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The pathogenesis of <i>C. jejuni</i> in broiler chickens is still poorly understood despite the importance of poultry meat as a source of infection in humans. The overall objective of this project was to understand the role of flagella and Campylobacter invasion antigens in mucosal and systemic colonization, and to evaluate the vaccine potential of <i>C. jejuni</i> paralyzed flagella mutants. As a first step to track <i>C. jejuni in vivo</i>, a Green Fluorescent Protein (GFP) reporter system that is constitutively expressed was constructed. The system was transformed into different <i>C. jejuni</i> strains and isolates, and their mucosal and systemic spreading was studied over the period of 7 days. <i>C. jejuni</i> NCTC11168V1 and V26 share the same background but differ in their ability to colonize chickens. <i>C. jejuni</i> 81-176 and K2-55 share the same genetic background but K2-55 has an insertion mutation in <i>pflA</i> gene that produced paralyzed flagella. Although the K2-55 flagella remained intact structurally, it did not secret <i>Campylobacter</i> invasion antigens (Cia). The reporter system was stable in all of these strains both <i>in vitro</i> and <i>in vivo</i>. Fluorescent bacteria were visualized successfully using fluorescent and confocal microscopes. C. jejuni NCTC11168V1 and 81-176 were detected in the intestinal tract and in the liver and spleen of more than 30% of the challenged birds, while V26 and K2-55 were only detected in the intestinal tract. <i>C. jejuni</i> 81-176 and K2-55 did not spread systemically to the spleen and liver of BALB/c mice challenged using the same approach, although they colonized the ceca.<p> A live attenuated vaccine based on <i>C. jejuni</i> K2-55 protected broiler chickens from <i>C. jejuni</i> 81-176 challenge in chickens following streptomycin treatment of drinking water. The same vaccine had no significant protection against a heterolgous <i>C. jejuni</i> NCTC11168V1 strain challenge. The vaccine was a poor stimulator of secretory IgA.<p> Macrophage-like HD11 cells inflammatory response to the presence of <i>C. jejuni</i> K2-55 was not significantly different from their response to wild-type 81-176 when measured by qRT-PCR. The lack of Cia secretion and motility had no effect on expression of IL-1â, IL-2, IL-6, IL-8, IL, IL-10, IL-12â, or TLR5. A <i>flgK</i> mutant expressing the flagella up to the hook had a significantly lower expression of these genes.

The pathogenesis of <i>C. jejuni</i> in broiler chickens is still poorly understood despite the importance of poultry meat as a source of infection in humans. The overall objective of this project was to understand the role of flagella and Campylobacter invasion antigens in mucosal and systemic colonization, and to evaluate the vaccine potential of <i>C. jejuni</i> paralyzed flagella mutants. As a first step to track <i>C. jejuni in vivo</i>, a Green Fluorescent Protein (GFP) reporter system that is constitutively expressed was constructed. The system was transformed into different <i>C. jejuni</i> strains and isolates, and their mucosal and systemic spreading was studied over the period of 7 days. <i>C. jejuni</i> NCTC11168V1 and V26 share the same background but differ in their ability to colonize chickens. <i>C. jejuni</i> 81-176 and K2-55 share the same genetic background but K2-55 has an insertion mutation in <i>pflA</i> gene that produced paralyzed flagella. Although the K2-55 flagella remained intact structurally, it did not secret <i>Campylobacter</i> invasion antigens (Cia). The reporter system was stable in all of these strains both <i>in vitro</i> and <i>in vivo</i>. Fluorescent bacteria were visualized successfully using fluorescent and confocal microscopes. C. jejuni NCTC11168V1 and 81-176 were detected in the intestinal tract and in the liver and spleen of more than 30% of the challenged birds, while V26 and K2-55 were only detected in the intestinal tract. <i>C. jejuni</i> 81-176 and K2-55 did not spread systemically to the spleen and liver of BALB/c mice challenged using the same approach, although they colonized the ceca.<p> A live attenuated vaccine based on <i>C. jejuni</i> K2-55 protected broiler chickens from <i>C. jejuni</i> 81-176 challenge in chickens following streptomycin treatment of drinking water. The same vaccine had no significant protection against a heterolgous <i>C. jejuni</i> NCTC11168V1 strain challenge. The vaccine was a poor stimulator of secretory IgA.<p> Macrophage-like HD11 cells inflammatory response to the presence of <i>C. jejuni</i> K2-55 was not significantly different from their response to wild-type 81-176 when measured by qRT-PCR. The lack of Cia secretion and motility had no effect on expression of IL-1â, IL-2, IL-6, IL-8, IL, IL-10, IL-12â, or TLR5. A <i>flgK</i> mutant expressing the flagella up to the hook had a significantly lower expression of these genes.

TSG101 exhibits multiple functions, including vesicular trafficking, cell growth, differentiation, and transcriptional regulation. However, the cellular signaling that regulates TSG101 functions remains unexplored. Our previous result indicates that TSG101 can be phosphorylated by PKC and GSK3£]. In this thesis, we further investigate the detail phosphorylated amino acid residues by using in vitro kinase assay in conjunction with MALDI-TOF and peptide array analysis. The results indicate that S13, S48, S103 and S367, T383 residues could be phosphorylated by PKC, S182 and S212 by GSK3£]. Coiled-coil domain of TSG101, which contains 2 consensus CK¢º phosphorylation sites,could be phosphorylated by CK¢º. These results indicate the functions of TSG101 might be regulated by these kinases. Proteasome mediated protein degradation is important to maintain proper cellular functions including cell growth, differentiation and signaling associated with cell cycle control. Impaired function of this system has been implicated in human diseases associated with neurodegeneration and cancer. The inhibitor of proteasome has been successfully used in treatment of these related diseases. In this thesis, we successfully established Ub-X-GFP reporter cell lines, which could be used in the future study on the functional role of TSG101, an E2 variant, in the proteasome mediated degradation pathway. Furthermore, these cell lines will serve a useful cellular platform for screening new proteasome inhibitors.

Medicago truncatula (M. truncatula) has been proposed as a model legume for molecular and genetic studies of legumes. While many genetic resources have been developed for this model legume, genetic transformation of the M. truncatula line A17 proved to be a problem. A reproducible transformation method is described for M. truncatula A17. Procedures are detailed that yielded an average regeneration frequency of 35% for recovery of transgenic shoots from cotyledonary node explants. Previously, rooting of transgenic shoots of this line has proven difficult, but media and culture procedures are described that yielded an average frequency of 39% for root induction from 419 phosphinothricin-resistant shoots. Fertile M. truncatula A17 plants transgenic for 35S-GFP, phas-GUS or phas-GFP were obtained. The presence of transgenes was confirmed by expression of transgenes and by genomic DNA blots. Interestingly, although GUS and GFP driven by the phas promoter were very strongly and uniformly expressed in seed cotyledons of most transgenic M. truncatula lines, silencing of the GUS expression from the phas promoter was observed in several lines, indicating the occurrence of novel epigenetic events. The diminution of GFP fluorescence in transgenic M. truncatula occurs despite the presence of GFP transcript and protein. To evaluate the generality and causes of this phenomenon, fluorescence during leaf development from the same 35S-GFP transgene was compared in M. truncatula, rice and Arabidopsis. A substantial decrease in fluorescence early in the development of M. truncatula and rice leaves was found to correlate with chlorophyll accumulation. Several approaches showed that chlorophyll is causally involved in the loss of GFP fluorescence. Removal of chlorophyll from leaves of transgenic M. truncatula, rice or Arabidopsis through etiolation or by extraction with ethanol yielded up to a tenfold increase in fluorescence. Direct evidence that chlorophyll is implicated in the loss of fluorescence from GFP was obtained by mixing solutions of chlorophyll and GFP. At low concentration, fluorescence loss was fourfold greater for chlorophyll b than for chlorophyll a, reflecting their relative interference with GFP excitation and emission. Thus, substantial errors in estimating promoter activity from GFP fluorescence can occur if pigment interference is not considered.

Lysine is one of the more limiting amino acids in protein sources for chickens. Since lysine is also an essential amino acid for animals, it is an important component of animal dietary formulation. Therefore, an accurate pre-determination of bioavailable lysine in feedstuffs is important. An optical density (OD) based microbiological assay for lysine determination using E. coli lysine auxotroph has been previously developed. However, because the assay is based on bacterial growth response to extracellular lysine measured as OD, it can be relatively time consuming (10-12h). Therefore, more rapid assays are needed if pre-formulation estimates are required. In this dissertation whole cell fluorescent biosensors for the quantification of bioavailable and total lysine in feed protein sources were developed. The biosensor for quantification of bioavailable lysine was based on the growth response of E. coli to an external source of lysine and lysinecontaining small peptides. Green fluorescent protein (GFP) was inserted in the genome of E. coli lysine auxotroph as a part of a mini-Tn5- transposon by conjugation. Bacterial growth response to external lysine and small peptides was monitored and recorded by measuring the fluorescence emitted by GFP. The second type biosensor developed was designed for the quantification of total lysine. It was based on the measurement of a promoter activity, which was induced and modulated by extracellular concentration of lysine. Cad promoter was amplified from E. coli K-12 genome and was cloned into promoterless gfp plasmid. The construct was electroporated into electrocompetent E. coli cells. The promoter activity was induced under the conditions of low pH and graded concentrations of lysine. Lysine-dose response was measured by the fluorescence of GFP. Both methods were characterized as having a high potential for practical application.

The cellular process of programmed cell death, or apoptosis, is critical in homeostasis and development. In addition it's misfunction is implicated in an array of disease states from cancer to neurodegeration, making it an attractive pathway for drug targeting. A family of proteases, known as caspases, plays a central role in the apoptotic cascade resulting in the ultimate destruction of the cell. We report a genetically encoded dark-to-bright reporter of caspase activity used in E.coli, mammalian cells, and whole organisms which can be used to monitor apoptosis. This reporter, caspase activatable green fluorescent protein (CA-GFP) consists of GFP fused through a flexible linker containing the caspase-3 and -7 recognition sequence, DEVD, to a hydrophobic peptide derived from the influenza A viral M2 protein. This fusion reporter shows a significant fluorescent response in the presence of active caspase. CA-GFP is unique in its ability to hold GFP in a dark state prior to cleavage by active protease. We investigate the mechanism of quenching, examining the structural characteristics which lead to the inability of the GFP chromophore to mature in the presence of the peptide. In better understanding the mechanism of quenching we can engineer CA-GFP to ultimately be used in transgenic animal models. This requires the development of a palette of protease-activatable fluorescent proteins (PrA-FP) which would enable the monitoring of multiple proteolytic events within a cell or organism in real time. Our development of this palette of reporters, varying in their fluorescence and proteolytic response shows that CA-GFP has the potential to be a powerful tool for the study of the role of apoptosis during development in whole organism models and could be an important tool in understanding the role of individual proteases within the complex biochemical environment in the cell.

A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS. / Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS.

A produção de animais transgênicos possui aplicações que envolvem desde a pesquisa básica à produção agropecuária. O recente progresso na clonagem animal por transferência nuclear (TN) possibilitou a produção de animais transgênicos utilizando linhagens de células doadoras de núcleo previamente modificadas geneticamente. A possibilidade de manipulação genética, estudo da expressão gênica e adequada seleção da célula doadora de núcleo na TN não somente pode garantir a presença da construção gênica em toda a prole, como também pode evitar a produção de animais portadores de modificações indesejáveis resultantes da inserção do inserto em regiões codificantes do genoma, em decorrência da inserção aleatória das técnicas de transferência gênica mais comuns. Este trabalho teve como objetivo geral produzir animais transgênicos a partir de transferência nuclear utilizando como células doadoras de núcleo fibroblastos modificados geneticamente por transdução lentiviral. Objetivos específicos foram a produção e caracterização de linhagens de fibroblasto fetal portadores do gene da Proteína Fluorescente Verde (eGFP) quanto à seleção da expressão do transgene, passagem celular e posição da inserção do transgene e sua utilização na técnica de transferência de núcleos para a análise da competência de desenvolvimento a blastocisto e estabelecimento de gestações. Para tal, fibroblastos fetais bovinos foram transduzidos pelo sistema lentiviral. Células expressando o gene da eGFP foram selecionadas por citometria de fluxo e utilizadas como doadoras de núcleo na TN. Foram analisados o efeito do período de cultivo dos fibroblastos, assim como o efeito da reclonagem na competência de desenvolvimento a blastocisto e estabelecimento de gestações. O cultivo celular submetido à reclonagem foi analisado quanto à posição de inserção do transgene, sendo constatado nos fetos produzidos neste experimento a presença de uma inserção única em região não transcrita do cromossomo 14. Não houve efeito neste experimento do tempo de cultivo na competência de desenvolvimento a blastocisto, mas houve efeito benéfico da reclonagem celular. Além disso, foram obtidos 4 fetos, sendo 3 transgênicos, dos embriões transferidos provenientes das TNs que utilizaram células transgênicas de inserção aleatória em baixas e altas passagens e 6 fetos dos 37 embriões transferidos provenientes das TN que utilizaram células reclonadas, sendo todos transgênicos. Conclui-se que a produção de células transgênicas mediante mecanismo de transdução lentiviral, pode resultar, após TNCS, em embriões geneticamente idênticos à células doadora capazes de sustentar o desenvolvimento in vitro a blastocisto e o estabelecimento de gestações. Finalmente, são discutidos fatores ligados ao processo de seleção e reclonagem que aumentam a eficiência da produção de gestações por TNCS. / Genetically modified animals have numerous applications ranging from basic research to agriculture production. Recent progress in animal cloning by nuclear transfer (NT) has made possible the production of transgenic animals using previously genetically modified cell lineages. The possibility of genetic manipulation, gene expression studies and adequate selection of the nuclei donor cell for NT not only can guarantee the presence of the gene construction in the offspring, but also can avoid the production of animals that carries undesirable characteristics, often as a result of the random insertion of transgenes in transcripted areas of the genome. General objective of this study was to produce transgenic animals by nuclear transfer using lentivirus-genetically modified nuclei donor fibroblasts. Specifically, objectives were the production and characterization of fetal fibroblasts lineages expressing eGFP (enhanced Green Fluorescent Protein, eGFP) gene in different cell passages regarding transgene insertion position and its use for the nuclear transfer procedure. The potential of blastocyst development and pregnancy establishment were analyzed in embryos reconstructed with late and early passages and with clonal or random insertion of transgenes (recloning). For that, bovine fetal fibroblasts were transduced with lentiviruses. eGFP expressing cells were selected by flow citometry sorting and used as nuclei donor cells for NT. Transgene integration site of the cell culture submitted to recloning was analised. It was observed that an unique insertion in a non-transcribed área of chromossome 14 was present in the fetuses recovered in this recloning experiment. No effect of culture time on development of blastocysts was observed, however, there was a beneficial effect of the cell recloning Besides, 4 fetuses (3 of them were transgenic) were obtained when 64 embryos reconstructed with random transgene position cells in late and early passages were transferred and 6 fetuses were obtained when 37 embryos reconstructed with recloned cells were transferred (all of them were transgenic). In conclusion, lentivirus transduction was able to produce transgenic cells with a stable expression of the transgene. These cells, when used for SCNT, can be reprogrammed and genetically identical embryos able to sustain in vitro culture, pregnancy establishment and recognition. Finally, recloning and cell selection procedures are discussed as a possible approach to increase pregnancy efficiency after TNCS.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-12T16:05:41Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação ADALUCIA DA SILVA PPGG 2016.pdf: 2174365 bytes, checksum: e3305d6246515bbc247cd29783f8b265 (MD5) / Made available in DSpace on 2017-07-12T16:05:41Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação ADALUCIA DA SILVA PPGG 2016.pdf: 2174365 bytes, checksum: e3305d6246515bbc247cd29783f8b265 (MD5) Previous issue date: 2016-03-07 / FACEPE / Os tripanossomatídeos são parasitas flagelados causadores de diversas doenças humanas e que apresentam algumas características moleculares bem distintas. Nestes organismos, o controle da sua expressão gênica é quase exclusivamente pós-transcricional e dados obtidos até o momento sugerem que a etapa de iniciação da tradução tem um papel importante neste controle. Durante a iniciação da tradução de eucariotos, o complexo trimérico eIF4F (formado pelas subunidades eIF4A, eIF4G e eIF4E) tem uma participação importante no reconhecimento do mRNA, facilitando o recrutamento ribossomal para iniciar a síntese proteica. Múltiplos homólogos das subunidades do complexo eIF4F foram identificados em tripanossomatídeos, mas não foi possível elucidar a função específica de cada um deles. Desta forma, este trabalho teve como objetivo o desenvolvimento de um ensaio in vivo para a avaliação do efeito da superexpressão desses homólogos de Trypanosoma brucei na tradução de um mRNA repórter, o qual codifica a proteína GFP (green fluorescent protein). Três linhagens repórter foram obtidas (4212, 4213 e 4235) e sete homólogos das subunidades do complexo eIF4F e um de PABP foram testados nestas linhagens. A análise da superexpressão foi feita através de ensaios de Western blot, seus perfis de crescimento foram avaliados por curvas de crescimento e a expressão de GFP foi avaliada através de citometria de fluxo. Os resultados apresentados mostram que as linhagens repórter 4213 e 4235 são viáveis para serem utilizadas na caracterização das proteínas envolvidas no controle da expressão gênica de tripanossomatídeos. Foi observado que as proteínas EIF4E1 e EIF4E2 provocam diminuição do crescimento, enquanto a superexpressão de EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 e PABP1 não alteram o crescimento celular dos parasitas. Apesar da detecção de variações na expressão de GFP durante a superexpressão de alguns dos homólogos testados, não foi possível, contudo, confirmar que estas proteínas aumentam ou diminuem a tradução. Ensaios complementares ainda precisam ser feitos para confirmar a real função destes. / The trypanosomatids are flagellated parasites responsible for several human diseases and which display unique molecular characteristics. Control of gene expression in these organisms is almost exclusively post-transcriptional and the data generated so far suggest that the initiation stage of translation plays an important role in this control. During translation initiation in eukaryotes, the trimeric complex eIF4F (formed by the eIF4A eIF4G and eIF4E subunits) has a relevant role in mRNA recognition and facilitates ribosomal recruitment to start the protein synthesis. Multiples homologues for the eIF4F subunits have been identified in trypanosomes, but it has not been possible to elucidate their specific functions. Thus, this study aimed to develop an in vivo assay to evaluate the effect of the overexpression of these Trypanosoma brucei homologues during the translation of a reporter mRNA encoding for the green fluorescent protein (GFP). Three reporter strains were generated (4212, 4213 and 4235) in which seven homologues of eIF4F subunits and one of their PABP partner were overexpressed. Confirmation of overexpression was carried out by Western blot assays, growth profiles were evaluated by cell counting and GFP expression was assessed by flow cytometry. The results generated show that the reporter lines 4213 and 4235 are feasible for use in the characterization of proteins involved in the control of trypanosomatids gene expression. Overexpression of EIF4E1 and EIF4E2 was seen to induce a reduction in cell growth, while EIF4E3, EIF4E4, EIF4E4W279A, EIF4G3, EIF4G4 and PABP1 did not interfere with growh. Despite the detection of variations in GFP expression during overexpression of some of the homologues tested it was not possible to confirm if these proteins increase or decrease translation. Complementary assays still need to be done to confirm the true function of these factors.