Le récepteur de la progestérone (PR) est un cible pharmacologique majeure pour la contraception, et pour le traitement de certaines pertubations endocriniennes ainsi que des cancers hormono-dépendants de l’utérus et du sein. Chez la femme, PR est exprimé sous deux isoformes majeures PRA et PRB qui sont des facteurs de transcription fonctionnellement distincts. L’expression de PRA vs PRB est souvent altérée dans certaines situations pathologiques selon des mécanismes encore mal identifiés. Dans cette thèse, nous démontrons que des phosphorylations clés regulées par des MAPK distincts contrôlent la stabilité de PRB et PRA. PRA est sélectivement stabilisée par la p38 MAPK tandis que PRB est préférentiellement stabilisé par la p42/44 MAPK. Ces mécanismes différentiels régulent donc le rapport d’expression PRA/PRB de façon ligand-dépendente et mettent les fonctions progestatives sous le contrôle de l’activité des facteurs de croissance et des cytokines pro-inflammatoires. Or, dans les cellules cancéreuses, la suractivité de certains stimuli extracellulaires provenant de telles signalisations et activant préférentiellement p42/44 et/ou p38 MAPK, pourrait être à l’origine des pertubations du rapport PRA/PRB observées dans les tumeurs du sein. Afin d’explorer la contribution différentielle des isoformes du PR dans la signalisation cellulaire, nous avons élaboré un modèle cellulaire original permettant de contrôler l’expression de PRA et/ou PRB de façon conditionnelle, réversible et dose-dépendante. Par une approche transcriptomique, nous avons identifiés les gènes régulés de façon différentielle par PRA et/ou PRB en absence ou présence de l’hormone. Nous montrons que plusieurs aspects de la signalisation de PR comme la sélectivité de la régulation transcriptionnelle, la dialogue-croisée avec des facteurs de croissance ainsi que l’efficacité antiproliférative des antiprogestatifs dépendent de l’expression differentielle des isoformes du PR. Une nouvelle approche thérapeutique ou préventive possible dans les cancers hormono-dépendants pourrait consister à administrer des antagonistes du PR. Cependant, la plupart des antiprogestatifs disponibles comme la mifépristone présentent des effets agonistes partiels et ne sont pas sélectifs du PR, produisant ainsi des effets indésirables majeurs. Dans un projet collaboratif, et sur la base d’études cristallographiques de PR, nous avons synthétisé et caractérisé plusieurs dizaines de molécules antagonistes du PR, nommés APRn. L’étude des relations structure-fonctions de ces APRn a permis d’identifier les substitutions introduites dans la structure stéroïdienne qui sont responsables des propriétés agonistes/antagonistes de ces molécules. Plusieurs APRn sélectionnés sont dépourvus d’effets agonistes partiels, sont spécifiques du PR et inhibent son activité transcriptionnelle par un nouveau mécanisme d’action dit « passif », en raison de leur capacité particulière à inhiber le recrutement des corégulateurs transcriptionnels. Ces antagonistes sélectifs de PR offrent des perspectives thérapeutiques intéressantes dans les maladies de la reproduction et des cancers hormono-dépendants de l’utérus et du sein. L’ensemble de nos résultats apportent des informations nouvelles sur les mécanismes impliqués dans la sélectivité fonctionnelle des isoformes du PR en physiopathologie, ainsi que sur la possibilité d’un ciblage pharmacologique spécifique par de nouveaux antagonistes utilisables dans le traitement du cancer du sein. / Progesterone receptor (PR) is an essential pharmacological target for contraception, female reproductive disorders as well as for hormone-dependent breast and uterine cancers. Human PR is expressed as two major isoforms PRA and PRB which behave as distinct transcriptional factors. PRA vs PRB expression is often altered under pathological conditions notably breast cancer through unknown mechanisms. In this thesis we demonstrate that down-regulations of PRB and PRA proteins are negatively controlled by key phosphorylation events involving distinct MAP kinase signaling. PRA is selectively stabilized by p38 MAPK whereas p42/44 MAPK specifically controls PRB stability leading to unbalanced PRA/PRB ratios in a ligand sensitive manner. In cancer cells, elevated extracellular stimuli such as epidermal growth factors or pro-inflammatory cytokines that preferentially activate p42/44 or p38 MAPK respectively may result in opposite variations in PRA/PRB expression ratio. These results may explain altered PRA/PRB ratios often associated with breast tumors. To get a mechanistic understanding of how varied PRA/PRB ratio contributes in cell signaling, we generated an original bi-inducible PR-isoform cell model allowing selective, reversible and dose-dependent expression of PRA and/or PRB, enabling fine-tune adjustment of PRA/PRB ratio in the same cells. Using this cell-based system, we undertook genome-wide transcriptomic studies to investigate transcriptional regulation driven by unliganded and liganded PR isoforms. We report that several aspects of PR signaling such as target gene selection/transcriptional regulation, cross-talk with growth factors and antiproliferative efficacy of antiprogestin are highly dependent upon variation in PRA/PRB ratio. A new potential therapeutic strategy in PR-dependent pathological conditions may rely on the use of PR antagonists. Most of the currently available antiprogestins such as mifepristone present partial agonist activity and are not selective to PR leading to undesirable side effects. Therefore, in a collaborative project we have synthesized and characterized several new PR antagonist compounds named as APRn. Structure-activity relationship studies allowed identification of the key substitutions in steroidal skeleton responsible for agonist/antagonist character of these molecules. Several selected APRn lack partial agonist effect, are PR specific and inhibit PR transcriptional properties through a new passive mechanism of action i.e. impaired recruitment of transcriptional coregulators. Such PR selective antagonists devoid of partial agonist character might provide important therapeutic perspectives for various reproductive tract abnormalities and hormone-dependent uterine and breast cancers. Altogehter, our results provide mechanistic insights into the functional selectivity of PR isoforms and their pharmacological targeting by the use of PR antagonists.

Made available in DSpace on 2014-12-17T15:32:31Z (GMT). No. of bitstreams: 1 RosileneCS.pdf: 376311 bytes, checksum: 8a0459347381c3b197002ddd72f02225 (MD5) Previous issue date: 2005-11-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Oral squamous cell carcinoma (OSCC) is the most common malignancy in oral cavity and human papillomavirus (HPV) may have an important role in its development. The aim of this experiment was to investigate the HPV DNA and viral types in 90 cases of OSCC. Moreover, a comparative analysis between the cases of OSSC with and without HPV DNA was performed by using cell cycle markers p21 and pRb in order to detect a possible correlation of these proteins and HPV infection. DNA was extracted from paraffin embedded tissue and amplified by PCR (polymerase chain reaction) with primers PCO3+ e PCO4+ for a fragment of human β-globin gene. After this procedure, PCR for HPV DNA detection was realized using a pair of generic primers GP5+ e GP6+. Immunohistochemical study was performed by streptoavidin-biotin technique and antibodies against p21 and pRb proteins were employed. Eighty-eight cases were positive for human β-globin gene and HPV DNA was found in 26 (29.5%) of then. It could not be detected significant correlation between HPV and age, sex and anatomical sites of the lesion. The most prevalent viral type was HPV 18 (80.8%). Regarding the immunohistochemical analysis, it was detected significant association between HPV presence and pRb immunoexpression (p=0,044), nevertheless, the same was not observed in relation to p21 protein (p =0,416). It can be concluded that the low detection of HPV DNA in OSCC by the present experiment suggests a possible role of the virus in the development and progression in just a subset of this disease / O carcinoma epiderm?ide oral ? a neoplasia maligna mais freq?ente da cavidade oral e o papilomav?rus humano (HPV) parece ter um relevante papel na indu??o desta les?o. Neste trabalho investigou-se o DNA do HPV e tipos virais em 90 casos de carcinoma epiderm?ide oral (CEO). Realizou-se tamb?m uma an?lise comparativa entre os grupos de CEO com DNA do HPV e sem o DNA do v?rus, empregando-se os marcadores do ciclo celular p21 e pRb, a fim de estabelecer poss?vel correla??o entre a express?o imuno-histoqu?mica dessas prote?nas e a infec??o pelo HPV. O DNA foi extra?do de tecido emblocado em parafina e amplificado por PCR (rea??o em cadeia da polimerase) com um par de primers designados PCO3+ e PCO4+ para um fragmento do gene da β-globina humana. Posteriormente, realizou-se PCR para detec??o do DNA de HPV utilizando-se um par de primers gen?ricos designados GP5+ e GP6+. A tipagem viral foi realizada pela hibridiza??o dot blot. No m?todo imuno-histoqu?mico utilizou-se a t?cnica da streptavidina-biotina com um painel de anticorpos monoclonais para as prote?nas p21 e pRb. Dos 88 casos positivos para o gene da β-globina humana, em 26 (29,5%) foi detectado o DNA do HPV. N?o houve associa??o significativa entre o HPV e as vari?veis idade e sexo dos pacientes e localiza??o anat?mica da les?o. O tipo viral prevalente foi o HPV 18 (80,8%). Quanto ? an?lise imuno-histoqu?mica, foi observada associa??o estatisticamente significativa entre a presen?a do HPV e a express?o imunohistoqu?mica de pRb (p=0,044), entretanto, n?o houve qualquer diferen?a estatisticamente significativa entre a express?o da prote?na p21 e a presen?a do v?rus (p =0,416). P?de-se concluir que o baixo percentual de detec??o do DNA do HPV no carcinoma epiderm?ide oral no presente trabalho, sugere uma poss?vel participa??o do HPV no desenvolvimento e progress?o de apenas um subgrupo dessas les?es

Les concentrations d’oxygène (O2) dans la moelle osseuse hématopoïétique, sont très inférieures à celle de l’air (20% d’O2) puisqu’elles vont de 4% dans les zones juxta-vasculaires à 0,1% près de l’endoste, où siègent les cellules souches hématopoïétiques (CSH), essentiellement quiescentes. Ce paramètre physiologique, rarement pris en compte, est un élément important dans la régulation de l’hématopoïèse. Les effets bénéfiques des faibles concentrations d’oxygène sur le maintien des cellules souches hématopoïétiques sont maintenant bien établis. Par contre, la réponse du compartiment des progéniteurs aux faibles concentrations d’oxygène est moins examinée mais très discutée, certains montrant une différenciation associée à un blocage de la prolifération alors que d’autres montrent leur disparition de la culture probablement par apoptose. C’est dans ce contexte que se place ces travaux qui visent à approfondir les effets des faibles concentrations en oxygène (de 3 à 0,1%) sur ce compartiment. La culture pendant 72h à 0,1% O2 de la lignée murine de progéniteurs hématopoïétiques non leucémiques FDCP-Mix entraîne leur arrêt progressif en quiescence (Ki-67 négatif) de ces cellules sans induction d’apoptose. Cet arrêt est associé à la différenciation granulocytaire d’une majorité de la population. Dans ces mêmes conditions de culture persiste une population restreinte de cellules qui s¹auto-renouvellent lentement et qui sont capables après repiquage en culture à 20% d’O2 de repeupler une culture liquide et de former des colonies en milieu semi-solide. Ces changements fonctionnels sont associés aux modifications de protéines du cycle cellulaire impliquées dans la quiescence cellulaire : p27KIP1, pRb et CDK. Cette caractérisation permet désormais d’utiliser cette lignée comme modèle pour l’étude des équilibres fondamentaux au maintien de l’homéostasie hématopoïétique. / Oxygen concentrations (O2) in hematopoietic bone marrow vary from 4% in capillaries to less than 0.1% in subendosteum, where hematopoietic where mostly quiescent stem cells reside. This physiological factor, rarely investigated, is an essential piece of hematopoiesis regulation. The beneficial effects of low oxygen concentrations on the maintenance of hematopoietic stem cells are now well established. In contrast, the effects of low O2 concentration on the progenitors compartment, were much less explored and are then more controversial: some articles evidence a pro-differentiative effect related to a cell proliferation blockade while others observe their rapid disappearance from cultures probably due to apoptosis. In this particular context takes place this work which aims to investigate the low oxygen concentration effect (from 3% to 0.1%) on this precise compartment. Culture of the murine non-leukemic hematopoietic progenitor cell line FDCP-Mix line at 0.1% O2 during 72h induces a progressive G0 quiescence blockade (Ki-67 negative) without apoptosis increase. This G0 cell cycle arrest is correlated with the granulo-monocytic differentiation of most cells. In the mean time a minor population of self-renewing cells continues to cycle slowly as evidenced by their 5-FU sensitivity in primary culture and by their capacity to give rise to colonies and to repopulate liquid cultures when replated in cultures at 20% O2. G0 quiescence and granulocytic differentiation induced by low O2 concentrations is associated with cell cycle protein modifications: p27KIP1, pRb, CDK. This characterization allows FDCP-Mix usage as model to investigate fundamental balances responsible for hematopoietic long-term maintenance.

This dissertation is devoted to the study of the molecular biology of major tumor suppressors, defined as those that prevent the cellular processes identified as the hallmarks of cancer. Specifically, the major tumor suppressors pRb and STK11 are explored in the context of osteosarcoma and lung cancer, respectively. RB1 was the first tumor suppressor gene discovered. Over four decades of work have revealed that the Rb protein (pRb) is a master regulator of biological pathways influencing virtually every aspect of intrinsic cell fate including cell growth, cell-cycle checkpoints, differentiation, senescence, self-renewal, replication, genomic stability and apoptosis. While these many processes may account for a significant portion of RB1's potency as a tumor suppressor, a small, but growing stream of evidence suggests that RB1 also significantly influences how a cell interacts with its environment, including cell-to-cell and cell-to-extracellular matrix interactions. Chapter 2 highlights pRb's role in the control of cell adhesion and how alterations in the adhesive properties of tumor cells may drive the deadly process of metastasis. Chapter 3 defines a role for pRb as a suppressor of the progression to metastasis by upregulating integrin α10. Transcription of this integrin subunit is herein found to be pRb-dependent in mouse osteoblasts. Classic pRb partners in cell cycle control, E2F1 and E2F3, do not repress transcription of integrin α10 and phosphorylation of pRb is not necessary for activation of the integrin α10 promoter. Promoter deletion revealed a pRb responsive region between -108bp to -55bp upstream of the start of the site of transcription. pRb activation of transcription also leads to increased levels of integrin α10 protein and a greater concentration of the integrin α10 protein at the cell membrane of mouse osteoblasts. These higher levels of integrin α10 correspond to increased binding to collagen substrate. Consistent with our findings in mouse osteoblasts, we found that integrin α10 is significantly underexpressed in multiple solid tumors that have frequent inactivation of the pRb pathway. Bioinformatically, we identified data consistent with an 'integrin switch' that occurs in multiple solid tumors consisting of underexpression of integrins α7, α8, and α10 with concurrent overexpression of integrin β4. pRb promotes cell adhesion by inducing expression of integrins necessary for cell adhesion to a substrate. We propose that pRb loss in solid tumors exacerbates aggressiveness by debilitating cellular adhesion, which in turn facilitates tumor cell detachment and metastasis. Lung cancer is the leading cause of cancer-related death in the U.S. and additional targeted therapies are desperately needed to treat these patients. STK11 is the third most frequently mutated gene in lung adenocarcinoma following only KRAS and TP53, yet its mutational status is not currently clinically evaluated and no therapies have been approved to specifically target its pathway. A deep understanding of the complex pathways controlled by STK11 and their alterations in cancer are required to develop effective therapies for patients with loss-of-function mutations. In Chapter 4 we present the current understanding of STK11, focusing on its molecular biology and therapeutic implications, including a compilation of studies evaluating STK11 somatic mutations in human lung cancer tissue and how the frequency of these mutations varies across histological subtypes and patient populations. Finally, we review the strategies being used to target STK11-deficient cancers at the clinical trial, pre-clinical, and basic science levels as well as proposing potential new therapies that might benefit this patient population. STK11 is a tumor-suppressor commonly mutated in lung adenocarcinoma (LuAd). There are a number of agents that may selectively target the deregulated pathways in STK11 mutated tumors, and thus, identifying the subset of adenocarcinomas that harbor these mutations could have significant clinical benefit. In Chapter 5, we characterized a cohort of 442 adenocarcinoma patients with respect to STK11 mutation status and subset of this cohort using immunochemistry, gene expression, and western blotting. We found that measuring STK11 mutation status is complicated by the fact that many STK11 mutations lead to expression of a stable protein that is indistinguishable from wild type (WT) via immunohistochemistry. To circumvent this, we used published cell line mutation and gene expression data to derive a signature correlating with STK11 mutation status. This signature was validated in the cohort of 442 lung adenocarcinomas and strongly correlates with mutation status (ROC curve AUC = 85.29). These data suggest that STK11; mutation status may be best assessed by measuring the downstream targets included in our signature.

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

The retinoblastoma tumor suppressor (pRb) is thought to orchestrate terminal differentiation by inhibiting cell proliferation and apoptosis and stimulating lineage-specific transcription factors. In this thesis I have shown that in the absence of pRb, differentiating primary myoblasts fused to form short myotubes that never twitched and degenerated via a non-apoptotic mechanism. The shortened myotubes exhibited an impaired mitochondrial network, mitochondrial perinuclear aggregation, autophagic degradation and reduced ATP production. Bcl-2 and autophagy inhibitors restored mitochondrial function and rescued muscle degeneration, leading to twitching myotubes that expressed normal levels of muscle-specific proteins and eventually exited the cell-cycle. A hypoxia-induced glycolytic switch also rescued the myogenic defect after chronic or acute inactivation of Rb in a HIF-1-dependent manner. These results demonstrate that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program. I next tested the effect of retinoblastoma protein family members – p107 and p130 – on skeletal myogenesis in the absence of Rb. Chronic or acute inactivation of Rb plus p130 or Rb plus p107 increased myoblast cell death and reduced myotube formation, yet expression of Bcl-2, treatment with autophagy antagonist or exposure to hypoxia extended myotube survival, leading to long, contracting myotubes that appeared indistinguishable from control myotubes. Triple mutations in Rb family genes further accelerated cell death and led to elongated myocytes or myotubes containing two nuclei, some of which survived and twitched under hypoxia. Whereas nuclei in Rb-/- myotubes were unable to stably exit the cell-cycle, myotubes lacking both p107/p130 became permanently post-mitotic, suggesting that pRb, but not p107 or p130 may be lost in cancer because of the unique requirement for cell-cycle exit during terminal differentiation. This thesis demonstrates that pRb is required to inhibit apoptosis in myoblasts and autophagy in myotubes but not to activate the differentiation program, and reveal a novel link between pRb and cell metabolism.

Darbo tikslas – įvertinti pienarūgščių bakterijų įtaką akrilamido formavimuisi pusruginės duonos gaminiuose, kurių gamybai naudotos skirtingos PRB (Lactobacillus delbruecki, Pediococcus acidilactici, Pediococcus pentosaceus ir Lactobacillus sakei), bei įvertinti RS 190 deaktyvuotų mielių įtaką kvietinių miltų technologinėms savybėms ir kvietinių kepinių kokybei. Nustatyta, kad akrilamido kiekis kepiniuose kito nuo 37,87 ± 0,55 iki 74,64 ± 0,36 µg/kg. Pusruginės duonos kepiniuose, fermentuotose L. delbruecki nustatytas mažiausias akrilamido kiekis, lyginant su kitais pusruginės duonos kepiniais, kurių gamyboje buvo naudotos PRB. PRB proteolitinis aktyvumas yra tiesiogiai susijęs su akrilamido kiekio mažinimu duonos kepiniuose. Kepiniuose, pagamintuose su L. delbruecki (su didžiausiu proteolitiniu aktyvumu) susidarė mažiausias akrilamido kiekis (37,87 ± 0,55 µg/kg), o kepiniuose fermentuotuose P. pentosaceus ir P. acidilactici (su mažiausiu proteolitiniu aktyvumu) nustatytas didžiausias akrilamido kiekis, atitinkamai, 74,64 ± 0,36 ir 63,38 ± 0,98 µg/kg. Atlikus kvietinių miltų su deaktyvuotų mielių priedu ir be priedo farinografinį tyrimą, nustatyta, kad didžiausiu vandens įgėrimu (59,1 ± 1,18 %) ir tešlos stabilumu (5 kartus didesnis) pasižymėjo kvietiniai miltai su priedu, tačiau jų tešlos susidarymo trukmė 1,3 karto ilgesnė. Deaktyvuotų mielių priedas turi teigiamos įtakos kepinių savitajam tūriui, minkštimo akytumui ir drėgniui. Pagal gautus tyrimo rezultatus galima... [toliau žr. visą tekstą] / The aim of this study was to investigate acrylamide formation in semi rye bread fermented with Lactobacillus delbruecki, Pediococcus acidilactici, Pediococcus pentosaceus and Lactobacillus sakei, and, to investigate the influence of RS 190 deactivated yeast on wheat flour technological properties and wheat bread quality. Results show that the amount of acrylamide in bread samples ranged from 37.87 ± 0.55 to 74.64 ± 0.36 µg/kg. Lactofermentation reduce acrylamide formation in semi rye bread. L. delbruecki was found to have a higher effect on acrylamide reduction in bread samples in compare with P. acidilactici, P. pentosaceus and L. sakei. Also, proteolytic activity of LAB have a influence on acrylamide content reduction in bread. Bread made with L. delbruecki (the highest proteolytic activity) contained lower acrylamide content (37.87 ± 0.55 µg/kg), while breads prepared with P. pentosaceus and P. acidilactici (lower proteolytic activity) contained higher acrylamide contents (74.64 ± 0.36 and 63.38 ± 0.98 µg/kg, respectively). Pharinographic analysis of wheat flour with deactivated yeast and without deactivated yeast results shows that the higher water absorbtion have wheat flour with deactivated yeast (59.1 ± 1.18 %) and higher dough stability (5 times higher) but longer dough formation time (1,3 times longer). The addition of deactivated yeast in wheat flour have a significant effect of bread specific volume, porosity and moisture content. In conclusion we could say... [to full text]

Made available in DSpace on 2014-06-12T15:52:08Z (GMT). No. of bitstreams: 2 arquivo2820_1.pdf: 8386047 bytes, checksum: f165dd7cc5f2eedad7f74c08f159ecde (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Faculdade de Amparo à Ciência e Tecnologia do Estado de Pernambuco / O câncer cervical é o segundo tipo de neoplasia que mais acomete as mulheres no Brasil, com alta prevalência em Pernambuco. A associação desta patologia como o papilomavírus humano (HPV) já está bem estabelecida e atualmente, sabe-se que o HPV pode ser encontrado em vários outros locais de infecção, além da região genital. Com o intuito de apontar a distribuição corpórea do HPV pelo corpo humano foi realizada uma revisão da literatura buscando por sítios corpóreos em que o DNA viral já tinha sido identificado. Dentre os tipos virais de alto risco que mais acometem a população brasileira, sabe-se que o HPV16 aparece associado a mais de 50% dos achados. Baseado nisso, uma busca pela presença do DNA do HPV e pelos variantes virais do HPV16 foi realizada em mulheres de Pernambuco que apresentavam lesões genitais. Complementarmente, sabe-se que sistemas de expressão são amplamente utilizados para produção de diversas moléculas biológicas, sendo o bacteriano o mais rápido e fácil de ser utilizado. Visando melhor utilização do sistema de expressão em bactéria, desenvolvemos um método para detectar a produção de -galactosidase em cepas heterólogas de Escherichia coli. Esse sistemas bacterianos vem sendo utilizados para produção de diversas moléculas virais, como oncoproteínas virais. Baseado no levantamento bibliográfico realizado foi possível identificar DNA viral nos mais diferentes sítios corpóreos, inclusive com ausência de lesões clínicas, apontando para a possibilidade do HPV agir como um oportunista. Da população estudada, mais de 50% foram positivas para o HPV, com achados de múltiplas infecções com tipos virais distintos, onde o variante Europeu foi o mais frequente nos casos de HPV16 positivo. A linhagem Origami (DE3) de E.coli demonstrou-se eficiente no ensaio colorimétrico expressando o gene da -galactosidase com baixa produção de proteínas bacterianas. Baseado nisso, esse modelo bacteriano foi utilizado no processo de sub-clonagem do gene E7 do HPV16 permitindo após indução do promotor visualizar uma banda de 15 KDa na eletroforese de proteínas totais, banda provavelmente referente a oncoproteína viral. No entanto, faz-se necessário emprego de testes imunológicos com anticorpos específicos para confirmar sua produção e posterior purificação. A tipagem da população a respeito do variante do HPV mais predominante e produção da proteína E7 permitem o aumento nos conhecimentos dos diferentes mecanismos de interação do vírus com o hospedeiro e favorecem ao desenvolvimento de métodos diagnósticos e terapêuticos mais eficientes e específicos para as diferentes regiões do mundo

Indiana University-Purdue University Indianapolis (IUPUI) / Human papillomaviruses (HPVs) are one of the most common causes of sexually transmitted disease in the world. HPVs are divided into high-risk (HR) or low-risk (LR) types based on their oncogenic potential. HPVs 16 and 18 are considered HR types and can cause cervical cancer. HPVs 6 and 11 are classified as LR and are associated with condyloma acuminata (genital warts). Viral proteins of both HR and LR HPVs must be able to facilitate a replication competent environment. The E7 proteins of LR and HR HPVs are responsible for maintenance of S-phase activity in infected cells. HR E7 proteins target all pRb family members (pRb, p107 and p130) for degradation. LR E7 does not target pRb or p107 for degradation, but does target p130 for degradation. Immunohistochemistry experiments on HPV 6 infected patient biopsies of condyloma acuminata showed that detection of p130 was decreased in the presence of the whole HPV 6 genome. Further, the effect of HR HPV 16 E7 and LR HPV 6 E7 on p130 intracellular localization and half-life was examined. Experiments were performed using human foreskin keratinocytes transduced with HPV 6 E7, HPV 16 E7 or parental vector. Nuclear/cytoplasmic fractionation and immunofluorescence showed that, in contrast to control and HPV 6 E7-expressing cells, a greater amount of p130 was present in the cytoplasm in the viii presence of HPV 16 E7. The half-life of p130, relative to control cells, was decreased in the cytoplasm in the presence of HPV 6 E7 or HPV 16 E7, but only decreased by HPV 6 E7 in the nucleus. Inhibition of proteasomal degradation extended the half-life of p130, regardless of intracellular localization. Experiments were also conducted to detect E7-binding partners. Cyclin C and cullin 5 were identified as proteins capable of binding to both HPV 6 E7 and HPV 16 E7. Preliminary experiments showed that decreasing protein levels of p600, a binding partner of both HPV 6 E7 and HPV 16 E7, by RNA interference might affect p130 stability. Elucidating the mechanisms of p130 degradation may identify potential targets for preventing degradation of p130 and allowing restoration of cell cycle control.

Les facteurs d’ADP-ribosylation (ARFs) sont des petites GTPases impliquées dans le transport vésiculaire, la synthèse des lipides membranaires et la réorganisation du cytosquelette d’actine. Les isoformes 1 (ARF1) et 6 (ARF6) sont les plus étudiées. ARF1 est connue pour être distribuée à l’appareil de Golgi, alors qu’ARF6 est confinée principalement à la membrane plasmique. Récemment, il a été démontré qu’ARF6 est hautement exprimée et activée dans plusieurs cellules de cancer du sein invasif et que celle-ci contrôle les processus de migration et d’invasion. Cependant, le rôle d’ARF1 dans ces processus biologiques impliqués dans la formation de métastases du cancer du sein demeure méconnu. Dans la présente étude, nous avons utilisé comme modèle d’étude pour ARF1 les MDA-MB-231, une lignée de cellules invasives du cancer du sein exprimant de haut niveau de récepteurs au facteur de croissance épidermique (EGFR). Afin d’évaluer le rôle d’ARF1 dans la migration, dans la transition épithéliale mésenchymateuse (EMT) et dans la prolifération cellulaire, nous avons procédé à deux types d’approches expérimentales, soit l’inhibition de l’expression endogène d’ARF1 par l’interférence à l’ARN de même que la surexpression de formes mutantes dominante négative (ARF1T31N) et constitutivement active d’ARF1 (ARF1Q71L), qui miment les formes inactive et active de la GTPase, respectivement. De manière intéressante, la suppression d’ARF1 et la surexpression de la forme inactive d’ARF1 induisent l’arrêt de la migration et de la prolifération des MDA-MB-231 de manière dépendante à l’activation de l’EGFR et ce, en bloquant l’activation de la voie PI3Kinase. De plus, nous démontrons qu’ARF1, de même que les ARF GEFs Cytohésine-1 et Cytohésine-2, contribuent au phénotype invasif des cellules tumorales de cancer du sein. Dans les mêmes approches expérimentales, nous montrons que l’inactivation d’ARF1 dans les MDA-MB-231 déclenche un arrêt de croissance irréversible associé à l’induction de la sénescence et ce, en régulant la fonction de la protéine du rétinoblastome pRb. Enfin, cette étude a permis de mettre en évidence le rôle physiologique d’ARF1 dans les processus de migration et de prolifération cellulaire, deux événements biologiques responsables de la progression du cancer du sein. / The ADP-ribosylation factors (ARFs) are small GTPases involved in vesicular transport, lipids synthesis and cytoskeleton remodelling. The isoforms 1 (ARF1) and 6 (ARF6) are the most studied. ARF1 is classically distributed at the Golgi apparatus whereas ARF6 is found at the plasma membrane and onto recycling endosomes. It was recently demonstrated that ARF6 is highly expressed and activated in several breast cancer cell lines and is associated with enhanced migration and invasiveness. However, the role of ARF1, in these biologicals processes necessary for metastasis formation, remains unclear. In this study, we used MDA-MB-231 cells, an invasive breast cancer cell line, that expressed high levels of EGFR (Epidermal Growth Factor Receptor) to investigate the role of ARF1 in migration and proliferation. To further establish the role of ARF1 in cell migration, EMT and proliferation, we used two experimental approaches. First, we decreased endogenous ARF1 expression by RNA interference and second we overexpressed the dominant negative (ARF1T31N) and constitutively active (ARF1Q71L) ARF1 mutants, which mimick the inactive and active forms of ARF1, respectively. We demonstrated that depletion of ARF1 as well as overexpression of the inactive form of ARF1 blocked EGFR-mediated cell migration and proliferation by inhibiting the activation of PI3Kinase. Moreover, we showed, using invasive and non invasive breast cancer cell lines, that ARF1 and both Cytohesin-1 and Cytohesin-2 are required for invasivness. Using similar approaches, we reported that inactivation of ARF1 in MDA-MB-231 cells promotes cell growth arrest associated to senescence program by regulating the function of the Retinoblastoma protein pRb. Altogether, these findings demonstrate a physiological role for ARF1 in cell migration and proliferation. These two biologicals events are necessary for breast cancer progression.