Made available in DSpace on 2016-06-02T20:21:25Z (GMT). No. of bitstreams: 1 3461.pdf: 3248917 bytes, checksum: b02925f28a9fc50ea71c3af6beebca5c (MD5) Previous issue date: 2011-03-04 / Financiadora de Estudos e Projetos / The desintegrinas are cys-rich polypeptides without enzymatic activity, some found in snake venoms with an adhesive motif to integrin, unleashing distinct responses. Several research groups have been studying snake venom disintegrins with focus in pharmacological applications in tumor progression and proliferation, angiogenesis, blood clotting, and osteoporosis. This work shows the results of the construction of cDNA library of Rhinocerophis alternatus venom gland, cloning and expression of ALT-C, a disintegrina-like protein, in bacterial system. The library was comprised of 812 sequences that were classified into: (i) coding for hypothetical protein or mismatched in databases (unknown) corresponding to 20.94%; (ii) transcripts for cellular process 17.86%; and (iii) transcripts coding for toxic protein with 61.21%. Among toxic proteins the SVMPs were the most abundant transcripts 58.59%. Other toxic transcripts were C-type lectins (15.56%), bradykinin potentiating peptides (11.8%), serineproteases (5.18%), cys-rich secretory proteins (2.28%), vascular endothelial growth factors (2.28%), L-amino acid oxidases (1.86%) and phospholipases A2 (1.45%). The percentages correspond to this snake venom effects, low enzymatic activity and hemorrhagic and clotting activity as main action, derived from the SVMPs, C-type lectin and serineproteases. One of the SVMPs contigs were chosen based on the similarity with ALT-C, the defined sequence was called ALT-Ch (ALT-C homologue) since there has 4 conservative substitutions compared to the previously determined sequence. The ALT-C-h sequence was used in PCR to inclusion the restriction site for BamHI and EcoRI, cloned in pGEX4T-1 and transformed into E. coli AD494 (DE3) for expression. The cell culture was grown until the log fase and induced with IPTG for 3 hours, the expressed protein's identity was confirmed by Western Blotting using Polyclonal antibody Anti-ALT-C. The construction of the R. alternatus venom gland cDNA library provides a source of interesting active biomolecules, such as desintegrinas, to pharmacological applications in pathologies involving integrins like angiogenesis, tumor progression and metastasis. / As desintegrinas são polipeptídeos sem atividade enzimática, ricos em cisteína, geralmente encontrados no veneno de serpentes, possuidoras de um motivo adesivo reconhecedor de integrinas antagonizando ou agonizando a atividade das mesmas. Diversos grupos de pesquisa vêm estudando desintegrinas de venenos de serpentes com o foco em aplicação farmacológica nos processos de progressão e proliferação tumoral, angiogênese, coagulação sanguínea, e osteoporose. Este trabalho apresenta os resultados da construção da biblioteca de cDNA da glândula venenífera de Rinocerophis alternatus e a clonagem e expressão da ALT-C, uma tipo desintegrina, em sistema bacteriano. A biblioteca foi composta por 812 sequências que foram classificadas em: (i) codificantes para proteínas hipotéticas ou sem equivalência nos bancos de dados (unknown) correspondendo a 20,94%; (ii) transcritos para produtos celulares 17,86%; e (iii) transcritos codificantes para proteínas tóxicas 61,21%. Dentre as proteínas tóxicas as SVMPs foram os transcritos mais abundantes com 58,59% dos compostos tóxicos do tecido. Os demais transcritos tóxicos encontrados foram as lectinas do tipo-C (15,56%), peptídeos potenciadores de bradicinina (11,8%), serinoproteases (5,18%), proteínas secretórias ricas em cisteína (2,28%), fatores de crescimento endotelial vascular (2,28%), L-aminoácido oxidases (1,86%) e fosfolipases A2 (1,45%). As percentagens encontradas correspondem aos efeitos do veneno dessa serpente, que possui baixa atividade enzimática e predominate ação hemorrágica e de alteração na coagulação sanguínea derivada da ação das SVMPs, lectinas do tipo-C e serinoproteases. Um dos contigs para SVMPs foi eleito pela similaridade com a ALT-C. A sequência determinada por esse contig foi denominada ALT-C-h (homóloga da ALT-C) por apresentar 4 substituições conservativas da sequência determinada anteriormente. A sequência foi utilizada em PCR para inclusão de sítio de restrição para BamHI e EcoRI, clonado em pGEX4T-1 e transformados em E. coli AD494(DE3) para ensaios de expressão. O cultivo foi crescido até a fase log e a indução expressão realizada com IPTG por 3 horas, a identidade da proteína expressa foi confirmada por western blotting utilizando anticorpo policlonal anti-ALT-C. A construção da biblioteca de cDNA da glândula venenífera de R. alternatus fornece uma fonte de estudo interessante de biomoléculas ativas nos venenos como, por exemplo, as desintegrinas visando aplicações farmacológicas para patologias envolvendo integrinas como angiogênese, progressão e metástase tumoral.

Resumo: Sob condições de estresse hídrico, a cana-de-açúcar pode sofrer mudanças fisiológicas e bioquímicas, tais como diminuição nas atividades fotoquímicas, redução da fixação de CO2 e acúmulo de osmólitos e osmoprotetores. O objetivo deste trabalho foi identificar, através da técnica de macroarranjo de cDNA, o perfil de expressão de genes promotores das diferentes vias metabólicas em palmitos da variedade de cana-de-açúcar SP80-3280 submetidas ao estresse hídrico nos dias 5, 9, 13 e 17 após o início da condição de supressão de água, sendo considerado o dia 1 como controle. Os resultados do macroarranjo mostraram que as proteínas mais expressas sob déficit hídrico pertencem a quatro categorias das quais as ESTs mais importantes foram selecionadas. As quatro categorias descritas abaixo estão discutidas neste trabalho. As ESTs da via do metabolismo de açúcar e amido (invertase de parede celular (INV), sacarose fosfato sintase (SFS), sacarose fosfato fosfatase (SFF), trealose fosfato sintase (TFS), trealose fosfato sintase/fosfatase (TFS/F) e hexoquinase (HXQ)) pertencem a categoria de bioenergética. Colina monooxigenase (CMO) e betaína aldeído desidrogenase (BADH) as quais pertencem a via do metabolismo de glicina betaína, foram selecionadas a partir da categoria do metabolismo secundário. A terceira categoria compreende as ESTs do metabolismo de aminoácido (D-pirrolina-5- carboxilato sintase (P5CS), ornithina -aminotransferase (OAT) e prolil 4-hidroxilase (PH)) as quais pertencem a via da biossíntese de prolina, e a quarta categoria, resposta ao estresse, compreende as ESTs aleno oxido sintase (AOS), aleno oxido ciclase (AOC) e lipoxigenase (LOX), pertencentes a via de biossíntese do jasmonato. / Abstract: Under water deficit, sugarcane undergoes physiological and biochemical alterations such as photochemical activity reduction, CO2 fixation reduction and the accumulation of osmolytes and osmoprotectants. This work was undertaken to identify the gene expression profile in sugarcane (var. SP80-3280) under water deficit through the cDNA macroarray technique. Leafroll tissues were collected from plants subjected to 5, 9, 13 and 17 days of water restriction, and day 1 was used as control. Macroarray results showed that most proteins expressed under water restriction belong to four categories, from which the most important ESTs were selected. The four categories described below are discussed in this work. Sugar and starch metabolism pathway ESTs (cell wall invertase (CWI), sucrose phosphate synthase (SPS), sucrose phosphate phosphatase (SPP), trehalose phosphate synthase (TPS), trehalose phosphate synthase phosphatase (TSP/P) and hexoquinase (HXQ) were selected from the bioenergetics category. Choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH), which belong to the glycine betaine metabolism pathway, were selected from the secondary metabolism category. The third category comprised the amino acid metabolism ESTs D- pyrroline-5-carboxylate synthase (P5CS), Ornithine - aminotransferase (OAT) and Prolyl 4-hydroxylase (PH), which belong to the proline biosynthesis pathway, and the fourth category, stress response, comprised the ESTs for Allene oxide synthase (AOS), Allene oxide cyclase (AOC) and Lipoxygenase (LOX), which belong to the jasmonate biosynthesis pathway. / Orientador: Maria Inês Tiraboschi Ferro / Coorientador: Sonia Marli Zingaretti / Coorientador: Roberto Willians Noda / Banca: João Suzuki / Banca: Poliana Fernanda Giachetto / Mestre

Workshop cluster 1 (WC1) molecules are exclusively expressed on the surface of gamma delta T cells and act as co-receptors and bind pathogens thus also functioning as pattern recognition receptors. The aim was to obtain cDNA evidence to support the recent caprine genome annotation of the WC1 multigene family conducted by a colleague. To get cDNA sequences three strategies were used. Strategy 'I' was used to obtain three clones that corresponded to WC1 SRCR domain d9 through the intracytoplasmic tail sequence. Strategy 'II' was used to obtain 6 clones. A PCR was conducted using SRCR domain b7 through the intracytoplasmic tail sequence. A third strategy obtained full-length WC1 transcripts. The three sequences that extended from SRCR domain d9 to the intracytoplasmic tail matched closely with predicted goat Gene 1 or 14. Another 3 sequences that extended from the SRCR b7 domain through SRCR domain d11 or through the intracytoplasmic tail matched with the predicted Genes 1, 2 and 14, respectively. Two additional full-length cDNA clones CH-MA-03 and 41 were completely sequenced in stages which involved a PCR amplication of the internal domains to complete the sequencing. The a1 domain of CH-MA41 was 100% similar to the annotated and predicted Gene 4 while CH-MA03 also was closest to Gene 4 with a 99% similarity. However, the intracytoplasmic tail sequence of these two cDNA clones was a Type II tail while Gene 4 had a Type I tail. Because of this difference in tails these two cDNA clones had a greater overall similarity with Genes 7 and 15 which had Type II tails. These results suggest that the genome assembly may have errors.

Japanese encephalitis virus (JEV) is the causative agent of Japanese encephalitis (JE), the leading cause of vaccine-preventable neurological disease. JEV is a flavivirus that is primarily transmitted through the bite of infected mosquitoes, similar to dengue virus (DENV), St. Louis encephalitis virus (SLEV), West Nile virus (WNV), and Zika virus (ZIKV). The two viral characteristics that dictate virulence are (1) neuroinvasiveness, the ability of the virus to invade the central nervous system(CNS), and (2) neurovirulence, the capacity of the virus to kill resident cells in the CNS. The clinically proven live-attenuated JEV vaccine, SA14-14-2, lacks both pathogenic characteristics unlike its virulent parental virus, SA14. Previous work has revealed the viral E gene as the main determinant of these two pathogenic properties, though the molecular mechanisms behind their attenuation remain unclear. The E gene encodes for the viral envelope glycoprotein that is involved in viral entry into susceptible host cells. The E protein of SA14-14-2 differs from SA14 by nine amino acids. To investigate the role of these mutations in JEV virulence, we created a series of SA14E mutants using infectious cDNA technology. Here, we report the independent function of domains I (DI) and II (DII) of the viral E protein in JEV neurotropism. We reveal that an individual mutation in DI, E138K,and synergism between two mutations in DII, E244G and K279M,are independently sufficient for the attenuation of JEV neuroinvasion. Also, we report that multiple E mutations are required for full attenuation of JEV neurovirulence. Overall, our findings show the direct relationship between genetic factors and JEV neuroinvasion. These results provide a solid foundational base for the logical development of other, currently non-existing, live-attenuated neurotropic flavivirus vaccines and antivirals.

Safeners are the chemicals used to protect plants from detrimental effects of herbicides, but their mode of action at the molecular level is not well understood. As an initial step towards understanding the molecular mechanism of safener action in trees, homologous genes in hybrid poplar (Populus nigra x Populus maximowiczii) that were induced by a safener were identified. We here describe the identification of differentially expressed genes in Populus that are induced by Concep-III, a herbicide safener. Expressed sequence tags (ESTs) enriched for transcriptionally induced genes were isolated by suppressive subtractive hybridization (SSH). The SSH library cDNA inserts were used to construct a cDNA microarray for high-throughput validation of the up-regulated expression of safener-induced genes. Single-pass and partial sequences of 1,344 safener-induced ESTs were assembled into 418 single-tons and 328 clusters, but the putative functions of almost 53% of the ESTs are not known. Genes encoding proteins involved in all three different phases of safener action, viz., oxidation, conjugation, and sequestration, were found in the SSH library. Almost 75% of genes that showed greater than 2-fold expression upon safener treatment were redundant in the SSH library. The expression pattern for selected genes was validated by reverse transcription-polymerase chain reaction. A few safener-induced genes that were not previously reported to be induced by safeners, but which may have a role in herbicide metabolism, were identified. The newly identified genes could have potential for application in genetic engineering of plants for herbicide detoxification and tolerance.

Reverse genetics is a discipline that involves the use of genetic manipulation and modification to study an organism's altered phenotype. In this study, infectious recombinant viruses were rescued from altered cDNA clones encoding the antigenome of human parainfluenza virus type 3 and the resulting phenotypes were examined. In one clone, the gene for the enhanced green fluorescent protein was inserted into the virus antigenome to be expressed during viral replication, resulting in infected cells emitting green fluorescence. Viral titers, mRNA replication, and genomic replication for the virus expressing the enhanced green fluorescent protein were reduced when compared to the human parainfluenza virus type 3 wild-type strain. In addition, the sensitivity of the virus expressing the enhanced green fluorescent protein to antiviral compounds is increased when compared to the wild-type strain, which may lead to the identification of false positive antiviral compounds. An assay that measures the enhanced green fluorescent protein as a direct indicator of virus replication can be shortened to 3 days in duration and is a more robust assay compared to assays that measure cellular viability. In other clones, mutations were introduced into the phosphoprotein gene to eliminate the expression of the D domain of the PD protein in order to understand its function. The titers of two recombinant knockout viruses that are deficient in the expression of the D domain are reduced when compared to the wild-type strain in both MA-104 and A549 cells. In MA 104 cells, viral mRNA transcription and genomic replication of the two knockout viruses are reduced when compared to the wild-type strain. In A549 cells, cellular expression and secretion of antiviral cytokines infected with the two knockout viruses are either reduced or remain unchanged when compared to the wild-type strain. These results suggest that the D domain may play a role in viral RNA synthesis and not in counteracting the host cell's antiviral response. The results of these studies shed light on the influence an additional gene has on viral replication and possible functions of the D domain.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19016号 / 農博第2094号 / 新制||農||1029(附属図書館) / 学位論文||H27||N4898(農学部図書室) / 31967 / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 保川 清, 教授 河田 照雄, 教授 谷 史人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19967号 / 医博第4157号 / 新制||医||1017(附属図書館) / 33063 / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 斎藤 通紀, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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This thesis includes two studies: The first examined patterns of neutral genetic diversity within Bromus tectorum L. across the IMW region, and uses patterns of microsatellite (SSR) genotype distribution to make inferences about the respective roles of adaptively significant genetic variation, adaptive phenotypic plasticity, and facultative outcrossing in the ongoing invasion and recent range expansion of B. tectorum. It has been previously demonstrated that, due to extremely low outcrossing rates, it is possible to characterize individual genotypes of this species using four SSR loci. We sampled 20 individuals from each of 96 B. tectorum populations (classified by region and habitat) from throughout the IMW and used these SSR markers to characterize each individual. We found 131 four-locus SSR genotypes; however, the 14 most common genotypes collectively accounted for 79.2% of the individuals sampled. Individuals with certain SSR genotypes sorted strongly into warm or salt desert habitats (stringent habitats) and flowered earlier than individuals with genotypes from more mesic habitats, providing evidence of adaptively significant genetic variation associated with these genotypes. Other SSR genotypes were found across a wide range of habitats though they tended to be less prevalent in stringent habitats, providing evidence that adaptive phenotypic plasticity may be important for the distribution of some common genotypes. We observed very few heterozygous individuals, consistent with the highly inbreeding reproductive strategy of B. tectorum. Because specialist genotypes dominating recently invaded areas within the IMW region contained unique alleles, they are not likely to have resulted from recombination, leading us to doubt the role of facultative outcrossing as a significant mechanism facilitating the current range expansion of B. tectorum in the IMW.Previous research investigating the population and ecological genetics of Bromus tectorum L. in the North American invaded range has relied on either allozyme or microsatellite (SSR) genetic analyses, both of which have proven to have shortcomings. In order to overcome the issues associated with these other marker types, in the second study of this thesis we developed single nucleotide polymorphism (SNP) markers for B. tectorum by 1) obtaining normalized cDNA, 2) sequencing normalized cDNA using 454 sequencing, 3) aligning resultant contigs and looking for SNPs, 4) designing assays for SNP validation and genotyping using KASPar, 5) converting working KASPar assays for use with the Fluidigm EP1 platform using the 96.96 Dynamic ArrayTM IFC. Sequencing resulted in 1258041 reads, which assembled into 65486 contigs (20782 large contigs exceeding 500 base pairs). Using selection criteria of at least 10x coverage and 30% of the minor allele, 3333 putative SNPs were identified. We developed KASP assays for 255 putative SNPs, which resulted in 101 working polymorphic assays. Ninety-six assays were then successfully converted for use with KASP on the Fluidigm EP1 genotyping platform using 96.96 dynamic arrays.