Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l’histoire naturelle de la maladie. Nous nous sommes intéressés à l’association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d’optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l’ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l’inhibition de PARP. L’apoptose est mesurée en cytométrie de flux par l’intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l’iodide de propidium. Le test de clonogénicité permet d’évaluer la capacité des cellules à échapper à la mort et à former une colonie. L’activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L’immunofluorescence sur cellules fixées a permis d’étudier les dommages de l’ADN (γH2AX), la voie intrinsèque de l’apoptose (l’activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l’expression et l’activité de PARP (PAR) ainsi que l’expression d’acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l’association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l’inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l’activation plutôt qu’avec l’expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l’hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d’autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l’ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l’expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l’inhibition de PARP par le PJ en diminuant l’expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l’association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l’élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP. / Introduction Driven by the facts that non small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide and that NSCLC patients often develop resistance against Cisplatin (CDDP)-based therapies, we addressed the question of the combination therapy of CDDP with poly(ADP-ribose) polymerases (PARP) inhibitors. Inhibitors of PARP have raised great expectations for the treatment of a variety of cancers, either as monotherapeutic agent against DNA repair-deficient tumours or combined to DNA-damaging compounds.Material and methods We generated nine CDDP-resistant clones by prolonged exposure to low dose CDDP of the A549 NSCLC parental cell line. Two distinct PARP inhibitors, CEP8983 (CEP) and PJ34 (PJ) as well as PARP1 knockdown with small interfering RNAs (siRNAs) were used for PARP inhibition. Apoptosis was measured by the simultaneous assessment for the loss of the mitochondrial transmembrane potential (m) and the breakdown of the plasma membrane using the m-sensitive fluorochrome DiOC6(3) and the vital dye propidium iodide, respectively. Moreover clonogenic survival was assessed. In vitro assessments of the enzymatic activity of cells were based on the reduction of the colorless tetrazolium salt. Immunofluorescence microscopy determinations were performed with antibodies specific for DNA damage (γH2AX), intrinsic apoptosis (cleaved Caspase-3 and cytochrome c), and homologous recombination (RAD51 and BRCA1). Immunoblotting was assed for PARP1 expression and activity (PAR) and base excision repair (BER) effectors (XRCC1 and polymerase β). We developed an immunohistochemical staining method that specifically detects PAR on paraffin-embedded cell pellets and tissue sections.Results We found that PARP inhibitors and PARP1 siRNAs synergized with CDDP in the killing of NSCLC cells in vitro. Unexpectedly, CDDP-resistant NSCLC cell clones developed addiction to PARP hyperactivation, thereby becoming susceptible to apoptosis induction by PARP inhibition. We showed that these cisplatin-resistant clones, exhibited high PARP protein levels and increased PARP activity, leading to an increased poly-ADP ribosylation of cellular proteins, as compared to their parental, cisplatin-sensitive counterparts. These cisplatin-resistant cells become susceptible to cell death as induced by PARP inhibition, correlating with the hyperactivity of PARP (elevated PAR levels) more accuratly than with the overexpression of PARP. Suggesting that PAR levels may constitute a more accurate biomarker than PARP to predict the sensitivity of cells to PARP inhibition. We expanded the observation that cisplatin resistance causes PARP upregulation and hyperactivation and subsequent sensitization to PARP inhibition to additional five human cancer cell lines including two NSCLC (H1650 and H460), one mesothelioma (P31), one ovarian (TOV112D) and one cervical cancer (HeLa) cell line. To get further insight into this issue, we generated in vivo experiments. Tumors derived from CDDP-resistant cells were characterized by elevated levels of PAR suggesting that PAR levels are preserved during tumor formation. Those PAR-overexpressing tumors responded to the administration of PJ in vivo with a consistent reduction in PAR immunoreactivity. CDDP resistant clones that are specifically killed by PARP inhibitors assessed efficient homologous recombination repair however deficient BER elongation.Conclusion We showed a beneficial effect for the association therapy of PARP inhibitors with CDDP in several NSCLC cell lines. We have identified an addiction to PARP in CDDP resistant cell lines with deficient BER elongation. We postulate that PAR is a specific predictive biomarker for the response to PARP inhibitors.

Made available in DSpace on 2016-04-04T18:31:33Z (GMT). No. of bitstreams: 1 Gerson Roberto Luqueta.pdf: 6509270 bytes, checksum: ca46642c2f88e2b7c6526f32ab1af762 (MD5) Previous issue date: 2012-12-06 / This present dissertation aims to propose the use of a wireless sensor network for monitoring the lethality of sterilization process in biologically contaminated materials that uses heat as a sterilizing agent, to replace a wired system. The survey also assesses if sensors could be used as monitors of the parameter in theoretical thermic lethality of the process, and therefore serves as an assistance element in decision making for releasing the materials sterilized in hospitls and laboratories. For this purpose, a platform with sensor nodes was mounted for temperature measurement process, lethality calculation and quality determination of the radio signal and is results were compared with wired commercial equipment. / A presente disserta??o tem por objetivo propor o uso de uma rede de sensores sem fio para monitora??o da letalidade de processo de esteriliza??o de materiais biologicamente contaminados que utiliza o calor como agente esterilizante, em substitui??o a um sistema com fio. A pesquisa avalia tamb?m se os sensores poder?o ser utilizados como monitores do par?metro da letalidade t?rmica te?rica do processo, e assim servirem como elemento de aux?lio na tomada de decis?o para a libera??o dos materiais esterilizados em hospitais e laborat?rios. Para tanto, uma plataforma com n?s sensores foi montada para medi??o da temperatura de processo, c?lculo da letalidade e determina??o da qualidade do sinal de r?dio e os seus resultados foram comparados com um equipamento comercial com fio.

Le sarcome de Ewing est la seconde tumeur pédiatrique de l’os la plus fréquente. Elle est caractérisée par une translocation chromosomique résultant à la fusion de EWSR1 avec un membre de la famille ETS. Chez 85% des patients, cette fusion conduit à l’expression de la protéine chimérique EWS-FLI1 qui est l’oncogène majeur de ce sarcome. Ce dernier agit principalement par son action transcriptionelle sur des cibles qui lui sont propres. Au niveau thérapeutique, le sarcome d’Ewing est traité par chimiothérapie, chirurgie locale et par radiothérapie. La survie à long terme des patients est de l’ordre de 70%, mais beaucoup plus basse pour les patients métastatiques et quasi nulle lors d’une récidive. Parmi maintes caractéristiques, certains cancers présentent une dérégulation énergétique. L’influence d’EWS-FLI1 sur cet aspect n’a fait l’objet d’aucune étude dans le contexte du sarcome d’Ewing. Nous avons donc étudié par profilage métabolomique des cellules de sarcome d’Ewing en présence ou en absence d’EWS-FLI1. En comparant ces deux conditions, des modulations du profil énergétique relatif au cycle de Krebs, des précurseurs de le glycosylation ainsi que des métabolites de la voie de la méthionine et du tryptophane ont été observés. En parallèle, grâce à un crible de banque de shRNAs réalisé dans des conditions expérimentales similaires à l’étude métabolomique (lignée d’Ewing avec ou sans EWS-FLI1), nous avons pu identifier des gènes présentant des caractéristiques « synthétique létales », c'est-à-dire tuant uniquement les cellules du sarcome d’Ewing en présence de son oncogène. / Ewing sarcoma, the second most commonly occurring pediatric bone tumor, is most often characterized by a chromosomal translocation between EWSR1 and FLI1. The gene fusion EWS-FLI1 accounts for 85% of all Ewing sarcoma and is considered the major oncogene and master regulator of Ewing sarcoma. EWS-FLI1 is a transcriptional modulator of targets, both directly and indirectly. Ewing sarcoma is aggressively treated with chemotherapy, localized surgery and radiation and has an overall survival of about 70%, however, survival for metastasis or relapsed cases remains low. One of the cancer hallmarks, metabolic deregulation, is most likely partly dependent on EWS-FLI1 in Ewing sarcoma cells. In order to get a better understanding of Ewing sarcoma biology and oncogenesis, it might be of high interest to investigate the influence of EWS-FLI1 in Ewing sarcoma cells. We therefore performed a global metabolic profiling of Ewing sarcoma cells with or without inhibition of EWS-FLI1. Several changes in the energy metabolism were observed throughout this study; the observed changes were consistent with an energy profile that moved from a cancer cell energy metabolism towards the energy metabolism of a more normal cell upon EWS-FLI1 inhibition, primarily based on the TCA cycle. Levels of TCA intermediates, glycosylation precursors, methionine pathway metabolites and amino acids, especially changes in the tryptophan metabolic pathway, were altered upon EWS-FLI1 inhibition. Parallel to this study, we performed a high-throughput synthetic lethality screen, in order to not only identify essential genes for cell survival and proliferation, but also to identify new synthetic lethal targets that could specifically target Ewing sarcoma cells carrying the EWS-FLI1 fusion gene.

No description available.

Submitted by Aelson Maciera (aelsoncm@terra.com.br) on 2017-08-01T17:24:55Z No. of bitstreams: 1 TeseMCS.pdf: 2997068 bytes, checksum: 7f84184a2f58192e394eaee3ae05cdec (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-01T19:08:53Z (GMT) No. of bitstreams: 1 TeseMCS.pdf: 2997068 bytes, checksum: 7f84184a2f58192e394eaee3ae05cdec (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-08-01T19:09:03Z (GMT) No. of bitstreams: 1 TeseMCS.pdf: 2997068 bytes, checksum: 7f84184a2f58192e394eaee3ae05cdec (MD5) / Made available in DSpace on 2017-08-01T19:15:57Z (GMT). No. of bitstreams: 1 TeseMCS.pdf: 2997068 bytes, checksum: 7f84184a2f58192e394eaee3ae05cdec (MD5) Previous issue date: 2016-09-06 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Cette thèse analyse la relation entre les pratiques policières quotidiennes et la conformation avec la police militaire de l'Etat de São Paulo, responsable pour mantenir l’ordre. Issu d’entretiens et d’analyses de données officielles, le présent travail cherche à comprendre comment les policiers, lors de leurs opérations, sélectionnent les personnes qui subiront les actions de la police et, par conséquent, celles du système de justice criminelle. Partant de là, il a été possible de comprendre, de manière plus particulier, quels sont les publics et les crimes les plus surveillés par les policiers militarisés et, d'une manière globale, les caractéristiques du modèle de police ostensible de São Paulo. L'objectif des opérations policières ostensibles est de prendre en flagrant délit des suspects criminels, ce qui confère à la Police Militaire la responsabilité de sélectionner et « d’expulser » des rues tous ceux que les policiers identifient comme des criminels. Cela autorise la Police Militaire à avoir recours notamment à trois actions : a) l’arrestation ou b) l’élimination des personnes identifiées comme des criminelles et c) le « bon sens » du policier en tant qu’outil pour distinguer les criminels et les « bons citoyens ». Une des conclusions de cette recherche repose sur le constat d’un ample usage du « bon sens » policier lors des opérations de vigilance ostensible, ainsi que de son aspect racial. De ce fait, le « bon sens » policier, au même titre que la létalité et les arrestations, est devenu le responsable de l’accumulation de désavantages pour la population jeune et noire, en ce qui concerne le droit à la vie en sécurité ; en effet on observe que ce groupe risque beaucoup plus d’aller en prison que le reste de la population. De plus, nous avons constaté que pendant les vingt dernières années la politique sécuritaire menée dans São Paulo s’est focalisé sur le type de vigilance ostensible décrit ci-dessus, et ce en dépit de l’échec de l’opération pour ce qui concerne la diminution du nombre de crimes contre les biens à São Paulo. / This thesis analyzes the relation between daily police practices and the conformation with ostensible policing run by the Military Police of São Paulo State. This work started from interviews and analysis of official data to understand how the Military Police, during the ostensible policing, selects the people who will suffer the police approach and therefore the prosecution of the criminal justice system. Thenceforth it was possible to understand specifically which public and which crimes were most closely watched by the military police and, in an embracing way, the characteristics of the São Paulo ostensible policing model. The purpose of ostensible policing is to catch criminal suspects, implying to the Military Police the responsibility to select and remove from the streets those who the police itself identifies as “bandits”. It is also observed that this framework does not configure a public security policy, but a crime and violence management, marked by the "war" against certain types of crimes that are available to the Military Police, especially with three features: a) imprisonment; b) elimination of those identified as “bandits”, and c) the “police scent” as a differentiation tool to identify "bandits" and "good citizens". One of the study highlights is that the wide use of the “police scent” by the military police which has racial aspects in its composition added to the lethality and imprisonment, became responsible for the accumulation of disadvantages for the young black population, referring to the right to secure life and a higher risk of being arrested for property offenses in relation to the rest of the population. In all, it became clear that in the last twenty years there is an insistence from the state public security in an ostensible policing with such characteristics, even in the face of failure in the decrease of numbers of property offenses in the state of São Paulo. / A tese analisa a articulação entre práticas policiais cotidianas e a conformação do policiamento ostensivo militarizado protagonizado pela Polícia Militar. O presente trabalho partiu de entrevistas e análise de dados oficiais da segurança pública paulista para compreender como os policiais, durante o policiamento ostensivo, selecionam as pessoas que sofrerão as investidas da polícia e, por conseguinte, do sistema de justiça criminal. A partir daí foi possível entender, de forma específica, quais são os públicos e os crimes mais vigiados pelos policiais militares e, de forma abrangente, as características do modelo de policiamento ostensivo paulista. Constatou-se que o objetivo do policiamento ostensivo é flagrar suspeitos criminais, o que incute à PM a responsabilidade de selecionar e “retirar” das ruas aqueles que os policiais identificam como “bandidos”. Para tal estão disponíveis à PM, sobretudo, três recursos: a) o aprisionamento ou b) a eliminação daqueles identificados como bandidos e c) o tirocínio policial enquanto ferramenta para diferenciar “bandidos” e “cidadãos de bem”. Uma das conclusões da pesquisa é a constatação da ampla utilização do tirocínio pelos policiais que atuam no policiamento ostensivo e de seu aspecto racializado; isto significa que, para a fundamentação da suspeita policial, são utilizados marcadores raciais. Desta forma, o tirocínio, ao lado da letalidade policial e do aprisionamento se tornaram responsáveis pelo acúmulo de desvantagens para a população jovem e negra, no que tange ao direito à vida segura e a um maior risco de serem presos por crimes patrimoniais em relação ao restante da população. No mais, constatou-se que nos últimos vinte anos há uma insistência por parte da segurança pública paulista num policiamento ostensivo com tais características, mesmo diante do insucesso na diminuição no número de crimes patrimoniais no estado de São Paulo.

The present work describes the phytochemistry study and of biological activity of the species Scutellaria racemosa Pers (Labiatae) and Pfaffia tuberosa Spreng (Amaranthaceae). Four compounds were isolated of the S. racemosa Pers: lupeol (14), oroxilin A (10), dinatin (12) and oroxyloside (11). The antimicrobial and cytotoxic activities and the capacity of inhibition of enzymes prolyl oligopeptidase (POP), dipeptidyl peptidase IV (DPP IV) and acetylcholinesterase (AChE) of the crude extract (EB), fractions and isolated compounds of the S. racemosa Pers were evaluated. The n-hexane (FH) and ethyl acetate (FA) fractions were the most active against Staphylococcus epidermidis, Bacillus subtilis and Pseudomonas aeruginosa. The evaluation of the crude extract and fractions using the Brine Shrimp Lethality Test indicated that this plant does not present toxicity. About the tests of enzymatic inhibition, the ethyl acetate (FA) and n-butanol (FB) fractions of S. racemosa Pers and the compounds dinatin (12) and oroxyloside (11) demonstrated significant capacity of inhibition of the POP. The inhibition promoted for the dinatina (12) (100 μM) corresponded 43% and for the oroxyloside (11) (100 μM) corresponded 34% of the total enzyme tested. The crude extract (EB) and the respective fractions of the Pfaffia tuberosa Spreng were also evaluated about the antimicrobial and cytotoxic activities and of enzymatic inhibition of the POP, DPP IV and AChE. Through these assays, it was verified that the crude extract (EB) and the fractions of the Pfaffia tuberosa Spreng do not present important antimicrobial and cytotoxic activity. In relation to the tests of inibitory activity of the POP, the dichloromethane (FD) and ethyl acetate (FA) fractions present IC50 of 21.4 and 28.5 μg/mL against of POP, respectively. Dichloromethane, ethyl acetate and n-butanol fractions (FD, FA and FB) presented low activity against DPP IV (< 20%). The ethyl acetate (FA) and n-butanol (FB) fractions showed significant inhibition of the AChE in the amounts, 6.25 and 25 μg, respectively. / O presente trabalho descreve o estudo fitoquímico e a atividade biológica das espécies Scutellaria racemosa Pers (Labiatae) e Pfaffia tuberosa Spreng (Amaranthaceae). Quatro compostos foram isolados da S. racemosa Pers: lupeol (14), oroxilina A (10), dinatina (12) e oroxilosídeo (11). A atividade antimicrobiana, citotóxica e a capacidade de inibição das enzimas prolil oligopeptidase (POP), dipeptidil peptidase IV (DPP IV) e acetilcolinesterase (AChE) do extrato bruto (EB), frações e compostos isolados da S. racemosa Pers foram avaliadas. As frações n-hexano (FH) e acetato de etila (FA) foram as mais ativas contra Staphylococcus epidermidis, Bacillus subtilis e Pseudomonas aeruginosa. A avaliação do extrato bruto (EB) e frações através do teste de letalidade frente a Artemia salina indicou que esta planta não apresenta toxicidade significativa. Quanto aos testes de inibição enzimática, as frações acetato de etila (FA) e n-butanol (FB) de S. racemosa Pers e os compostos dinatina (12) e oroxilosídeo (11) demonstraram significativa capacidade de inibição da POP. A inibição promovida pela dinatina (12) (100 μM) correspondeu a 43% e pelo oroxilosídeo (11) (100 μM) correspondeu a 34% do total de enzima testada. O extrato bruto (EB) e as respectivas frações da Pfaffia tuberosa Spreng também foram avaliadas quanto a atividade antimicrobiana, citotóxica e de inibição enzimática da POP, DPP IV e AChE. Através destes ensaios, verificou-se que o extrato bruto (EB) e a frações da Pfaffia tuberosa Spreng não apresentam importante atividade antimicrobiana e citotóxica. Em relação aos testes de atividade inibitória da POP, as frações diclorometano (FD) e acetato de etila (FA) foram as que apresentaram os melhores resultados, com IC50 de 21,4 e 28,5 μg/mL, respectivamente. As frações diclorometano, acetato de etila e n-butanol (FD, FA e FB) apresentaram baixa capacidade de inibição da DPP IV (< 20%). As frações acetato de etila (FA) e n-butanol (FB) apresentaram significativa inibição da AChE, nas quantidades de 6,25 e 25 μg, respectivamente.

Made available in DSpace on 2014-07-29T15:07:43Z (GMT). No. of bitstreams: 1 Dissertacao Duanne A da Silva.pdf: 1367420 bytes, checksum: 2450c56c8175888ad1aaa259e17f1059 (MD5) Previous issue date: 2011-02-24 / Herpetic meningoencephalitis is an infection of the central nervous system caused by bovine herpesvirus 5 (BoHV-5) despite BoHV-1 also being associated with this pathology. The course of the disease is usually fatal. Meningoencephalitis has caused significant economic burden to the Brazilian cattle industry due to its high mortality rate, especially in young animals. Because of cross-reactivity and the absence of an effective serological test that differentiates types 1 and 5 of herpesvirus, the actual prevalence of infection with both viruses is unknown. It is estimated that some animals, supposedly infected with type 1, may be seropositive for type 5. For a better understanding of the molecular epidemiology of this virus in Goiás, 110 brain samples of young cattle, that died with neurological signs, referred to the reference laboratory of the State of Goiás for rabies diagnosis in 2008, were analyzed by multiplex PCR to amplify the glycoprotein C (gC) region of the BoHV-1 and -5 DNA. Of the 110 samples, 53.6% were positive for bovine herpesvirus. Of these, 22.0% were positive for BoHV-1, whereas 52.5% were positive for BoHV-5. Furthermore, 13.6% of samples showed co-infection for types 1 and 5. Among these, one sample came from a buffalo. Positivity for both bovine herpesvirus and rabies virus was observed in 53.3% of samples. This study showed that BoHV-1 and -5 are circulating among cattle in the State of Goiás and that herpesvirus type 1 can also be encephalitogenic. Although there was no evidence of viral replication, after inoculation of all 20 brain samples in cell culture, the possibility of viruses being associated with neurological disease cannot be ruled out. This was the first study to demonstrate the presence of BoHV-1 and BoHV-5 in samples obtained from herds in Goiás. / A meningoencefalite herpética é uma infecção do sistema nervoso central de curso geralmente fatal causada pelo herpesvírus bovino tipo 5 (BoHV-5), todavia, também o BoHV-1 pode estar associado a essa patologia. A enfermidade tem gerado muitos prejuízos à pecuária brasileira por apresentar elevada letalidade, principalmente, entre animais jovens. Devido a reatividade cruzada e a falta de um teste sorológico eficaz que diferencie os tipos 1 e 5 de herpesvírus bovino, não se sabe a real prevalência das infecções por ambos os vírus. Estima-se que uma parcela dos animais supostamente infectados pelo tipo 1 possa ser soropositiva para o tipo 5. Para um melhor entendimento da epidemiologia molecular desses vírus em Goiás, 110 amostras de cérebro de bovinos jovens que vieram a óbito com sinais neurológicos, encaminhadas ao laboratório de referência do Estado para diagnóstico de raiva no ano de 2008, foram analisadas por PCR multiplex para amplificação da região da glicoproteína C (gC) do DNA do BoHV-1 e -5. Das 110 amostras, 53,6% foram positivas para herpesvírus bovino. Destas, 22,0% foram positivas para o BoHV-1, enquanto 52,5% foram positivas para o BoHV-5. Além disso, encontrou-se 13,6% de coinfecção para os tipos 1 e 5. Entre estas, uma amostra foi proveniente de um bubalino. Foi observada positividade simultânea para herpesvírus bovino e o vírus da raiva em 53,3% das amostras. Este estudo permitiu concluir que o BoHV-1 e o -5 circulam no Estado de Goiás e que o BoHV-1 apresenta potencial encefalitogênico. Apesar de não ter ocorrido multiplicação viral em nenhuma das 20 amostras de cérebro inoculadas em cultura de célula, não se pode descartar a possibilidade da doença neurológica estar associada aos vírus nas outras amostras analisadas. Este foi o primeiro estudo com amostras de Goiás a comprovar a presença dos dois vírus nos rebanhos do Estado.

Les extrémités des chromosomes contiennent des répétitions de séquences d’ADN appelées télomères qui empêchent l’activation inopportune de la réponse aux dommages de l'ADN afin de préserver l'intégrité génomique. Les télomères raccourcissent à chaque cycle de réplication d’ADN et la télomérase a pour fonction de contrebalancer cette érosion en allongeant les télomères. Les cellules somatiques n’expriment pas la télomérase, donc leur durée de vie est normalement limitée par ce raccourcissement progressif des télomères qui conduit à l'activation de la voie p53 entraînant un arrêt de la croissance cellulaire. En revanche, les cellules cancéreuses acquièrent l'immortalité cellulaire principalement en réactivant la télomérase ou en utilisant des méthodes alternatives d'allongement des télomères basées sur la recombinaison d’ADN. Auparavant, dans notre laboratoire, un criblage CRISPR à l'échelle du génome a été réalisé dans la lignée cellulaire pré-B NALM-6 traitée avec la molécule BIBR1532, un inhibiteur de la télomérase. Ces résultats suggéraient que cinq sous-unités du complexe de remodelage de la chromatine INO80, lorsque supprimées indépendamment, réduisaient la prolifération des cellules ayant un raccourcissement des télomères induit par le BIBR1532. Mon objectif était d'étudier cette interaction génétique afin de comprendre les processus biologiques impliqués dans cette létalité synthétique. Après l'élimination des gènes codant à la fois pour la sous-unité enzymatique de la télomérase humaine (hTERT) ainsi que les sous-unités spécifiques du complexe INO80 humain, nous avons constaté que les cellules double-négatives avaient une capacité proliférative réduite, ce qui démontre que l’interaction génétique mesurée par criblage CRISPR est bel et bien spécifique. Étant donné le rôle du facteur de transcription p53 dans la réponse cellulaire au raccourcissement télomérique, nous avons exploré l’importance de cette voie de signalisation pour l’interaction entre le complexe INO80 humain et la télomérase. Après l’activation de p53 avec un traitement avec la molécule nutlin-3a, les niveaux d'expression de plusieurs cibles de p53 tels que MDM2 et CDKN1A ont augmenté dans les cellules ayant une délétion du gène NFRKB, codant pour une sous-unité du complexe INO80 humain. Les cellules ayant une délétion du gène UCHL5, codant pour le partenaire d’interaction de NFRKB, ont également montré une augmentation de l’expression de MDM2 lorsque traitées avec nutlin-3a. Enfin, la perte de télomérase (hTERT) modifie les niveaux d'expression des composants de la 2 voie p53 CDKN1A, BAX et MDM2. En conclusion, la suppression des gènes codant pour des sous-unités du complexe INO80 telles que NFRKB ou UCHL5 est nuisible aux cellules ayant une délétion de la télomérase. Le complexe INO80 humain peut être impliqué dans l'inhibition de la voie p53, en réponse à l'activation de p53 soit par des télomères courts ou avec un traitement avec nutlin-3a. Des recherches plus approfondies sur cette interaction génétique pourraient mener au développement de nouvelles thérapies combinatoires afin d’inhiber la croissance des cellules cancéreuses. / The ends of chromosomes contain telomeric repeats that prevent the DNA damage response from being activated in order to preserve genomic integrity. Telomerase functions to alleviate incomplete DNA replication at telomeres, and to repair those telomeres damaged by various means including oxidative damage. The lifespan of telomerase negative somatic cells is normally restricted by gradual telomere shortening which can lead to the activation of the p53 pathway resulting in cellular growth arrest. Cancer cells often elongate their telomeres in order to acquire cellular immortality predominantly by reactivating telomerase or by using recombination-based, alternative telomere lengthening methods. Previously in our lab, a genome-wide CRISPR screen was conducted in the pre-B cell line NALM-6 treated with a small molecule inhibitor of telomerase, BIBR1532. These previous results suggested that five subunits of the INO80 chromatin-remodeling complex, when independently deleted, reduced cellular proliferation in cells with BIBR1532 induced telomere shortening. My goal was to investigate this genetic interaction in order to understand the biological processes implicated in this synthetic lethal relationship. After the knockout of the genes encoding both the enzymatic subunit of human telomerase (hTERT) and specific subunits of the human INO80 complex, I found that the proliferative capacity of NALM-6 cells was reduced. This result indicates the genetic interaction identified by CRISPR screening is in fact specific. In addition, after p53 stimulation with nutlin-3a treatment, expression levels of the p53 pathway component MDM2 were altered after the knockout of the genes encoding specific subunits of the human INO80 complex, NFRKB and UCHL5, individually. CDKN1A expression was also altered after nutlin-3a treatment and NFRKB knockout. Finally, the loss of telomerase (hTERT) alters the expression levels of the p53 pathway components CDKN1A, BAX and MDM2. In conclusion, the deletion of the genes encoding specific subunits of the INO80 complex, including NFRKB and UCHL5, is harmful to cells after hTERT knockout. The human INO80 complex may be involved in inhibiting the p53 pathway, in response to p53 activation by short telomeres or nutlin-3a treatment. Further investigation into this synthetic lethal relationship may shed light on new combinatorial therapeutics in cancer.

Mutations in the hereditary breast/ovarian cancer genes BRCA1/2 were shown to be synthetic lethal with poly(ADP-ribose) polymerase inhibitors (PARPi). This toxicity is assumed to derive from PARPi-induced DNA double strand breaks (DSBs) that necessitate BRCA function in homologous recombination (HR) and/or fork protection (FP). However, PARPi accelerates replication forks. While high-speed replication could cause DSBs, the finding that PARPi leads to single-stranded DNA (ssDNA) gaps/nicks suggests replication gaps could also or alone be the cause of synthetic lethality. Here, we demonstrate that PARPi toxicity derives from replication gaps. Isogenic cells deficient in BRCA1 or the BRCA1-associated FANCJ, with common DNA repair defects in HR and FP, exhibit opposite responses to PARPi. Deficiency in FANCJ, a helicase also mutated in hereditary breast/ovarian cancer and Fanconi anemia, causes aberrant accumulation of fork remodeling factor HLTF and limits unrestrained DNA synthesis with ssDNA gaps. Thus, we predict replication gaps as a distinguishing factor and further uncouple HR, FP and fork speed from PARPi response. BRCA-deficient cells display excessive gaps that are diminished upon resistance, restored upon re-sensitization and when targeted augment synthetic lethality with PARPi. Furthermore, we define the source of gaps to defects in Okazaki fragment processing (OFP). Unchallenged BRCA1-deficient cells have elevated poly(ADP-ribose) and chromatin-associated PARP1 but aberrantly low XRCC1 indicating a defective backup OFP pathway. Remarkably, 53BP1 loss resuscitates OFP by restoring XRCC1-LIG3 that suppresses the sensitivity of BRCA1-deficient cells to drugs targeting OFP or generating gaps. Collectively, our study highlights unprotected lagging strand gaps as a determinant of synthetic lethality, providing a new paradigm and biomarker for PARPi toxicity.

No description available.