Orientador: Fernando Eduardo Torres Orihuela / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Matematica, Estatistica e Computação Cientifica / Made available in DSpace on 2018-08-12T08:34:02Z (GMT). No. of bitstreams: 1 VillanuevaZevallos_JuanElmer_D.pdf: 1127069 bytes, checksum: 8ac303abd191b4c264038dcd1ce40be1 (MD5) Previous issue date: 2008 / Resumo: Os principais tópicos aqui considerados são do tipo aritmético. Introduzimos e estudamos semigrupos que generalizam os chamados semigrupos de Arf. Além de seu interesse particular, eles podem ser usados para esclarecer a estrutura de anéis de semigrupos no sentido de Lipman. Também calculamos os valores exatos dos pesos de semigrupos usando o número de lacunas pares. Isto está relacionado ao recobrimento duplo de curvas e tem interesse no estudo de moduli e constelação de curvas. / Abstract: The main topics considered here are of arithmetical type. We introduce and study semigroups that generalize the so-called Arf semigroups. Apart from being interesting by their own, they may be used to clarify the structure of semigroup rings in the sense of Lipman. We also compute the true value of the weights of semigroups by using the number of even gaps. This is related to double covering of curves and is useful to the study of moduli and constellation of curves. / Doutorado / Geometria Algebrica / Doutor em Matemática

La migration cellulaire joue un rôle essentiel dans le développement des organismes multicellulaires et dans certaines pathologies comme le cancer, où elle permet la formation de métastases. Le trafic vésiculaire est un régulateur clé de la migration cellulaire, notamment en contrôlant la localisation de protéines impliquées dans la migration telles que les intégrines, les cadhérines et les récepteurs transmembranaires. En particulier, notre laboratoire a montré que l'endocytose contrôle l'orientation et la communication cellulaire durant la migration cellulaire collective. Notre hypothèse est que d'autres événements du trafic vésiculaire pourraient aussi être impliqués dans ce type de migration. Ainsi, le but de cette thèse a été de déterminer la fonction des petites GTPases Arf, importantes pour la formation de vésicules et le tri de cargo dans ces vésicules et de leurs régulateurs dans la migration cellulaire collective. Un modèle pour étudier la migration cellulaire collective est les chambres d’œufs de Drosophila melanogaster. En effet, lors de l’ovogénèse, des cellules folliculaires appelées cellules de bordure migrent à travers les cellules nourricières pour atteindre l’ovocyte. Conformément à notre hypothèse, un fort défaut de migration est observé lorsque les Arfs sont déplétées spécifiquement dans les cellules de bordure. De plus, un constat similaire est observé après la déplétion de certains régulateurs des Arfs (ArfGAPs et ArfGEFs). Notamment, nous avons démontré que l’ArfGAP Drongo et sa fonction d'activation de l’activité GTPase sont essentielles pour le détachement initial des cellules de bordure du tissu folliculaire. Drongo promeut le détachement en contrôlant la localisation de la myosine phosphatase afin de réguler l’activité de la myosine II à l’arrière des cellules. De plus, nous avons montré que Drongo agit sur l’Arf de classe III (Arf51F) de manière antagoniste à l’ArfGEF Steppke pour déplacer la myosine phosphatase de l’arrière du groupe de cellules. D’un autre côté, nous avons aussi démontré qu’une autre GAP, ArfGAP1, contrôle la directionnalité de migration. Cette ArfGAP agit potentiellement en régulant la localisation de certains déterminants de la migration tels que l’E-cadhérine et les récepteurs tyrosine kinase. Ainsi, nos recherches ont démontré un rôle essentiel des Arfs ainsi que des rôles spécifiques de deux ArfGAPs dans la migration cellulaire collective. / Cell migration is implicated in various important biological processes, notably it is central for the dissemination of cancer cells. Vesicular trafficking is a key regulator of cell migration, notably by controlling the localisation of proteins involved in migration such as integrins, cadherins and transmembrane receptors. In particular, our laboratory has shown that endocytosis controls orientation and cellular communication during collective cell migration. Our hypothesis is that other events of vesicular trafficking might be implicated in collective cell migration. Thus, the purpose of this thesis was to assess the function of small GTPases Arf, important for vesicle formation and cargo sorting into those vesicles, and their regulators in collective cell migration. A powerful model to study collective cell migration is the migration of follicular cells named border cells during oogenesis in Drosophila melanogaster. Border cells (BCs) detach from the follicle epithelium surrounding the egg chambers and form a small cluster of six to ten cells that migrates invasively between the giant nurse cells that compose the center of the egg chamber, toward the oocyte. Accordingly to our hypothesis, a strong migration defect is observed when the Arfs are depleted specifically in the border cells. Moreover, a similar finding is observed after depletion of some Arfs regulators (ArfGAPs and ArfGEFs). In particular, the ArfGAP Drongo and its GTPase-activating function are essential for the initial detachment of the border cell cluster from the basal lamina. We demonstrated through protein localization and genetic interactions that Drongo controls the localisation of the myosin phosphatase in order to regulate myosin II activity at the back of the cluster and promote border cells detachment. Moreover, we showed that Drongo acts on the class III Arf (Arf51F) antagonistically to the guanine exchange factor Steppke to displace myosin phosphatase from the back of the cluster. On the other hand, we have also demonstrated that the GAP ArfGAP1 controls the directionality of migration. This ArfGAP potentially acts by regulating the localization of certain determinants of migration such as E-cadherin and receptors tyrosine kinase. Thus, our research has demonstrated an essential role for Arfs in collective cell migration and specific contributions of two ArfGAPs in this migration process.

Im ersten Teil dieser Arbeit wurden mittels molekular-zytogenetischer Methoden die chromosomalen Aberrationen in c-myc aktivierten ARFnull- und INK4a/ARFnull-Mauslymphomen untersucht. Die zytogenetischen Ergebnisse wurden mit dem Therapieverlauf der Mäuse nach Cyclophosphamid-Behandlung verglichen. In den ARFnull-Lymphomen erkannten wir den Gewinn des Chromosoms 14 als einen Marker für gute und den Gewinn des Chromosoms 6 als Marker für schlechte Behandlungserfolge. Auf den Chromosomen 6 und 14 der Maus liegen demnach bisher unbekannte Gene, welche für die Wahl der Behandlungsmethode ARF-defizienter Tumore von entscheidender Bedeutung sind. Der zweite Teil der Arbeit befaßt sich mit Common Fragile Sites (CFS), die als "hot spots" für chromosomale Brüche und Umbauten in Tumorgenese und Karyotypevolution diskutiert werden. CFSs treten als seltene Lücken im Chromatin oder als partiell deletierte bzw. rearrangierte Chromosomen auf. Ihre Zahl wird durch Zugabe von Replikations-hemmenden Chemikalen, wie Aphidicolin (APC), erheblich gesteigert. Wir untersuchten die CFS-Expression in Lymphozytenkulturen der Mausstämme BALB/c und C57BL/6. Die APC-induzierten CFS-Häufigkeiten der Chromosomenbanden wiesen im Vergleich zwischen beiden Mausstämmen eine signifikante Korrelation und damit eine starke Konservierung auf. Ebenfalls wurde zwischen Maus / Mensch-syntenischen Bereichen eine Konservierung der CFS-Häufigkeiten detektiert. Die Tendenz zur CFS-Bildung ist also ein spezifisches Merkmal jedes chromosomalen Abschnitts, das evolutionär konserviert ist. Weiterhin wurden einzelne CFSs mit molekular-zytogenetischen Methoden genauer kartiert. Auf diese Weise beschrieben wir erstmals eines der zehn häufigsten CFSs menschlicher Lymphozyten, FRA7K, und erweiterten somit die Anzahl molekular charakterisierter humaner CFSs auf insgesamt 13. Für fünf dieser CFSs analysierten wir mittels FISH-Analyse die homologen Bereiche im Mausgenom hinsichtlich ihrer CFS-Expression. Die beobachteten Läsionen traten jeweils in exakt den entsprechenden Sequenzen auf. Vereint man diese fünf Beispiele (FRA2G/Fra2D, FRA7G/Fra6A3.1, FRA7H/Fra6B1, FRA7K/Fra12C1 und FRA9E/Fra4C2) mit bekannten Homologen aus der Literatur, so wurde für insgesamt acht CFSs eine Konservierung zwischen Mensch und Maus auf molekularer Ebene gefunden. Trotz zahlreicher Untersuchungen ist der zelluläre Mechanismus, der die Ausbildung von CFSs an spezifischen Stellen des Genoms bewirkt, bis heute weitgehend unklar. Bekannt ist, daß CFSs durch Replikationsinhibition induziert werden und in Metaphase-Zellen sichtbar werden, welche DNA-Replikations-Checkpoints unterlaufen haben.Sämtliche jüngeren Studien beschrieben Inseln erhöhter DNA-Helix-Flexibilität (Schwankungen im Biegungswinkel des Moleküls) in den Bereichen von CFSs. Wir berechneten die DNA-Helix-Flexibilität entlang der Sequenz für alle molekular kartierten humanen und Maus-CFSs sowie für stabile Kontrollregionen. Anders als in der Literatur beschrieben, fanden wir für Mensch und Maus, daß sich CFSs und Kontroll-DNAAbschnitte in ihrer Dichte an Inseln mit erhöhter DNA-Helix-Flexibilität nicht unterschieden. Nahezu alle Regionen der häufigen molekular charakterisierten CFSs umfassen große Gene, deren Exons mehr als 650 kb genomische Sequenz überspannen. Im Gegensatz dazu traten große Gene in den stabilen Kontrollregionen deutlich seltener auf. Öffentlich zugängliche RNA-Expressionsdaten dieser Gene zeigten, daß CFSs bevorzugt in Bereichen mit transkriptionell aktiven, großen Genen entstehen. Darauf und auf dem Wissen aus der Literatur begründen wir die Hypothese, daß eine Blockierung der DNAReplikationsgabel, vor allem in Bereichen großer transkriptionell aktiver Gene - eventuell begründet durch deren offenere Chromatinstruktur - und das anschließende Unterlaufen von Zellzyklus-Checkpoints an der Bildung von CFSs und damit in einem Anstieg von Doppelstrangbrüchen beteiligt sind.

Candida albicans est une levure pathogène opportuniste de l’homme. La capacité de C. albicans à changer de forme en réponse à des stimuli externes, passant d’une croissance bourgeonnante à filamenteuse, est associée à sa virulence. Cette morphogenèse requiert une réorganisation du cytosquelette d’actine et un trafic membranaire ciblé. Chez Saccharomyces cerevisiae, les petites protéines G de type Arf jouent un rôle important dans le trafic membranaire et la polarité cellulaire. Le rôle de ces protéines chez C. albicans est largement méconnu. C. albicans a 3 protéines Arf, Arf1-Arf3 et 2 Arf-like, Arl1 et Arl3. Nos résultats indiquent que seule Arf2 est nécessaire à la viabilité et à la résistance aux antifongiques, et qu’Arf2 et Arl1 sont critiques pour la croissance filamenteuse hyphale; le mutant arl1/arl1 en particulier forme des hyphes 2 fois plus courtes que la souche sauvage. Les mutants Δ/pTetARF2 et arl1/arl1 ont un défaut de virulence dramatique et ARL1 est particulièrement critique pour la candidose oropharyngée. Nos résultats indiquent que les défauts du mutant Δ/pTetARF2 seraient dus à une altération du Golgi, et ceux d’arl1/arl1 de l’incapacité de ce mutant à restreindre sa croissance à un site unique. Ce défaut de croissance polarisée du mutant arl1/arl1 n’est pas lié à la mislocalisation de son effecteur Imh1, ni à une misrégulation de la phosphatidylsérine flippase Drs2. Par contre, nos données suggèrent que le défaut de croissance hyphale de ce mutant résulterait d’une hypersécrétion. Cette étude nous a permis d’identifier Arf2 et Arl1 comme protéines clés du trafic membranaire, critiques pour la croissance filamenteuse et la virulence de C. albicans. / The human fungal pathogen Candida albicans switches from budding to filamentous growth. This dramatic morphogenesis is critical for its virulence and requires sustained polarized growth, via exocytosis and endocytosis, as well as reorganization of intracellular compartments. In the yeast Saccharomyces cerevisiae, Arf G-proteins and their regulators function at the interface of membrane traffic and cell polarity. The roles of this class of proteins during the transition to filamentous growth and virulence in C. albicans are largely unknown. In C. albicans there are 3 Arf proteins, Arf1-Arf3 and 2 Arf-like proteins, Arl1 and Arl3. Our results reveal that only Arf2 is required for viability and sensitivity to antifungal drugs and that both Arf2 and Arl1 are required for hyphal growth, with arl1/arl1 hyphal filaments being 2-fold shorter than that of the wild-type strain. Furthermore, both Δ/pTetARF2 and arl1/arl1 mutants have drastically reduced virulence, with ARL1 particularly critical for oropharyngeal candidiasis. We show that the defects in Δ/pTetARF2 is due to an alteration of Golgi integrity, while the defects in the arl1 mutant are likely to result from the inability of this mutant to restrict growth to a single site. Further analyses of the arl1/arl1 mutant revealed that this defect does not result from a misregulation of the GRIP-domain golgin coiled-coil tethering protein Imh1 nor of the phosphatidylserine flippase Drs2. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion. Together our work identifies Arf2 and Arl1 as key regulators of membrane traffic, critical for hyphal growth and virulence.

Biological tubes possessing a curvilinear form and a hollow interior exist in most multicellular eukaryotes. In Eumetazoa, the tubes usually comprise an eminently complex network and enable the transport and exchange of fluids and gases between tissues and organs, but also between organisms and their environment. Thus, tubular structures are both morphologically and physiologically integral parts of the animals. Based on a genetic screen for novel factors involved in heart tube differentiation and morphogenesis in Drosophila, the identified mutants were subdivided into several classes: cardiac hyperplasia (kuz and mam, both involved in the Notch-dependent cardiomyocyte specification, Publication 1); impaired cytokinesis (pav and tum, both components of the centralspindlin complex); a single ptc mutant showing a “truncated” heart (Publication 2); and a single loss-of-function mutant displaying reduced lumen diameter in epithelial tubes and perturbed secretion of ECM-components. The latter allele was mapped to the gene locus gartenzwerg (garz) that encodes a large ArfGEF. Due to its novelty, garz was selected as a central part of the thesis (Publication 3). Although garz seems to be expressed ubiquitously, its transcripts are abundant in active secreting cells of tubular structures. Moreover, mutations of garz abolish Golgi-integrity, cause massive retention of secretory cargo in the ER and arrest the apical transport of lipids and ECM molecules. As a consequence, lumen of the salivary glands and trachea fail to expand and show a decreased diameter. The observed phenotypes in tracheal network and salivary glands phenocopy those of COPI/COPII-subunits as well as actin-dependent secretion mutants, suggesting the underlying mechanism might be common. Thus, it is supposed that proper tubulogenesis needs Garz for initiation of the Arf1-COPI machinery. Furthermore, Golgi-based post-translational modifications, targeted sorting of vesicles, outward transport of proteins, or directed membrane delivery all depend on the secretory pathway, and such processes are essential in establishing polarized cells which build the tubular structures. In conclusion, this mechanism seems to be neither restricted to tubulogenesis nor specific to Drosophila. Due to the presence of garz homologues in every eukaryotic genomes, the Arf1/COPI based secretory pathway may play a universal role in metazoan development.

MDM2 and MDMX are two major negative regulators of tumor suppressor p53. Both MDM2 and MDMX can inactivate p53 and play important roles in mouse embryonic development in a p53 dependent manner. MDM2 possesses ubiquitin E3 ligase activity and mediates self-ubiquitination as well as ubiquitination and degradation of p53 by proteasome. We identify MDMX as another ubiquitin E3 ligase substrate of MDM2. MDM2 promotes the ubiquitination and degradation of MDMX through proteasome pathway. The RING domains of both MDMX and MDM2 are required and sufficient for MDM2-mediated MDMX ubiquitination. ARF overexpression, DNA damage or MDM2 overexpression can all stimulate MDMX ubiquitination and degradation. We present evidence that MDMX is also sumoylated. The sumoylation sites on MDMX are identified. ARF N-terminus is required for stimulating both MDMX ubiquitination and sumoylation. We also demonstrate that MDMX binds to ARF in an MDM2-dependent fashion.

MDM2 regulates p53 predominantly by promoting p53 ubiquitination. However, ubiquitination-independent mechanisms of MDM2 have also been implicated. Here we show that MDM2 inhibits p53 DNA binding activity in vitro and in vivo. MDM2 binding promotes p53 to adopt a mutant-like conformation, losing reactivity to antibody Pab1620, while exposing the Pab240 epitope. The acidic domain of MDM2 is required to induce p53 conformational change and inhibit p53 DNA binding. ARF binding to the MDM2 acidic domain restores p53 wild type conformation and rescues DNA binding activity. Furthermore, histone methyl transferase SUV39H1 binding to the MDM2 acidic domain also restores p53 wild type conformation and allows p53-MDM2-SUV39H1 complex to bind DNA. These results provide further evidence for an ubiquitination-independent mechanism of p53 regulation by MDM2, and reveal how MDM2-interacting repressors gain access to p53 target promoters and repress transcription. Furthermore, we show that the MDM2 inhibitor Nutlin cooperates with the proteasome inhibitor Bortezomib by stimulating p53 DNA binding and transcriptional activity, providing a rationale for combination therapy using proteasome and MDM2 inhibitors.

No description available.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.

Retinoblastoma development is a multistep process, and inactivation of the RB1 gene is not sufficient for tumorigenesis. Previous studies suggest that the p53-tumor suppressor is inactivated due to overexpression of p53-antagonists MDM4 and MDM2. This thesis evaluates the importance of ARF, a p53-activator that inhibits MDM2. In retinoblastomas, ARF protein is nearly undetectable despite robust mRNA expression. Chemical inhibition of the proteasome, which regulates ARF protein-turnover, did not result in ARF accumulation in retinoblastoma cells, indicating that ARF protein was not aberrantly degraded by the proteasome. During mouse retinoblastoma development, Arf protein was expressed at low level, and p53-target genes involved in cell cycle arrest and autoregulation were not activated. Overexpression of ARF in retinoblastoma cells led to growth inhibition, accompanied by increased expression of p53 and p53-transcriptional targets. Taken together, our data suggests that low ARF protein is an important factor in silencing of the p53-pathway during retinoblastoma development.