M. Tech. (Biotechnology, Faculty of Applied and Computer Science), Vaal University of Technology / Tulbaghia violacea Harv. (wild garlic) has been used in traditional medicine in Southern Africa for the treatment of various ailments. Despite the widespread use and popularity of this medicinal plant as a herbal medicine, there is contradictory evidence regarding the safety and toxicity of the plant. The phytochemical profiling of the plant has also been neglected in research. The determination of chemical constituents present in plant material as well as the potential toxicity found in plants are preliminary steps necessary for the discovery and development of novel therapeutic agents with improved efficacy. The aim of this study was to evaluate the cytotoxic and genotoxic potential of crude extracts from the leaves, stems and roots of T. violacea. This was performed in vitro using aqueous and ethanol extracts of the leaves, stems and roots. The aim of the study was achieved by three major objectives; (1) to identify the active phytocompounds present in the leaves, stems and roots, (2) to assess the cytotoxicity using the MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) cell proliferation assay, and (3) to evaluate the genotoxic potential of the leaf, stem and root water extracts using the Allium cepa assay. A total of 14 phytochemicals were each extracted separately with distilled water and 70% ethanol by maceration from the leaves, stem and roots of T. violacea. The results of the qualitative phytochemical analysis showed that pharmacologically active compounds such as tannins, terpenoids, flavonoids, saponins, proteins, steroids, cardiac glycosides, phenols and coumarins were present in some organs of T. violacea. However, phlobatannins, leucoanthocyanins, alkaloids, carbohydrates and anthocyanins were absent in all plant parts. Overall, the leaves of the plant contained more active compounds than those present in the stems and roots when both water and 70% ethanol were used as the extractants. The quantitative phytochemical analysis for the Total Flavonoids Content (TFC) and Total Phenolic Contents (TPC) was also assessed. The water (0.027 mg/g) and 70% ethanol (0.053 mg/g) were most effective in extracting flavonoids from the leaves while the least amounts were obtained from the stems and roots. This observation was similar to the TFC were the water extracts of the leaves were the most effective in extracting phenols followed by the stems and roots. The MTT assay was conducted using two cell lines RAW 264.7 and C2C12. The experiment was conducted in triplicates for the leaf, stem and root extracts (water and ethanol) of T. violacea. The experimental design employed a 23 factorial design where three independent variables (concentration, incubation time and type of extracts) were selected using two levels for each variable (high (+) and low (-)). The results illustrated that both the water and ethanol vi extracts only showed a significant reduction in the number of viable cells at the concentration higher than 250 μg/ml treatment for both RAW 264.7 and C2C12 cells. The ethanol extracts from the leaves, stems and roots were found to be toxic towards the RAW 264.7 cells even at lower concentrations at both 24 and 48 h incubation periods (% cell viability < 50%). The water extracts were non-toxic to RAW 264.7 cells except for the water stem extract which showed toxicity after 48 h incubation (IC50 = 9.475 (4.061 to 23.39)). For the C2C12 cells, the lowest potent toxic concentration was 250 μg/ml for the ethanol extract of the stem after 48 h incubation. Overall, the T. violacea plant extracts were non-toxic as percentage cell viability greater than 50% was noted for both extraction solvents in all the plant parts of T. violacea. No cytotoxic activity was observed in all T. violacea plant parts with the C2C12 cell line (IC50 > 30 μg/ml). For the Allium cepa assay, only the water crude extracts of the leaves, stems and roots of T. violacea were used. A similar trend of potent genotoxic activity in the water stem extracts compared to the leaf and root extracts at the concentration ranges studied. Similar to the MTT assay, it is clear from the study that at higher concentrations, the water crude extracts from the leaves, stems and roots of T. violacea is toxic. From this study, it can be concluded that the extraction of compounds using water is more efficient than using ethanol. Overall, the T. violacea leaf extracts extracted the most phytocompounds and showed the highest percentage of viable cells as well as desirable IC50 values. However, preparation of herbal remedies using T. violacea plant extracts should be done with caution due to their possible genotoxic and cytotoxic potential at higher concentrations. This study raises a need to further conduct in vivo cytogenetic studies to ascertain the possible toxic effects of T. violacea crude extracts.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Made available in DSpace on 2014-06-11T19:32:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-20Bitstream added on 2014-06-13T20:23:16Z : No. of bitstreams: 1 maschio_lr_dr_sjrp.pdf: 1208225 bytes, checksum: 581a26de1a4603e41d2d07020f15f18d (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / Devido às crescentes expansões demográficas e industriais observadas nas últimas décadas, o meio ambiente tem recebido uma carga significativamente crescente de efluentes domésticos, industriais e agrícolas, causando impactos severos nos ecossistemas e um potencial comprometimento à saúde humana. Dentre os efluentes domésticos, podemos citar uma gama de poluentes, tais como químicos de diversas categorias, além de contaminações por agentes biológicos diversos. Já os efluentes industriais contêm poluentes orgânicos e/ou inorgânicos, dependendo da atividade industrial. Baseando-se nestes dados, este trabalho teve como objetivo investigar, por meio de ensaios biológicos com dois organismos-teste, a possível presença de contaminantes com potencial citotóxico, genotóxico e mutagênico, que são despejados ao longo do rio Preto, inclusive na Represa Municipal de São José do Rio Preto. O material biológico utilizado neste estudo constituiu-se de sementes de Allium cepa (cebola) e peixes da espécie Oreochromis niloticus (Tilápia). Coletas de águas foram realizadas, sazonalmente, nos meses de agosto de 2006 e 2007 (estação seca) e março de 2007 e 2008 (estação chuvosa), em seis pontos distintos: Ponto 1 (P1), 8 km antes do represamento; Ponto 2 (P2), 1 km antes do represamento; Ponto 3 (P3), local de despejo do esgoto; Ponto 4 (P4), margem oposta do despejo do esgoto; Ponto 5 (P5), saída do represamento; Ponto 6 (P6), 1 km após o represamento. Análises químicas foram realizadas para todas as coletas realizadas. Para a realização do estudo, 100 sementes de Allium cepa foram submetidas à germinação, em placa de Petri, em amostras de águas coletadas nos seis diferentes pontos do rio Preto, em água ultra pura (controle negativo) e em uma substância reconhecidamente aneugênica (Trifluralina - controle positivo), sempre à temperatura ambiente... / Due to increasing population and industrial expansion observed in recent decades, the environment has received a significant increased burden of domestic industrial and agricultural sewerage, which can cause severe impacts on ecosystems, and a potential damage to human health as well. A wide range of harmful pollutants can be found in domestic effluent, such as chemicals from various categories, in addition to contamination by various biological agents. On the other hand, industrial effluents contain organic and / or inorganic pollutants, depending on industrial activity. Based on these data, this study aimed to investigate, by means of biological tests with two test-organism, the possible presence of contaminants with cytotoxic, genotoxic and mutagenic potential, which are dumped along the Preto river, an important river that flows in the region of Sao Jose do Rio Preto/SP. The biological material used in this study consisted of seeds of Allium cepa (onion) and one specie of fish (Tilapia: Oreochromis niloticus). Water samples were taken seasonally in August 2006 and 2007 (dry season) and March 2007, and 2008 (rainy season), in six distinct sites: Site 1 (S1), 8 km before the damming, Site 2 (S2), 1 km before the damming, Site 3 (S3), place of sewerage discharge; Site 4 (S4), opposite margin of sewage discharge, Site 5 (S5), end of the damming; Site 6 (S6) 1 km after damming. Chemical analyses were performed for all collected samples. For the study, 100 seeds of A. cepa were submitted to germination in Petri dishes with samples water from six different sites of the Preto river, Ultra pure water (negative control), and with an aneugenic substance (Trifluralin - positive control). For most of collection points and periods studied, root meristems cells of A. cepa, exposed to water samples collected along the Preto river, showed no significant differences... (Complete abstract click electronic access below)

Submitted by Deyse Queiroz (deysequeirozz@hotmail.com) on 2018-05-22T15:00:44Z No. of bitstreams: 1 JESSÉ MALVEIRA GOMES - DISSERTAÇÃO PPGHT - 2017.pdf: 1522466 bytes, checksum: cc3a2a2cb3b9b312f031097d4500a8df (MD5) / Made available in DSpace on 2018-05-22T15:00:44Z (GMT). No. of bitstreams: 1 JESSÉ MALVEIRA GOMES - DISSERTAÇÃO PPGHT - 2017.pdf: 1522466 bytes, checksum: cc3a2a2cb3b9b312f031097d4500a8df (MD5) Previous issue date: 2017-03 / O milho é uma das culturas mais produzida e importante no mundo, sendo utilizada na alimentação humana e animal. O Brasil é um dos maiores produtores desse grão, sendo o terceiro maior produtor do mundo. Apesar de uma produção elevada, a produtividade é comprometida pela ação de insetos pragas, sendo a Spodoptera frugiperda Smith a de maior atuação nessa cultura. Em meio a esse desafio, o uso constante de inseticidas é utilizado com intuito de minimizar as perdas nas lavouras, porém, ocasionando grandes impactos ambientais, bem como a contaminação de trabalhadores rurais. Uma das estratégias viáveis e promissoras na supressão de pragas tem sido o manejo integrado de pragas (MIP), que tem como alicerce o uso isolado ou consorciado de técnicas de controles de pragas, sendo o controle biológico uma das ferramentas mais sustentáveis do ponto de vista ecológico. Desta forma, o presente trabalho teve como objetivo avaliar a toxicidade da cepa bacteriana 358.1 isolada do petróleo e o sobrenadante da cultura de células sobre o controle de lagartas de 2º e 3º instares de S. frugiperda, comparando as taxas de mortalidade e efeitos sobre seu desenvolvimento e com inseticidas químicos comerciais. O experimento consistiu de duas etapas, o primeiro ensaio foi realizado com lagartas de 3º instar com três diluições diferentes da cepa 358.1, sobrenadante da cultura de células, solução salina como controle negativo e o inseticida fenpropatrina como controle positivo. No segundo ensaio utilizou-se lagartas de 3º e 2º instares, a cultura celular da cepa 358.1 em sua concentração total (4 x 108 UFC/mL), seu sobrenadante da cultura, como controle negativo água destilada e testemunha positiva o produto comercial flubendiamida. Os experimentos com a cepa 358.1, sobrenadante da cultura de células e os produtos químicos testados não resultaram em uma taxa de mortalidade de S. frugiperda significante, porém, a cepa 358.1 e seu sobrenadante da cultura resultaram no aumento da duração da fase larval e em distúrbios morfofisiológicos do inseto, ocasionando, inclusive, no surgimento de adultos com asas atrofiadas, sendo desta maneira, promissores para à realização de novos testes. / Corn is one of the most produced and important crops in the world, utilized for human and animal feeding. Brazil is one of the top producers of this grain, being the third biggest producer worldwide. Despite the great production, the productivity is compromised due to the activity of insect pest. Because of this challenge, the continuous use of insecticides is done aiming to minimize lost in the crops. However, it causes major environmental impact and it is also contaminating rural workers. One of the viable and promising strategies to the pest suppression has been the pest integrated management (PIM), which is based on the use of isolated or consortium of pest controlling techniques, the biological control being one of the most sustainable tools in an ecological perspective. Therefore, the present study had the goal of evaluating the toxicity of the 358.1 bacterium strain isolated from the oil and supernatant of the cell culture under control of caterpillars from 2nd and 3rd instars of S. frugiperda, comparing the death rates and effects on their development, and also comparing with commercial chemical insecticides. The experiment consisted in two stages; the first assay was done with 3rd instar caterpillar with three different dilutions from the 358.1 strain, supernatant of the cell culture, saline solution as negative control, and fenpropatrina insecticide as positive control. On the second assay, 3rd and 2nd instars caterpillars were used, the cell culture from the 358.1 strain on its total concentration (4 x 108 UFC/mL), supernatant of the cell culture, distilled water as negative control and flubendiamida, a commercialized product, as the positive control. The experiments with the 358.1 strain, supernatant of the cell culture and the tested chemical products didn’t show a significant death rate for S. frugiperda, however, the 358.1 strain and supernatant of the cell culture resulted in an increase in the larval phase duration and on morphophysiological disturbs on the insect, resulting yet in the emergence of adults with atrophied wings, thus being promising to the realization of new tests.

Submitted by Johnny Rodrigues (johnnyrodrigues@ufcg.edu.br) on 2018-05-15T17:22:01Z No. of bitstreams: 1 JOANA DARC PAZ DE MATOS - DISSERTAÇÃO PPGSA PROFISSIONAL 2015..pdf: 3814977 bytes, checksum: b0004fe2b930dc37c724b5922f77b462 (MD5) / Made available in DSpace on 2018-05-15T17:22:01Z (GMT). No. of bitstreams: 1 JOANA DARC PAZ DE MATOS - DISSERTAÇÃO PPGSA PROFISSIONAL 2015..pdf: 3814977 bytes, checksum: b0004fe2b930dc37c724b5922f77b462 (MD5) Previous issue date: 2015 / A cebola (Allium cepa L.) é uma hortaliça com grande demanda de consumo no Brasil, principalmente, na forma in natura. Seu preparo é o maior motivo de objeção pelo consumidor, devido a compostos voláteis que irritam os olhos. O processamento mínimo torna assim, o produto mais atrativo e pronto para consumo, por eliminar as etapas de manipulação. O presente trabalho objetivou avaliar as alterações na composição fitoquímica e microbiológica em formatos de cebola amarela IPA-11 minimamente processadas. Os bulbos foram cultivados de outubro de 2013 a março de 2014, numa área experimental do Centro de Ciências e Tecnologia Agroalimentar da Universidade Federal de Campina Grande, Campus de Pombal – PB; colhidos; curados durante seis dias à temperatura ambiente (30±2°C) e minimamente processados: seleção; remoção do catáfilos externos; cortes; sanitização e enxágue; drenagem das cebolas inteiras; centrifugação das rodelas e cubos; embalagem em bandeja de isopor recoberta com filme de PVC em porções de 200 g de cebolas; e, conservação a 4±1ºC, sob 70±5% UR, durante 10 dias. O delineamento experimental foi inteiramente casualizado, em esquema fatorial 3 x 6, correspondente a tipos de cortes (inteira, rodela e cubo) e tempos de conservação (0, 2, 4, 6, 8 e 10 dias), com cinco repetições. Os bulbos obtiveram rendimento maior que 90% no processamento mínimo para todos os cortes. Observou-se durante o período de conservação aumento na perda de massa fresca, na concentração de íons H+, nos teores de açúcares solúveis e redutores e no extravasamento de eletrólitos. Os teores de ácido ascórbico e a relação sólidos solúveis/acidez titulável reduziram com o tempo de conservação. Nos compostos bioquímicos, os flavonóides reduziram em todos os tratamentos. As clorofilas e os carotenóides oscilaram durante a conservação, independente do corte. Verificou-se que os compostos fenólicos e a pungência mantiveram-se praticamente constantes durante a conservação. Obteve-se baixa contagem de aeróbios psicrotróficos, bolores e leveduras. A contagem de coliformes totais e termotolerantes resultou valores de ˂3 NMP/g. O tipo de corte e o tempo de conservação não comprometeram a qualidade final das cebolas amarelas IPA-11 minimamente processadas. / The onion (Allium cepa L.) is a vegetable with great demand in Brazil, mainly in natura. Its preparation is the biggest consumer objection due to volatile compounds that irritate the eyes. The product becomes more attractive and ready to be used by eliminating manipulation steps. This study aimed to evaluate changes in the phytochemical and microbiological composition of yellow onion formats IPA-11 minimally processed. The bulbs were grown from October 2013 to March 2014 on an experimental area of the Center of Agrifood Science and Technology - Federal University of Campina Grande, Campus of Pombal - PB; harvested; cured for six days at room temperature (30 ± 2 ° C) and minimally processed: selection; removal of external cataphylls; cuts; sanitizing and rinsing; draining the whole onions; centrifugation of slices and cubes; packing styrofoam trays covered with PVC film in portions of 200 g of onions; and conservation at 4 ± 1 ° C under 70 ± 5% RH for 10 days. The experimental design was completely randomized in a factorial 3 x 6, corresponding types of cuts (whole, slice and cube) and storage time (0, 2, 4, 6, 8 and 10 days), with five replications. The bulbs have gained more than 90% yield in minimum processing for all cuts. It was observed a loss increase of weight during the retention period , the concentration of H + ions in soluble sugars and reducing and electrolyte leakage. The ascorbic acid content and the soluble solids / titratable acidity decreased with storage time. In the biochemicals compounds, flavonoids decreased in all treatments. The chlorophylls and carotenoids varied during storage, regardless the cut. It was found that the phenolic compounds and pungency remained virtually constant during storage. Obtained low aerobic psychrotrophic count, molds and yeasts. The total coliforms and thermotolerant resulted values below 3 MPN / g. The kind of cut and the storage time did not affect the final quality of yellow onions IPA-11 minimally processed.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

No description available.

The overall aim of this research was to improve the understanding of the ecology and diversity of Trichoderma species within the soil and rhizosphere of onion (Allium cepa L.) and potato (Solanum tuberosum L.) under intensive management in New Zealand. The indigenous Trichoderma population was measured in a field trial at Pukekohe over a three year period under six different crop rotation treatments. The treatments included two continuous onion and potato rotations (intensive), two onion/potato mixed rotation (conventional), and two green manure rotations (sustainable). Results showed that Trichoderma populations were stable in both the rhizosphere and bulk soil (1.5 x 10² to 8.5 x 10³ CFU g⁻¹ ODS). The planting and incorporation of an oat (Avena sativa L.) green manure in the sustainable rotations positively increased Trichoderma colony forming unit (CFU) numbers in the rhizosphere soil from 3.4 x 10² to 2.5 x 10³ g⁻¹ ODS. A Trichoderma species identification method was developed based on colony morphology. Representative isolates were verified using restriction fragment length polymorphism (RFLP) and DNA sequencing. The method allowed for rapid and reliable identification of isolated Trichoderma species. Five species were identified in the Pukekohe soil: T. asperellum, T. atroviride, T. hamatum, T. harzianum and T. koningii. Results showed identical species diversity between the rhizosphere, rhizoplane and bulk soil. The species did not strongly compete between each other for the rhizosphere ecological niche and differences in species proportions seemed to be caused by environmental factors rather than the rotation treatments. The incorporation of oat green manure in pots did not significantly promote the indigenous Trichoderma population size and diversity in the rhizosphere of onion plants up to 4 months old. The identified species were the same as in the field trial. The incorporation of onion scale residues was shown to result in low Trichoderma and high Penicillium CFU numbers and a reduction in plant size. Additionally, the presence of high levels (6.0 x 10⁵ CFU g⁻¹ ODS) of Penicillium CFU was negatively correlated with the presence of Trichoderma CFU. The effect of oat incorporation on Trichoderma saprophytic growth was also investigated in a soil sandwich assay and revealed no significant differences. A series of experiments indicated that onion extract obtained from dry onion scale residues had no antifungal activity against either Trichoderma or Penicillium and instead tended to promote their hyphal growth and sporulation. It also showed that competition between Penicillium and Trichoderma isolates was limited despite the ability of Penicillium to produce a wide range of inhibitory substances. Four indigenous Trichoderma species (T. atroviride, T. hamatum, T. harzianum and T. koningii) were shown to be rhizosphere competent in a split tube experiment over a 6 week period. The results of this experiment revealed that, the Trichoderma species clearly displayed differences in their ability to colonise the rhizosphere of young onion seedlings. Species such as T. koningii had the greatest rhizosphere colonising ability regardless of soil depth while T. harzianum displayed the weakest ability. Results also indicated that when inoculated as a mixture the four species competed with one another to colonise the rhizosphere. Overall, this research indicated that the studied crop rotation treatments and the use of oat as a green manure did not strongly promote indigenous Trichoderma populations. Species diversity was constant throughout the research with T. hamatum and T. koningii being the most frequently isolated species.

The research presented in this thesis investigated the functional morphology in root<br />systems in relation to their role in providing anchorage and stability for the plant. The<br />anchorage of different types of root systems was investigated as well as the influence of<br />several environmental factors on their development. The research presented in this study<br />was completed by carrying out a series of modelling, glasshouse and field experiments<br />using physical models and real plants.<br />Model experiments showed that solid shapes like bulbs are very well suited to resist<br />vertical upward forces, i.e. uprooting, and shed some light on the mechanism of<br />anchorage in bulbs. The results of this laboratory study showed that the concept of<br />optimal bulb shape for resisting uprooting is viable. Uprooting tests on real bulb plants<br />confirmed the theoretical predictions about it, and showed the importance of bulbs in<br />anchorage. This study also proved that the soil type is very important when considering<br />the anchorage of solid forms such as the bulbs.<br />A second model study showed that the simplest models of tap root-dominated root<br />systems increase their resistance to overturning with the third and second power of the<br />embedment depth in cohesionless and in cohesive soil respectively. Anchorage strength<br />of a root system dominated by a tap root will be maximised with minimum investment<br />in structural material if the rigid tap root is extended to the largest possible depth.<br />Glasshouse experiments investigated the effects of soil compaction and temperature,<br />two of the most important environmental factors, on the axial and lateral development<br />and growth of the root systems of two species of young pines. It was shown that the rate<br />of root axial development in both investigated species decreased with an increase in soil<br />compaction whereas the lateral proliferation of their roots systems was not significantly<br />affected by soil consistency. A temperature of around 15°C seemed to be optimal for the<br />root elongation rate since the increase in axial length of the roots of both species was<br />largest at this temperature.<br />The effect of mechanical stimulation as a factor in shaping the root systems of plants<br />was also investigated. Apart from the changes caused to the parts of the tree above<br />ground, unidirectional periodical flexing induced an increase in total root CSA and<br />larger biomass allocation to the roots parallel to the plane of flexing which, in turn,<br />resulted in a larger number of major lateral roots with larger CSA in the plane of<br />flexing.<br />Mechanical and morphological field studies on two Pinus species investigated the<br />anchorage of plate root systems and showed that lateral roots in older trees are not the<br />major source of root anchorage in either of the species; although in both species a<br />certain asymmetry in the distribution of major lateral root CSA was recorded, it was not<br />significantly correlated to the asymmetry in anchorage.

No description available.