Οι πρωτεογλυκάνες και οι αλυσίδες γλυκοζαμινογλυκανών τους αλληλεπιδρούν με αυξητικούς παράγοντες, διαμεμβρανικούς υποδοχείς όπως οι ιντεγκρίνες, ένζυμα, αναστολείς πρωτεασών και με άλλα μόρια της εξωκυττάριας ύλης όπως η ινονεκτίνη, η λαμινίνη και η tenascin. Στην παρούσα διατριβή μελετήσαμε τη χωροχρονική κατανομή των πρωτεογλυκανών versican και CD44 με τη μέθοδο της RT-PCR και του ανοσοφθορισμού από το στάδιο ΧΙ-ΧΙΙ (μορίδιο) έως το στάδιο ΗΗ16+ (28-29 ζεύγη σωμιτών) και τον ρόλο της versican και της CD44 με τη χρήση μονοκλωνικών αντισωμάτων έναντι αυτών κατά την ανάπτυξη του πρώιμου εμβρύου. Η versican είναι πρωτεογλυκάνη θειικής χονδροϊτίνης και αλληλεπιδρά με αυξητικούς παράγοντες, με διάφορες πρωτεΐνες της εξωκυττάριας ύλης και με διαμεμβρανικούς υποδοχείς όπως η CD44. Τα αποτελέσματα της RT-PCR έδειξαν ότι το mRNA της versican εκφράζεται σε όλα τα αναπτυξιακά στάδια που μελετήσαμε. Ενδιαφέρον παρουσιάζουν τα προϊόντα εναλλακτικής ωρίμανσης της versican που ανιχνεύσαμε ακόμα και στο στάδιο του μοριδίου και που η έκφρασή τους ρυθμίζεται αναπτυξιακά. Η παρουσία του mRNA της versican σε υψηλά επίπεδα στο στάδιο του μοριδίου (ΧΙ-ΧΙΙ) υποδεικνύει ότι το mRNA της versican είναι ωογενετικής προέλευσης στο στάδιο αυτό. Τα πειράματα μας του ανοσοφθορισμού έδειξαν ότι η versican πρωτεΐνη ανιχνεύεται στο στάδιο του μοριδίου και εκφράζεται έντονα στην επιβλάστη και στην υποβλάστη στο στάδιο του προχωρημένου βλαστιδίου (στάδιο ΧΙΙΙ). Στο στάδιο ΗΗ3+ (ενδιάμεσο γαστρίδιο / intermediate streak) παρατηρήσαμε μεγάλη ένταση φθορισμού στα κύτταρα που μεταναστεύουν μέσα από την πρωτογενή αύλακα και στα μεσεγχυματικά κύτταρα που θα σχηματίσουν το μεσόδερμα και το ενδόδερμα. Στο στάδιο ΗΗ4 (προχωρημένο γαστρίδιο / definitive streak) ένταση φθορισμού της versican ανιχνεύθηκε κύτταρα που μεταναστεύουν μέσα από την πρωτογενή αύλακα καθώς και στα κύτταρα που έχουν αρχίσει να σχηματίζουν το μεσόδερμα και το ενδόδερμα. Στο στάδιο που το έμβρυο έχει σχηματίσει 4 ζεύγη σωμιτών (στάδιο ΗΗ8), ανιχνεύσαμε υψηλή ένταση φθορισμού της versican στη νευρική πλάκα και στις νευρικές πτυχές καθώς ανασηκώνονται να σχηματίσουν το νευρικό σωλήνα. Στο στάδιο ΗΗ12 (16 ζεύγη σωμιτών), ισχυρή ένταση φθορισμού της versican ανιχνεύσαμε στο νευρικό σωλήνα, στο γειτονικό του εξώδερμα, στα κύτταρα της νευρικής ακρολοφίας (neural crest), στα κύτταρα του σωμίτη, στο μεσονέφρο και στο γειτονικό του πλάγιο μεσόδερμα που θα σχηματίσει τους μεσονεφρικούς σωληνίσκους. Αργότερα στην ανάπτυξη, ισχυρή ένταση φθορισμού της versican ανιχνεύσαμε επίσης στο διεγκέφαλο, στον οπτικό μίσχο, στο μεσεγκέφαλο, στο μυελεγκέφαλο, στα τοιχώματα του φάρυγγα και της ραχιαίας αορτής, στο ραχιαίο μεσοκάρδιο, στο μυοκάρδιο και στο ενδοκάρδιο, στο μυοτόμο και σκληροτόμο στους σωμίτες, στα τοιχώματα του εντέρου, καθώς και στην εξωκυττάρια ύλη των εμβρυϊκών κοιλοτήτων. Πειράματα σε έμβρυα που εκτέθηκαν στο αντίσωμα έναντι της versican σε διαφορετικά στάδια ανάπτυξης από το μορίδιο ως το προχωρημένο γαστρίδιο έδειξαν ότι η versican πιθανόν να συμμετέχει στα μονοπάτια σηματοδότησης που καθοδηγούν τη μετανάστευση των κυττάρων της νευρικής ακρολοφίας, στο μοριακό δίκτυο για το σχηματισμό του νευρικού σωλήνα, στον καθορισμό ή / και στη διαμερισματοποίηση των προκαρδιακών κυττάρων κατά το σχηματισμό της καρδιάς και στη διατήρηση της αρχιτεκτονικής των εμβρυϊκών κοιλοτήτων. Η CD44 είναι πρωτεογλυκάνη της κυτταρικής επιφάνειας και είναι ο κύριος υποδοχέας του υαλουρονικού. Ανιχνεύσαμε τη CD44 πρωτεΐνη ακόμα και στο στάδιο του μοριδίου. Η παρουσία του mRNA της CD44 σε υψηλά επίπεδα στο στάδιο του μοριδίου μπορεί να δείχνει ότι το mRNA είναι ωογενετικής προέλευσης. Με τη μέθοδο του ανοσοφθορισμού ανιχνεύσαμε έντονο φθορισμό της CD44 στα κύτταρα της επιβλάστης και ειδικότερα σε αυτά που γειτονεύουν με το βλαστόκοιλο και στην υποβλάστη στο στάδιο του προχωρημένου βλαστίδιου (ΧΙΙΙ), ενώ στο στάδιο ΗΗ3+ καθώς συνεχίζονται οι μορφογενετικές κινήσεις της γαστριδίωσης, η CD44 παρουσιάζει ισχυρή ένταση φθορισμού στα κύτταρα της επιβλάστης ,στα μεσεγχυματικά κύτταρα και στα κύτταρα του ενδοδέρματος. Στο στάδιο ΗΗ8 (4 ζεύγη σωμίτες), ανιχνεύσαμε ένταση φθορισμού της CD44 στη νευρική πλάκα και στις νευρικές πτυχές με πρότυπο έκφρασης παρόμοιο με αυτό της έκφρασης της versican. Αργότερα στην ανάπτυξη, στο στάδιο ΗΗ16+ (28-29 ζεύγη σωμιτών) ανιχνεύσαμε ισχυρή ένταση φθορισμού της CD44 ανιχνεύθηκε στα τοιχώματα του διεγκεφάλου, του μεσεγκεφάλου και του νευρικού σωλήνα, στα τοιχώματα του φάρυγγα, στις ραχιαίες και κοιλιακές αορτές, στα αορτικά τόξα, στη νωτοχορδή, στην εξωκυττάρια ύλη στην κοιλότητα του μεσεγκεφάλου και στην κοιλότητα του φάρυγγα, στο ακουστικό κυστίδιο και στην κοιλότητα του ακουστικού κυστιδίου και στα κύτταρα της νευρικής ακρολοφίας που μεταναστεύουν προς το ακουστικό κυστίδιο. Υψηλή ένταση φθορισμού της CD44 ανιχνεύσαμε επίσης στο σκληροτόμο και στο μυοτόμο στους σωμίτες, στα κύτταρα της νευρικής ακρολοφίας, στο ήπαρ, στο μυοκάρδιο, στο ενδοκάρδιο, στον αγωγό φλέβας και στο μεσονέφρο. Έμβρυα που εκτέθηκαν στο αντίσωμα έναντι της CD44 σε διαφορετικά στάδια ανάπτυξης από το μορίδιο ως το πρώιμο νευρίδιο έδειξαν το σημαντικό ρόλο της CD44 στην μορφογένεση του εγκεφάλου, στη διατήρηση της αρχιτεκτονικής της κοιλότητας του εγκεφάλου και των άλλων εμβρυϊκών κοιλοτήτων, στην μετανάστευση των κυττάρων της νευρικής ακρολοφίας, στον σχηματισμό της καρδιάς και του αγγειακού συστήματος και στη μορφογένεση των σωμιτών. Τα αποτελέσματα μας έδειξαν τη συνεργιστική δράση των CD44 και versican κατά την ανάπτυξη του πρώιμου εμβρύου. / Proteoglycans and their associated glycosaminoglycans can bind growth factors, integrin and non-integrin cell surface molecules, enzymes, protease inhibitors and other extracellular matrix components including fibronectin, laminin and tenascin. We studied the expression and spatiotemporal distribution of versican and CD44 by RT-PCR and immunofluorescence in the chick embryo from the morula stage (stage XI-XII) to early organogenesis (stage HH16+, 28-29 somites). We also studied the versican and CD44 role by using blocking antibodies in the early chick embryo. Versican is a chondroitin sulfate proteoglycan that binds growth factors and interacts with various extracellular matrix proteins and cell surface molecules including the CD44. Combined RT-PCR and immunohistochemistry demonstrated the expression of versican as early as the morula stage. Interestingly, we detected splice variants of versican at the morula stage, and their expression was developmentally regulated. The presence of versican mRNA at the morula stage may indicate that it is of oogenetic origin. Versican fluorescence was strong in the epiblast and the hypoblast at the late blastula stage (XIII). At stage HH3+ (intermediate streak), versican expression was intense in the cells ingressing through the primitive streak and the migrating mesenchymal cells which will form the mesoderm and endoderm. By the definitive streak stage (HH4), versican fluorescence was intense in the cells ingressing through the primitive streak and in the mesenchymal cells that have already started to form the mesoderm and endoderm. At stage HH8 (4 somite pairs), versican expression was strong in the neural plate, the elevated neural folds and the ectoderm neighboring the neural folds. At stage HH12 (16 somite pairs), versican fluorescence was intense in the neural tube and its adjacent ectoderm, the neural crest cells, the somite and in the mesonephros and in the adjacent lateral mesoderm that will form the mesonephric tubules. Later in development, versican fluorescence was intense in the diencephalon, the optic stalk, mesencephalon, myelencephalon. Versican fluorescence was also intense in the dorsal mesocardium, myocardium and endocardium, dorsal aorta and aortic arches, in the myotome and sclerotome in somites, gut and in the extracellular matrix of embryonic cavities. Inhibition of the function of versican by blocking antibodies showed that versican is crucial for the neural tube closure, neural crest migration, formation of the heart tube, for the architecture of embryonic cavities and consequently tissue and organ morphogenesis. CD44 is a transmembrane part-time proteoglycan and the main receptor for hyaluronan. We detected CD44 protein even at the morula stage. The presence of high levels of CD44 mRNA at the morula stage indicated that this is an oogenetic mRNA. CD44 fluorescence was strong in the epiblast cells, especially those neighboring the blastocoele, and in the hypoblast at the late blastula stage (XIII). At stage HH3+, during gastrulation, CD44 was expressed strongly in the epiblast cells, in mesenchymal cells and in endoderm cells. At stage HH8 (4 somite pairs), strong CD44 expression was detected in the neural plate and neural folds and their adjacent ectoderm and this expression pattern was similar to that of versican. Later in development, CD44 expression was intense in the diencephalon, optic stalks, mesencephalon, myelencephalon, metencephalon, auditory vesicles and the neural crest cells migrating towards the auditory vesicle and the neural tube. CD44 fluorescence was also intense in the dorsal mesocardium, myocardium, endocardium, aortae and aortic arches, sclerotome and myotome in somites, mesonephros, liver, gut and in the migrating neural crest cells that will form the sympathetic and enteric ganglia. Inhibition of CD44 function by blocking antibodies showed that CD44 is crucial for the architecture of the embryonic cavities such as the brain lumen, neural tube closure, neural crest cell migration, cardiac and cardiovascular formation and somite morphogenesis. Our results showed a synergistic role of CD44 and versican during the development of the early embryo.

FEITOSA, Neli Patricia Pereira. Imunoexpressão de marcadores de células -tronco, CD44 e CD133, no câncer gástrico primário e metástases linfonodais. 2015. 68 f. Dissertação (Mestrado em Patologia) - Faculdade de Medicina, Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by denise santos (denise.santos@ufc.br) on 2016-03-29T13:47:51Z No. of bitstreams: 1 2015_dis_nppfeitosa.pdf: 1566891 bytes, checksum: c867b84a46d19b6c755515c32880e5be (MD5) / Approved for entry into archive by denise santos(denise.santos@ufc.br) on 2016-03-29T13:50:03Z (GMT) No. of bitstreams: 1 2015_dis_nppfeitosa.pdf: 1566891 bytes, checksum: c867b84a46d19b6c755515c32880e5be (MD5) / Made available in DSpace on 2016-03-29T13:50:03Z (GMT). No. of bitstreams: 1 2015_dis_nppfeitosa.pdf: 1566891 bytes, checksum: c867b84a46d19b6c755515c32880e5be (MD5) Previous issue date: 2015 / Gastric cancer is the fourth most common cancer worldwide, accounting for the second leading cause of cancer mortality. Despite treatment with surgery and chemotherapy, the overall five-year survival of patients with gastric cancer remains low. One possible explanation for the ineffectiveness of therapy is the presence of cancer stem cells, a subpopulation of tumor cells that have stem cell characteristics. It has been reported that these, as well as embryonic stem cells, are immortal, can self-renew and to differentiate to be transformed in any cell type in the body. Several markers, including CD44 and CD133, have been reported as stem cell markers in both normal and cancerous cells and have been used to isolate cancer cells from solid tumors. The aim of this study was to evaluate the expression of CD44 and CD133 in primary gastric cancer and lymph node metastases by immunohistochemical and to relate it to clinicopathologic variables as histological type, gender, age, anatomical site, tumor size, angiolymphatic invasion, infiltration perineural, TNM classification (TN) and lymph node involvement. This study was developed from a set of 72 cases of gastric adenocarcinoma, from the Archives of Pathology and Forensic Medicine of the Federal University of Ceará Service (DPML-UFC). Tissue microarray and immunohistochemistry were utilized, with anti-CD44 monoclonal antibody and polyclonal anti-CD133. The cases with one or more cells with cytoplasmic and / or membrane immunostaining were considered positives. It was observed that 30% of the samples were positive for CD44. No statistically significant differences were found between the clinical and pathological variables studied and the immunoreactivity of CD44. Regarding the immunoreactivity of CD133, the present study showed that 24% of samples were positive. The degree of tumor invasion presented data showing a statistically significant trend (p = 0.0505), in reverse. The other variables, we found no statistically significant difference. The immunoreactivity of CD133 in histologically normal mucosa was higher than in the primary tumor and intestinal metaplasia, with statistically significant difference (p = 0.0159 and p = 0.0058, respectively). The CD133 immunostaining in intestinal type gastric carcinoma was significantly higher than in the histologically normal mucosal metaplasia (p = 0.0260). The frequency of expression of these markers is highly variable, and even in the samples considered positive, the stained cells percentage is also variable, and very low overall. / O câncer gástrico é a quarta neoplasia mais comum em todo o mundo, representando a segunda causa de mortalidade por câncer. Apesar do tratamento com cirurgia e quimioterapia, a sobrevida global em cinco anos de pacientes com câncer gástrico permanece baixa. Uma possível explicação para ineficácia da terapia é a presença de células-tronco cancerosas, uma subpopulação de células tumorais que apresentam características de células-tronco. Estas células, assim como as células tronco embrionárias, são consideradas imortais, podem se auto-renovar e se transformar em qualquer célula do corpo. Vários marcadores, incluindo CD44 e CD133, têm sido relatados como marcadores de células-tronco, normais e cancerosas, e utilizados para isolar células cancerosas de tumores sólidos. O objetivo desse trabalho foi avaliar a expressão de CD44 e de CD133, no câncer gástrico primário e metástases linfonodais, através de imunohistoquímica, e relacioná-la com as variáveis clínico-patológicas de tipo histológico, sexo, idade, localização anatômica, dimensão do tumor, invasão angiolinfática, infiltração perineural, classificação TNM (TN) e acometimento linfonodal. Este estudo foi desenvolvido a partir de um conjunto de 72 casos de adenocarcinoma gástrico, dos Arquivos do Serviço de Patologia e Medicina Legal da Universidade Federal do Ceará (DPML-UFC). Utilizou-se a técnica de tissue microarray asociada à imunohistoquímica com anticorpo monoclonal anti-CD44 e policlonal anti-CD133. Foram considerados positivos os casos que apresentaram uma ou mais células com imunomarcação citoplasmática e/ou membranar. Foi observado que 30% das amostras foram positivas para o CD44. Não foram encontradas diferenças estatisticamente significantes entre as variáveis clínico-patológicas estudadas e a imunoexpressão de CD44. A imunoexpressão do CD133 foi positiva em 24% das amostras. O grau de invasão do tumor apresentou dados com tendência estatisticamente significante (p=0,0505), de forma inversa. Nas demais variáveis, não foi encontrada nenhuma diferença estatisticamente significante. A imunoexpressão de CD133 na mucosa histologicamente normal foi maior do que no tumor primário e na metaplasia intestinal, com diferença estatística significante (p=0,0159 e p=0,0058, respectivamente). A imunoexpressão de CD133 no adenocarcinoma gástrico tipo intestinal foi significativamente maior na mucosa histologicamente normal do que na metaplasia (p=0,0260). A frequência da expressão desses marcadores é muito variável, e mesmo nas amostras consideradas positivas, o percentual de células coradas também é variável, e em geral muito baixo.

Acute myeloid leukemia (AML) is a subset of leukemia that is characterized by the clonal expansion of cytogenetically and molecularly abnormal myeloid blasts. These blasts are highly proliferative accumulating in bone marrow and blood which leads to severe infections, anemia, and bone marrow failure. The poor prognosis of AML patients caused by the low tolerance to intensive chemotherapy has encouraged the pursuit of alternative therapeutic approaches. Differentiation therapy which involves the use of agents that can release the differentiation block in these leukemic blasts has emerged as a promising therapeutic approach. The use of All-trans retinoic acid (ATRA) represents a successful example of such an approach, nonetheless its efficacy is restricted to one subtype of AML. Efforts have been focused on finding differentiation agents which are effective for the other more common AML subtypes. Anti-CD44 targeted antibodies that activate the CD44 cell surface antigen are a promising candidate. Previous studies have shown that anti-CD44 treatment has been able to release the differentiation block in AML1 through AML5 subtypes. The exact mechanism by which anti-CD44 treatment is able to induce its effects has not been fully elucidated. Recent studies highlight the role that epigenetic mechanisms play during haematopoiesis and leukemogenesis and therefore, in this work we investigated the epigenetic mechanisms associated with anti-CD44 induced differentiation. Using AML cell lines from different subtypes, we demonstrated that anti-CD44-induced differentiation results in an extensive change of histone modification levels. We found that inhibiting enzymes responsible for the H3K9ac, H3K4me, H3K9me, and H3K27me modifications, attenuated the anti-proliferative and differentiation promoting effects of antic-CD44 treatment. Taken together, these data highlight the promising potential of using anti-CD44 as a therapeutic agent across multiple subtypes in AML

In recent years, magnetic nanowires (NWs) have been widely used for their therapeutic potential in biomedical applications. The use of iron (Fe) NWs combines two important properties, biocompatibility and remote manipulation by magnetic fields. In addition the NWs can be coated and functionalized to target cells of interest and, upon exposure to an alternating magnetic field, have been shown to induce cell death on several types of adherent cells, including several cancer cell types. For suspension cells, however, using these NWs has been much less effective primarily due to the free-floating nature of the cells minimizing the interaction between them and the NWs. Leukemic cells express higher levels of the cell surface marker CD44 (Braumüller, Gansauge, Ramadani, & Gansauge, 2000), compared to normal blood cells. The goal of this study was to functionalize Fe NWs with a specific monoclonal antibody towards CD44 in order to target leukemic cells (HL-60 cells). This approach is expected to increase the probability of a specific binding to occur between HL-60 cells and Fe NWs. Fe NWs were fabricated with an average diameter of 30-40 nm and a length around 3-4 μm. Then, they were coated with both 3-Aminopropyl-triethoxysilane and bovine serum albumin (BSA) in order to conjugate them with an anti-CD44 antibody (i.e. anti-CD44-iron NWs). The antibody interacts with the amine group in the BSA via the 1-Ethyl-3-3-dimethylaminopropyl-carbodiimide and N-Hydroxysuccinimide coupling. The NWs functionalization was confirmed using a number of approaches including: infrared spectroscopy, Nanodrop to measure the concentration of CD44 antibody, as well as fluorescent-labeled secondary antibody staining to detect the primary CD44 antibody. To confirm that the anti-CD44-iron NWs and bare Fe NWs, in the absence of a magnetic field, were not toxic to HL-60 cells, cytotoxicity assays using XTT (2,3-Bis-2-Methoxy-4-Nitro-5-Sulfophenyl-2H-Tetrazolium-5-Carboxanilide) were performed and resulted in a high level of biocompatibility. In addition, the internalization of the coated NWs have been studied by coating them with a pH dependent dye (pHrodoTM Red) that showed a signal once the NWs were internalized by the cell.

Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

Diese Arbeit beschäftigt sich mit den Biomarkern Osteopontin und CD44 Standard, sowie CD44 Isovariante 6 beim Rektumkarzinom. Wir konzentrierten uns auf die prognostische Bedeutung von Osteopontin und CD44 Standard, sowie CD44 Isovariante 6. In einigen Vorgängerarbeiten zeigten sich Zusammenhänge vor allem bei der Tumorinduktion, Metastasierung und Überleben. In unserer Arbeit konnten wir bestätigen, dass sich hohe Serumkonzentrationen von OPN bei Patienten mit Rektumkarzinom hochsignifikant negativ auf das Gesamtüberleben auswirken. Niedrigere Serumkonzentrationen sind daher mit einer günstigeren Prognose assoziiert. Dies zeigte sich auch in der durchgeführten multivariaten Analyse. Wir kommen daher zu dem Schluss, dass sich OPN als prognostischer Marker eignet. In der Literatur zeigte sich CD44v6 mit verstärkter Metastasierung assoziiert. Dies konnten wir nicht bestätigen. Wir sahen CD44std und auch CD44v6 weder mit Gesamtüberleben, noch mit Tumorstadium und Metastasierung assoziiert. Auch wenn wir CD44 mit OPN gemeinsam auf das Gesamtüberleben untersuchten, fanden wir keinen signifikanten Einfluss. Als mögliche Schlussfolgerung dieser Arbeit könnte man die aktuelle Therapie des Rektumkarzinoms bei hohen OPN Werten reevaluieren. Bei hohen Osteopontin Werten wären dann ggfs. aggressivere Therapieprotokolle vorstellbar. / This thesis deals with the biomarkers osteopontin and CD44 standard, as well as CD44 isovariant 6 in rectal carcinoma. We focused on the prognostic importance of osteopontin and CD44 standard, as well as CD44 isovariant 6. In some previous studies, correlations were found, especially with tumor induction, metastasis and survival. In this thesis, we were able to confirm that high serum concentrations of osteopontin have a highly significant negative effect on overall survival in patients with rectal cancer. Lower serum concentrations are therefore associated with a better prognosis. This was also reflected in the multivariate analysis that was carried out. We therefore conclude that osteopontin is useful as a prognostic marker. In the literature, CD44v6 is shown to be associated with increased metastasis. We could not confirm this. We saw CD44std and CD44v6 associated neither with overall survival nor with tumor stage and metastasis. Even when we tested CD44 with OPN together on overall survival, we found no significant impact. As a possible conclusion of this thesis, therapy for rectal carcinoma could be re-evaluated with high OPN values. In the case of high OPN values, more aggressive therapy protocols might be conceivable.

Die Identifizierung endogener Stammzellen mit kardiogenem Potenzial und die Möglichkeit, deren Differenzierung zu steuern, würde einen Meilenstein in der kardioregenerativen Therapie darstellen. Innerhalb der Gefäßwand konnten unterschiedliche Stamm- und Vorläuferzellen identifiziert werden, die sog. Gefäßwand-residenten Stammzellen (VW-SCs). Zuletzt konnten aus CD34(+) VW-SCs, ohne genetische Manipulation, Kardiomyozyten generiert werden. Zusätzlich fungiert die Gefäßwand als Quelle inflammatorischer Zellen, die essenziell für die kardiogene Differenzierung der VW-SCs zu sein scheinen. Ziel dieser Arbeit war es, das Verhalten von CD44(+) VW-SCs zu untersuchen, um herauszufinden, inwieweit dieser Stammzelltyp eine endogene Generierung von Kardiomyozyten unterstützen könnte. Dabei wurde mit infarzierten Mäuseherzen, dem Aortenringassay (ARA) und dem kardialen Angiogeneseassay (CAA) gearbeitet. Sowohl in vivo in ischämischen Arealen infarzierter Mäuseherzen als auch ex vivo im CAA kam es zu einem signifikanten Anstieg von CD44(+) Zellen. Mittels Färbungen auf CD44 und Ki-67 konnte die Teilungsfähigkeit dieser Zellen demonstriert werden. Ex vivo ließen sich aus CD44(+) Zellen F4/80(+) Makrophagen generieren. Die CD44(+) VW-SCs können sich dabei sowohl zu pro-inflammatorischen iNOS(+) M1- als auch zu anti-inflammatorischen IL-10(+) M2-Makrophagen differenzieren. Eine Modulation der kardialen Inflammation könnte einen entscheidenden Einfluss auf die Kardiomyogenese haben. Unter VEGF-A kam es im CAA zu einer deutlichen Zunahme von CD44(+) Zellen. Unter Lenvatinib blieb das kardiale Sprouting gänzlich aus, die Anzahl der CD44(+) Zellen stagnierte und die VW-SCs verblieben in ihren physiologischen Nischen innerhalb der Gefäßwand. Warum es nach einem MI kaum zu einer funktionellen Herzmuskelregeneration kommt, ist weiterhin unklar. Die therapeutische Beeinflussung koronaradventitieller CD44(+) VW-SCs und inflammatorischer Prozesse könnte dabei zukünftig eine wichtige therapeutische Option darstellen. / The identification of endogenous stem cells with cardiogenic potential and the possibility to control their differentiation would represent a milestone in cardioregenerative therapy. Within the vascular wall, different stem and progenitor cells could be identified, the so-called vascular wall-resident stem cells (VW-SCs). Most recently, cardiomyocytes could be generated from CD34(+) VW-SCs, without genetic manipulation. In addition, the vascular wall acts as a source of inflammatory cells which appear to be essential for cardiogenic differentiation of VW-SCs. The objective of this work was to investigate the behavior of CD44(+) VW-SCs to see to what extent this stem cell type could support endogenous generation of cardiomyocytes. This was done using infarcted mouse hearts, the aortic ring assay (ARA), and the cardiac angiogenesis assay (CAA). There was a significant increase in CD44(+) cells in vivo in ischemic areas of infarcted mouse hearts and ex vivo in the CAA. A double staining for CD44 and Ki-67 demonstrated the ability of these cells to proliferate. Ex vivo, F4/80(+) macrophages could be generated from CD44(+) cells. Thereby, the CD44(+) VW-SCs can differentiate into both pro-inflammatory iNOS(+) M1 and anti-inflammatory IL-10(+) M2 macrophages. Modulation of cardiac inflammation may have a critical impact on cardiomyogenesis. Under VEGF-A, there was a clear increase in CD44(+) cells in the CAA. Under lenvatinib, cardiac sprouting was completely absent, the number of CD44(+) cells stagnated, and VW-SCs remained in their physiological niches within the vessel wall. Why there is little functional myocardial regeneration after MI remains unclear. Therapeutic manipulation of coronary adventitial CD44(+) VW-SCs and inflammatory processes may represent an important therapeutic option in the future.

Acute myeloid leukemia (AML) is characterized by a blockage in the differentiation of myeloid cells at different stages. CD44-ligation using anti-CD44 monoclonal antibodies (mAbs) has been shown to reverse the blockage of differentiation and to inhibit the proliferation of blasts in most AML-subtypes. However, the molecular mechanisms underlying this property have not been fully elucidated. Here, we sought to I) analyze the effects of anti-CD44 mAbs on downstream signaling pathways, including the ERK1/2 (extracellular signal-regulated kinase 1 and 2) and mTOR (mammalian target of rapamycin) pathways and II) use state-of-the-art Nuclear Magnetic Resonance (NMR) technology to determine the global metabolic changes during differentiation induction of AML cells using anti-CD44 mAbs and other two previously reported differentiation agents. In the first objective (Chapter 4), our studies provide evidence that CD44-ligation with specific mAbs in AML cells induced an increase in ERK1/2 phosphorylation. The use of the MEK inhibitor (U0126) significantly inhibited the CD44-induced differentiation of HL60 cells, suggesting that ERK1/2 is critical for the CD44-triggered differentiation in AML. In addition, this was accompanied by a marked decrease in the phosphorylation of the mTORC1 and mTORC2 complexes, which are strongly correlated with the inhibition of the PI3K/Akt pathway. In the second objective (Chapter 5), 1H NMR experiments demonstrated that considerable changes in the metabolic profiles of HL60 cells were induced in response to each differentiation agent. These most notable metabolites that significantly changed upon CD44 ligation were involved in the tricarboxylic acid (TCA) cycle and glycolysis such as, succinate, fumarate and lactate. Therefore, we sought to analyze the mechanisms underlying their alterations. Our results revealed that anti-CD44 mAbs treatment induced upregulation in fumarate hydratase (FH) expression and its activity which was accompanied by a decrease in succinate dehydrogenase (SDH) activity. Interestingly, our results indicated that FH induced by anti-CD44 mAb is regulated through the activation of the ERK1/2 pathway. Therefore, our findings highlight new elements in support for the use of anti-CD44 mAbs in AML therapies and open new perspectives to use metabolic profiling as a tool to support the potential possibilities for the development of CD44-targeted therapy of AML.

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L’infection par Helicobacter pylori touche environ la moitié de la population mondiale et est responsable de plusieurs pathologies gastro-intestinales incluant l’adénocarcinome gastrique. Les mécanismes de la carcinogénèse induite par H. pylori ne sont pas clairement élucidés. Mais, l’oncoprotéine CagA que possèdent certaines souches est très impliquée dans la carcinogénèse gastrique ; elle induit l’apparition d’un phénotype particulier, dit colibri, qui mime une transition épithélio-mésenchymateuse (EMT). De plus, CagA déstabilise les jonctions cellulaires en perturbant la E-cadhérine. Les objectifs de ces travaux ont été de déterminer si H. pylori induit une véritable EMT et si cette EMT est à l’origine de l’émergence de cellules souches cancéreuses (CSC). De plus, nous avons étudié le rôle joué par la protéine IQGAP1, protéine assurant le maintien des jonctions cellulaires, dans la carcinogénèse gastrique induite par H. pylori. Ces travaux ont montré que H. pylori induit une EMT in vitro. Cette EMT est à l’origine de l’émergence de cellules CD44high présentant les caractéristiques de CSC. L’étude du rôle de IQGAP1 au cours de la carcinogénèse gastrique liée à H. pylori a permis de déterminer son implication dans l’apparition de lésions néoplasiques dans un modèle de souris transgéniques hétérozygotes pour IQGAP1. En outre, IQGAP1 apparaît comme une protéine dont l’expression est modifiée par l’infection à H. pylori et par l’EMT induite par cette bactérie in vitro. Nos résultats permettent de mieux comprendre le mécanisme physiopathologique de l’adénocarcinome gastrique et seront potentiellement utiles au développement de nouvelles thérapeutiques anti-cancéreuses. / Helicobacter pylori infection is found in about half of the world population and is responsible for several gastrointestinal pathologies, including gastric adenocarcinoma. The mechanisms of the carcinogenesis due to H. pylori remain unclear. However, the link with gastric adenocarcinoma is partly due to the H. pylori CagA oncoprotein. CagA is responsible for a particular cell phenotype in vitro, the “hummingbird” phenotype which corresponds to an elongation of the cells, mimicking an epithelial to mesenchymal transition (EMT). EMT participates to carcinogenesis, and is involved in the generation of cancer stem cells (CSC). Moreover, CagA destabilize the cell junctions. This study aimed to determine wether H. pylori induces a true EMT, and if so, wether this EMT can generate CSCs. The role of IQGAP1, which is a scaffold protein involved in cell adhesion, was also studied in cases of gastric carcinogenesis due to H. pylori. We demonstrated that H. pylori induces an EMT in vitro. Moreover, we showed that this EMT is responsible for the emergence of CD44high cells which have the same characteristics as the CSCs. IQGAP1 has been identified as a protein implicated in neoplastic lesion development in a transgenic mouse model heterozygous for IQGAP1. Moreover, in vitro, the expression of IQGAP1 was modified by H. pylori infection and more specifically by the EMT induced by H. pylori. Our results allow a better understanding of gastric adenocarcinoma pathophysiology and will be helpful in developing new cancer chemotherapies.