The Epidermal Growth Factor Receptor (EGFR) is associated with a variety of cancers, including brain, lung, cervix, renal and breast. It is part of a family of receptors known as the ErbB receptors (ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), transmembrane proteins found on epithelial cells responsible for a multitude of signaling events. In cancers, EGFR is frequently mutated or improperly expressed, upregulated in more than 50 percent of basal-like cancers. Mutations commonly promote constitutive activation or increase receptor recycling. In basal-like breast cancers such as triple negative breast cancer (TNBC), named for the lack of hormone receptors (estrogen and progesterone) and the HER2 receptor, EGFR is highly upregulated and associated with a variety of oncogenic activity, including increased proliferation and migration, and inhibition of cell death. Changes in these pathways are predicated on altered trafficking and activation of EGFR, events driven by variation in stimuli and interacting partners, such as other ErbB family members or oncogenic adaptor proteins such as MUC1, a member of the mucin family. In TNBC, upon stimulus with epidermal growth factor (EGF), EGFR colocalizes with MUC1 in intracellular vesicles distributed throughout the cytoplasm. These intracellular vesicles are associated with early endosomes, as indicated by the presence of early endosome antigen 1 (EEA1). Association with MUC1 prolongs the presence of EGFR in these vesicles, as EGFR's stay is significantly reduced in cells lacking MUC1. Retention in these vesicles by MUC1 inhibits trafficking of EGFR to the lysosome for degradation and is also associated with an increase in EGF-dependent migratory ability. Introduction of late endosome inhibitors (thereby preventing lysosomal targeting) increases migration in the absence of MUC1, the same effect as in the presence of MUC1. Further, inhibition of retrograde trafficking significantly decreases the rate of migration and changes cellular distribution of filopodia corresponding to migratory ability in MUC1-containing cells. Taken together, these data indicate that MUC1 is responsible for altering EGFR trafficking by retaining EGFR in EEA1-positive vesicles for prolonged periods, allowing for increased signal transduction through retrograde trafficking of EGFR and structural reorganization promoting a migratory phenotype. Loss of the polarity protein Llgl1 is associated with alterations in EGFR trafficking, promoting highly diffuse EGFR distribution throughout the cytoplasm versus along basolateral membranes. These changes in trafficking are also associated with increases in AKT and dual-phosphorylated-ERK signal transduction, both downstream targets of activated EGFR. Altering localization of EGFR to other membranes and intracellular vesicles without inducing polarity loss through a point mutation at amino acid 667 was found to also upregulate the AKT pathway. Mislocalization driven by polarity loss or point mutation in the basolateral targeting domain is sufficient to increase migration speeds of non-cancerous epithelial cell lines in vitro. This increased oncogenic activity is likely attributed to increased nuclear localization of the transcription factor TAZ (transcription co-activator with a PDZ-binding domain), whose nuclear translocation is associated with increased stem-like properties such as migration and survival. Together, these data reveal the oncogenic potential caused by alterations in EGFR trafficking that occur when polarity is lost or EGFR is improperly associated with proteins that promote changes to canonical EGFR localization and degradation, such as MUC1.

No description available.

No description available.

La mucine MUC1 est une oncoprotéine transmembranaire dont la surexpression dans 90% des adénocarcinomes pancréatiques a été associée à un mauvais pronostic. MUC1 est impliquée dans la transduction des signaux intracellulaires et dans les interactions cellulaires permettant de conférer aux cellules tumorales des propriétés accrues en termes de prolifération et d’invasion cellulaire. De plus il a été montré un rôle important de MUC1 dans la chimiorésistance à la gemcitabine, et dans la transition épithélio-mésenchymateuse des cellules cancéreuses pancréatiques. De manière intéressante il a pu être montré que MUC1 pouvait être internalisée et localisée dans le compartiment nucléaire afin d’agir comme un co-activateur transcriptionnel permettant de moduler l’expression de nombreux gènes comme ceux de la voie Wnt/β-caténine, les cibles de Stat1/3 ou le cluster miR-200c/miR141. De plus, il a été démontré que MUC1 pouvait être régulé de façon épigénétique, par méthylation de son promoteur, par acétylation des histones et par les miARN.Notre objectif a été d’étudier l'inhibition de MUC1 par des miARN dérégulés dans le cancer pancréatique, afin de proposer une nouvelle stratégie thérapeutique innovante dans le but de ralentir la progression de ce cancer. Nous avons sélectionné des miARN pouvant cibler la mucine MUC1 aussi bien au niveau de son 3’UTR, de son 5’UTR ou de sa région codante par l'utilisation des bases de données miRanda, miRWalk et TargetScan. Afin d’affiner cette sélection, nous avons ensuite retenu seulement ceux étant dérégulés dans le cancer pancréatique en étudiant leur expression dans des lignées cellulaires cancéreuses pancréatiques humaines ainsi que dans un modèle murin transgénique de cancérogenèse pancréatique et dans les tissus de patients atteints d'adénocarcinome pancréatique.Nous avons dans un premier temps mis en évidence que parmi les miARN sélectionnés, la surexpression de miR-29a, miR-183, miR-200a, miR-330-5p, miR-876-3p et miR-939 entraînait une diminution de l'expression protéique de MUC1. En établissant le profil d'expression des miARN dans les trois modèles de cancer pancréatique dont nous disposions, nous avons pu mettre en évidence une dérégulation globale de miR-29a et miR-330-5p dans les lignées cellulaires cancéreuses pancréatiques humaines ainsi que chez les patients atteints d'adénocarcinome pancréatique, et une dérégulation plus spécifique pour les autres miARN. Nous avons alors pu mettre en évidence que parmi l'ensemble des miARN sélectionnés, seuls les miARN miR-29a et miR-330-5p avaient la capacité d’interagir avec l'ARNm de MUC1 au niveau de son 3’UTR. Nous avons donc entrepris dans un second temps d’étudier le rôle de miR-29a et miR-330-5p dans le cancer du pancréas. Pour cela, nous avons utilisé une stratégie transitoire de surexpression et d'inhibition des miARN et une stratégie stable en réalisant des lignées surexprimant les miARN ainsi qu’une lignée déficiente en MUC1. Nous avons pu mettre en évidence que la surexpression de miR-29a et miR-330-5p, permettait de ralentir la prolifération cellulaire, la migration, l'invasion cellulaire, la croissance tumorale et augmentait la chimiosensibilité des cellules cancéreuses pancréatiques à la gemcitabine. En conclusion, l'ensemble de ces données a permis de mettre en évidence un ensemble de miARN dérégulés dans le cancer du pancréas ayant la capacité de réguler négativement l'expression protéique de la mucine MUC1. Nous avons également montré que miR-29a et miR-330-5p étaient les seuls à réguler directement l'expression de MUC1 et qu’ils agissaient comme des suppresseurs de tumeurs en altérant les propriétés biologiques des cellules cancéreuses pancréatiques, in vitro et in vivo. Ces données nous permettent de proposer ces deux miARN comme une nouvelle piste thérapeutique potentielle pour le traitement de ce cancer. / The mucin MUC1 is a transmembrane oncoprotein overexpressed in 90% of pancreatic adenocarcinoma and associated with a poor prognosis. MUC1 is involved in cell signaling and cell interaction to enhanced tumor cell properties like cell proliferation and invasion. Furthermore it has been shown an important role of MUC1 in chemoresistance to gemcitabine, the basic treatment of pancreatic cancer, and in the epithelial-mesenchymal transition of pancreatic cancer cells. Interestingly it has been shown that MUC1 could be internalized and localized in the nuclear compartment to act as a transcriptional coactivator to modulate the expression of many genes such as the Wnt/β-catenin, the targets of Stat1/3 or the miR-200c/miR141 cluster. Furthermore, it has been shown that MUC1 is regulated by epigenetics: by methylation of the promoter, histone acetylation and by miRNAs in breast and ovarian cancer.Our aim was to study the inhibition of MUC1 by miRNAs deregulated in pancreatic cancer, to propose a new innovative therapeutic strategy to slow down progression of this cancer.We selected miRNAs targeting the mucin MUC1, in its 3\\\'UTR, its 5\\\'UTR or its coding region by using databases such as Miranda, miRWalk and TargetScan. To refine this selection, we then selected only those being deregulated in pancreatic cancer by studying their expression in human pancreatic cancer cell lines, tissues from patients with pancreatic adenocarcinoma and transgenic mouse model of early pancreatic carcinogenesis.We initially demonstrated that among the selected miRNAs, overexpression of miR-29a, miR-183, miR-200a, miR-330-5p, miR-and miR-939 876-3p led to a decrease of MUC1 protein expression. By establishing the miRNA expression profile in the three models of pancreatic cancer that we had, we were able to demonstrate an overall deregulation of miR-29a and miR-330-5p in human pancreatic cancer cell lines and in patients with a pancreatic adenocarcinoma, and a more specifically deregulation for the other miRNAs.We were then able to show that among the selected miRNAs, only miRNAs miR-29a and miR-330-5p had the ability to interact with MUC1 mRNA on its 3\\\'UTR. We therefore undertook to study the role of miR-29a and miR-330-5p in pancreatic cancer. For this, we used a transient strategy to overexpress or inhibit miRNAs and stable cell lines overexpressing the miRNA as well as a deficient cell line for MUC1. We were able to show that overexpression of miR-29a and miR-330-5p slowed down cell proliferation, migration, cell invasion, tumor growth and increased chemosensitivity of pancreatic cancer cells to gemcitabine.In conclusion, all these data allowed us to identify a set of deregulated miRNAs in pancreatic cancer which have the ability to decrease the mucin MUC1 protein expression level. We also showed that miR-29a and miR-330-5p were the only ones that can regulate the expression of MUC1 directly and act as tumor suppressors by altering the biological properties of pancreatic cancer cells in vitro and in vivo. These data allow us to propose these two miRNAs as a new potential therapeutic approach for the treatment of this cancer.

A MUC1 (MUC1) e a osteopontina (SPP1) são glicoproteínas expressas na superfície luminal uterina com funções na proteção e adesão celular. A MUC1 possui função antiadesiva, enquanto a osteopontina desempenha função adesiva. A expressão de ambas é regulada pelo hormônio esteróide progesterona. Durante a fase receptiva do útero, a MUC1 é inibida por este hormônio, enquanto a osteopontina é estimulada. O objetivo deste trabalho é caracterizar o polimorfismo genético destas moléculas e analisar a associação entre o polimorfismo, o diagnóstico de gestação e as diferenças esperadas na progênie (DEP) de várias características em novilhas da raça Nelore (Bos taurus indicus). A amostra foi constituída por 309 novilhas procedentes de duas fazendas participantes do Programa de Melhoramento Genético da Raça Nelore (PMGRN). A amostra da primeira fazenda incluiu 56 novilhas prenhas e foi utilizada para caracterização do polimorfismo e da estrutura do número variável de repetições em tandem (VNTR) MUC1 na raça Nelore. A segunda amostra foi constituída por 76 novilhas com resultado positivo de gestação e 156 com resultado negativo de gestação e foi utilizada para caracterização do polimorfismo dos genes MUC1 e SPP1 na raça Nelore, assim como para análise da associação entre os polimorfismos e os fenótipos. O gene MUC1 apresentou 5 alelos constituídos por um VNTR formado por uma seqüência de 60 nucleotídeos. A seqüência da repetição consensus foi idêntica às seqüências descritas em caprinos e em Bos taurus taurus. Descrevemos a seqüência de uma terceira repetição consensu na raça Nelore. O alelo 1 apresentou 10 repetições, o alelo 2 apresentou 12 repetições, o alelo 3 apresentou 15 repetições, o alelo 4 foi formado por 18 repetições, e o alelo 5 apresentou 24 repetições. O alelo com menor número de repetições apresentou a maior freqüência, sendo 0,70 na amostra do 1º grupo e 0,80 na amostra do 2º grupo. Os alelos 2, e 3 tiveram a segunda e terceira maior freqüência, sendo seguidos pelos alelos 4 e 5. A análise estatística não evidenciou associação entre o polimorfismo do gene MUC1 e o diagnóstico de gestação e entre o polimorfismo e os valores das diferenças esperadas na progênie das características consideradas economicamente importantes. Na amostra referente às novilhas da segunda fazenda, não identificamos o polimorfismo de nucleotídeo simples no íntron 4 (T/C) do gene SPP1 da osteopontina, como descrito na raça Holandesa. Sugerimos que a ausência deste polimorfismo possa constituir uma característica específica em Bos taurus indicus. Porém, dada a associação já descrita entre polimorfismo do gene SPP1 e características de crescimento, não descartamos a hipótese de que, assim como o MUC1, tenha ocorrido uma seleção indireta devido aos critérios aplicados pelo Programa de Melhoramento Genético da Raça Nelore. / MUC1 (MUC1) and osteopontin (SPP1) are glycoproteins expressed in the uterine luminal surface with predict functions in protection and cell adhesion. MUC1 has anti-adhesive role and osteopontin has adhesive role. The expression of both molecules is regulated by the steroid hormone, progesterone. During the receptive period for embryo implantation in the uterus, MUC1 is inhibited by progesterone and the osteopontin is stimulated. The objective of this work is to characterize the genetic polymorphism of these molecules, and to characterize associations between the polymorphisms, gestation rate and the expected progeny difference (EPD) in the Nelore breed (Bos taurus indicus). The study group comprised 309 heifers derived from two participating farms of the Nelore Cattle Breeding Program (PMGRN). The first farm provided 56 fertile female for the study for the characterization of the MUC1 polymorphism and the variable number of tandem repeats (VNTR) genomic structure in the Nelore breed. The second farm contributed 76 heifers with confirmed gestation and 156 no-pregnant female that were used to characterize both gene polymorphisms and to analyze the association between the MUC1 and SPP1 polymorphisms and the phenotypes. The MUC1 gene presented five alleles caused by length differences generated by a VNTR of 60 nucleotides. The consensus repeat sequence was identical to the caprine and Bos taurus taurus sequence. In this study we described the sequence of the third consensus repeat in the Nelore breed. Allele 1 presented with 10 repeats, allele 2 presented with 12 repeats, allele 3 presented with 15 repeats, allele 4 had 18 repeats and allele 5 had 24 repeats. Allele 1 had the smallest size and was the most frequent (0.70) in the group 1 and 0.80 in the group 2. Alleles 2 and 3 were the next most frequent followed by alleles 4 and 5. The ?2 test showed that the polymorphism was not significantly associated with the diagnostic of gestation and the EPD values. In this Nelore study group it was not possible to identify the single nucleotide polymorphism (T/C) of the SPP1 gene previously described in Holstein breed. The absence of this polymorphism could be specific to the Nelore cattle. However, considering the correlation between the SPP1 polymorphism and growth parameters, an indirect selection could happen due to the established criteria within the Nelore Cattle Breeding Program.

Peptide aus intrazellulären Pathogenen sowie Tumorantigene aus mutierten oder überexprimierten Proteinen werden MHC I-restringiert auf der Oberfläche von Mammaliazellen präsentiert und dabei von cytotoxischen T-Lymphozyten erkannt. Die Peptidgenerierung erfolgt vorwiegend durch das Ubiquitin/26S Proteasomensystem, das eine zentrale Rolle bei der antitumoralen Immunantwort spielt. In der vorliegenden Arbeit wurde eine Polyepitopvakzine in Form von Plasmid-DNA entwickelt und auf ihre Effizienz untersucht. Als Modellantigen diente MUC1, ein von verschiedenen Adenokarzinomen und hämatologischen Tumoren überexprimiertes Glykoprotein. Ein aus dem MUC1-Protein bekanntes HLA-A2.1-abhängiges Epitop wurde als Polyepitop in verschiedenen Variationen synthetisiert, einer 20S proteasomalen in vitro Degradation unterzogen und seine Verdauprodukte massenspektrometrisch analysiert. Als optimale Sequenz für die Prozessierung des Polyepitops in die gewünschten Einzelepitope erwies sich deren einfache Verknüpfung ohne intervenierende Sequenzen. Zur Untersuchung des Effekts einer Ubiquitinfusion auf die Stabilität des MUC1-Polyepitops wurden anschließend verschiedene Strategien der linearen Fusion von Ubiquitin an das Polyepitop innerhalb eines Plasmids getestet. Durch transiente Transfektion der Plasmide konnte die Stabilität ihrer Translationsprodukte mittels Immunoblotanalysen quantifiziert werden. Die Ergebnisse demonstrieren, daß das Polyepitop durch die N-terminale Fusion von Wildtyp-Ubiquitin am effizientesten degradiert wird, wobei eine Polyubiquitinierung nicht stattzufinden scheint. Die Auswertung der folgenden in vitro Cytotoxizitätsassays sowie der Immunisierungsstudien wurde durch die schwache Affinität des gewählten subdominanten MUC1-Epitops zum HLA-A2.1-Komplex beeinträchtigt. Die vorliegende Arbeit unterstreicht die Bedeutung einer umfassenden Analyse intra- und extrazellulärer Vorgänge bei der Entwicklung einer Plasmidvakzine unter Verwendung subdominanter Epitope. / Peptides from intracellular pathogens as well as tumor antigens from mutated or overexpressed proteins are presented in an MHC class I restricted manner on the surface of mammalian cells and are thereby recognized by cytotoxic T lymphocytes. Peptide generation is mainly carried out by the ubiquitin/26S proteasome system which plays a crucial role in the antitumor immune response. In the present study, a polyepitope vaccine was generated in the form of plasmid DNA and analyzed for its efficiency. As model antigen MUC1, a glycoprotein overexpressed in various adenocarcinomas and hematological tumors, was used. A known HLA-A2.1 restricted epitope from the MUC1 protein was synthesized as a polyepitope in different variations, subjected to a 20S proteasomal in vitro degradation, and its digestion products were analyzed by means of mass spectrometry. The optimal sequence for the processing of the polyepitope into the desired single epitopes was shown to be their simple conjunction without spacer sequences. Different strategies of linear fusion of ubiquitin to the polyepitope within a plasmid were subsequently tested to analyze the effect of ubiquitin fusion on the stability of the MUC1 polyepitope. After transient transfection of the plasmids, the stability of their translation products was quantified by means of immunoblot analyses. The results demonstrate that the polyepitope is degraded most efficiently through N-terminal fusion of wild type ubiquitin, while a polyubiquitination does not seem to occur. The evaluation of the subsequent in vitro cytotoxicity assays and immunization studies was impaired by the low affinity of the selected subdominant MUC1 epitope to the HLA-A2.1 complex. The present study emphasizes the importance of an extensive analysis of intra- and extracellular processes when generating a plasmid vaccine using subdominant epitopes.

Les métastases sont responsables de 90 % des décès causés par le cancer. Les métastases sont des foyers cancéreux secondaires qui se forment à distance de la tumeur d’origine. Des cellules cancéreuses quittent la tumeur primaire, rejoignent la circulation sanguine puis colonisent des organes voisins par migration à travers l’endothélium vasculaire. Ce phénomène d’adhésion à l’endothélium et de migration à travers l’endothélium appelé l’extravasation est une étape clé du processus métastatique. L’identification des molécules impliquées constitue une priorité dans le but d’élaborer de nouvelles drogues anticancéreuses. Nous avons précédemment montré que la molécule d’adhésion cellulaire InterCellular Adhesion Molecule-1 (ICAM-1) exprimée par les cellules endothéliales, est impliquée dans l’interaction des cellules de cancer de la vessie (BCs) avec l’endothélium. Cependant les ligands d’ICAM-1 n’ont pas été étudiés. Dans cette étude, nous utilisons des tests d'adhésion cellulaire et la microscopie à force atomique (AFM) afin d’identifier les ligands d’ICAM-1 et de mesurer les forces impliquées dans l’interaction ligand-ICAM-1. Nous avons identifié que les protéines MUC1 et CD43 exprimées par les BCs les plus invasives se lient à ICAM-1 en développant des forces d’intensité différente selon le couple considéré. Une analyse détaillée des événements de rupture suggère que CD43 est fortement lié au cytosquelette et que son interaction avec ICAM-1 correspond principalement à des sauts brusques. Au contraire, MUC1 semble être lié faiblement au cytosquelette et ses interactions avec ICAM-1 sont principalement associées à la formation de filaments membranaires ou « tethers ». Les forces mises en jeu lors de la migration des cellules cancéreuses à travers l'endothélium ont été étudiées par microscopie de forces de traction (TFM). Les résultats préliminaires montrent que les tractions exercées par les cellules cancéreuses lors de l’extravasation sont mesurables par TFM. / Cancer metastasis is associated with 90% cancer-associated deaths, when cancer cells escape from the primary tumor and form metastatic colonies in secondary sites. Extravasation is an important step in cancer metastasis, where cancer cells carried in blood, adhere and transmigrate through the endothelium. Therefore identifying the key molecules involved during the adhesion process could enable to develop new anticancer cancer drugs able to inhibit the adhesion of cancer cells to the endothelium. We have previously shown that InterCellular Adhesion Molecule-1 (ICAM-1) expressed by endothelial cells is involved in the interactions of bladder cancer cells (BCs) with the endothelium. However the ICAM-1 ligands have never been investigated. In this study, we combined adhesion assays and Atomic Force Microscopy (AFM) to identify the ligands involved and to quantify the forces relevant in such interactions. We report the expression of MUC1 and CD43 on BCs and demonstrate that these ligands interact with ICAM-1 to mediate cancer cell-endothelial cell adhesion in the case of the more invasive BCs. AFM experiments were performed to quantify the force ranges involved by MUC1 and CD43 during their interaction with ICAM-1. AFM measurements combined with a Gaussian Mixture Model showed distinct force ranges for the interaction of ICAM-1 with MUC1 and ICAM-1 with CD43. Furthermore, a detailed analysis of the rupture events suggests that CD43 is strongly connected to the cytoskeleton and that its interaction with ICAM-1 mainly corresponds to force ramps followed by sudden jumps. On the contrary, MUC1 seems to be weakly connected to the cytoskeleton as its interactions with ICAM-1 are mainly associated with the formation of tethers. The forces involved during the transmigration of cancer cells through the endothelium was investigated using Traction Force Microscopy (TFM). Preliminary results showed that tractions exerted by cancer cells during transmigration can be studied and quantified using TFM.

A MUC1 (MUC1) e a osteopontina (SPP1) são glicoproteínas expressas na superfície luminal uterina com funções na proteção e adesão celular. A MUC1 possui função antiadesiva, enquanto a osteopontina desempenha função adesiva. A expressão de ambas é regulada pelo hormônio esteróide progesterona. Durante a fase receptiva do útero, a MUC1 é inibida por este hormônio, enquanto a osteopontina é estimulada. O objetivo deste trabalho é caracterizar o polimorfismo genético destas moléculas e analisar a associação entre o polimorfismo, o diagnóstico de gestação e as diferenças esperadas na progênie (DEP) de várias características em novilhas da raça Nelore (Bos taurus indicus). A amostra foi constituída por 309 novilhas procedentes de duas fazendas participantes do Programa de Melhoramento Genético da Raça Nelore (PMGRN). A amostra da primeira fazenda incluiu 56 novilhas prenhas e foi utilizada para caracterização do polimorfismo e da estrutura do número variável de repetições em tandem (VNTR) MUC1 na raça Nelore. A segunda amostra foi constituída por 76 novilhas com resultado positivo de gestação e 156 com resultado negativo de gestação e foi utilizada para caracterização do polimorfismo dos genes MUC1 e SPP1 na raça Nelore, assim como para análise da associação entre os polimorfismos e os fenótipos. O gene MUC1 apresentou 5 alelos constituídos por um VNTR formado por uma seqüência de 60 nucleotídeos. A seqüência da repetição consensus foi idêntica às seqüências descritas em caprinos e em Bos taurus taurus. Descrevemos a seqüência de uma terceira repetição consensu na raça Nelore. O alelo 1 apresentou 10 repetições, o alelo 2 apresentou 12 repetições, o alelo 3 apresentou 15 repetições, o alelo 4 foi formado por 18 repetições, e o alelo 5 apresentou 24 repetições. O alelo com menor número de repetições apresentou a maior freqüência, sendo 0,70 na amostra do 1º grupo e 0,80 na amostra do 2º grupo. Os alelos 2, e 3 tiveram a segunda e terceira maior freqüência, sendo seguidos pelos alelos 4 e 5. A análise estatística não evidenciou associação entre o polimorfismo do gene MUC1 e o diagnóstico de gestação e entre o polimorfismo e os valores das diferenças esperadas na progênie das características consideradas economicamente importantes. Na amostra referente às novilhas da segunda fazenda, não identificamos o polimorfismo de nucleotídeo simples no íntron 4 (T/C) do gene SPP1 da osteopontina, como descrito na raça Holandesa. Sugerimos que a ausência deste polimorfismo possa constituir uma característica específica em Bos taurus indicus. Porém, dada a associação já descrita entre polimorfismo do gene SPP1 e características de crescimento, não descartamos a hipótese de que, assim como o MUC1, tenha ocorrido uma seleção indireta devido aos critérios aplicados pelo Programa de Melhoramento Genético da Raça Nelore. / MUC1 (MUC1) and osteopontin (SPP1) are glycoproteins expressed in the uterine luminal surface with predict functions in protection and cell adhesion. MUC1 has anti-adhesive role and osteopontin has adhesive role. The expression of both molecules is regulated by the steroid hormone, progesterone. During the receptive period for embryo implantation in the uterus, MUC1 is inhibited by progesterone and the osteopontin is stimulated. The objective of this work is to characterize the genetic polymorphism of these molecules, and to characterize associations between the polymorphisms, gestation rate and the expected progeny difference (EPD) in the Nelore breed (Bos taurus indicus). The study group comprised 309 heifers derived from two participating farms of the Nelore Cattle Breeding Program (PMGRN). The first farm provided 56 fertile female for the study for the characterization of the MUC1 polymorphism and the variable number of tandem repeats (VNTR) genomic structure in the Nelore breed. The second farm contributed 76 heifers with confirmed gestation and 156 no-pregnant female that were used to characterize both gene polymorphisms and to analyze the association between the MUC1 and SPP1 polymorphisms and the phenotypes. The MUC1 gene presented five alleles caused by length differences generated by a VNTR of 60 nucleotides. The consensus repeat sequence was identical to the caprine and Bos taurus taurus sequence. In this study we described the sequence of the third consensus repeat in the Nelore breed. Allele 1 presented with 10 repeats, allele 2 presented with 12 repeats, allele 3 presented with 15 repeats, allele 4 had 18 repeats and allele 5 had 24 repeats. Allele 1 had the smallest size and was the most frequent (0.70) in the group 1 and 0.80 in the group 2. Alleles 2 and 3 were the next most frequent followed by alleles 4 and 5. The ?2 test showed that the polymorphism was not significantly associated with the diagnostic of gestation and the EPD values. In this Nelore study group it was not possible to identify the single nucleotide polymorphism (T/C) of the SPP1 gene previously described in Holstein breed. The absence of this polymorphism could be specific to the Nelore cattle. However, considering the correlation between the SPP1 polymorphism and growth parameters, an indirect selection could happen due to the established criteria within the Nelore Cattle Breeding Program.

Mayer-Rokitansky-Kuster-Hauser Syndrome is a congenital disorder of the female reproductive tract due to impaired Müllerian duct development. There are three known categorical presentations: isolated, atypical, and MURCS association. Several developmentally significant factors including inappropriate AMH/AMHR interaction, and mutations in the WNT gene family and HOXA7-13 cluster have been studied. There has also been investigation into an autosomal dominant pattern of inheritance in families with multiple cases of the syndrome. Due to the presence of multiple subsets of patients with similar genetic abnormalities, it seems unlikely that a single etiology will be discovered.

Le travail effectué au cours de cette thèse se situe dans le cadre d'une approche anti-tumorale par immunothérapie. Le cœur de ce projet a consisté à développer une méthodologie permettant de synthétiser des mimes de la forme tumorale de la glycoprotéine MUC1 par multiligation chimique sur support solide. La stratégie de synthèse repose sur une approche convergente basée sur la condensation par liaison oxime entre un peptide aminooxy (Aoa) et un peptide aldéhyde. Le développement d'une approche utilisant la déprotection sélective du groupe Aoa sur le fragment médian a permis de coupler plusieurs motifs MUC1 par ligation chimique sur support solide et d'obtenir deux longs peptides comportant deux unités et demi de répétition de la protéine MUC1 pour l'un et un épitope T-auxiliaire supplémentaire pour l'autre composé.<br /> Ce travail ouvre la voie à la synthèse de chimères de MUC1 qui seront glycosylées par voie chimioenzymatique. Ces petites glycoprotéines synthétiques, bien que calquées sur des glycoprotéines naturelles, seront élaborées dans le but d'étudier différentes présentations des antigènes pour optimiser leur immunogénicité.