Le virus de la maladie de Marek (GaHV-2), est un "-herpèsvirus induisant des lymphomes T chez le poulet. L’étude des pri-miARN des régions RL à partir des miR-M4, -M11, M31 et -M1 a montré des initiations de transcription dispersées et internes aux longs pri-miARN initialement décrits. Les tests de fonctionnalité des régions en amont ont permis de conclure quant à l’absence d’activité promotrice suggérant une régulation par le prmiR-M9M4, caractérisé dans cette étude, ou par le prMeq. Le gène de 7,5 kpb d’un ARNlnc (ERL, Edited Repeat Long) épissé et antisens à ces transcrits a été caractérisé. Une hyper-édition A-en-G des transcrits ERL par ADAR1 a été mis en évidence. Principalement lié au cycle lytique, l’édition indique une répression fonctionnelle durant cette phase. Les régulations de l’expression d’ADAR1 chez l’humain, positives par la voie de réponse aux interférons et négatives par SOCS1, ont été validées chez le poulet. L’inhibition de SOCS1 par le gga-miR-155 et son orthologue viral mdv1-miR-M4-5p a été montré fonctionnelle chez le poulet comme chez l’humain et mène à une surexpression d’ADAR1 lors de l’activation des voies de réponse aux interférons. / Marek’s disease virus (GaHV-2) is an "-herpesvirus that induces T-cell lymphoma in chickens. In this study, we have shown that some pri-miRNAs, which are specific of miR-M4, -M11, M31 et -M1, initiated at dispersed transcription start sites. They are located in internal positions from previously described pri-miRNAs but upstream sequences lack promoter activity, indicated a potential regulation by the upstream prmiR-M9M4, characterised during this study, or the prMeq. The 7.5 kbp gene of the ERL (Edited Repeat Long) lncRNA, which is alternatively spliced et antisense of these pri-miRNAs was defined. An extensive A-to-G hyperediting of it sequence was observed strongly linked to the lytic phase, indicated a functional repression during this phase. We showed that, like the human one, the chicken ADAR1 expression was positively controlled by the IFN response pathway et negatively by the suppressor of cytokine signaling 1 (SOCS1). Like the human et murine miR-155-5p, the chicken gga-miR-155-5p et the GaHV-2 analog mdv1-miR-M4-5p deregulate this pathway by targeting et repressing expression of SOCS1, leading to the upregulation of ADAR1.

RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.

Les eIF2B-pathies représentent un groupe de leucodystrophies de transmission autosomique récessive du à des mutations du facteur ubiquitaire eIF2B. Celui-ci intervient dans l’initiation de la traduction et ses régulations, particulièrement en cas de stress cellulaires, grâce à son activité d’échange de guanine (GEF). Un large spectre clinique et mutationnel a été décrit pour cette pathologie.La diminution de l’activité GEF a pu être validée comme marqueur diagnostique spécifique des eIF2B-pathies dans les lymphoblastes de patients atteints avec un seuil d’activité à 77,5% pour une spécificité de 100% et une sensibilité de 89%.La compréhension des mécanismes moléculaires en cause a ensuite été recherchée selon trois approches :- une première focalisée sur l’étude de la réponse au stress du réticulum endoplasmique (RE) dans les lymphoblastes de patients eIF2B-mutés. L’hyper-activation transcriptionnelle et traductionnelle des gènes de la réponse au stress du RE, observée dans d’autres études et sur d’autres types cellulaires n’a pas été retrouvée dans cette étude.- une approche globale d’étude transcriptomique différentielle dans des fibroblastes primaires de patients eIF2B-mutés soumis ou non à un stress cellulaire. La comparaison du transcriptome avec celui de contrôles sains et de patients porteurs d’une autre leucodystrophie n’a pas permis de mettre en évidence un effet spécifique du stress dans les fibroblastes eIF2B-mutés. En revanche, il a pu être montré une dérégulation de l’expression de 70 gènes spécifiquement dans ces fibroblastes ainsi que l’implication de voies métaboliques telles que l’épissage et la stabilité des ARNm, importantes au cours du développement du système nerveux central. Ces gènes trouvés dérégulés dans les fibroblastes, appartenant notamment à la famille des hnRNP, ont été ensuite validés dans les cerveaux de patients eIF2B-mutés et une anomalie d’épissage de certains transcrits importants pour les cellules gliales a également été identifiée.- enfin, pour valider l’hypothèse d’une anomalie développementale des cellules gliales, le modèle des cellules souches embryonnaires (ESC) a été utilisé et un défaut génétique a été introduit dans ces cellules afin de mimer les mutations eIF2B. Une anomalie de différentiation de ces ESC en cellules gliales a pu être mise en évidence dans ce modèle qui pourrait alors constituer un outil de choix pour tester des molécules pouvant potentiellement améliorer la différenciation de ces cellules, principales en cause dans cette pathologie. / EIF2B-related disorders are an autosomal recessive leukodystrophy caused by mutations in the ubiquitary eIF2B factor. This one is involved in the translation initiation step and its regulation, particularly upon cellular stresses, thanks to its guanine nucleotide exchange factor (GEF) activity. A wide continuum clinical and mutational spectrum has been described for this pathology.The decrease of eIF2B GEF activity has been validated as an eIF2B-pathies specific biomarker in affected patients’ lymphoblasts with 100% specificity and 89% sensibility using a threshold at 77.5%.Functional molecular mechanisms involved in the physiopathology of eIF2B-related disorders have been searched by three approaches:- the first one focalized on the study of the endoplasmic reticulum stress response in lymphoblasts from eIF2B-mutated patients. The translational hyper-induction of specific genes involved in the unfolded protein response, identified in other cell types, was not observed in this study.- a global approach using a differential transcriptomic study of primary fibroblasts from eIF2B-mutated patients submitted or not to a cellular stress. The comparison with the transcriptomic profile of fibroblasts from healthy controls and patients presenting with other types of leukodystrophies not allowed us to identify a specific stress effect in eIF2B-mutated fibroblasts. On the other hand, it has been shown 70 genes specifically differentially deregulated in eIF2B-mutated fibroblasts as well as metabolic pathways implication, like splicing and mRNA stability, that are critical during the central nervous system development. We then validated that these genes, belonging the the hnRNP family, were also deregulated in brains from eIF2B-mutated patients and a splice abnormality of genes implicated in glial cells network has also been identified.- finally, in order to validate the hypothesis of an abnormal glial cell development, the embryonic stem cells (ESC) model has been used and a genetic default has been introduced in these cells to mimic eIF2B mutations. We identified an abnormal differentiation of these ESC into glial cells. Therefore, this model would provide a unique tool to search therapeutic agents that would improve glial cell differentiation, the major cells implicated in this pathology.

Après la transcription, le pré ARNm subit des étapes de maturation avant de sortir du noyau pour être traduit. Une des étapes de maturation est l'épissage. Il permet de souder les séquences codantes de l'ARNm entre elles (les exons) et d'exclure les régions non codantes (les introns).Des mutations génétiques sont à l'origine de défaut d'épissage. Elles peuvent conduire à des rétentions d'intron, des sauts d'exon et des inclusions de séquences introniques appelées pseudo exons.Ma thèse a porté sur l'utilisation du saut d'exon pour corriger ces inclusions. Je me suis intéressé à deux pathologies : la myopathie à cores et le syndrome de LowePour le premier cas, je me suis intéressé à une mutation dans l'intron 101 du gène RyR1. Cette mutation est à l'origine de la création d'un site donneur d'épissage qui active un site accepteur provoquant l'inclusion d'un pseudo exon de 99 nucléotides. Cette inclusion induit une baisse de la quantité du canal calcique RyR1 dans les cellules du patient. Ce canal permet le couplage entre l'excitation et la contraction musculaire. Ses défauts conduisent à diverses myopathies dont la myopathie à cores. Le patient présentait une hypotonie néonatale, une scoliose et des défauts respiratoires, et n'a jamais acquis la marche. J'ai dessiné des oligonucléotides, je les ai transfectés dans les cellules du patient en culture et ainsi montré par RT PCR que le saut du pseudo exon était possible. Afin d'optimiser l'efficacité pour pouvoir évaluer la restauration au niveau protéique et fonctionnel, j'ai développé un lentivirus exprimant une cassette U7 SmOPT avec les AON choisis. Après transduction des cellules, j'ai pu montrer que le saut du pseudo exon permettait le retour de la protéine et de sa fonctionnalité, cette approche pourrait donc permettre une correction chez le patient.Pour le deuxième cas, j'ai tenté de corriger une mutation du gène OCRL. Cette mutation crée un site donneur d'épissage dans l'intron 4 du gène OCRL et active un site accepteur d'épissage 66 nucléotides en amont. L'inclusion du pseudo exon induit la chute du taux de transcrit OCRL par un mécanisme de "Non sense mediated mRNA Decay". OCRL est une phosphatidyl inositol 5 Phosphatase permettant de réguler la quantité de Ptd Ins(4,5)P2 dans la cellule. Les défauts dans OCRL sont responsables d'une pathologie multisystémique, le syndrome de Lowe. J'ai pu obtenir des fibroblastes cutanés du patient. J'ai transfecté ces cellules avec des AONs choisis pour permettre un saut de l'exon pathogène. J'ai ensuite intégré la séquence des AONs efficaces dans un lentivirus U7. J'ai transduit les cellules du patient en culture et observé un retour de la protéine et un retour de l'activité enzymatique, cette approche pourrait donc théoriquement permettre une correction chez le patient.Ces deux travaux sont les premières preuves de principes de thérapie par modulation de l'épissage pour les myopathies congénitales et pour le syndrome de Lowe. Ils ouvrent la voie à des perspectives de traitement pour ces maladies génétiques. / After transcription, the pre mRNA will undergo different maturation step before getting out of the nucleus for translation. One of these step of maturation is the splicing. It allows to concatenate the coding sequences of the mRNA (the exons) and induces the exclusion of the non coding sequences (the introns).Genetics mutations can lead to splicing defects. These defects could be intron retention, exon skipping and exonisation of intronic sequences called pseudo exons.My thesis work was to evaluate the exon skipping therapy to correct these exonisation. I focuses on two diseases: core myopathy and Lowe syndrome.For the first one, my interest was on a mutation in the 101 th intron of RYR1 gene. This mutation creates a splicing donor site wich unveils a cryptic acceptor site. This leads to the inclusion of a 99 nucleotides pseudo exon. This inclusion induces a decrease of the quantity of the calcium channel RyR1 in the patient cells. This channel allows the excitation-contraction coupling, and therefore the muscular contraction. Defects in this channel lead to different myopathies (eg. core myopathy). The patient present at birth a major neonatal hypotonia, scoliosis and respiratory defects. He has never walked. I designed oligonucleotides (AON), transfected them in the cultured patient cells and showed by RT PCR that exon skipping was possible. In order to optimise the efficiency and to evaluate the rescue at a protein level and at a fonctionnal level, I devellopped a lentivirus which express a U7 Sm OPT cassette with the choosen AONs. After cell transduction, I have shown that exon skipping allowed the rescue of the protein and of its functionnality. This approach could permit a genetic correction for the patientFor the second case, I have tried to correct an OCRL mutation. This upstream creates a splicing donor site and unveils an acceptor site 66 nucleotides before. This leads to the inclusion of a pseudo exon which induces a severe decrease of OCRL transcripts level due to a "non sense mediated mRNA Decay". OCRL is a phosphatidyl inositol 5 Phosphatase, which regulates the Ptd Ins(4,5)P2 pool in the cell. OCRL defects induces a multi systemic disease the Lowe syndrome. I obtained patient cutaneous fibroblasts. I transfected these cells with choosen AONs to correct the splicing defect. I integrated the AONs sequence into a U7 lentiviral cassette. I transduced the cultured patient cells and observed a rescue of the protein with a rescue of its activity. This approach could, theoritically permit a correction in the patient.These two studies are the first proof of concept of splicing modulation therapy for congenital myopathy and for Lowe syndrome. This work offers a lot of perspective for the tratment of these genetic illness

BACKGROUND: The inactivation of the tumor suppressor gene BRCA1 is a predisposing factor for a breast/ovarian cancer development. Formation of cancer-specific alternative splicing variants with aberrant biological properties can represent additional mechanism decreasing the overall BRCA1 activity in DNA double strand break (DDSB) repair. In this study, we analyzed BRCA1 alternative splicing variants BRCA114-15 and 17-19 ascertained previously during the screening of high-risk breast cancer individuals. METHODS: We established a stable MCF-7 cell line-based model system for an in vitro analysis of BRCA1 variants. Using this system, we analyzed the impact of BRCA114-15 and 17-19 variants on DNA repair kinetics using comet assay and confocal immunomicroscopy. The capacity of DNA repair was assessed directly by an in vitro NHEJ assay and indirectly by a mitomycin C sensitivity test. The proliferation activities were determined by a clonogenic assay and growth curves. RESULTS: Overexpression of BRCA114-15 and 17-19 increases the endogenous level of DNA damage, slows down the DDSB repair, and decelerates the initial phase of radiation-induced foci formation and prolongs their persistence. Moreover, BRCA114-15 and 17-19 differentially influence the activity of HR and NHEJ and sensitivity of MCF-7 cells to ionizing...

L’homéostasie cellulaire est intimement liée au trafic membranaire, processus dynamique qui permet les échanges de lipides et de protéines entre les compartiments cellulaires mais aussi entre la cellule et le milieu extracellulaire. L’intersectin-1 (ITSN1) est une protéine d’échafaudage multifonctionnelle, impliquée dans les processus d’endocytose, d’exocytose, diverses voies de signalisation ainsi que dans la survie cellulaire. L’ensemble de mes travaux de doctorat a permis d’identifier deux nouveaux partenaires de l’ITSN1, RhoU et l’OPHN1, et de montrer leur implication dans le trafic membranaire. De plus je démontre que les variants d’épissage de l’ITSN1 pourraient avoir une spécificité d’interactiondifférente vis-à-vis de ses partenaires. Nous montrons aussi que l’ITSN1 est capable de former des complexes entre ses différentes isoformes. Ainsi, l'ensemble de ces données apportent de nouvelles connaissances sur l’interactôme d’ITSN1. / The cellular homeostasis is tightly linked to the membrane trafficking, a dynamic process which allows lipid and protein exchange between the cellular compartments as well as the cell and the environment. Intersectin1 (ITSN1) is a multifunctional scaffold protein implicated in the processes of endocytosis and exocytosis, different signaling pathways and cell survival. In present study I have identified two new partners of ITSN1, RhoU and OPHN1, and demonstrated their implication in membrane trafficking. Surprisingly, I have also found that the alternative splicing of ITSN1-L can lead to the change of the specificity of its interaction with binding partners. In addition, I have shown that different ITSN1 isoforms are capable to form complexes with each other. All together these data add new knowledge to ITSN1 interactome.

A programação epigenética do genoma por metilação do DNA, modificação das histonas e remodelamento da cromatina é crucial para o desenvolvimento e o crescimento normal dos mamíferos e alterações nesses mecanismos contribuem diretamente para a transformação maligna. Em função do papel relevante da metilação do DNA na carcinogênese, da importância e da necessidade de identificação de marcadores moleculares em tumores de cabeça e pescoço e dos resultados já obtidos pelo grupo, o presente trabalho teve por objetivo geral investigar o perfil de metilação de ilhas CpG em carcinomas primários de cabeça e pescoço (CECP), bem como identificar e iniciar estudos funcionais de biomarcadores candidatos para diagnóstico e prognóstico desses tumores. Alguns genes previamente descritos com padrões anormais de metilação em neoplasias humanas (CDH1, CDH13, DAPK, CDKN2A, RASSF1A, SOCS3 e TIMP3), além de outros dois genes (MX1 e SLC15A3) identificados em nosso estudo anterior, foram analisados em amostras normais e margens cirúrgicas de CECP por meio da técnica de pirosequencimento de DNA após tratamento com bissulfito de sódio. As análises estatísticas não mostraram diferenças significativas para a maioria dos genes analisados, com exceção do gene SLC15A3, que apresentou diferença significativa (p<0,05) entre tumores e amostras de sangue de doadores saudáveis. Diferentemente, os resultados obtidos com a metodologia de PCR-MSP em nosso trabalho anterior mostraram que o gene MX1 está metilado em CECP. Os estudos funcionais do MX1, por RNA de interferência, ensaios de migração e citometria de fluxo mostraram que seu produto contribui para migração e proliferação celular, e talvez para resistência à apoptose. Os resultados sugeriram que o nível de expressão do MX1 pode ser um preditor do potencial metastático em carcinoma epidermóide / The genome epigenetic programming by DNA methylation, histone modification and chromatin remodeling is crucial for normal growth and development in mammals and changes in these mechanisms contributes directly to malignant transformation. Regarding the role of DNA methylation in carcinogenesis, the importance and necessity for the identification of molecular markers in head and neck tumors, and the results already obtained by the group, this study was aimed at investigating the methylation profile of CpG islands in primary head and neck squamous cell carcinomas (HNSCC), and to identify and initiate functional studies of candidate biomarkers for diagnosis and prognosis of these tumors. Therefore, some genes previously described with abnormal methylation pattern in humans tumors (CDH1, CDH13, DAPK, CDKN2A, RASSF1A, and TIMP3 SOCS3), and two other genes (MX1 and SLC15A3) identified in our previous study were analyzed in normal samples, surgical margins, and in HNSCC by pyrosequencing after sodium bisulfite treatment. The statistical analysis showed no significant differences for most of analyzed genes, except for the SLC15A3, which showed significant difference (p <0.05) between tumors and blood samples from healthy donors. However, our earlier results showed a higher frequency of MX1 hypermethylation in primary HNSCC using the PCR-MSP methodology. To gain a better understanding of the role MX1 in cancer biology we investigated whether a downregulation of MX1 by interference RNA contribute to apoptosis resistance and cell migration during cancer development. Wound healing and flow cytometry assays were performed to determine changes in cell motility, death and cell cycle in SCC cells. The results indicated that low levels of MX1 could regulate the cell cycle, increase proliferation, and enhance tumor cell migration in HNSCC cell lines, but it might not contribute to apoptosis resistance. It also suggests that the level of MX1 expression may be a predictor of metastatic potential in HNSCC

Orientador: Jorg Kobarg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto e Biologia / Made available in DSpace on 2018-08-14T00:02:08Z (GMT). No. of bitstreams: 1 Bressan_GustavoCosta_D.pdf: 16510013 bytes, checksum: 30f3887b16b18caf89b81d091caca8d7 (MD5) Previous issue date: 2009 / Resumo: A proteína Ki-1/57 foi descoberta através da reação cruzada do anticorpo monoclonal Ki-1 em células do linfoma de Hodgkin. Foi demonstrado previamente que Ki-1/57 sofre fosforilação por PKCs e metilação por PRMT1, uma arginino metiltransferase que modula diversas proteínas ligadoras a RNA. Nesse trabalho, é mostrada a interação de Ki-1/57 com sondas de RNA e com proteínas envolvidas no controle de splicing de pré-mRNA. O seu envolvimento no controle de splicing foi confirmado em ensaios de cotransfecção em células de mamíferos. Análises de microscopia de confocal mostraram a localização da construção EGFP-Ki-1/57 em diferentes corpúsculos nucleares de forma dependente da metilação celular. Essas regiões compreendem nucléolos, speckles, corpos de Cajal e GEMS, conhecidamente envolvidas na biogênese, maturação ou armazenamento de complexos de processamento de RNA/pré-RNA no núcleo. Análises a partir de construções truncadas sugeriram o N-terminal de Ki-1/57 como importante para a interação com proteínas reguladoras de splicing e localização nos corpúsculos nucleares, enquanto o C-terminal como necessário e suficiente para a ligação a RNA poliuridina e localização citoplasmática. Por outro lado, essas duas regiões pareceram atuar em conjunto no processamento do gene E1A. Similarmente a hnRNPQ, Ki-1/57 e outras proteínas funcionalmente relacionadas, SFRS9 é mostrada como alvo de metilação por PRMT1. A inibição da metilação resultou em um aumento do número de células apresentando localização da construção EGFP-SFRS9 no interior de nucléolos, mostrando a importância dessa modificação para a localização subnuclear de SFRS9. As características estruturais de Ki-1/57 também foram investigadas através de diferentes abordagens. Análises por SAXS, gel filtração analítica e ultracentrifugação analítica indicaram uma estrutura bastante alongada e flexível para a construção C-terminal 6xhis-(122-413)Ki-1/57. Ensaios de proteólise limitada também sugeriram uma baixa composição de núcleos hidrofóbicos estáveis e compactos. A capacidade de Ki-1/57 em sofrer enovelamento induzido após a interação com ligantes também foi monitorada em experimentos de dicroísmo circular. Embora não tenha sido observada nenhuma alteração estrutural após a incubação de 6xhis-(122-413)Ki-1/57 com o RNA poliuridina, a adição de TFE foi capaz de promover pequenos ganhos de elementos de estrutura secundária regular. Esses dados, juntamente com predições computacionais, sugerem que Ki-1/57 é uma nova proteína intrinsecamente desordenada, o que pode explicar o elevado número de diferentes proteínas parceiras que ela é capaz de interagir. / Abstract: The Ki-1/57 protein has been discovered through the cross reactivity of the monoclonal antibody Ki-1 in Hodgkin lymphoma cells. Previously, it was demonstrated that Ki-1/57 undergoes phosphorylation by PKCs and methylation by PRMT1, an arginine methyltransferase that modulates many RNA binding proteins. Here, the interaction of Ki-1/57 with RNA polyuridine and proteins involved in pre-mRNA splicing control are shown. Its involvement in splicing regulation was confirmed by cotransfection assays in mammalian cells. Confocal microscopy analyses revealed the localization of EGFP-Ki-1/57 at different nuclear bodies, depending on the cellular methylation status. These regions include nucleoli, speckles, Cajal bodies and GEMS, which are all known to be involved in biogenesis, maturation or storing of RNA/pre-mRNA processing complexes in the nucleus. Analysis from experiments with truncated forms of Ki-1/57 suggested its N-terminus as important for its interaction with splicing proteins and localization at nuclear bodies. In turn, its C-terminus was seen as necessary and sufficient for the cytoplasmic localization and polyuridine RNA binding. However, these two regions seemed to be required working together for an efficient splicing activity on E1A gene. Similarly to hnRNPQ, Ki-1/57 and others functionally related proteins, SFRS9 is shown here as a target for methylation by PRMT1. The inhibition of this activity resulted in increase in the number of cells showing EGFP-SFRS9 in the nucleoli, suggesting the importance of methylation for the subnuclear localization of SFRS9. The structural characteristics of Ki-1/57 also have been investigated through different approaches. Analyses by SAXS, analytical gel filtration and analytical ultracentrifugation techniques suggested a very elongated and flexible structure for the C-terminal construct (122-413)Ki-1/57. Also, limited proteolysis analysis suggested a low composition of stable and compact hydrophobic cores. The ability of Ki-1/57 in suffering binding-induced folding was also investigated. Although no structural modification has been observed after incubating (122-413)Ki-1/57 with a polyuridine RNA, the addition of the TFE probe was able to promote a small gain of regular secondary structural elements. These findings, together with different computational predictions, pointed out that Ki-1/57 is a novel intrinsically unstructured protein. This could explain the wide array of protein partners with which it is able to interact. / Doutorado / Bioquimica / Doutor em Biologia Funcional e Molecular

Em virtude dos pobres resultados obtidos no tratamento do Câncer de Pulmão, seja em estágios iniciais ou na doença avançada localmente, há a necessidade de se desenvolver marcadores moleculares e imunohistoquímicos que possam prever o comportamento tumoral. Ácido Hialurônico (HA) é um componente da matriz extracelular, responsável pela hidratação e manutenção do equilíbrio osmótico tecidual. Concentrações de HA estão elevadas em vários tipos de cânceres, incluindo pulmão. Hialuronidases (HAases), são uma família de enzimas relacionadas com a propagação de infecções bacterianas, toxinas de venenos e progressão tumoral. A quebra do HA em pequenos fragmentos (3-25 dissacarídeos) promovidos pela ação das HAases tipo Hyal1, Hyal2 e Hyal3, está relacionada à promoção do câncer através da indução da angiogênese e estímulo a proliferação através de ativação da via tirosina quinase. Algumas isoformas de HAases, descritas como produto de splicing alternativo, possuem atividade enzimática diversificada. A heterogeneidade de expressão das HAases foi identificada em alguns tipos de câncer e pode ser correlacionada com o comportamento diferenciado dos tumores. Em uma primeira instância, o perfil de expressão das HYAL foi avaliado em tecidos pulmonares tumorais e normais de 69 tumores ressecados de pacientes com adenocarcinomas (ADC) e carcinomas de células escamosas (CCE) oriundos do Hospital das Clinicas e Hospital do Câncer AC. Camargo. A expressão da HYAL1- selvagem (wt) e variantes 1 a 5, HYAL2-wt, HYAL3-wt e variantes 1 a 3 foi identificada por PCR e seqüenciamento direto. Diferentes proporções de HYAL3-wt e variantes foram expressas em tecidos pulmonares tumorais e controles. HYAL1-wt esteve associada com prognóstico desfavorável e HYAL3-v1 com prognóstico favorável. Diante dos resultados obtidos dos tumores de pacientes do Hospital das Clínicas e Hospital AC. Camargo, prosseguimos a investigação para estudar a imunoexpressão das Hyal 1 e 3 e HAS 1, 2 e 3 nos CCE e ADC. Observamos que a intensidade de expressão de Hyal 3 foi maior pelas células tumorais quando comparada aos controles, porém esta diferença foi marginalmente significante. Já o resultado da análise da freqüência de imunoexpressão das Hyal 1 e 3, e HAS1, 2 e 3 demonstrou expressão na maioria dos espécimes tumorais e controles. A associação entre as variáveis foi testada e evidenciou imunoexpressão concomitante de HYAL e HAS nos tumores. O modelo matemático de sobrevida , controlado para sexo, idade e estadiamento mostrou risco de morte associado com adenocarcinoma sólido e imunoreatividade para HAS2 e HAS3. Para validar os resultados obtidos, sobretudo com a imunoexpressão das Hyal e HAS nos CCE e ADC, estudamos a população de pacientes do Hospital Universitário de Coimbra. Documentamos pela primeira vez uma via pela qual a hiperexpressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, podem favorecer a invasão nos ADC e CCE. Surpreendentemente, demonstramos que a imunoexpressão de HAS1 e 3 pelas células epiteliais neoplásicas confere mais agressividade aos ADC acinares e papilares, mas uma expressão negativa de HAS1 pelas células mesenquimais confere um papel protetor a MEC auxiliando-a a evitar a invasão pelas células tumorais em ambos os tipos subtipos histológicos. A interação entre a expressão das hialuronidades e sintases do àcido hialurônico foi avaliada em relação à expressão de proteínas da transição epitélio-mesênquimal nos tumores de pacientes do Hospital Universitário de Coimbra. Hyal, HAS, E-caderina e TGF- modularam uma via invasiva tumorinduzida nos ADC e CCE de pulmão, e estiveram associados a um espectro diferente de agressividade, uma vez que houve uma relação inversa entre a expressão de biomarcadores epiteliais e mesenquimais. Enquanto a hiperexpressão de HAS1 e HAS3 provê uma agressividade aos CCE e ADC, uma hiperexpressão de TGF- e E-caderina, confere um efeito protetor à MEC ao evitar a invasão por células tumorais em ambos os tipos histológicos. Comparamos os níveis de imunoexpressão das Hyal1 e 3 e HAS 1, 2 e 3 nos tumores ressecados no Hospital das Clínicas e Hospital AC. Camargo com os níveis obtidos em tumores do Hospital Universitário de Coimbra. Verificamos que a imunoexpressão das HAS 1, 2, 3 e Hyal1 foi significativamente maior nos tumores de pacientes do Hospital Universitário de Coimbra, enquanto que a imunoexpressão de Hyal 3 foi significativamente maior nos tumores de pacientes brasileiros. Por todas essas razões, nossos resultados sugerem que estratégias direcionadas à modulação dos níveis de HYAL1-wt e HYAL3-v1, da hiper imuno expressão de HAS3 e Hyal 3 respectivamente por células epiteliais neoplásicas e mesenquimais, da alta síntese de HAS3 e Hyal 1, ou a resposta local baixa de TGF- e E-caderina, poderão ter grande impacto no câncer de pulmão / Given the poor results obtained in the treatment of Lung Cancer, in early stages or locally advanced disease, there is a need to develop molecular markers and immunohistochemical studies that can predict tumor behavior. Hyaluronic Acid (HA) is a component of extracellular matrix is responsible for hydration and maintenance of tissue osmotic equilibrium. Concentrations of HA are elevated in several types of cancers, including lung. Hyaluronidases (HAases) are a family of enzymes involved in the spread of bacterial toxins, poisons and tumor progression. The breakdown of HA into small fragments (3-25 disaccharides) promoted by the action of type HAases Hyal1, Hyal 2 and Hyal 3 is related to the promotion of cancer by inducing angiogenesis and stimulate proliferation through activation of the tyrosine kinase. Some isoforms HAases, described as the product of alternative splicing, have diverse enzymatic activity. The heterogeneity of expression of HAases was identified in some cancers and can be correlated with the different behavior of tumors. In a first instance, the expression profile of Hyal spliced forms was evaluated in tumor and normal lung tissue of 69 tumors resected from patients with adenocarcinomas(ADC) and squamous cell carcinomas (SqCC) from the Hospital das Clínicas and Hospital AC. Camargo. Gene expression of HYAL1 wild-type (wt) and variants 1 to 5 HYAL2-wt, and HYAL3-wt and variants 1 to 3 was identified by PCR and direct sequencing. Different proportions of HYAL3-wt and variants were expressed in tumor and normal lung tissue. HYAL1-wt was associated with unfavorable prognosis and HYAL3-v1 with favorable prognosis. Given the genetic abnormalities found in tumors of patients from Hospital das Clinicas and Hospital AC. Camargo, we continued our research to study the expression of Hyal 1.3 and HAS 1, 2, 3 in squamous cell carcinomas and adenocarcinomas. We observed that the intensity of expression of Hyal 3 was higher in tumor cells compared to controls, but this difference was marginally significant. Since the result of frequency analysis of immunoreactivity of Hyal 1 and 3, and HAS1, 2 e 3 showed expression in the majority of tumor samples and controls. The association between variables was tested and showed concomitant immunoexpression of the HAS and HYAL in tumors. The mathematical model of survival, adjusted for sex, age and staging showed risk of death associated with adenocarcinoma and solid and HAS3 HAS2 immunoreactivity.To validate the results, especially with the immunostaining of Hyal and HAS in squamous cell carcinomas and adenocarcinomas of the lung, the patient population studied at the University Hospital of Coimbra. Documented for the first time a route by which the overexpression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells may favor the invasion in ADC and SqCC, respectively. Surprisingly, we demonstrated that hyper HAS1 and 3 immunoreactivity by neoplastic epithelial cells confers more aggressiveness to the ADC acinar and papillary, but a negative expression of HAS1 by mesenchymal cells confers a protective role ECM-helping to prevent the invasion by tumor cells in both types histological subtypes.The interaction between the expression of hialuronidades and hyaluronic acid synthases was evaluated for protein expression of epithelial-mesenchymal transition in tumor patients at the University Hospital of Coimbra. Hyaluronidase, hyaluronan synthase, Ecadherin, and TGF- modulated via an invasive tumor-induced in the ADC and SqCC lung, and were associated with a different spectrum of aggressiveness, since there was an inverse relationship between the expression of epithelial biomarkers and mesenchymal cells. While overexpression of HAS1 and HAS3 provides an aggressiveness to SqCC and ADC, an overexpression of TGF- and E-cadherin confers a protective effect by preventing the ECM invasion by tumor cells in both histological types. We compared the levels of immunostaining Hyal 1, 3 and HAS1, 2 and 3 in tumors resected at the Hospital AC. Camargo, and the levels obtained in tumors of the Hospital Universitário de Coimbra. We found that the immunostaining of HAS 1, 2, 3 and Hyal1 was significantly higher in tumors from patients of Coimbra, while Hyal 3 immunoreactivity was significantly. higher in tumors of patients in Brazil. For all these reasons, our results suggest that strategies directed at modulating the levels of HYAL1-wt and HYAL3-v1, the immunohistochemical expression of HAS3 and Hyal 3 respectively by neoplastic epithelial and mesenchymal cells, the synthesis of HAS3 and Hyal1 or the local response of low TGF- and E-cadherin, may have great impact on lung cancer

Le cancer de la prostate est le cancer le plus fréquent chez les hommes et la capacité des tumeurs à développer une résistance face aux thérapies anti-androgéniques vient souvent compromettre le pronostic des patients. Le développement de nouvelles approches thérapeutiques afin de circonvenir à la progression de ces tumeurs représente un besoin important la gestion de ce type de cancer. Plusieurs démonstrations récentes établissent l’implication de la famille des proprotéines convertases dans la progression tumorale. Ces enzymes ont pour fonctions biologiques de cliver une variété de précurseurs protéiques jouant des rôles importants dans la tumorigénèse. Dans le cancer de la prostate, la proprotéine convertase PACE4 est fortement surexprimée dans les cellules cancéreuses et joue un rôle dans la prolifération et la capacité à former des tumeurs, ce qui en fait une cible thérapeutique d’intérêt. En ce sens, des inhibiteurs peptidomimétiques ont été développés dans l’optique de la thérapie ciblée contre la PACE4. Toutefois, dans le but de développer une approche thérapeutique optimale, il convient néanmoins de comprendre le niveau de redondance fonctionnelle entre les différents membres de la famille des convertases, qui sont connus pour partager plusieurs de leurs substrats, ainsi que les mécanismes moléculaires régissant l’activité de la PACE4 et de ses substrats sous-jacents. L’utilisation d’une approche de répression génique stable envers les différentes convertases a permis de mettre en lumière les fonctions uniques de la PACE4 dans la progression tumorale. De plus, grâce à une approche de protéomique comparative, le premier substrat de la PACE4 dans le cancer de la prostate; le growth differenciation factor 15, a été découvert. Ce substrat permet de commencer à dresser l’implication de PACE4 dans le paysage moléculaire du cancer de la prostate. Grâce à des modalités d’imagerie moléculaire, l’emploi de versions radiomarquées des inhibiteurs peptidiques a également permis de démontrer que les composés s’accumulent dans les cellules cancéreuses en fonction des niveaux de PACE4 présents, et ce, tant in cellulo qu’in vivo. Ces données suggèrent un potentiel pour le développement d’un examen théranostique pour prédire la réponse tumorale à la pharmacothérapie anti-PACE4. Finalement, l’analyse de l’épissage alternatif de l’ARNm de PACE4 a permis l’élucidation des caractéristiques biochimiques et des fonctions spécifiques d’une nouvelle isoforme; la PACE4-altCT, qui est exprimée chez les cellules cancéreuses de la prostate, mais aussi d’autres types de cancer. Cette découverte a permis de redéfinir le modèle de travail en intégrant le concept de la rétention intracellulaire de cette isoforme qui semble médier la plupart de l’activité pro-proliférative reliée à l’activité PACE4, ce qui en fait la cible pharmacologique principale des inhibiteurs peptidiques dans le cancer de la prostate, mais aussi un biomarqueur potentiel. / Abstract: Prostate cancer is the most common cancer among men. The capabilities of tumors to adapt and overcome antiandrogenic therapy is persistently worsening patient’s prognostic and the development of novel therapeutic approaches to circumvent tumor progression therefore represents an unmet need. Many reports now demonstrate the implication of the enzymes from the proprotein convertase family in the progression of tumor from many cancer types. These enzymes are responsible for the processing of various protein precursors playing important roles in tumorigenesis. In prostate cancer, the proprotein convertase PACE4 is strongly overexpressed in cancer cells and plays a role in cell proliferation and tumor formation thus making a strong case for its use as a pharmacological target. For this reason, PACE4 peptidomimetic inhibitors were generated to develop PACE4-targeted therapies. However, to develop an optimal therapeutic approach regarding the inhibition of this enzyme, a complete understanding of the level of functional redundancy between the different convertases in prostate cancer is needed. Moreover, understanding the molecular mechanisms both upstream and downstream of PACE4 in prostate cancer cells would allow a better understanding of the considerations underneath such a therapeutic strategy. Using a stable gene silencing approach to knockdown all co-expressed member of the convertase family in prostate cancer cells, the roles of PACE4 in tumor progression were found to be unique and non-redundant among the other family member. Through a comparative proteomic approach, the first PACE4-specific substrate in prostate cancer; growth and differentiation factor 15, was identified. With this substrate growth factor, it is now possible to initiate the dissection of PACE4 biochemical functions in the prostate cancer molecular landscape. Using a radiolabelled version of the PACE4 peptide inhibitors, it was possible to demonstrate using molecular imaging that when applied in cellulo and in vivo, the compound is uptaken by cancer cells as well as by tissues according to their PACE4 expression levels. These data suggest that such PACE4 molecular imaging with pharmacological inhibitor could be developed as a theranostic assay to predict which tumor could be treated by PACE4-targetted therapy. Lastly, PACE4 mRNA alternative splicing analysis permitted the discovery of a new PACE4 isoform; named PACE4-altCT, which is strongly overexpressed by prostate cancer cells as well as other cancer types. As this isoform displays specific biochemical features and functions, notably being intracellularly retained and mediating most of the PACE4-associated cell growth capabilities, this discovery further redefined our working model, pointing to PACE4-altCT as the pharmacological target of inhibitory peptides in prostate cancer as well as a potential biomarker.