Submitted by GABRIELA DE FÁTIMA SANTANA-MELO (gabrieladsantana@yahoo.com.br) on 2016-05-15T23:21:48Z No. of bitstreams: 1 Tese Gabriela F. Santana-Melo.pdf: 2618239 bytes, checksum: 2bf460d87225bef3f4ce02747616a638 (MD5) / Rejected by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br), reason: Solicitamos que realize uma nova submissão seguindo as orientações abaixo: No campo “Versão a ser disponibilizada online imediatamente” foi informado que seria disponibilizado o texto completo porém no campo “Data para a disponibilização do texto completo” foi informado que o texto completo deverá ser disponibilizado apenas 6 meses após a defesa. Caso opte pela disponibilização do texto completo apenas 6 meses após a defesa selecione no campo “Versão a ser disponibilizada online imediatamente” a opção “Texto parcial”. Esta opção é utilizada caso você tenha planos de publicar seu trabalho em periódicos científicos ou em formato de livro, por exemplo e fará com que apenas as páginas pré-textuais, introdução, considerações e referências sejam disponibilizadas. Se optar por disponibilizar o texto completo de seu trabalho imediatamente selecione no campo “Data para a disponibilização do texto completo” a opção “Não se aplica (texto completo)”. Isso fará com que seu trabalho seja disponibilizado na íntegra no Repositório Institucional UNESP. Por favor, corrija esta informação realizando uma nova submissão. Agradecemos a compreensão. on 2016-05-18T18:11:13Z (GMT) / Submitted by GABRIELA DE FÁTIMA SANTANA-MELO (gabrieladsantana@yahoo.com.br) on 2016-07-08T17:12:34Z No. of bitstreams: 1 Tese Gabriela F. Santana-Melo.pdf: 2618239 bytes, checksum: 2bf460d87225bef3f4ce02747616a638 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-11T14:52:41Z (GMT) No. of bitstreams: 1 santanamelo_gf_dr_sjc.pdf: 2618239 bytes, checksum: 2bf460d87225bef3f4ce02747616a638 (MD5) / Made available in DSpace on 2016-07-11T14:52:41Z (GMT). No. of bitstreams: 1 santanamelo_gf_dr_sjc.pdf: 2618239 bytes, checksum: 2bf460d87225bef3f4ce02747616a638 (MD5) Previous issue date: 2016-02-01 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A necessidade da fabricação de novos biomateriais que possam, além de mimetizar o tecido ósseo, fornecer resistências mecânicas favoráveis próximas às do tecido ósseo natural têm despertado interesse de pesquisadores com o objetivo de melhorar a qualidade de vida de pessoas que sofreram algum tipo de lesão. Scaffolds de nanofibras poliméricas fabricados por eletrofiação apresentam características tridimensionais (3D) e poros interconectados que permitem a colonização de toda a superfície 3D por células com a consequente formação de tecidos. O scaffold de poli (butileno adipato-co-tereftalato) (PBAT) mostra-se um biomaterial promissor para regeneração óssea, porém tem sido pouco explorado até a data. Embora do uso da HA seja consagrado para uso biomédico, sua utilização em polímeros ainda é pouco estudada, principalmente em associação ao PBAT. Desta forma, o objetivo deste estudo foi avaliar a efetividade in vitro de scaffolds poliméricos (PBAT) com incorporação de nanopartículas de HA (nHAp) em diferentes concentrações, produzidos por eletrofiação, por meio da bioatividade celular e expressão gênica de osteoblast-like MG63. Células (MG63) foram cultivadas sobre scaffolds de PBAT; PBAT/3%nHAp e PBAT/5% nHAp e sem a presença dos mesmos (controle) e avaliadas pelos testes qualitativo (MEV) e quantitativo de adesão e proliferação celular (1 e 7 dias e aos 1, 3, 7, 14 e 21 dias, respectivamente), citotoxicidade celular (1, 3 e 7 dias), corante vermelho de alizarina e formação de mineralização (14 dias) e análise da expressão de genes relacionados à osteogênese por qRT-PCR aos 7, 14 e 21 dias de cultura celular. Os dados foram analisados estatisticamente por variância (ANOVA) e Tukey (p<0,05). Os scaffolds de PBAT e PBAT/nHAp não apresentaram efeito citotóxico e sua arquitetura tridimensional influenciou positivamente na adesão e proliferaçãocelular, formação de matriz mineralizada bem como em alguns períodos na expressão dos genes ALP, Col I, Runx2, OC e OPN em relação ao grupo controle. O efeito osteocondutor e osteoindutor da nHAp promoveu melhor resposta celular nos scaffolds de PBAT/nHAp, independente da concentração. Esses resultados demonstram a efetividade in vitro dos scaffolds de PBAT e PBAT/nHAp, apresentando grande potencial para aplicação biomédica. / The need for the manufacture of new biomaterials that may, in addition to mimic to bone tissue, providing favorable mechanical strength close to natural bone have aroused the interest of researchers in order to improve the quality of life of people who have suffered some kind of injury. Scaffolds polymer nanofibers fabricated by electrospinning have three dimensional features (3D) and interconnected pores that allow the colonization of the entire 3D surface of cells with the consequent formation of tissue. Poly (butylene adipate-co-terephthalate) (PABT) scaffold showed to be a promising biomaterial for bone regeneration, however, has been underexplored to date. Although the use of HA is consecrated to biomedical use, their use in polymers is not well known, especially in association with PBAT. The aim of this study was evaluating in vitro effectiveness of polymeric (PABT) scaffolds with incorporated HA (nHAp) nanoparticles, obtained by electrospinning, through cellular bioactivity and osteoblast-like MG63 gene expression. MG63 cells were grown on PABT; PABT/3%nHAp and PABT/5%nHAp scaffolds and without their presence (control), and evaluated by qualitative (MEV) and quantitative tests of cell adhesion and proliferation (1 and 7 days and at 1, 3, 7, 14 and 21 days, respectively), cell cytotoxicity (1, 3 and 7 days), alizarin red dye and mineralization formation (14 days) and expression of genes related to osteogenesis by qRT-PCR to 7, 14 and 21 days of cell culture. Data were statistically analyzed by variance (ANOVA) and Tukey test (p<0.05). The PBAT and PBAT/nHAp scaffolds showed no cytotoxic effect and its three-dimensional architecture influenced positively in the cell adhesion and proliferation, mineralized matrix formation as well as in some periods the expression of genes ALP, Col I, Runx2, OC and OPN in relation the control group. The osteoconductive and osteoinductive effect of nHAp promoted better cellular response in scaffolds of PABT/nHAp independent of concentration. These results demonstrate the in vitro effectiveness of PABT and PABT/nHAp scaffolds, presenting great potential for biomedical application.

Made available in DSpace on 2014-06-12T15:51:26Z (GMT). No. of bitstreams: 2 arquivo3091_1.pdf: 1433589 bytes, checksum: cee30d09ca85980508dbf3f0ac6de0cd (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2009 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Constituindo fontes ricas de compostos farmacologicamente ativos, com um amplo espectro de atividades biológicas, as toxinas e enzimas presentes nos venenos das famílias Crotalidae e Viperidae estão associadas a diversas atividades biológicas, como hemorrágica, fibrinolítica, inibidora da agregação plaquetária, proteolítica, neurotóxica e miotóxica. Dentre os constituintes de venenos de serpentes, merece destaque o grupo formado pelas metaloproteases, que compreende uma grande família de toxinas, com aproximadamente 200 membros catalogados, envolvendo uma marcante diversidade de estruturas e funções biológicas. Em meio a essa diversidade estrutural e funcional, um subgrupo de metaloproteses do tipo PIII, que apresenta atividade indutora de apoptose, vêm sendo alvo de inúmeras pesquisas. Ainda assim, muito pouco se conhece a respeito dessas proteínas. Em torno de doze exemplares dessas metaloproteases, como VAP1 e 2 de venenos de Crotalus atrox, foram identificadas. Desde o isolamento e purificação dessas proteínas, toxinas com alta similaridade às VAPs têm sido caracterizadas e purificadas do veneno de outras espécies de Viperídeos, que habitam diferentes lugares na Terra. Baseado no importante papel que essas metaloproteases indutoras de apoptose vascular podem desempenhar na angiogênese, através da inibição do crescimento de células neoplásicas, devido à indução da apoptose das células vasculares que nutrem o tumor, impedindo o crescimento acelerado e descontrolado das células tumorais, nós investigamos a expressão de metaloproteases VAP-like da glândula de venenos de três viperídeos, representantes do território brasileiro. Por clonagem molecular e reação em cadeia da polimerase em tempo real quantitativa, um gene calibrador de Crotalus durissus terrificus, homólogo a VAP1, nomeado crotastatin, e demais genes homólogos VAP1/crotastatin-like da glândula de veneno de Bothrops atrox, Crotalus durissus cascavella e Lachesis muta rhombeata foram expressos em diferentes níveis. O batroxstatin, o precursor crotastatin-like, de B. atrox, é expresso 87 vezes mais que crotastatin-1, de C. d. cascavella, e 7.5 vezes mais que lachestatins, de L. m. rhombeata. Além do mais, análises estruturais, in silico, das sequências de aminoácidos indicam que batroxstatin-2, crotastatin e lachestatin-1 e -2 apresentam domínios estruturais e sítio de ligação ao metal, iguais aos de VAP1, sendo dessa forma inclusos em um ramo filogenético que compreende as toxinas indutoras de apoptose

Submitted by Maria Beatriz Vieira (mbeatriz.vieira@gmail.com) on 2017-03-06T13:55:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-09T20:15:32Z (GMT) No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-03-09T20:17:03Z (GMT) No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-09T20:17:13Z (GMT). No. of bitstreams: 2 dissertacao_mariana_peil_da_rosa.pdf: 780579 bytes, checksum: 32fb27eef72fa6d21ed64bfe1f7c5af1 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2014-03-31 / Sem bolsa / O objetivo deste trabalho foi avaliar a expressão do gene HPS no tegumento de quatro genótipos de soja, em épocas distintas de coleta após a antese. O experimento foi conduzido em casa de vegetação da Estação Terras Baixas da Embrapa Clima Temperado e no Laboratório de Sementes e Biotecnologia (UFPel/FAEM), em delineamento completamente casualizado, com três repetições. Utilizou-se duas cultivares de tegumento amarelo, COODETEC 202 e BMX Potência RR (POT) e duas linhagens de tegumento preto, Tegumento Preto (TP) e IAC 222. Efetuou-se sete coletas dos tegumentos, em intervalos de 5 dias cada, durante o desenvolvimento das sementes de soja entre os períodos de 25 a 55 dias após a antese. Procedeu-se a extração de RNA dos tegumentos, seguido da obtenção do cDNA para posterior análise de qRT-PCR, visando quantificar o acúmulo relativo do gene HPS. A cultivar IAC 222 apresentou valores de quantificação relativa superior em todas as épocas avaliadas, sendo o maior valor encontrado na coleta de 35 dias após a antese. O acúmulo relativo de transcritos do gene HPS no tegumento de sementes de soja difere entre as épocas avaliadas, bem como entre os genótipos estudados. / The objective of this study was to evaluate the gene expression of HPS in the seed coat of four soybean genotypes at different sampling times after anthesis. The experiment was conducted in greenhouse at Estação Terras Baixas of Embrapa Clima Temperado and Laboratório de Sementes e Biotecnologia (UFPel / FAEM) in Capão do Leão city - RS, in a randomized complete block design with three replicates, and treatments arranged in a factorial model. Were used four contrasting genotypes, two cultivars of yellow coat (COODETEC 202 and BMX Potência RR) and two genotypes of black coat (Tegumento Preto and IAC). Seed coat samples were collected along the developing soybean seed, intervals for 5 days, between the periods 25-55 days after anthesis. Proceeded the RNA extraction from the seed coats, followed by obtaining the cDNA for further analysis of qRT-PCR, to quantify the relative accumulation of gene transcripts HPS. IAC 222 showed higher values of relative quantification in all periods, with the highest value found in the collection of 35 days after anthesis. The relative accumulation of transcripts of the HPS gene in the seed coat of soybean differs between the periods evaluated, as well as between genotypes.

Made available in DSpace on 2014-08-20T13:44:34Z (GMT). No. of bitstreams: 1 tese_caroline_borges_bevilacqua.pdf: 1981019 bytes, checksum: 797e667e01edbc569a7b08d3268c701b (MD5) Previous issue date: 2013-10-30 / Cold stress adversely modifies their physiology, metabolism plant growth and development, as well as, it limits crop productivity. The responses of rice (Oryza sativa L.) subjected to low temperatures are still poorly understood. A better understanding of stress tolerance mechanism in rice plants will help to develop rice germplasm with improved field level tolerance under variable temperature and sowing depth conditions. To characterize rice genotypes with variation in sensitivity to cold, these are the following objectives: to evaluate the applicability of different Stress Indices using seedling lengthas parameter; classify accessions cultivated rice and red rice as Indica or Japonica; compare response to rice cultivars cold-tolerant and cold-sensitive to cold stress according to the dry matter accumulation and possible changes in chlorophyll content; categorize different genotypes with regard to sensitivity to cold and to sowing depth stresses and, analyze the expression of cold-responsive genes, and also genes submergence-responsive. The seeds after seven days at 25°C were exposed at 4°C for 24h and after that, photosynthesis was measured later, the plants were 72h at 25°C (recovery period) to assess the dry mass and chlorophyll. For the other experiments, the seedlings were collected 7 and/or 14 days maintained at 25°C or 18/13°C day/night and different sowing depths (1.5cm, 5cm, 10cm and 15cm), differential gene expression were performed with those seedlings using different genes induced by cold. To evaluated gene expression using different genes induced by cold and anoxia, samples were collected after exposure to 10 ° C for 6, 24 and 96 h at 1.5 cm and 10 cm deep sowing. The results showed that is possible to identify superior genotypes for tolerance to these abiotic stresses based on the Tolerance Index (STI) and Media Geometric (GM) to select genotypes tolerant to cold or sowing depth, using as a parameter the seedling shoot length measurement. Japonica and Indica subspecies respond differently to abiotic stresses, however for some of these stress-responsive genes, these subspecies responded similarly. Furthermore, the analysis at the molecular level of cold tolerance and sowing depth indicated the importance of ABA- dependent and ABA-independent signal transduction pathways in plants under abiotic stress. / O estresse causado pelo frio interfere negativamente na fisiologia, metabolismo, crescimento e desenvolvimento das plantas e, portanto, limita a produtividade em lavouras de arroz. As respostas em nível de crescimento em arroz (Oryza sativa L.) submetido a baixas temperaturas ainda são pouco compreendidas. Um melhor entendimento do mecanismo de tolerância ao estresse em plantas de arroz pode ajudar na identificação, no germoplasma de arroz, de plantas com tolerância submetidas à temperatura variável, além de ser útil para outros estresses abióticos, como diferentes profundidades de semeadura. Para caracterizar genótipos de arroz, com variação na sensibilidade ao frio, tiveram-se como objetivos:avaliar a aplicabilidade de diferentes índices de estresse utilizando-se como parâmetro o comprimento de plântula; classificar acessos de arroz cultivado e vermelho como Japonica ou Indica; comparar a resposta ao frio de cultivares de arroz tolerante e sensível a esse estresse, com relação ao acúmulo de massa seca e possíveis alterações no teor de clorofila;categorizá-los com relação à sensibilidade ao frio e à profundidade de semeadura; e analisar a expressão de genes que respondem a frio, assim como genes responsivos a submersão, sob condições de frio e/ou tratamento constituídos por diferentes profundidades de semeadura. Para avaliar o acúmulo de massa seca e o teor de clorofila, as sementes, após sete dias a 25°C, foram expostas a 4°C durante 24 h e logo após, foi medida a fotossíntese e,posteriormente, as plantas ficaram 72 h a 25°C para sua recuperação. Já para os demais experimentos,as plântulas foram coletadas 7 e/ou 14 dias mantidas a 25°C ou 18/13°C dia/noite e diferentes profundidades de semeadura (1.5cm, 5cm, 10cm e 15cm); as avaliações da expressão gênica diferencial foram realizadas com essas amostras coletadas, para 4 diferentes genes induzidos pelo frio e também em amostras coletadas após exposição a 10°C durante 6, 24 e 96 h a 1.5 cm e 10 cm de profundidade de semeadura.Os resultados indicaram que é possível a identificação de genótipos superiores para a tolerância a esses estresses abióticos com base em seus índices de estresse, utilizando como parâmetro o comprimento da parte aérea, devido a habilidade das plantas tolerar estresses abióticos afetar a morfologia assim como a fisiologia da planta de arroz. Assim como é possível a utilização do Índice de Tolerância (STI) e da Média Geométrica (GM) para selecionar genótipos tolerantes ao frio ou profundidade de semeadura, baseado no comprimento de parte aérea de plântula. As subespécies Japonica e Indica respondem diferentemente aos estresses abióticos, no entanto, para alguns genes responsivos a esses estresses, essas subespécies apresentam o mesmo respondem semelhantemente. Além disso, as análises a nível molecular da tolerância ao frio e a profundidade de semeadura indicaram a importância das vias ABA-dependente e ABA-independente como vias de transdução do sinal em plantas sob estresse abiótico.

With a continuous development of molecular-biology methods more attention has been paid to molecular detection of minimal residual diseases in solid tumors. In our study we focused on detection of MRD in neuroblastoma. Neuroblastoma is one of the peripheral neuroblastic tumors (pNTs) that accounts approximately for10 percent of all childhood cancers. The question raised however not answered until this day is whether evidence of MRD in bone marrow may be used as independent prognostic factor in diagnosis of neuroblastoma. Furthermore, it is important to establish what kind of testing technique should be used and what values to look at. There exist various methodologies in detection of MRD evidence in neuroblastoma. These methods differ in cost and complexity, but mainly some of them are more specific and sensitive than the other. Cancer cells may be detected in the blood as well as in the bone marrow. Very often it is the bone marrow that is affected by the metastasis in neuroblastoma, therefore 85% of all high risk neuroblastomas show positive results in the standard cytomorphology tests of bone marrow. Low numbers of cancer cells in bone marrow or peripheral blood (especially during or after the end of treatment) are below the standard values of detection limit in most of the classic methodologies...

Philosophiae Doctor - PhD / The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene. / South Africa

Mucosa associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH andt(14;18)(q32;q21)/IGH-MALT1, which commonly activate the NF-κB pathway. Gastric MALT lymphomas harbouring such translocation do not respond to Helicobacter pylori eradication, while those without translocation can be cured by antibiotics. To understand the molecular mechanism of MALT lymphoma with and without chromosome translocation, 24 cases (15 translocation-positive and 9 translocation-negative) of MALT lymphomas together with 7 follicular lymphomas and 7 mantle cell lymphomas were analysed by Affymetrix gene expression microarray platform. Unsupervised clustering showed that cases of MALT lymphoma were clustered as a single branch. However, within the MALT lymphoma group, translocation-positive cases were intermingled with translocation-negative cases. Gene set enrichment analysis (GSEA) of the NF-κB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions showed that translocation-positive MALT lymphomas were characterized by an enhanced expression of NF-κB target genes, particularly TLR6, CCR2, CD69 and BCL2, while translocation-negative cases were featured by active inflammatory and immune responses, such as IL8, CD86, CD28 and ICOS. Separate analyses of the genes differentially expressed between translocation-positive and negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. The differential expression of these NF-κB target genes between MALT lymphoma with and without translocation was confirmed by quantitative RT-PCR and immunohistochemistry or Western blot. Expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10 mediated NF-κB activation in vitro. In addition, there was cooperation between expression of BCL10, MALT1 or API2-MALT1, and stimulation of the antigen receptor or CD40 or TLR in NF-κB activation as shown by both reporter assay and IκBα degradation. Interestingly, expression of BCL10 but not API2-MALT1 and MALT1, in the presence of LPS stimulation, also triggered IκBβ degradation, suggesting activation of different NF-κB dimers between these oncogenic products. Study by co-immunoprecipitation showed that BCL10 directly interacts with MALT1. Sub-cellular localisation experiments in BJAB B-cells, showed that BCL10 localisation was affected by MALT1. When BCL10 was over-expressed, the protein was predominantly expressed in the nuclei, but when MALT1 was over-expressed, BCL10 was mainly localised in the cytoplasm. When both BCL10 and MALT1 were over-expressed, BCL10 was expressed in the cytoplasm in the early hours when the protein level was low, but in both the cytoplasm and nuclei after 9 hours when the protein level was high. Over-expression of API2-MALT1 did not shown any apparent effect on BCL10 sub-cellular localisation in vitro. Finally, comparison of MALT lymphoma expression microarray with other lymphomas showed lactoferrin to be highly expressed in MALT lymphoma. This was confirmed by qRT-PCR, showing lactoferrin to be significantly over-expressed in MALT lymphoma compared to FL and MCL. Thus lactoferrin may be a potential marker for MALT lymphoma.

This study represents the first comprehensive analysis of gene regulation underlying the distinct categories of resistance afforded to wheat (Triticum aestivum, L.) by different Dn genes. Russian wheat aphid (Diuraphis noxia, Mordv.) feeding on susceptible wheat cultivars causes leaf rolling, chlorosis and the eventual death of the plant. Plants expressing Dn genes are resistant to D. noxia infestation, but different Dn genes afford phenotypically distinct modes of resistance: the Dn1 gene confers an antibiotic effect to lower aphid fecundity; Dn2 confers tolerance to high aphid pressure; and Dn5 confers antixenosis, and aphids do not prefer such plants as hosts. Little is known about the components involved in establishing a successful defence response against D. noxia attack and how these differ between the distinct resistance categories. It is assumed that the Dn genes function as classic R genes in plant defence, being receptors for elicitors in aphid saliva. Upon recognition, defence response signalling is initiated, but the exact mechanics of subsequent cellular events in aphid resistance have only recently come under investigation. Evidence from cDNA microarray and subtractive hybridization experiments indicated the involvement of kinase signalling cascades and photosynthetic proteins in the response against D. noxia. However, expression analysis describing how these processes differ between plants carrying different Dn genes and how these differences account for antibiosis, antixenosis or tolerance had not been conducted. We consequently investigated the downstream components involved in or affected by the generation of these resistance mechanisms by comparing the responses in transcript regulation of Tugela near-isogenic lines with different Dn genes to D. noxia infestation. cDNA-AFLP analysis was selected as an appropriate functional genomics tool, since it is semi-quantitative, does not require prior sequence information and allows for the discovery of novel genes. cDNA-AFLP analysis yielded 121 differentially regulated transcript-derived fragments (TDFs) grouped into eight expression clusters. We cloned and sequenced 49 representative TDFs, which were further classified into five broad functional categories based on inferred similarity to database sequences. Transcripts involved in such diverse processes as stress, signal transduction, photosynthesis, metabolism and gene regulation were found to be differentially regulated during D. noxia feeding. Many TDFs demonstrated homology to proteins with unknown function and several novel transcripts with no similarity to previously published sequences were also discovered. Detailed expression analysis using quantitative RT-PCR and RNA hybridization provided evidence that the time and intensity of induction of specific pathways is critical for the development of a particular mode of resistance. This includes: the generation of kinase signalling cascades and the induction of several ancillary processes such as ubiquitination, leading to a sustained oxidative burst and the hypersensitive response during antibiosis; tolerance as a passive resistance mechanism countering aphid-induced symptoms through the repair or de novo synthesis of photosystem proteins; and the possible involvement of ethylene-mediated wounding pathways in generating volatile organic compounds during antixenosis. This is the first report on the involvement of KCO1, a vacuolar K+ channel, in assisting cytosolic Ca2+-influx and preventing leaf rolling, as well as on the role of iron homeostasis as a gene regulatory mechanism for sustaining the oxidative burst during the antibiotic defence response. This study opens up several areas of investigation heretofore unexplored in cereal-aphid interaction research. Of particular interest is the induction of genes involved in photosynthetic compensation during Dn2 tolerance responses, since these constitute a novel, passive resistance mechanism exclusive to aphid defence as opposed to the active resistance triggered in the presence of the Dn1 gene in the form of a general hypersensitive response. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Nitrogen cycling processes can be tracked using quantitative Polymerase Chain Reaction (qPCR) to determine the presence and qReverse Transcriptase-PCR (qRT-PCR) to determine expression of key genes, or ‘biological markers’, for nitrogen metabolism. Nitrification is catalyzed in part, by two enzymes: ammonia monooxygenase (AMO; NH3 NH2OH) and nitrite oxidoreductase (NXR; NO2- NO3-). For denitrification, four enzymes act sequentially: nitrate reductase (NAR/NAP; NO3- NO2-), nitrite reductase (NIR; NO2- NO), nitric oxide reductase (NOR; NO  N2O), and nitrous oxide reductase (NOS; N2O  N2). A principle of wastewater treatment (WWT) is to remove excess nitrogen by taking advantage of natural nitrogen cycling or biological nitrogen removal (BNR). This process involves using microorganisms to bring influent ammonia through nitrification and denitrification to release nitrogen gas, which does not contribute to eutrophication. A novel shortcut nitrogen removal configuration could increase nitrogen removal efficiency by promoting nitritation/denitritation, reducing the classic nitrogen cycle by removing the redundant oxidation/reduction step to nitrate (NO3-). Here, three nitrogen transformations were used to track the three main phases in the nitrogen cycle; ammonia monooxygenase for nitrification, nitrite oxidoreductase for shortcut, and nitrous oxide reductase for denitrification. Primers for qPCR and qRT-PCR were designed to capture as much sequence diversity as possible for each step. Genes from bacteria known to perform the nitrogen transformations of interest (amoA, nxrB, nosZ) were used to BLAST-query the Integrated Microbial Genomes & Microbiomes database (img.jgi.doe.gov) to find homologs from organisms commonly found in WWT. These sequences were then aligned to find regions sufficiently conserved for primer design. These PCR primers were tested against standards for each gene and used to track nitrogen transformation potential and expression in a novel lab-scale algal photo-sequencing batch reactor which promotes shortcut nitrogen removal from wastewater across three solids retention times (SRT, or mean cell residence time); 5, 10 and 15 days. SRT 15 had the greatest total nitrogen removal with nitritation and denitritation observed. Nitrate was not detected in the first cycle and shortcut nitrogen removal was supported by low levels of nxrB genes and transcripts. Simultaneous nitrification/denitrification was supported by elevated concentrations of nosZ during the light period and less nitrite produced than ammonium consumed. Nitritation was predominantly performed by Betaproteobacteria amoA and nitrous oxide reduction was predominantly from nosZ group I (Proteobacteria-type).

No description available.