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Construção e manipulação de clone infeccioso de uma amostra brasileira do vírus da diarreia viral bovina / Construction and manipulation of infectious clone from a brazilian bovine viral diarrhea virus isolateArenhart, Sandra 29 March 2012 (has links)
Conselho Nacional de Desenvolvimento Científico e Tecnológico / Bovine viral diarrhea virus (BVDV) is a worldwide pathogen associated with
important losses to livestock production. Most of these losses come from reproductive
disorders and from the ability of the virus to produce persistent infections following in utero
infection of the fetus. A number of reverse genetics methodologies have been used for BVDV
in order to better understand the biology of the virus, which allowed the elucidation of a
number of biological features including virus replication, host-virus interaction, immune
response, and the pathogenesis of fetal infection. The present study describes the construction,
characterization and manipulation of an infectious clone out of a non-cytophatic Brazilian
BVDV strain IBSP4-ncp. The cDNA recombinant clone was constructed by yeast
homologous recombination with a low-copy vector, from three genomic fragments
comprising the open reading frame (ORF). The two untranslated regions (5' and 3' UTR) were
replaced by the respective UTRs of the reference strain NADL. The constructed vector was
transcribed in vitro and the resulting RNA was transfected on MDBK cells to rescue
infectious virus. The rescued viruses (IC-pBSC_IBSP4-ncp#2 and #3) were maintained for
ten passages in tissue culture and characterized in vitro, showing replication dynamics, focus
size and morphology similar to those of the parental IBSP-4. Genomic analysis revealed five
point mutations in the gene coding for Npro protein, resulting in amino acid changes. These
mutations probably reflect an adaptation of the virus to the heterologous UTRs. The infectious
clone IC-pBSC_IBSP4-ncp#2 was further used for the construction of a recombinant virus
expressing the Gaussia luciferase (Gluc) reporter gene. The reporter gene was inserted
between the Npro and Core genes, being flanked by an upstream linker and a downstream
sequence of the Foot and Mouth Disease virus protease (FMDV2Apro) for accurate protein
processing. The recombinant vector was in vitro transcribed and the RNA was transfected on
MDBK cells. Recombinant infectious viruses were rescued (IC-pBSC_IBSP4-ncpGluc#3 and
#4) and characterized in vitro, showing replication dynamics, focus size and morphology
similar to those of the parental IBSP-4 clone. The Gluc reporter gene was accurately
expressed and processed by the recombinant virus during 15 passages in tissue culture. These
studies revealed that the infectious clone constructed herein can be easily manipulated and is
able to carry in its genome heterologous genes up to 555 base pairs in length in a stable
fashion and without interference with its replication efficiency. Thus, the constructed clone
may be very useful for genetic manipulation towards studying different aspects of the BVDV
biology and its interactions with the host, and for the development of vaccine strains with
attenuated phenotype and/or with antigenic markers. / O vírus da diarreia viral bovina (BVDV) é um patógeno de bovinos distribuído
mundialmente, associado com importantes perdas econômicas. As maiores perdas devem-se
aos problemas reprodutivos causados pela infecção, e pela capacidade do vírus de causar
persistência após infecção fetal no terço inicial da gestação. Para entender melhor a biologia
desse vírus, sistemas de genética reversa foram desenvolvidos e tem permitido a elucidação
de vários aspectos da replicação viral, interação vírus hospedeiro, resposta imune e
patogenia da infecção fetal. O presente estudo relata a construção, caracterização e
manipulação de um clone infeccioso, a partir da cepa brasileira não-citopática IBSP4-ncp. O
clone de DNA recombinante foi construído pela técnica de recombinação homóloga em
levedura, utilizando um vetor de baixo número de cópias, construído a partir de três
fragmentos genômicos, que compreendiam a fase aberta de leitura (open reading frame, ORF)
do vírus. As duas regiões não traduzidas (5 e 3 UTR) foram substituídas pelas respectivas
UTRs da cepa de referência NADL. O vetor construído foi transcrito in vitro e o RNA obtido
foi transfectado em células MDBK para recuperação de vírus infecciosos. Os vírus
recuperados (CI-pBSC_IBSP4-ncp#2 e #3) foram mantidos por 10 passagens em cultivo
celular e caracterizados in vitro, apresentando dinâmica de replicação, tamanho e morfologia
de focos similares ao vírus parental IBSP-4. A análise do genoma por sequenciamento revelou
cinco mutações pontuais no gene Npro, com trocas de aminoácidos, provavelmente refletindo
uma adaptação do vírus às UTRs heterólogas. O clone infeccioso construído CIpBSC_
IBSP4-ncp#2, foi então utilizado para a construção de um vírus recombinante
expressando o gene repórter Gaussia luciferase (Gluc). O gene repórter foi inserido entre os
genes Npro e Core do vírus. Para o processamento da proteína repórter, uma sequência ligante
foi adicionada anteriormente ao gene, e a sequência da protease do vírus da Febre Aftosa
(FMDV2Apro) foi inserida após o gene. O vetor recombinante construído foi transcrito in vitro
e o RNA obtido foi transfectado em células MDBK. Vírus recombinantes infecciosos foram
recuperados (CI-pBSC_IBSP4-ncpGluc#3 e #4) e caracterizados in vitro, apresentando
dinâmica de replicação, tamanho e morfologia de focos similares ao vírus obtido do clone
infecioso. O gene repórter Gluc foi corretamente expresso e processado pelo vírus
recombinante durante 15 passagens em cultivo celular. Com os resultados obtidos nestes
estudos, conclui-se que o clone infeccioso construído pode ser facilmente manipulado e é
capaz de carrear em seu genoma, e expressar de forma estável, genes heterólogos com até 555
pares de base, que parecem não interferir com sua capacidade replicativa. Dessa forma, o
clone obtido pode ser muito útil para manipulação genética visando estudar diferentes
aspectos da biologia do BVDV e de suas interações com o hospedeiro, assim como para a
produção de cepas vacinais com fenótipo atenuado e/ou com marcadores antigênicos.
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Análises da epidemiologia molecular e evolução de pestivírusWeber, Matheus Nunes January 2016 (has links)
O gênero Pestivirus da família Flaviviridae, compreende vírus de genoma RNA fita simples e polaridade positiva que comumente estão associados a infecções em ruminantes e suídeos. Possui quatro espécies reconhecidas: o vírus da diarreia viral bovina tipo 1 (BVDV-1), o BVDV-2, o vírus da doença da fronteira (BDV) e o vírus da peste suína clássica (CSFV). Além disso, possui possíveis espécies atípicas, sendo o vírus ‘HoBi’-like a mais reconhecida na literatura. Com o objetivo de gerar mais informações acerca da epidemiologia molecular de pestivírus no País e dos mecanismos na evolução de vírus do gênero, o presente trabalho será apresentado sob a forma de seis artigos científicos. Os trabalhos visam a caracterização de pestivírus detectados em bovinos e javalis no Rio Grande do Sul, a identificação de cepas resultantes de provável recombinação homóloga, a análise das variantes virais intrahospedeiro (quasispécies) de animais infectados pelo vírus ‘HoBi’-like, e a comparação da multiplicação in vitro de diferentes cepas de pestivírus em cultivos celulares oriundos de diferentes raças bovinas. No primeiro trabalho, amostras de soro bovino de animais do Rio Grande do Sul foram obtidas junto ao serviço veterinário oficial e submetidas a RT-PCR para detecção de pestivírus seguida de sequenciamento e análise filogenética das amostras positivas, onde 57,6% foram classificadas como BVDV-1 e 42,4% como BVDV-2, sendo constada frequência elevada de BVDV-2 quando comparada a outras regiões no mundo. No segundo trabalho, 40 amostras de javalis cativos obtidas de abatedouro foram testadas utilizando metodologia semelhante a anteriormente mencionada, e foi possível a detecção de uma amostra positiva, que foi classificada como BVDV-2, sendo a primeira evidência molecular do BVDV em javalis na literatura. No terceiro artigo científico, foi pesquisado a ocorrência de recombinação homóloga, utilizando metodologia in silico, em sequências de pestivírus presentes em banco de dados públicos, onde foram descritos novos possíveis eventos de recombinação entre BVDV-1a e BVDV-1b, BVDV-2a e BVDV-2b e CSFV-2.2 com outro genogrupo indefinido. No quarto e quinto trabalhos, animais persistentemente infectados com o vírus ‘HoBi’-like foram gerados experimentalmente e, utilizando RT-PCR seguida de clonagem, sequenciamento e análises in silico, a composição e variações intrahospedeiro das quasispécies virais em circulação foram avaliadas, onde foi possível observar que características individuais do hospedeiro são importantes na seleção de variantes virais e que a nuvem de mutantes aumenta com o passar do tempo, não havando quasispécie dominante. Por fim, no sexto artigo científico, a multiplicação de cepas de BVDV-1a e 1b, BVDV-2a e vírus ‘HoBi’-like foram avaliadas in vitro em células de cultivo primário obtidas de gado europeu, zebuíno e raça mista e comparadas utilizando modelos estatísticos, onde foi possível a observação de ausência de diferenças entre as raças (P=0,88), mas presença de diferença entre indivíduos da mesma raça (P˂0,05). Os resultados aqui apresentados somam informações acerca da epidemiologia molecular, biologia e evolução dos pestivírus, sendo importantes para embasar futuras campanhas de controle e erradicação das doenças causadas por pestivírus. / The genus Pestivirus of the family Flaviviridae, comprises single stranded RNA-viruses that are commonly associated with infections in ruminants and swine. It has four recognized species: bovine viral diarrhea virus 1 (BVDV-1), BVDV-2, border disease virus (BDV) and classical swine fever virus (CSFV). It also has putative atypical species where the ‘HoBi’-like virus is the most highlighted in the literature. In order to generate more information about molecular epidemiology of pestiviruses and important mechanisms in the evolution of the virus genus, this work will be presented in six scientific articles form. The works aimed in the characterization of pestivirus detected in cattle and wild boar in Rio Grande do Sul state, identification of strains resulting from putative homologous recombination, analysis of intrahost viral variants (quasispecies) of animals infected with ‘HoBi’-like viruses, and the comparison of in vitro growth of different pestivirus strains in cell cultures derived from different cattle breeds. In the first study, bovine serum samples of cattle from Rio Grande do Sul state were collected by the official veterinary service and submitted to RT-PCR for detection of pestivirus followed by DNA sequencing and phylogenetic analysis of the positive samples, where 57.6% were classified as BVDV-1 and 42.4% as BVDV-2, which revealed high frequency of BVDV- 2 when compared to other regions worldwide. In the second work, 40 captive wild boar lung samples obtained from slaughterhouse were tested using similar methodology described above, and one sample resulted positive and was classified as BVDV-2, which represented the first molecular evidence of BVDV in wild boars. In the third scientific article, the occurrence of homologous recombination was investigated using in silico methods applied to sequences available in public databases, which are described putative new events between BVDV-1a and BVDV-1b, BVDV-2a and 2b and CSFV-2.2 and undetermined genogroup. In the fourth and fifth studies, animals persistently infected with the ‘HoBi’-like viruses were experimentally generated and using RT-PCR followed by cloning, sequencing and in silico analysis, the composition and intrahost variations of viral quasispecies in circulation were evaluated, where it was observed that individual characteristics of the host are important in the selection of viral variants and the mutant clouds increased overtime. Finally, in the sixth scientific article, the growth of BVDV-1a and 1b, and BVDV-2b, ‘HoBi’-like virus strains were evaluated in vitro using primary cell cultures obtained by taurine, indicine and mixed breed cattle using statistical models, it was possible to observe the absence of differences between cattle breeds (P=0.88), but presence of difference between individuals within the breed (P˂0.05). The results presented herein add information about the molecular epidemiology, biology and evolution of pestiviruses, being important to support future control and eradication programs of diseases caused by pestiviruses.
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Estudos sobre vacinologia e evolução do vírus da cinomose caninaBudaszewski, Renata da Fontoura January 2017 (has links)
O vírus da cinomose canina (CDV) é um importante patógeno de cães domésticos e carnívoros selvagens. A infecção pelo CDV é relevante a nível mundial e está associada com alta morbidade e mortalidade. Em diversos países a cinomose é considerada controlada pelo uso de vacinas, no entanto, no Brasil ainda é endêmica, principalmente devido ao grande número de animais não domiciliados. Além disso, surtos em cães e várias espécies de animais silvestres ocorrem com frequência, dizimando populações ameaçadas. As vacinas vivas atenuadas são seguras para cães, mas seu uso não é aconselhado em espécies altamente suscetíveis à infecção pelo CDV. Também os relatos de surtos de cinomose em cães supostamente vacinados levantam a hipótese de que as vacinas disponíveis no mercado podem não ser eficientes frente a algumas cepas de campo. Com o objetivo de gerar dados acerca dos mecanismos de evolução do CDV e desenvolver e testar a eficácia de uma vacina bivalente inativada contra o vírus da raiva (RABV) e CDV a presente tese será apresentada na forma de dois artigos científicos. Ainda, um artigo de revisão sobre os modelos animais utilizados para obtenção de informações sobre o vírus do sarampo utilizando a infecção de CDV em furões e cães foi publicada e será apresentada na presente tese. No primeiro artigo, foi analisada a ocorrência de recombinação homóloga em genomas de CDV e detectou-se oito possíveis vírus recombinantes, incluindo um evento de recombinação entre uma cepa de campo e uma cepa vacinal atenuada, sugerindo que o uso de vacinação com vírus vivo atenuado pode influenciar a evolução do CDV. No segundo trabalho, uma vacina recombinante bivalente inativada baseada em RABV expressando as glicoproteínas do envelope do CDV, hemaglutinina e proteína de fusão, mostrou-se eficiente na proteção contra infecção por CDV em furões quando utilizado um protocolo prime/boost. Finalmente, foi publicada uma revisão de literatura sobre os modelos animais utilizados para obtenção de informações sobre a patogênese do vírus do sarampo utilizando a infecção com o vírus da cinomose. / Canine distemper virus (CDV) is an important pathogen of domestic dogs and wild carnivores. CDV infection is globally relevant and it is associated with high morbidity and mortality. In several countries, distemper is considered controlled by vaccination, however, in Brazil it is still endemic, mainly due to the large number of non-domiciliated animals. In addition, outbreaks in dogs and various species of wild animals occur frequently, decimating threatened populations. Live attenuated vaccines are safe for dogs, but their use is not advised in species that are highly susceptible to CDV infection. Also, reports of canine distemper in supposedly vaccinated dogs raise the hypothesis that commercially available vaccines may not be effective against some wild type strains. In order to investigate the mechanisms of CDV evolution and to develop and assess the efficacy of an inactivated bivalent vaccine against rabies virus and CDV, this thesis will be presented in the form of two scientific papers. Furthermore, a review article on the animal models used to gain information on measles virus using CDV infection in ferrets and dogs has been published and will be presented in this thesis. In the first paper, the occurrence of homologous recombination in CDV genomes was analyzed and eight possible recombinant viruses were detected, including a recombination event between a wild type strain and an attenuated vaccine strain, suggesting that the use vaccines based on attenuated live virus may influence CDV evolution. In the second study, an inactivated bivalent recombinant vaccine based on RABV expressing CDV envelope glycoproteins, hemagglutinin and fusion protein, proved to be effective in protecting against CDV infection in ferrets when using a prime/boost protocol. Finally, a literature review was published on the animal models used to obtain information on the pathogenesis of measles virus using infection with canine distemper virus.
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Funkční in vitro analýza alternativních sestřihových variant genu BRCA1 / The functional in vitro analysis of the BRCA1alternative splicing variantsŠevčík, Jan January 2012 (has links)
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...
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Influência do gene PTEN na expressão de RAD51 e suas parálogas, RAD51C e RAD51B, em linhagens de glioblastoma multiforme tratadas com etoposídeo / PTEN gene Influence in expression of RAD51 and its Paralogs RAD51C and RAD51B, in Glioblastoma strains treated with EtoposideAna Clara Oliveira 12 May 2016 (has links)
O Glioblastoma Multiforme (GBM) é o tipo de tumor cerebral maligno com maior incidência na população. A perda do gene PTEN (fosfatase e tensina homóloga) é uma alteração comum associada ao GBM (até 60%) e esse gene codifica uma enzima que antagoniza a ação de PI3K, inibindo a fosforilação de AKT e, desse modo, regulando vias de sinalização relativas à sobrevivência celular e proliferação. Mutações em PTEN têm sido associadas à instabilidade genômica e ao aumento no número de quebras de fita dupla, além de serem relacionadas também à redução da expressão de RAD51, a qual é uma proteína-chave da via de reparo por recombinação homóloga (HR). Diante disso, o objetivo deste estudo foi avaliar se o status de PTEN interfere na expressão de RAD51 e proteínas parálogas (RAD51C e RAD51B) e, consequentemente, se PTEN é capaz de influenciar a eficiência de HR. Com o objetivo de induzir a formação de quebras de fita duplas (DSBs) no DNA, as células foram tratadas com a droga antitumoral etoposídeo, que produz quebras no DNA, principalmente duplas (DSBs). Duas linhagens de GBM com status diferentes de PTEN foram utilizadas: T98G (PTEN mutado) e LN18 (PTEN tipo selvagem). As células de GBM foram tratadas com etoposídeo em diferentes experimentos ou ensaios: proliferação celular, quantificação da necrose e apoptose, cinética do ciclo celular, imunofluorescência da proteína ?- H2AX, quantificação dos níveis de expressão de RAD51 e parálogas e o silenciamento de PTEN na linhagem LN18. Os resultados mostraram que a linhagem LN18 foi mais sensível à droga nos tempos iniciais (24 e 72 h) (até 61,2% de redução), em comparação com a T98G (até 12,3% de redução); no tempo mais tardio de análise (120 h), ambas as linhagens sofreram redução acentuadana proliferação. Adicionalmente, a LN18 exibiu maior porcentagem de células apoptóticas e necróticas, em comparação com a linhagem T98G, nos tempos de24, 72 e 120 horas após o tratamento. O ensaio de imunofluorescência revelou maior indução de células positivas para ?-H2AX na linhagem LN18 em relação à T98G (p =<0,001), após tratamento com etoposídeo (50 e 75 ?M). Nessas concentrações, a análise da cinética do ciclo celular mostrou um bloqueio na fase G2 em ambas as linhagens (p<0,01) nos tempos analisados (24, 48 e 72h), mas apenas a linhagem LN18 revelou bloqueio na fase S. A expressão de RAD51, RAD51B e C foi mais elevada em LN18 em comparação com a T98G e U87MG, nas células tratados (75?M) e controles. PTEN foi silenciado (siRNA-PTEN) na linhagem LN18 para verificar se a redução da expressão desse gene reduziria também a expressão de RAD51 e parálogas. Após 72 horas de silenciamento, com 69,9% de inibição de PTEN, a expressão de RAD51 e RAD51C também se mostrou reduzida em relação ao grupo controle. Em conjunto, os resultados obtidos no presente estudo indicam que o status de PTEN é crucial para as vias de sobrevivência, controle do ciclo celular e indução de apoptose nas células de GBM, indicando a relação entre PTEN e RAD51 e parálogas nas células de GBM tratadas com um indutor de quebras no DNA. Adicionalmente, outras ferramentas de estudo são requeridas para investigar as vias moleculares e possíveis interações e complexos proteicos envolvendo a participação de PTEN e RAD51 e suas proteínas parálogas / Glioblastoma multiforme (GBM) is the most common malignant brain tumor. Loss of PTEN (Phosphatase and tensin homolog deleted on chromosome 10) gene is the most frequent alteration associated with GBM and encodes a phosphatase enzyme that antagonizes the PI3K, by inhibiting AKT phosphorylation thereby regulating signaling pathways related to cell survival and proliferation. PTEN deficiency has been associated with genomic instability and increased endogenous DSBs, as well as reduced expression of RAD51, which is a key gene with crucial role in HR. In this study, we aimed to evaluate whether the PTEN status in GBM cell lines can affect RAD51 expression and HR efficiency under conditions of treatment with the antineoplastic drug etoposide, which targets the DNA topoisomerase II enzyme, thus leading to the production of DNA breaks. T98G (PTEN mutated) and LN18 (PTEN wild-type) cells were treated with etoposide, and several assays were carried out: cell proliferation, detection and quantification of necrosis and apoptosis, cell cycle kinetics, immunofluorescence staining, RAD51 (and paralogs) protein expression, and PTEN silencing in LN18 cell line, by using the siRNA method. LN18 cells showed a greater reduction in cell proliferation, compared to T98G after treatments (25, 50, 75 e 100 µM) at 24, 72 and 120h. Both cell lines showed a significant increase (p=<0.001) in cell death induction, but LN18 presented a greater percentage of apoptotic and necrotic cells than T98G (24, 72 and 120h). The induction of DSB was analyzed by immunostaining (with ?-H2AX antibody), and for the concentrations (50 and 75 µM) tested, LN18 showed higher levels of ?-H2AX positive cells than that observed for T98G (p=<0.001). The analysis of cell cycle kinetics performed for cells treated with etoposide (50 and 75 µM) and collected at 24, 48 and 72h, LN18 presented a greater G2-blockage, as compared to T98G; only LN18 showed a blockage at the S-phase. The expression of RAD51, RAD51B and C was higher in LN18 compared to T98G and U87MG cells treated with etoposide (75 µM) and controls. When we silenced PTEN in LN18 linage, to check if PTEN silencing may reduce the expression of RAD51 and its paralogs, we found a 69.9% reduction in PTEN protein expressions, and the expression of RAD51 and RAD51C was also found reduced, compared to the control group. Taken together, the results obtained in this study indicate that the status of PTEN is critical for survival pathways, cell cycle control and induction of apoptosis in GBM cells, confirming the relationship between PTEN and RAD51 and its paralogs in GBM cells treated with an inducer of DNA breaks. These results contribute with relevant information for further studies on molecular pathways underlying the interaction between PTEN and RAD51 and its paralogs
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Mechanismy reparace DNA v mechu Physcomitrella patens / Mechanisms of DNA repair in the moss Physcomitrella patensHolá, Marcela January 2015 (has links)
Over the course of an organism's life, its genome is exposed to endogenous and exogenous chemical, physical and biological agents - genotoxins. These genotoxins alter its basic structural components - sugar residues, phosphodiester bonds, and nitrogenous bases. Organisms have therefore evolved a plethora of different strategies to both repair DNA lesions and maintain genomic stability. These DNA repair pathways are linked with several other cell pathways, including chromatin remodelling, DNA replication, transcription, cell cycle control, apoptosis - programmed cell death (PCD), thereby providing a coordinated cellular response to DNA damage. Biochemical mechanisms of DNA repair are relatively well understood in yeast and mammals, however, far less so in plants. While these repair mechanisms are evolutionary conserved, significant differences still remain. Therefore, further investigation is required. This thesis summarises the introduction of a novel plant model - the moss, Physcomitrella patens (Physcomitrella). As a haploid gametophyte with unique characteristics of high frequency of homologous recombination (HR), and apical growth of filaments, it is an ideal organism to study DNA repair in plants. Previous research on Physcomitrella regarding mechanisms of DNA lesion repair induced by...
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Geração de linhagens de células CHO transfectadas com vetores para expressão de anticorpos monoclonais humanizados anti-determinantes leucocitários: anti-CD3 e anti-CD18. / Generation of CHO cell lines expressing humanized monoclonal antibodies anti-leukocytary determinants: anti-CD3 and anti-CD18.Flávia Serpieri 23 October 2009 (has links)
O projeto de obtenção de huAcMos (Anticorpos Monoclonais Humanizados) tinha como escopo a humanização de anticorpos murinos com potencial terapêutico, inserção das sequências em vetores de expressão e transfecção em células CHO (do inglês, Chinese Hamster Ovary). A expressão do huAcMo Anti-CD18 resultou em baixos níveis da proteína recombinante e inciamos o processo de expressão de isoformas do huAcMo Anti-CD3. As células foram transfectadas com seqüências codificadoras do fragmento FvFc Anti-CD3 e clonadas pelo equipamento ClonePix FL. O fragmento foi caracterizado e demonstrou uma menor afinidade quando comparada com a molécula murina original. Ulizamos o sistema de recombinação homóloga (CHO Flp-In, Invitrogen) para expressão da molécula inteira do huAcMo Anti-CD3. Os clones foram caracterizados e demonstrou, assim como o fragmento FvFc, uma menor afinidade pelo alvo. As diferenças nas propriedades de ligação são freqüentemente encontradas após processos de humanização; dependendo da função efetora esta diminuição de afinidade não é negativa para a molécula. / The humanized antibodies (huMab) project intent to use murine antibodies with therapeutic potencial to obtain more human sequences with maintened specificity. The sequences were inserted in expression vectors and transfected in CHO (Chinese Hamster Ovary) cells. Anti-CD18 huMab expression results in low levels of recombinant protein and lead us to try the expression of Anti-CD3 isoforms. The cells were transfected for the expression of a FvFc antibody fragment and cloned using ClonePix FL equipment. The fragment characterization demonstrate a lower affinity when compared with the murine molecule. We use the homologous recombination system (CHO Flp-In) for the expression of the whole molecule of huMab Anti-CD3; like the FvFc fragment, the whole molecule demonstrate a lower affinity for the target. The differences in the affinity properties are frequently found after humanization process and depending on the expected efector functions is not negatively characterized.
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Vývoj rychlé metody cílené mutageneze bakterie Streptococcus zooepidemicus / Development of a fast method for site-directed mutagenesis in Streptococcus zooepidemicusČerný, Zbyněk January 2016 (has links)
This diploma thesis is focused on development of a fast method for site-directed gene mutagenesis in Streptococcus zooepidemicus based on the mechanism of natural competence. Several genes were selected based on experimental data which highly probably influence hyaluronic acid synthesis. The deletion of the selected genes from genomic DNA was performed as proof of concept, and the resulting recombinant strains were characterized regarding changes of hyaluronic acid precursor concentrations (glucuronic acid and N-acetylglucosamin) in time of cultivation and the end production of hyaluronic acid.
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Fonctions et régulations des protéines PARP2 et de XRCC1 dans la réparation des dommages à l’ADN / Functions and Regulation of PARP2 and XRCC1 Proteins in DNA RepairFouquin, Alexis 15 September 2017 (has links)
Les modifications post-traductionnelles des protéines par des polymères d’ADP-ribose (PAR) ou par phosphorylation permet l’assemblage des complexes de la réparation de l’ADN à la chromatine endommagée dont les fonctions sont essentielles pour assurer le maintien de la stabilité du génome. En réponse aux lésions de l’ADN, l’activité de synthèse de PAR des protéines PARP1 et PARP2 est fortement stimulée. Les PAR servent de signalisation pour le recrutement de multiples protéines, dont la protéine plateforme XRCC1.Les études menées au cours de cette thèse ont porté sur l’étude de la régulation des fonctions des protéines PARP1, PARP2 dans la réparation des cassures double brins (CDB) et l’étude des modifications de XRCC1 par phosphorylation en réponse à des dommages de l’ADN. En utilisant des substrats permettant de mesurer l’efficacité des différentes voies de réparation des CDB, nous avons démontré que PARP2, et non PARP1, est impliqué dans la régulation du choix des voies de la réparation des CDB. Plus spécifiquement, nous avons montré que PARP2 stimule l’initiation de la résection des extrémités des CDB dépendante de CtIP, indépendamment de son activité catalytique. Par des approches de vidéo-microscopie, nous avons pu déterminer que PARP2 limite l’accumulation de 53BP1 aux sites de dommages induits par micro-irradiation laser. Nous proposons que la protéine PARP2, en limitant le recrutement de la protéine 53BP1 aux sites de dommages, favorise la réparation des CDB dépendante de la résection des extrémités d’ADN, au détriment de la voie canonique de jonction des extrémités. Ces résultats sont les premiers démontrant un rôle de PARP2 dans le choix des voies de réparation des CDB.En parallèle, nous avons analysé comment la phosphorylation régule les fonctions de la protéine XRCC1. Par des approches in vitro et in vivo, nous avons pu déterminer que l’interdomaine 1 de XRCC1 est phosphorylé par la kinase CDK5. En réponse aux dommages induits par un agent alkylant, XRCC1 est activement déphosphorylé in vivo. De plus, nous avons observé que lorsque l’interdomaine 1 ne peut pas être phosphorylé in vitro, l’interaction de XRCC1 avec les PAR synthétisés par PARP1 et PARP2 augmente, et le recrutement de XRCC1 aux sites de dommages de l’ADN est accru. Ces résultats indiquent pour la première fois que la déphosphorylation de XRCC1 en réponse à un stress génotoxique participe activement à son recrutement aux sites de dommages.Dans leur ensemble, ces travaux ont contribué à améliorer nos connaissances fondamentales des réseaux de protéines impliquées dans la prise en charge des dommages de l’ADN. La compréhension de ces mécanismes est essentielle non seulement car ils participent au maintien de la stabilité du génome mais aussi du fait du développement exponentiel de nouvelles stratégies anti-tumorales qui visent à inhiber les voies de la réparation dans la but de cibler spécifiquement les cellules cancéreuses. / Post-translational modifications of proteins by polymers of ADP-ribose (PAR) or by phosphorylation allow the assembly of DNA repair protein complexes at damaged chromatin and are crucial to ensure genome stability. In response to DNA insults, the synthesis of PAR by the PARP1 and PARP2 proteins is strongly induced. PAR act as a signaling platform for the recruitment of multiples proteins at the sites of DNA damages, including the scaffold protein XRCC1. Research conducted during this PhD have been focused on studying the regulation of PARP1 and PARP2 functions in double-strands break repair (DSBR), and in investigating the role of XRCC1 modifications by phosphorylation in response to DNA damage.Using DNA repair assay allowing us to assess the accuracy of the different DSBR pathways, we demonstrated that PARP2, and not PARP1, is involved in the regulation of DNA double-strands break repair pathway choice. More precisely, we showed that PARP2 stimulates CtIP dependent initiation of end-resection at DSB, independently of its catalytic activity. By live cell imaging, we were able to determine that PARP2 limit 53BP1 accumulation at DNA damage sites induced by laser-microirradiation. We propose that by limiting 53BP1 accumulation at DNA damage sites, PARP2 stimulate DSB repair pathway that depend on DNA end-resection, thus counteracting the canonical end-joining pathway. These results are the first demonstrating a role for PARP2 in DNA DBSR pathway choice.In addition, we analyzed how the functions of XRCC1 are regulated by phosphorylation. Using in vitro and in vivo approaches, we were able to demonstrate that the linker 1 region of XRCC1 is phosphorylated by the CDK5 kinase. XRCC1 is actively dephosphorylated in response to DNA damage induced by an alkylating agent in vivo. We also observed that when the linker 1 cannot be phosphorylated, the XRCC1 interaction between the PAR synthetized by PARP1 and PARP2 is stimulated, and XRCC1 recruitement at the sites of DNA damage is far more efficient. These evidences indicate for the first time that the dephosphorylation of XRCC1 actively participate in its recruitment at the site of DNA damage. Put together, this work contributed to strengthen our fundamental knowledge of the protein network involved in the DNA damage response. Knowledge of those mechanisms is crucial since they participate in maintaining genome stability, and because new antitumoral drugs targeting DNA repair pathways in the attempt to specifically killed tumor cells are exponentially released.
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Funkční in vitro analýza alternativních sestřihových variant genu BRCA1 / The functional in vitro analysis of the BRCA1alternative splicing variantsŠevčík, Jan January 2012 (has links)
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...
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