6 71,2%)] em relação ao grupo de pacientes sem linfonodos comprometidos pela neoplasia [mediana 1,49% (0,3 17,1%)] (p=0,0002). Conclusão: concluímos então que quando estudamos vários tumores mamários invasivos de mesma classificação histológica, podemos notar que existe uma variação na quantidade de células CD44+/CD24- entre eles. Nosso estudo mostrou que essa variação está relacionada à agressividade tumoral e à sua capacidade de gerar metástases já que, tumores com maior quantidade de células CD44+/CD24- apresentam maior taxa de comprometimento dos linfonodos axilares. / It is known that solid tumors are composed by a heterogeneous combination of cells and only a small portion of these cells has the capacity to proliferate and generate new tumors. Previous studies about the breast cancer initiation have been based on a combination of CD44 and CD24cell surface markers. It has been shown that this subpopulation of breast cancer cells with high expression of CD44 and low expression of CD24 (CD44+/CD24-) has a greater capacity to generate tumors when compared with the subpopulation of cells CD44- /CD24+. The study objective was to identify whether the rate of cells with CD44+/CD24- phenotype present in breast tumors is related with the rate of ipsilateral lymph node metastasis, in addition to evaluate its relationship with other risk factors known to be related with worst prognosis. Patients and methods: we prospectively evaluated 53 surgical specimens from 42 patients with histological diagnosis of breast cancer, quantifying CD44+/CD24- cells through flow cytometry. We list the percentage of these cells found in each sample with axillary lymph node status, hormone receptors and Her-2, patient age, histological grade, pathological tumor diameter and histological tumorclassification. Results: we find a significant increase of CD44+/CD24- population in the invasive ductal carcinomas, in patients with axillary metastasis [median 8.53% (3.6 - 71.2%)] than in the group of patients without lymph nodes metastasis [median 1.49% (0.3 - 17.1%)] (p = 0.0002). Conclusion: when we studied several invasive breast tumors of same histological classification, we note that there is variation in the number of CD44+/CD24- cells. Our study showed that this variation is related to tumor aggressiveness and their ability to generate metastasis, because tumors with high rate of CD44+/CD24- cells have a higher rate of lymph node metastasis.

Die vorliegende Habilitationsschrift "Neue Serummarker bei urologischen Malignomen mit dem Schwerpunkt Prostatakarzinom und Anwendung von Proteinase-Inhibitoren in der Therapie des Prostatakarzinoms" faßt Ergebnisse zusammen, die ich in den Jahren von 1996 bis 2000 als Erstautor in wissenschaftlichen Artikeln von peer reviewed Zeitschriften veröffentlicht habe. Zusätzlich werden 14 Arbeiten mit ihren Aussagen eingeschlossen, bei denen ich als Koautor beteiligt war. Gegenstand der Habilitationsschrift sind Untersuchungen zur diagnostischen Optimierung des Tumormarkers Prostataspezifisches Antigen (PSA) und zum Expressionsverhalten von CD44-Proteinen bzw. Matrix-Metalloproteinasen (MMPs) als potentielle, neue Marker bei urologischen Karzinomen. Die nachgewiesene Bedeutung der MMPs bei der Tumorprogression und -metastasierung hat mich dazu veranlaßt, tierexperimentelle Studien zur Hemmung der MMP-Aktivität mit synthetischen Inhibitoren in meine Arbeit aufzunehmen. Zielstellung war hierbei die Evaluierung neuer Therapieoptionen beim fortgeschrittenen Tumor. Im Mittelpunkt der Untersuchungen steht das Prostatakarzinom (PCa) als häufigster maligner Tumor des Mannes. 1. Das PSA ist ohne Zweifel der beste Tumormarker in der Diagnostik des PCa. Der Optimierung dieses Markers wird große Bedeutung zugemessen. Dabei werden verschiedene Konzepte verfolgt, wobei die Bestimmung der Isoformen des PSA die zur Zeit erfolgreichste Richtung zu sein scheint. Die Ergebnisse meiner u.a. im Rahmen von Multizenterstudien durchgeführten Untersuchungen belegen den diagnostischen Nutzen der zusätzlichen Bestimmung des f-PSA%, um PCa-Patienten früher zu erkennen und besser gegenüber Patienten mit benigner Prostatahyperplasie (BPH) abgrenzen zu können. Ein weiteres Ergebnis der Untersuchungen zur diagnostischen Validität anderer PSA-Isoformen ist die Feststellung, daß die Bestimmung des gebundenen PSA keinen Vorteil gegenüber dem f-PSA% hat. Widersprüchliche Angaben in der Literatur konnten damit ausgeräumt werden. Diese Ergebnisse veranlaßten die Firma Roche als Kooperationspartner, die Weiterentwicklung eines ACT-PSA Prototyp-Testsystems einzustellen. Die Ergebnisse meiner Untersuchungen zu den PSA-Isoformen haben inzwischen Eingang in den klinischen Alltag gefunden und werden in der Urologischen Klinik der Charité genutzt. Es wurden Entscheidungsgrenzen für den f-PSA%-Wert zur Indikationsstellung von Stanzbiopsien der Prostata als Klinikstandard erarbeitet. 2. Bei verschiedenen urologischen Tumoren wurde das Expressionsverhalten von CD44-Proteinen und MMPs bzw. deren Inhibitoren bestimmt, um eine mögliche Bedeutung bei der Tumorprogression zu erfassen. Diese Untersuchungen sind gleichzeitig Voraussetzung für eine mögliche Anwendung der Komponenten in der Diagnostik und Therapiekontrolle bei diesen Tumorentitäten. Im Gegensatz zu anderen menschlichen Tumoren konnten bei den untersuchten urologischen Tumoren keine veränderten Serumkonzentrationen der CD44-Proteine einschließlich der Varianten nachgewiesen werden. Daher habe ich weiterführende Studien zu den CD44-Proteinen nicht durchgeführt. 3. Im Tumorgewebe sowie im Plasma von Patienten mit PCa und Nierenzellkarzinom konnte ich signifikante Veränderungen von MMPs und deren Inhibitoren nachweisen. Die Situation im Gewebe spiegelt sich zum Teil im Blut der Patienten wider. Diese Beobachtungen beweisen die Bedeutung der MMPs bei der Tumorprogression und -metastasierung. Prinzipiell kann die Bestimmung einzelner MMPs bzw. TIMPs in der Diagnostik von urologischen Tumoren genutzt werden. Die niedrige Sensitivität in der individuellen Erfassung des einzelnen Tumorpatienten schränkt jedoch die praktische Anwendung ein. 4. Die veränderte MMP-Expression bzw. die Dysbalance zwischen MMPs und TIMPs hat mich veranlaßt, synthetische Inhibitoren zur Blockierung der MMP-Aktivität in einem Standardtiermodell des menschlichen PCa einzusetzen. In einer ersten Untersuchung am Dunning-Tumor der Ratte wurde nachgewiesen, daß dieser Tumor MMP9 exprimiert und die Serumkonzentration mit der Tumorgröße korreliert. In weiteren tierexperimentellen Untersuchungen wurde der Einfluß von Batimastat, einem Breitspektrum-Inhibitor der MMPs und einem neuentwickelten, selektiveren Inhibitor (Icol) auf das orthotope Tumorwachstum ermittelt. Beide Substanzen führten zu einer Hemmung des lokalen Tumorwachstums. Durch Applikation von Icol wurde eine Reduzierung des Tumorgewichtes um 90% im Vergleich zu den unbehandelten Kontrolltieren erreicht. Diese Beobachtungen haben eine doppelte Bedeutung. Zum einen beweist die Hemmwirkung von synthetischen MMP-Inhibitoren im Tiermodell die Funktion der MMPs bei der lokalen Tumorprogression. Zum anderen werden durch diese erfolgreichen tierexperimentellen Studien mit neuen Substanzen Voraussetzungen für die klinische Anwendung bei Patienten mit hormonrefraktärem PCa geschaffen. / The aim of my "habilitation thesis" was to evaluate the diagnostic validity of prostate-specific antigen (PSA) in serum and tissue, the serum pattern of CD44 proteins and of the matrix metalloproteinases (MMPs) in serum and tissue of urological malignancies. As MMPs seem to play an important role in tumor progression and metastasis, animal studies were additionally initiated in order to investigate the influence of synthetic inhibitors of MMPs on prostate cancer. 1. PSA is the most important and accurate tumor marker in prostate cancer diagnosis. However, PSA is an organ-specific marker, but is not tumor-specific. Elevated PSA concentrations are seen with non-malignant prostatic diseases like benign prostatic hyperplasia (BPH). Moreover, not all patients with prostate cancer have elevated PSA concentrations. In order to optimize the diagnostic validity of PSA, several concepts have been developed. Determination of the PSA isoforms in serum could help discriminate between prostate cancer and BPH. In various own studies, including a multicenter clinical trial, the determination of free PSA and the calculation the ratio of free PSA to total PSA (fPSA/tPSA) has proven to be a promising tool in prostate cancer diagnosis. Regarding the diagnostic validity of the complexed PSA conflicting data exist. Our results, using a newly developed alpha-1-antichymotrypsin-PSA (ACT-PSA) assay by Roche are contradictory to recent published data. Based on data of a multicenter trial, the determination of ACT-PSA as well as the ACT-PSA to tPSA ratio did not improve the differential diagnostic impact in patients undergoing evaluation for prostate cancer compared to the ratio fPSA/tPSA. 2. In various malignant diseases characteristic alterations in the expression of CD44 proteins and their variants have been observed. In contrast to those observations in other carcinomas, the determination of soluble CD44 proteins in serum is not suitable for detecting and staging patients with urological malignant tumors. Therefore, further investigation have not been performed. 3. Matrix-metalloproteinases (MMP) form a group of endogenous proteases with the common ability to degrade various components of the extracellular matrix. It could be demonstrated that increased levels of MMP are associated with the invasive and metastatic potential in human malignant tumors. However, little is known about the role of MMPs in renal cell carcinoma. In own study significant changes of MMP expression have been observed. Although changes in specific MMPs might be characteristic for renal carcinoma tissues and might be partly reflected in the blood, data shown that even MMP-9 as the best plasma marker, had a low sensitivity in detecting renal cell carcinoma. Increased concentrations of MMP-9 in tumor tissue may have important implications for the therapeutic potential of synthetic inhibitors of MMPs. 4. The importance of inhibitors of MMPs in cancer has been demonstrated in various studies. In own investigations, altered levels of MMPs and their specific inhibitors have been elucidated in prostate cancer. Therefore, a study to evaluate the efficacy of synthetic MMP inhibitors (batimastat, Icol) in a standard prostate cancer animal model was performed. Previously, the high expression of MMP-9 in this prostate cancer (Dunning tumor) compared with normal prostatic tissue could be demonstrated. Batimastat and the newly developed inhibitor Icol reduced the orthotopic tumor weights up to 90% in a dose-dependent manner. This results confirmed the importance of MMPs and their inhibitors in tumor progression. It can be concluded that selective inhibition of MMP activity is a novel therapeutic approach, which bears promise for studies in patients with hormone-refractory prostate cancer.

Im ersten Teil dieser Dissertation wurde der Einfluss des mitochondrialen Proteins TCAIM (T cell activation inhibitor, mitochondrial) auf die T-Zellaktivierung untersucht. Hierzu wurde eine transgene Mauslinie mit einem T-zellspezifischen knock-in (KI) von Tcaim in den Rosa26 Lokus generiert. Die Tcaim-Überexpression beeinflusste die Fission und Umverteilung von Mitochondrien und reduzierte die T-Zellrezeptor (TZR)-induzierte Bildung mitochondrialer, radikaler Sauerstoffspezies. In vitro stimulierte CD4+ Tcaim KI T-Zellen zeigten eine geringere Aktivierung, Proliferation und IL-2 Sekretion als Kontrollzellen. T-Zellen aus Tcaim KI Mäusen, die in Rag-1 knock-out Mäuse transferiert wurden, waren nicht fähig ein allogenes Haut-Transplantat abzustoßen und behielten einen naiven Phänotyp. Diese Ergebnisse zeigen, dass TCAIM als mitochondriales Protein wichtige Schritte in der Zellaktivierung und der Bildung von Gedächtnis-T-Zellen beeinflusst. Der zweite Teil der Dissertation beschäftigte sich mit dem Einfluss der CD44-Oberflächenexpression auf die Differenzierung von T-Helfer (TH)-Zellen. Eine hohe CD44-Expression unterscheidet Effektor- von naiven T-Zellen. Durch die allogene Stimulation von CD4+ T-Zellen bildeten sich drei verschiedene Populationen: CD44+, CD44++ und CD44+++. Sowohl in vitro als auch in vivo generierte alloreaktive TH17-Zellen wurden in der CD44+++ Population, TH1-Zellen hingegen in der CD44++ Population, detektiert. Es wurde beschrieben, dass sowohl eine geringe TZR- als auch eine geringe CD28-Stimulation eher die Bildung von TH17- als TH1-Zellen unterstützen. Unter genau diesen Bedingungen kann CD44 als kostimulatorisches Molekül die Signaltransduktion verstärken. Tatsächlich zeigten allogenreaktive CD44+++ TH-Zellen eine höhere ZAP-70-Phosphorylierung als CD44++ TH-Zellen. Diese Ergebnisse unterstützen die Annahme, dass CD44 durch die Verstärkung der Signaltransduktion die TH17-Differenzierung fördern kann. / Within the first part of this thesis, the influence of the mitochondrial Protein TCAIM (T cell activation inhibitor, mitochondrial) on T cell activation was investigated. Tcaim expression correlated negatively with the rejection of allografts and it is down-regulated during T cell activation. To study effects of TCAIM during T cell activation, we generated a T cell-specific mouse strain with a Tcaim knock-in (KI) targeted to the Rosa26 locus. Tcaim overexpression changed the mitochondrial morphology and reduced the T cell receptor (TCR)-induced mitochondrial reactive oxygen species production. In vitro activation of Tcaim KI CD4+ T cells resulted in a decreased activation, proliferation and cytokine release. Importantly, Rag-1 knock-out mice, reconstituted with Tcaim KI T cells, tolerated allogeneic skin grafts. Thus, by regulating TCR-induced mitochondrial distribution and ROS production, TCAIM controls important steps during T cell activation and memory formation. The second part dealt with the influence of CD44 surface expression level for T helper cell (Th cell) differentiation. By association with lymphocyte-specific protein kinase (LCK) it can enhance T cell signaling. Allogeneic stimulation of CD4+ T cells resulted in the formation of three distinguishable populations: CD44+, CD44++ and CD44+++. In vitro and in vivo generated allo-reactive TH17 cells were mainly CD44+++. This is in contrast to TH1 cells which were dominantly CD44++. Titration experiments revealed that low TCR- and co-stimulation supports TH17 rather than TH1 development. Under exactly these conditions it was reported that CD44 can act as co-stimulatory molecule and replace CD28. Indeed, CD44+++CD4+ T cells contained already more phosphorylated ZAP-70 as compared to CD44++ cells. Our results support the notion that CD44 enhances TCR signaling strength by delivering LCK, which is required to support TH17 development.

Les nanoparticules à base d’acide hyaluronique (AH) sont utilisées pour de nombreuses applications pharmaceutiques. Elles peuvent cibler les tumeurs par interaction avec le CD44,qui est un récepteur biologique surexprimé à la surface de certaines cellules cancéreuses. Dans ce projet nous explorons l’application potentielle de ces nanoparticules dans les domaines cosmétiques, car l’AH est aussi un ingrédient important pour l’hydratation et le renouvellement de la peau. Les copolymères à bloc à base de polypeptides et de polysaccharides ont été synthétisés par une combinaison de polymérisation par ouverture de cycle et de couplage par chimie « click ». Les nanoparticules ont été obtenues par l’auto assemblage de ces copolymères en utilisant un procédé de nano precipitation, dont la taille et la morphologie sont contrôlées par les paramètres expérimentaux. L’interaction entre les nanoparticules d’AH et le CD44 a été quantifiée par la résonance de plasmon de surface(RPS). En comparant avec l’AH libre en solution, les nanoparticules d’AH ont montré une interaction plus efficace avec le CD44, mettant ainsi en évidence un effet de multivalence des nanoparticules. Finalement, la dégradation enzymatique de ces nanoparticules d’AH a été évaluée avec deux types de hyaluronidases, HYAL1 et SPAM-1. La digestion des nanoparticules de l’AH a été significativement ralentie par rapport à l’AH libre. De manière très surprenante, ces nanoparticules de AH ont pu inhiber l’activité de l’enzyme HYAL1 et protéger l’AH libre dans la solution. Enfin, des ligands du récepteur TLR2 de type lipopeptide ont été synthétisés et leurs performances via TLR2 ont été évaluées par RPS. / Nanoparticles based on hyaluronic acid (HA) are widely used in pharmaceutics. They can target the tumor by the interaction with CD44, a biological receptor overexpressed in some cancer cells. In this project, we investigate the potential applications of these nanoparticles in cosmetics, since HA is also an important ingredient for the skin hydration and renewing. Block copolymers based on polypeptides and polysaccharides were synthesized using a combination of ring opening polymerization and “click chemistry”. The nanoparticles were formed by the self-assembly of these block copolymers using a nanoprecipitation process, and their size and morphology were controlled by the experimental conditions. The interaction between nanoparticles and CD44 were measured by surface plasmon resonance(SPR). Compared to free hyaluronic acid chains in solution, the HA-based nanoparticles could interact more efficiently with CD44, thus demonstrating a multivalent effect. The enzymatic degradation of these HA nanoparticles was then evaluated with twohyaluronidases: HYAL1 and SPAM-1. The digestion of the HA nanoparticles was significantly slower than that of free hyaluronic acid. Surprisingly, these HA nanoparticles could even inhibit the activity of the enzyme HYAL1 and protect free HA chains in the solution. Finally, lipopeptide-based ligands of the biological receptor TLR2 were also synthesized and their performances were evaluated by SPR.

O estudo das células-tronco cancerígenas (CTCs) durante o processo de malignização e no carcinoma epidermóide intra-bucal já instalado é essencial para um melhor entendimento de como essas células participam da formação e manutenção de uma neoplasia. Atualmente, a identificação de células com características de células tronco se dá principalmente através da expressão de marcadores celulares como o ALDH1 e o CD44. A proteína ALDH1 é responsável pela oxidação de aldeídos intracelulares e vem sendo utilizada para o isolamento de CTCs em inúmeros canceres incluindo casos de cabeça e pescoço. A proteína CD44 é uma glicoproteína envolvida na adesão e migração celular, também participa do processo de metástase e já foi associada às CTCs. Nesse trabalho, a expressão dessas proteínas foi analisada em 45 casos de displasias epiteliais e 13 casos de carcinomas epidermóide intra-bucais. As lesões displásicas foram classificadas em casos leves (19), moderados (18) e intensos (8) e foram também divididas em casos de baixo risco (22) e alto risco de transformação maligna (23). A expressão imunohistoquímica para a ALDH1 foi encontrada predominantemente na camada basal em 16 casos de displasias epiteliais e em 7 carcinomas epidermóides, com a marcação difusa pela epitélio neoplásico. A expressão imunohistoquímica de CD44 foi encontrada em 42 displasias epiteliais e em 12 carcinomas epidermóides, sendo que nas displasias, a expressão ocorreu predominantemente na camada basal do epitélio e no carcinoma epidermóide a expressão foi disseminada. Ambos marcadores exibiram aumento de expressão com a evolução do grau das displasias. / The study of cancer stem cells (CTCs) in the process of malignant transformation and intra-oral squamous cell carcinoma already installed is essential for a better understanding of how these cells participate in the formation and maintenance of a neoplasm. Currently, identification of cells with characteristics of stem cells is primarily through the expression of cell markers such as CD44 and ALDH1. The ALDH1 protein is responsible for the oxidation of intracellular aldehydes and has been used for the isolation of CTCs in numerous cancers including head and neck cases. The CD44 protein is a glycoprotein involved in cell adhesion and migration, also participates in the process of metastasis and has been associated with CTCs. In this work, the expression of these proteins was analyzed in 45 cases of epithelial dysplasia and 13 cases of intraoral squamous cell carcinomas. The dysplastic lesions were classified as mild (19), moderate (18) and intense (8) cases and were also divided into low-risk cases (22) and high risk of malignant transformation (23). The immunohistochemical expression for ALDH1 was found predominantly in the basal layer in 16 cases of epithelial dysplasia and squamous cell carcinoma in 7, with diffuse labeling by neoplastic epithelium. Immunohistochemical expression of CD44 was found in 42 epithelial dysplasias and 12 squamous cell carcinomas, and in dysplasias, the expression occurred predominantly in the basal layer of the epithelium and in squamous cell carcinoma expression was widespread. Both markers showed increased expression with the evolution of the degree of dysplasia.

O estudo das células-tronco cancerígenas (CTCs) durante o processo de malignização e no carcinoma epidermóide intra-bucal já instalado é essencial para um melhor entendimento de como essas células participam da formação e manutenção de uma neoplasia. Atualmente, a identificação de células com características de células tronco se dá principalmente através da expressão de marcadores celulares como o ALDH1 e o CD44. A proteína ALDH1 é responsável pela oxidação de aldeídos intracelulares e vem sendo utilizada para o isolamento de CTCs em inúmeros canceres incluindo casos de cabeça e pescoço. A proteína CD44 é uma glicoproteína envolvida na adesão e migração celular, também participa do processo de metástase e já foi associada às CTCs. Nesse trabalho, a expressão dessas proteínas foi analisada em 45 casos de displasias epiteliais e 13 casos de carcinomas epidermóide intra-bucais. As lesões displásicas foram classificadas em casos leves (19), moderados (18) e intensos (8) e foram também divididas em casos de baixo risco (22) e alto risco de transformação maligna (23). A expressão imunohistoquímica para a ALDH1 foi encontrada predominantemente na camada basal em 16 casos de displasias epiteliais e em 7 carcinomas epidermóides, com a marcação difusa pela epitélio neoplásico. A expressão imunohistoquímica de CD44 foi encontrada em 42 displasias epiteliais e em 12 carcinomas epidermóides, sendo que nas displasias, a expressão ocorreu predominantemente na camada basal do epitélio e no carcinoma epidermóide a expressão foi disseminada. Ambos marcadores exibiram aumento de expressão com a evolução do grau das displasias. / The study of cancer stem cells (CTCs) in the process of malignant transformation and intra-oral squamous cell carcinoma already installed is essential for a better understanding of how these cells participate in the formation and maintenance of a neoplasm. Currently, identification of cells with characteristics of stem cells is primarily through the expression of cell markers such as CD44 and ALDH1. The ALDH1 protein is responsible for the oxidation of intracellular aldehydes and has been used for the isolation of CTCs in numerous cancers including head and neck cases. The CD44 protein is a glycoprotein involved in cell adhesion and migration, also participates in the process of metastasis and has been associated with CTCs. In this work, the expression of these proteins was analyzed in 45 cases of epithelial dysplasia and 13 cases of intraoral squamous cell carcinomas. The dysplastic lesions were classified as mild (19), moderate (18) and intense (8) cases and were also divided into low-risk cases (22) and high risk of malignant transformation (23). The immunohistochemical expression for ALDH1 was found predominantly in the basal layer in 16 cases of epithelial dysplasia and squamous cell carcinoma in 7, with diffuse labeling by neoplastic epithelium. Immunohistochemical expression of CD44 was found in 42 epithelial dysplasias and 12 squamous cell carcinomas, and in dysplasias, the expression occurred predominantly in the basal layer of the epithelium and in squamous cell carcinoma expression was widespread. Both markers showed increased expression with the evolution of the degree of dysplasia.

Ο καρκίνος του παχέος εντέρου αποτελεί αναμφισβήτητα ένα παγκόσμιο πρόβλημα, όντας η δεύτερη αιτία θανάτου από καρκίνο. Τα γεγονότα που οδηγούν σε καρκίνο του παχέος εντέρου ακολουθούν στις περισσότερες περιπτώσεις συγκεκριμένη αλληλουχία. Έτσι, ξεκινώντας από υπερπλαστικό επιθήλιο, εξελίσσεται σε αδένωμα/δυσπλασία, σε ενδοεπιθηλιακή νεοπλασία και καταλήγει σε καρκίνο. Κατά την εξέλιξη σε καρκίνο σημαντικές αλλαγές λαμβάνουν χώρα στη σύσταση και οργάνωση του εξωκυττάριου χώρου του εντερικού τοιχώματος. Για το λόγο αυτό απαιτείται μελέτη του μεταβολισμού των εξωκυττάριων μακρομοριακών συστατικών, με στόχο την κατανόηση και διασαφήνιση της διηθητικής και μεταστατικής συμπεριφοράς των κακοήθων νεοπλασιών του παχέος εντέρου. Στην παρούσα εργασία κρίθηκε χρήσιμη η μελέτη των ενζύμων μεταβολισμού του υαλουρονικού οξέος (ΗΑ), συστατικό του εξωκυττάριου χώρου που φαίνεται να εμπλέκεται στο μηχανισμό της ανάπτυξης του όγκου, της διείσδυσης των καρκινικών κυττάρων και στη διάδοση των μεταστάσεων. Για τον παραπάνω λόγο μελετήθηκε η δραστικότητα των υαλουρονιδασών (Hyals) με ζυμογράφημα-ΗΑ, σε εκχυλίσματα ιστών και σε ορούς, και των συνθασών του ΗΑ (HAS-1, HAS-2) καθώς και του υποδοχέα του ΗΑ, CD44, με RT-PCR ανάλυση. Τα αποτελέσματα που προκύπτουν από το ζυμογράφημα-ΗΑ σε εκχυλίσματα ιστών συναινούν σε αυξημένη δραστικότητα των Hyals σε καρκινικά σε σύγκριση με τα μακροσκοπικώς φυσιολογικά δείγματα στην πλειονότητα των σταδίων, ενώ και οι δύο δραστικότητες είναι σημαντικά αυξημένες σε σχέση με τα δείγματα υγιών ιστών. Όσον αφορά τα δείγματα ορών, τα επίπεδα των Hyals φαίνεται να μειώνονται αισθητά 7 ημέρες μετεγχειρητικά σε σχέση με την ημέρα πριν την εγχείρηση, ενώ 1, 3 και 6 μήνες μετά εμφανίζεται και πάλι σταδιακή αύξηση των επιπέδων τους. Σε γονιδιακό επίπεδο, τα επίπεδα της HAS-1, HAS-2 και του CD44 εμφανίζονται αυξημένα στα καρκινικά δείγματα έναντι των μακροσκοπικώς φυσιολογικών. / Colon cancer is indisputably a great problem, being the second cause of death by cancer. The facts that lead to colon cancer have certain concatenation. Thereby, starting most of the times as hyperplastic epithelium, it develops to endoepithelium adenoma, and it finally leads to cancer. During this development important changes happen at the constitution and organization of the extracellular matrix of the intestinal tract. For this purpose, study of the metabolism of the extracellular matrix constituents is required, so as to understand the metastatic behaviour of the malignant tumors of colon cancer. The present study focuses on the metabolism enzymes of hyaluronic acid (HA), a constituent of the extracellular matrix that seems to be involved in the tumor growth mechanism, the infiltration of the cancerous cells and metastasis. Particularly, hyaluronidases (Hyals) activity (HA-zymography) and hyaluronan synthases (HAS-1, HAS-2) as well as hyaluronan receptors (CD44) expression (RT-PCR analysis) are studied at this study. The results of the study show increased Hyals levels in cancerous samples compared to the macroscopically normal ones, in all anatomic sites of colon examined, while both activities remain significantly increased compared to healthy samples.. as far as it concerns Hyals levels in sera, they seem to decrease perceptibly 7 days postoperatively, while 1, 3 and 6 months afterwards gradually increased to reach the amount preoperatively. In gene level, HAS-1, HAS-2 and CD44 expression levels were increased in cancerous samples compared to the macroscopically normal ones.

Οι κακοήθεις όγκοι των όρχεων εμφανίζονται κυρίως σε άνδρες ηλικίας 15-35 ετών και παρουσιάζουν αυξητική τάση τα τελευταία 40 χρόνια. Περίπου 95% των νεοπλασιών προέρχονται από τα βλαστικά κύτταρα και οι περισσότεροι από τους όγκους των όρχεων είναι επιθετικοί και ικανοί για γρήγορη και ευρεία διασπορά της νόσου. Η πρόγνωση της κλινικής εξέλιξης της νόσου αποτελεί μια πρόκληση και ο ρόλος διαφόρων βιολογικών δεικτών έχει μελετηθεί προσπαθώντας να διευκρινιστούν τα ακριβή αίτια και οι παθογενετικοί μηχανισμοί της ανάπτυξης των όγκων στους όρχεις. Τόσο τα κύτταρα του στρώματος όσο και τα νεοπλασματικά κύτταρα εμπλέκονται στην αναδιοργάνωση του ECM έτσι ώστε να διευκολυνθεί ο πολλαπλασιασμός, η μετακίνηση και διήθηση των νεοπλασματικών κυττάρων. Οι PGs είναι βιοδραστικά μόρια και η έκφρασή τους τροποποιείται σημαντικά στο μικροπεριβάλλον του όγκου. Μεταβολές της έκφρασης των PGs από τα κύτταρα του όγκου και τα στρωματικά κύτταρα επηρεάζουν τα σηματοδοτικά μονοπάτια των νεοπλασματικών κυττάρων, την ανάπτυξη και επιβίωσή τους, την προσκόλληση και μετανάστευσή τους καθώς και τη δημιουργία νέων αγγείων στον όγκο. Σκοπός της διδακτορικής διατριβής ήταν να μελετηθεί η παρουσία των εξωκυττάριων PGs, versican και decorin καθώς και των μεμβρανικών PGs, syndecan-4 και CD44 (standard isoform) και να διερευνηθεί η πιθανή συσχέτιση της έκφρασής τους με τη βιολογική συμπεριφορά των όγκων όρχεων που προέρχονται από βλαστικά κύτταρα. Αρχικώς, η έκφραση και ο εντοπισμός τους μελετήθηκε με ανοσοϊστοχημεία σε αρχειακό υλικό 71 ασθενών με σεμινωματώδεις και μη-σεμινωματώδεις όγκους και τα αποτελέσματα επιβεβαιώθηκαν με ανοσοεντοπισμό ή με μελέτη της γονιδιακής έκφρασης μέσω RT-PCR σε περιορισμένο αριθμό δειγμάτων. Στη συνέχεια, η παρουσία των PGs συσχετίστηκε με τα κλινικοπαθολογικά χαρακτηριστικά των ασθενών και τη νεοαγγειογένεση που παρατηρείται στους όγκους. Διαπιστώθηκε ότι οι versican και decorin εκφράζονται σε μικρά ποσά στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων και παρατηρείται σημαντική αύξηση των ποσών και των δύο PGs κυρίως στο στρώμα που περιβάλλει τόσο τους σεμινωματώδεις όσο και τους μη-σεμινωματώδεις όγκους. Οι μη-σεμινωματώδεις όγκοι χαρακτηρίζονται από μεγαλύτερη αύξηση στην έκφραση της versican σε σχέση με τους σεμινωματώδεις. Η decorin παρουσιάζει σχεδόν παρόμοια επίπεδα έκφρασης και στις δύο κατηγορίες των όγκων όρχεων. Η αυξημένη εναπόθεση της decorin στο στρώμα των όγκων δεν συσχετίζεται στατιστικά με καμία από τις κλινικοπαθολογικές μεταβλητές που μελετήθηκαν. Αντιθέτως, η αυξημένη εναπόθεση της versican τόσο στα σεμινώματα όσο και στους μη-σεμινωματώδεις όγκους συσχετίζεται στατιστικά με την παρουσία αγγειακών και λεμφαγγειακών διηθήσεων, το μέγεθος του πρωτοπαθούς όγκου (στατιστικά σημαντική συσχέτιση παρατηρήθηκε μόνο στους μη-σεμινωματώδεις όγκους), την παρουσία μεταστάσεων στους λεμφαδένες, την ύπαρξη απομακρυσμένων μεταστάσεων (συσχετίστηκε μόνο στους μη-σεμινωματώδεις όγκους) και το στάδιο της νόσου. Επιπλέον, στην παρούσα μελέτη βρέθηκε ότι η συσσώρευση της versican στο στρώμα των όγκων όρχεων σχετίζεται με την παρουσία μεγαλύτερου αριθμού νέων αγγείων. Η μελέτη της διαμεμβρανικής PG syndecan-4 ανέδειξε ότι εκφράζεται από τα τα σπερματικά κύτταρα των σπερματικών σωληναρίων, κυρίως πλησίον του βασικού υμένα, στους φυσιολογικούς ιστούς των όρχεων. Η syndecan-4 εντοπίζεται τόσο στο κυτταρόπλασμα όσο και στη μεμβράνη των φυσιολογικών κυττάρων. Αντιθέτως, δεν διαπιστώθηκε εναπόθεση της syndecan-4 στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων. Τόσο οι σεμινωματώδεις όσο και οι μη σεμινωματώδεις όγκοι όρχεων εμφανίζουν αυξημένη έκφραση του γονιδίου της syndecan-4. Οι σεμινωματώδεις όγκοι χαρακτηρίζονται από σημαντικά πιο αυξημένη έκφραση του γονιδίου της syndecan-4 σε σχέση με τους μη-σεμινωματώδεις όγκους. Η υψηλή έκφραση της syndecan-4 στα κύτταρα του όγκου στα σεμινώματα επιβεβαιώθηκε με ανοσοϊστοχημική χρώση των ιστών, ενώ η έκφρασή της παρουσίαζε διακυμάνσεις στα κύτταρα των μη-σεμινωματωδών όγκων. Στους μη-σεμινωματώδεις όγκους αναγνωρίστηκαν περιπτώσεις ασθενών με ασθενέστερη έκφραση της syndecan-4 στα κύτταρα του όγκου. Η ελαττωμένη παρουσία της syndecan-4 στα κύτταρα των μη-σεμινωματωδών όγκων συσχετίστηκε με την παρουσία λεμφαδενικών μεταστάσεων, αγγειακής και λεμφαγγειακής διήθησης και με το στάδιο της νόσου στους ασθενείς. Επιπλέον, η syndecan-4 εντοπίστηκε στο ινώδες στρώμα και στα στρωματικά κύτταρα στους σεμινωματώδεις και μη-σεμινωματώδεις όγκους. Η παρουσία της syndecan-4 στο στρώμα συσχετίστηκε με την παρουσία λεμφαδενικών μεταστάσεων, αγγειακών και λεμφαγγειακών διηθήσεων και το στάδιο της νόσου μόνο στα σεμινώματα. Η έκφραση της syndecan-4, στο στρώμα κακοήθων όγκων, αναδεικνύεται για πρώτη φορά σε αυτή τη μελέτη και συσχετίζεται με τον αυξημένο αριθμό αγγείων στο στρώμα των όγκων όρχεων. Η μελέτη του υποδοχέα CD44 στην παρούσα διδακτορική διατριβή πραγματοποιήθηκε με ανοσοϊστοχημεία χρησιμοποιώντας το πολυκλωνικό αντίσωμα Η-300 που αναγνωρίζει την κύρια ισομορφή CD44s. Διαπιστώθηκε ότι η πρωτεΐνη του CD44 δεν εκφράζεται από τα φυσιολογικά κύτταρα των σπερματικών σωληναρίων των όρχεων. Επίσης, δεν παρατηρήθηκε χρώση για τον CD44 στο διάμεσο συνδετικό ιστό, στα μεσολοβιώδη διαφράγματα που περιβάλλουν τα σπερματικά σωληνάρια των όρχεων. Ο CD44 βρέθηκε ότι εκφράζεται στα κύτταρα του όγκου στο 27% των ασθενών με σεμινώματα και στο 36.8% των ασθενών με μη-σεμινωματώδεις όγκους. Επίσης, βρέθηκε ότι ο CD44 εκφράζεται στο στρώμα του όγκου στο 48.5% των ασθενών με σεμινώματα και στο 26.3% των ασθενών με μη-σεμινωματώδεις όγκους. Η έκφραση του CD44 στα κύτταρα του όγκου και στο στρώμα συσχετίστηκε με το μέγεθος του όγκου, την παρουσία μεταστάσεων σε λεμφαδένες, τη διήθηση αγγείων και λεμφαγγείων και το προχωρημένο στάδιο της νόσου μόνο στους ασθενείς με σεμινώματα. Δεν παρατηρήθηκε καμία συσχέτιση της παρουσίας του CD44 στους μη-σεμινωματώδεις όγκους με τα κλινικοπαθολογικά χαρακτηριστικά των ασθενών. Η παρουσία του CD44 στα κύτταρα και τον ECM του στρώματος των όγκων συσχετίστηκε με αυξημένο αριθμό αγγείων, εύρημα που προτείνει ότι ο υποδοχέας εμπλέκεται στη νεοαγγειογένεση. Τα ευρήματα της διδακτορικής διατριβής προτείνουν ότι οι PGs versican, syndecan-4 και CD44 εμπλέκονται στην εξέλιξη των όγκων όρχεων και μπορούν να αποτελέσουν προγνωστικούς δείκτες. Για να τεκμηριωθεί η κλινική χρησιμότητα της έκφρασής τους σε όγκους όρχεων που προέρχονται από βλαστικά κύτταρα απαιτείται περαιτέρω έρευνα σε μεγαλύτερο αριθμό δειγμάτων. / Testicular tumors present in men aged 15-35 years with increasing incidence in the last 40 years. Approximately 95% of these tumors arise from germ cells. Although most of these tumors are aggressive with rapid growth and spread, the chemotherapy is very efficient and the mortality has been markedly reduced. Prognosis is a challenging issue and the role of various biological markers has been investigated in order to elucidate the mechanisms of the development and progression of the disease. Tumor and stromal cells are implicated in the remodeling of the ECM that facilitates the growth, motility and invasion of tumor cells. PGs are bioactive molecules and their expression is markedly affected in the microenvironment of the tumor. Alterations in the expression of PGs by tumor and stromal cells modulate various signaling pathways in tumor cells affecting tumor cell growth and survival, adhesion and migration as well as neoangiogenesis. The aim of the present thesis was to study the distribution of matrix secreted PGs, versican and decorin as well as that of cell surface associated PGs, syndecan-4 and CD44 and to investigate the possible association of their expression with the biological behavior in testicular germ cell tumors. Their expression and localization was studied performing immunohistochemistry in archive material in 71 patients with seminomatous and non-seminomatous germ cell tumors and the results were confirmed by western blots and study of the gene expression in limited number of tissue from patients. The presence of PGs was correlated with the clinicopathological variables of the patients and with the number of microvessels present in the tissues. The study revealed that versican and decorin present in small amounts in the interstitial connective tissue in the interlobular septa surrounding the seminiferous tubules and are markedly increased in the peritumoral and sometimes in intratumoral stroma in seminomas and non seminomatous tumors. Non seminomatous tumors exhibited increased staining for versican as compared to seminomas, whereas decorin is almost equally distributed in both types of testicular germ cell tumors. The increased accumulation of decorin in the tumor stroma was not correlated with any clinicopathological variable of the patients. In contrast, the accumulation of versican in both seminomas and non-seminomatous tumors was correlated with the presence of lymphatic and vascular invasion, the size of the tumor (only in non-seminomatous tumors), the presence of metastases in lymph nodes, the presence of distant metastases (evaluated only in non-seminomas) and the stage of the disease. Furthermore, the accumulation of versican in tumor stroma of testicular germ cell tumors was correlated with the increased number of microvessels in the tissues. The study of transmembrane PG syndecan-4 revealed that it was expressed by germ cells in the seminiferous tubules in normal tissue. Syndecan-4 presents both in the cytoplasm and the cell membrane of the cells. The interstitial connective tissue in the interlobular septa surrounding the seminiferous tubules was negative for the presence of syndecan-4. Both seminomas and non-seminomatous tumors exhibit increased expression of syndecan-4 and the highest expression of syndecan-4 is detected in seminomas. The increased expression of syndecan-4 in tumor cells in seminomas was confirmed by immunohistochemistry, whereas its expression was varied considerably in tumor cells among non-seminomas. Some cases in non-seminomatous tumors demonstrated with lower expression of syndecan-4 in tumor cells. The weaker expression of syndecan-4 in tumor cells was correlated with the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease in the patients with non-seminomatous tumors. Furthermore, the presence of syndecan-4 in the stroma and in the stromal cells was demonstrated in both types of testicular germ cell tumors. The stromal presence of syndecan-4 was correlated with the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease only in seminomas. The stromal staining of syndecan-4 is demonstrated for first time and it correlates with the increased number of microvessels present in testicular germ cell tumors. The study of CD44 in this thesis was conducted by immunohistochemistry using the polyclonal antibody H-300 that recognizes the main isoform CD44s. Our data indicate that CD44 is not expressed by germ cells present in the seminiferous tubules or the connective tissue in the normal testis. CD44 was found to be expressed in 27% and 36.8% of patients with seminomas and non-seminomas, respectively. Furthermore, CD44 was found in the stromal cells and / or stroma in 48.5% and 26.3% of patients with seminomas and non-seminomas, respectively. The expression of CD44 in the tumor cells as well as the presence of CD44 in stromal cells and / or stroma was correlated with the tumor size, the presence of lymph node metastases, the presence of vascular and lymphatic invasion and the stage of the disease only in seminomas. Nor the presence of CD44 in tumor cells neither the stromal staining of CD44 was correlated with any clinicopathological variable examined in non-seminomatous tumors. Overall the stromal staining of CD44 in testicular germ cell tumors was correlated with the neoangiogenesis, suggesting the implication of CD44 in the formation of new blood vessels. The data of this thesis suggest that versican, syndecan-4 and CD44 are implicated in the development of testicular germ cell tumors and may serve as prognostic biological markers. Further studies in a larger number of patients should be performed to determine the clinical utility of the expression of these PGs in testicular tumors.

Submitted by Neusa Fagundes (neusa.fagundes@unioeste.br) on 2018-03-06T19:28:50Z No. of bitstreams: 2 Giovane_Zanin2014.pdf: 3169659 bytes, checksum: ca4852dea2558da8d3c74260c79fc413 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-03-06T19:28:50Z (GMT). No. of bitstreams: 2 Giovane_Zanin2014.pdf: 3169659 bytes, checksum: ca4852dea2558da8d3c74260c79fc413 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-09-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The polyvinyl acetate is a polymer used in the development of formulations for sustained release of drugs. Hyaluronic acid (HA) can interact with receptors on the plasma membrane, especially CD44 that overexpressed in tumor cells. This paper presents a pre-formulation study of isolated polyvinyl acetate films with the addition of HA to the application perspective in pharmaceutical dosage forms destined to modified drug release. The films were prepared using the solvent evaporation method and evaluated according to macroscopic and morphologic characteristics, thickness, Fourier transform infrared spectroscopy (FTIR), thermal analyses (thermogravimetry and differential scanning calorimetry), scanning electron microscopy, water vapor transmission, and swelling index. Addition of HA enabled the formation of isolated films, influencing transparency, flexibility, and thickness. FTIR spectra demonstrated that only physical mixing occurred. TG and DSC curves indicated that films are thermally stable up to 200C. Electron micrographs showed modifications in the polymer mesh structure in samples (85:15 and 80:20) previously immersed in simulated gastric fluid and more pronounced after immersion in simulated intestinal fluid. The HA concentration also influenced water vapor permeability and swelling increasing these indices. We propose that films with 95:05 and 90:10 compositions can be used for modified drug release, as they were similar to a reference film and maintained targeted delivery. / O acetato polivinílico (AcPV) é um polímero utilizado no desenvolvimento de formulações para liberação sustentadas de fármacos. O ácido hialurônico (AH) possui a habilidade de interagir com receptores nas membranas plasmáticas celulares, em especial o CD44 que esta super-expresso em células tumorais. O objetivo deste trabalho foi realizar um estudo de pré-formulação em filmes isolados de acetato polivinílico (AcPV) adicionados de AH na perspectiva de aplicação em formas farmacêuticas destinadas à liberação modificada de fármacos. Os filmes foram produzidos pelo método de evaporação do solvente e avaliados quanto as características macroscópicas e morfológicas, espessura, espectroscopia no infravermelho (FT-IR), análises térmicas (TG e DSC), microscopia eletrônica de varredura, transmissão de vapor de água e índice de intumescimento. Os resultados demonstraram que adição de AH permitiu a formação de filmes isolados adequados influenciando na transparência, flexibilidade e espessura. Espectro no FT-IR evidenciaram ocorrência apenas de mistura física entre os constituintes. As curvas TG e DSC indicaram que os filmes são termicamente estáveis até a temperatura de 200 ºC. Nas micrografias eletrônicas foi possível observar alterações na estrutural da malha polimérica nas composições 85:15 e 80:20 para as amostras previamente imersas em fluido de simulação gástrica, sendo ainda mais pronunciado após imersão em fluído de simulação intestinal. A concentração do AH também influenciou diretamente permeabilidade ao vapor de água e o intumescimento, aumentando estes índices. Na perspectiva de aplicação dos filmes avaliados, destacamos as composições 95:05 e 90:10, as quais sugerem maior potencial para aplicação em formas farmacêuticas para liberação modificada de fármacos uma vez que foram os mais semelhantes ao filme padrão e que mantiveram a possível capacidade de sítio alvo especificidade.