No description available.

The Photosystem II complex (PSII) contains besides D1, D2, CP43, CP47 and several lumenal extrinsic proteins also a large number of small intrinsic subunits. These subunits are found in all kinds of oxygenic phototrophs and their sequences are usually highly conserved. It is assumed that the small subunits are involved in stabilization, assembly or dimerization of the PSII complex but their exact function remains largely unknown. Thus, we chose four small PSII proteins: cytochrome b-559, PsbH, PsbI and Psb28, and we studied their function and location in PSII of the cyanobacterium Synechocystis sp. PCC 6803. We attempted: (i) to better define the role of cyt b-559 in the assembly of PSII using various mutant strains lacking one or more PSII core protein subunits; (ii) to clarify the location of the PsbH subunit using NTA gold labelling methodology; (iii) to improve the available knowledge on function of the PsbI protein in the assembly and repair of the cyanobacterial PSII; and (iv) to confirm the presence of the Psb28 protein in PSII, to localize it and to establish its importance for the cyanobacterial PSII.

No description available.

O grupo neuwiedi é composto por oito espécies amplamente distribuídas na diagonal das formações abertas da América do Sul: Bothrops diporus, B. erythromelas, B. lutzi, B. mattogrossensis, B. marmoratus, B. neuwiedi, B. pauloensis e B. pubescens. Foi utilizada uma amostra de 140 espécimes, com intuito de fornecer um teste independente, do limite das espécies e uma hipótese filogenética para os membros do grupo. As inferências filogenéticas realizadas a partir da máxima parcimônia, máxima verossimilhança e análise bayesiana, utilizando sequências de 1018 pb dos genes mitocondriais citocromo b e ND4 combinados, recuperaram topologias similares, nas quais as espécies do grupo neuwiedi formam um grupo monofilético com elevados valores de suporte. Foi recuperado o monofiletismo recíproco para as espécies B. erythromelas, B. lutzi e B. pubescens. Entretanto, B. diporus, B. mattogrossensis, B. neuwiedi, B. marmoratus e B. pauloensis apresentaram-se parafiléticas ou polifiléticas, como atualmente definidas. Haplótipos de Bothrops diporus e B. mattogrossensis foram recuperados em dois clados distintos, B. neuwiedi e B. marmoratus em três clados, e B. pauloensis em quatro clados. Os padrões parafiléticos e polifiléticos recuperados para os haplótipos podem ser decorrentes de um arranjo taxonômico inadequado, retenção de morfologia ancestral, convergência morfológica, plasticidade fenotípica, eventos de introgressão genética recente e antiga na história evolutiva do grupo, homoplasia molecular e retenção de polimorfismo ancestral, fenômenos que não são mutuamente exclusivos. O compartilhamento de haplótipos de DNA mitocondrial, simpatria e indivíduos de morfologia intermediária entre B. marmoratus e B. pauloensis são evidências que sugerem a ocorrência de introgressão recente e a existência de uma zona de hibridação na região central do Brasil. As inferências filogenéticas para o grupo neuwiedi sugerem a possível existência de espécies crípticas, demonstrando a necessidade premente de uma reavaliação da taxonomia corrente, a partir da análise conjunta dos dados morfológicos e DNA mitocondrial aqui apresentados. Também serão necessários estudos complementares a partir de marcadores nucleares. Esta pesquisa, quando comparada aos estudos filogenéticos prévios, representa um marcante aumento na quantidade e qualidade da amostragem para o grupo neuwiedi, incluindo todas as espécies atualmente reconhecidas e amostra de 140 indivíduos para 92 localidades de 13 Estados do Brasil, fornecendo importantes informações para o entendimento e conservação da biodiversidade das serpentes Neotropicais. / The neuwiedi group of species of the genus Bothrops is widespread in open areas of South America, and it is composed by eight current species: Bothrops diporus, B. erythromelas, B. lutzi, B. mattogrossensis, B. marmoratus, B. neuwiedi, B. pauloensis, and B. pubescens. A total of 140 samples were used to provide an independent test of the species boundaries and a hypothesis of phylogenetic relationships among the members of this group. Our combined data (1018 bp of cyt b and ND4 from mtDNA) recovered the neuwiedi group as a highly supported monophyletic group in Maximum Parsimony, Maximum Likelihood and Bayesian Analyses. Reciprocal monophyly for each B. erythromelas, B. lutzi and B. pubescens was recovered. However, B. diporus, B. mattogrossensis, B. neuwiedi, B. marmoratus and B. pauloensis showed to be paraphyletic and polyphyletic as currently defined. Haplotypes from each B. diporus and B. mattogrossensis were recovered in two different clades, B. neuwiedi and B. marmoratus were recovered in three clades, and B. pauloensis in four clades. The lack of monophyletic species could be explained by imperfect taxonomic analysis method, retention of ancestral morphology, convergent morphology, plasticity phenotipic, recent and ancient introgression, molecular homoplasy and incomplete lineage sorting, and these phenomena are not mutually exclusive. Sympatry, phenotipycally intermediate specimens and mtDNA haplotypes shared between B. marmoratus and B. pauloensis, provide evidences for recent introgression and the possibility of occurrence of a hybrid zone in Central Brazil. These phylogenetic inferences within neuwiedi group suggest the possible occurrence of cryptic species which were no detected morphologically, and the necessity of a current taxonomy review based on combining data presented here to morphological data. Compared to previous phylogenetic studies, the investigation presented here represents a remarkable increasing in quantity and quality concerning the group sampling, since it includes all the currently recognized species and samples of 140 individuals from 92 localities of 13 states of Brazil. It has never been done before, and supplies important information for understanding and to the conservation of Neotropical snake biodiversity.

O grupo neuwiedi é composto por oito espécies amplamente distribuídas na diagonal das formações abertas da América do Sul: Bothrops diporus, B. erythromelas, B. lutzi, B. mattogrossensis, B. marmoratus, B. neuwiedi, B. pauloensis e B. pubescens. Foi utilizada uma amostra de 140 espécimes, com intuito de fornecer um teste independente, do limite das espécies e uma hipótese filogenética para os membros do grupo. As inferências filogenéticas realizadas a partir da máxima parcimônia, máxima verossimilhança e análise bayesiana, utilizando sequências de 1018 pb dos genes mitocondriais citocromo b e ND4 combinados, recuperaram topologias similares, nas quais as espécies do grupo neuwiedi formam um grupo monofilético com elevados valores de suporte. Foi recuperado o monofiletismo recíproco para as espécies B. erythromelas, B. lutzi e B. pubescens. Entretanto, B. diporus, B. mattogrossensis, B. neuwiedi, B. marmoratus e B. pauloensis apresentaram-se parafiléticas ou polifiléticas, como atualmente definidas. Haplótipos de Bothrops diporus e B. mattogrossensis foram recuperados em dois clados distintos, B. neuwiedi e B. marmoratus em três clados, e B. pauloensis em quatro clados. Os padrões parafiléticos e polifiléticos recuperados para os haplótipos podem ser decorrentes de um arranjo taxonômico inadequado, retenção de morfologia ancestral, convergência morfológica, plasticidade fenotípica, eventos de introgressão genética recente e antiga na história evolutiva do grupo, homoplasia molecular e retenção de polimorfismo ancestral, fenômenos que não são mutuamente exclusivos. O compartilhamento de haplótipos de DNA mitocondrial, simpatria e indivíduos de morfologia intermediária entre B. marmoratus e B. pauloensis são evidências que sugerem a ocorrência de introgressão recente e a existência de uma zona de hibridação na região central do Brasil. As inferências filogenéticas para o grupo neuwiedi sugerem a possível existência de espécies crípticas, demonstrando a necessidade premente de uma reavaliação da taxonomia corrente, a partir da análise conjunta dos dados morfológicos e DNA mitocondrial aqui apresentados. Também serão necessários estudos complementares a partir de marcadores nucleares. Esta pesquisa, quando comparada aos estudos filogenéticos prévios, representa um marcante aumento na quantidade e qualidade da amostragem para o grupo neuwiedi, incluindo todas as espécies atualmente reconhecidas e amostra de 140 indivíduos para 92 localidades de 13 Estados do Brasil, fornecendo importantes informações para o entendimento e conservação da biodiversidade das serpentes Neotropicais. / The neuwiedi group of species of the genus Bothrops is widespread in open areas of South America, and it is composed by eight current species: Bothrops diporus, B. erythromelas, B. lutzi, B. mattogrossensis, B. marmoratus, B. neuwiedi, B. pauloensis, and B. pubescens. A total of 140 samples were used to provide an independent test of the species boundaries and a hypothesis of phylogenetic relationships among the members of this group. Our combined data (1018 bp of cyt b and ND4 from mtDNA) recovered the neuwiedi group as a highly supported monophyletic group in Maximum Parsimony, Maximum Likelihood and Bayesian Analyses. Reciprocal monophyly for each B. erythromelas, B. lutzi and B. pubescens was recovered. However, B. diporus, B. mattogrossensis, B. neuwiedi, B. marmoratus and B. pauloensis showed to be paraphyletic and polyphyletic as currently defined. Haplotypes from each B. diporus and B. mattogrossensis were recovered in two different clades, B. neuwiedi and B. marmoratus were recovered in three clades, and B. pauloensis in four clades. The lack of monophyletic species could be explained by imperfect taxonomic analysis method, retention of ancestral morphology, convergent morphology, plasticity phenotipic, recent and ancient introgression, molecular homoplasy and incomplete lineage sorting, and these phenomena are not mutually exclusive. Sympatry, phenotipycally intermediate specimens and mtDNA haplotypes shared between B. marmoratus and B. pauloensis, provide evidences for recent introgression and the possibility of occurrence of a hybrid zone in Central Brazil. These phylogenetic inferences within neuwiedi group suggest the possible occurrence of cryptic species which were no detected morphologically, and the necessity of a current taxonomy review based on combining data presented here to morphological data. Compared to previous phylogenetic studies, the investigation presented here represents a remarkable increasing in quantity and quality concerning the group sampling, since it includes all the currently recognized species and samples of 140 individuals from 92 localities of 13 states of Brazil. It has never been done before, and supplies important information for understanding and to the conservation of Neotropical snake biodiversity.

Made available in DSpace on 2014-06-11T19:32:16Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-26Bitstream added on 2014-06-13T19:42:46Z : No. of bitstreams: 1 costa_jrv_dr_jabo.pdf: 396090 bytes, checksum: 23a92262b1f5d4ef9aee14c8383cae79 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aedes aegypti (Linnaeus), principal vetor da dengue no Brasil, tem sido combatido com o uso maciço de produtos químicos, contribuindo com o desenvolvimento de resistência e inviabilizando o controle do inseto. Bioinseticidas à base de Bacillus thuringiensis vêm apresentando resultados satisfatórios no controle de dípteros, devido à produção de proteínas bioinseticidas denominadas Cry (cristal), Cyt (citolíticas) e Chi (quitinase) e os efeitos sinergísticos existentes entre elas. O presente trabalho objetivou a seleção de isolados de B. thuringiensis, portadores de genes cry, cyt e chi com alta eficiência no controle de A. aegypti. Uma coleção de 1073 isolados de B. thuringiensis, provenientes de diversas regiões brasileiras, foi submetida à Reação em Cadeia da Polimerase (PCR), com base nos iniciadores específicos para os genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. Os isolados dípteros-específico foram avaliados quanto à estruturação genética e através de bioensaios seletivos e quantitativos, para determinação da CL50 e CL90. Apenas 45 (4,19%) apresentaram amplificação para os genes cry e cyt e destes, 25 (54,34%) eram quitinolíticos. Foram definidos 21 haplótipos entre os 45 isolados de B. thuringiensis, os quais foram submetidos aos bioensaios seletivos, indicando 13 isolados que causaram 100% de mortalidade às larvas de A. aegypti. Os bioensaios quantitativos e análise de agrupamento permitiram selecionar quatro isolados altamente tóxicos às larvas de A. aegypti. A identificação dos genes cry, cyt e chi de B. thuringiensis e análise dos efeitos sinergísticos entre as toxinas, associadas à análise da toxicidade a insetos vetores permitiram a seleção de isolados que poderão ser utilizados em formulações de novos bioinseticidas brasileiros, podendo contornar possíveis problemas de resistência. / Aedes aegypti (Linnaeus), the main vector of dengue fever in Brazil, has been controlled with massive use of chemical products wich, contributed to the development of resistance decreasing the insect control efficacy. Bioinsecticides based on Bacillus thuringiensis is currently presenting satisfactory results on controlling dipterans, due to the production of Cry proteins (crystal proteins), Cyt (citolitic) and Chi (chitinase) with synergistic effects between them. The present work aimed to select B. thuringiensis isolates with cry, cyt and chi genes exhibiting high efficiency on A. aegypti larvae. A bacterial collection containing 1073 isolates of B. thuringiensis, obtained from different Brazilian locations, had their DNA isolated and submitted to PCR amplifications using specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. The dipterans-especific isolates were then evaluated for the genetic structure and through selective and latter to quantitative bioassays for the determination of LC50 and LC90. Forty five out of 1073 isolates (4.19%) presented amplicons for the cry and cyt genes and from these 25 (54.34%) were detected as chitinolitic. Twenty one haplotypes were identified among the 45 isolates of B. thuringiensis, which were submitted to the selective bioassays, indicating 13 isolates that caused 100% mortality of A. aegypti larvae. The quantitative bioassays and the statistical analysis have pointed four toxic isolates to A. aegypti larvae. The identification of the cry, cyt and chi genes of B. thuringiensis and the analysis of the synergistic effects among the toxins, along with the toxicity analysis towards the vector insects allowed the selection of a set of isolates that can be used for the formulation of new bioinsecticides from Brazilian origin, that would avoid or at least delay the appearance of resistance problems.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-26T18:53:54Z No. of bitstreams: 1 TeseLS.pdf: 6231137 bytes, checksum: 82ce7c68a05ca7eb79c0ea1da7fe74e9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:39:44Z (GMT) No. of bitstreams: 1 TeseLS.pdf: 6231137 bytes, checksum: 82ce7c68a05ca7eb79c0ea1da7fe74e9 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-27T19:39:56Z (GMT) No. of bitstreams: 1 TeseLS.pdf: 6231137 bytes, checksum: 82ce7c68a05ca7eb79c0ea1da7fe74e9 (MD5) / Made available in DSpace on 2016-09-27T19:40:04Z (GMT). No. of bitstreams: 1 TeseLS.pdf: 6231137 bytes, checksum: 82ce7c68a05ca7eb79c0ea1da7fe74e9 (MD5) Previous issue date: 2015-06-23 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / The penaeid shrimp (Crustacea, Decapoda, Penaeidae) are the most economically fishing resource of the greatest global importance both for fisheries and the aquaculture industry. Shrimp fishing is one of the pillars of national economy in Mozambique, where they provide around USD 80,000,000 per year in export earnings. In recent years there has been observing a reduction in the shrimp fishery industry probably due the over-exploitation or due the presence of other shrimp species that were not been observed on the coast Mozambique before which may competing for the ecological niche. Despite the great fishing economic importance of penaeid shrimps in Mozambique, there is no genetic scientific literature available thus far on their genetic characteristics. Therefore, this is the first work addressing the genetic diversity of penaeids species of greatest economic value from Mozambique coast F. indicus and M. monoceros, for the first time shrimp from this coast were identified using DNA Barcoding tool. In addition, for the first time sequences of penaeid shrimp from Mozambique coast were deposited in GenBank and Bold system. The objective of this study was to evaluate the genetic diversity and population structure of the species F. indicus and M. monoceros, and identify nine species of penaeids from the Mozambican coast. To assess the genetic diversity and structure of F. indicus and M. monoceros, 160 samples were collected along the Mozambique coast. Three mitochondrial genes (COI, Cyt b, and the control region D-loop) were analyzed. A great genetic diversity was observed, however, the D-loop presented higher values (F. indicus: Hd = 1, h = 13; Hi = 0.0133, M. monoceros Hd = 0,99; H-24; and Hi = 0.0092). D-loop showed unique haplotypes; the Tajima D test and Fu Fs values were negative and significant for the COI and Cyt b genes. The mismatch distribution curves suggested that the two espéceis undergone to a recent population expansion (10.397 to 28.418) years ago F. indicus and M. monoceros respectively. The AMOVA analysis showed that over 99% of the variation occurs within populations. The Fst Pairwise values pointed to a non structured population. The nature of ocean currents along the Mozambique channel as well as the complete absence of physical and / or environmental barriers may be the main factors that influence the non structure of these two species. To identify shrimp penaeids from Mozambique based on DNA barcondig, a total of 69 samples were collected in the Maputo Bay. The genetic distance tree grouped six species into two clades as to the place of origin, suggesting the presence of cryptic species. The intraspecific genetic distance ranged from (0 to 8.636) and interspecific distance from (3.897 to 21.558). The distance distribution analysis 10 of the nearest neighbor ranged from (3.897-18.971). The DNA Barcoding identification tool was efficient to identify the Penaeid species from mozambican coast. However the presence of cryptic species pointed the need for further studies which must be conducted using molecular analyzes; morphological taxonomy and the ecological treats to evaluate each of the sibling species to reach a correct decision of the taxonomic status. / Os camarões peneídeos (Crustacea, Decapoda, Penaeidae) constituem um recurso pesqueiro de grande importância econômica mundial tanto para a indústria pesqueira quanto para a aquicultura. Em Moçambique, a indústria pesqueira constitui uma das bases da sua economia, onde a pesca do camarão chega a render cerca de 80 milhões de dólares anuais. Nos últimos anos, tem-se verificado uma redução na pesca do camarão, provavelmente devido à sobrexplotação e à presença de espécies exóticas. Este é o primeiro estudo genético envolvendo as espécies de maior valor econômico de Moçambique. O objetivo deste estudo foi avaliar a diversidade genética e a estrutura populacional das espécies Fenneropeaneus indicus e Metapenaeus monoceros e identificar através do DNA barcoding nove espécies de camarão peneídeo. Para avaliar a diversidade e a estrutura genética de F. indicus e M. monoceros, foram coletadas 160 amostras ao longo da costa de Moçambique e analisados os genes mitocondriais COI (citocromo oxidase subunidade 1), Cyt b (citocromo oxidase subunidade b) e a região controle D-loop. Foi observada uma alta diversidade genética, sendo que o D-loop apresentou os valores mais elevados (F. indicus: Hd=1; h=13; Hi=0,0133; M. monoceros: Hd=0,99; h=24; e Hi=0,0092). O D-loop apresentou haplótipos únicos. Os valores do teste D de Tajima e Fs de Fu foram negativos e significativos para os genes COI e Cyt b. As curvas de distribuição mismatch sugeriram que as duas espécies passaram por uma expansão populacional recente de 10,397 e 28,418 anos para F. indicus e M. monoceros, respectivamente. A AMOVA referente aos genes COI e Cyt b mostrou que mais de 99% da variação ocorre dentro das populações. Os valores de FST par a par indicaram não haver estruturação populacional. A natureza das correntes marinhas ao longo do canal de Moçambique, bem como a ausência completa de barreiras físicas e/ou ambientais podem ser os principais fatores que influenciam a manutenção destas duas espécies como populações únicas. Para a identificação dos peneídeos de Moçambique com base no DNA barcondig, um total de 69 amostras foi coletado na Baía de Maputo. A árvore de distância genética agrupou seis espécies em dois clados com relação ao lugar de origem, sugerindo a presença de duas linhagens. A distância genética intraespecífica variou de 0 a 8,636 e a distância interespecífica variou de 3,897 a 21,558. A análise da distribuição de distância para o vizinho mais próximo variou de 3, 897 a 18,971. A ferramenta de identificação DNA barcoding, foi eficiente na identificação das espécies de peneídeos da costa moçambicana.

Cette thèse concerne le développement d’approches spectroscopiques infrarouge et Raman exaltées de surface: la spectroscopie infrarouge exaltée de surface (SEIRAS) combinée avec une cellule de perfusion et la spectroscopie Raman exaltée de surface (SERS) couplée avec l’électrochimie. Dans le cadre du premier projet, différentes protéines ont été étudiées : lactose perméase (LacY), complexe I et IM30. Nous avons déterminé le pKa de Glu325 dans LacY sauvage et dans différents mutants portant des mutations dans le centre actif de translocation des protons. Sauvage complexe I a été oxydé avec différents agents oxydants et réduit avec NADH. Spectres différentiels correspondants ont été analysés. Des changements conformationnels dans la protéine IM30, induits par la présence des ions Mg2+, ont été observés.Dans le cadre du deuxième projet, une cellule spectroélectrochimique contenant une grille d’or a été adaptée pour étudier des protéines redox actives. Cette grille d’or sert à la fois de substrat SERS et d’électrode de travail. Cyt c, Hb et Mb ont d'abord été utilisés pour valider la configuration, puis l'approche a été étendue pour étudier une protéine membranaire. / This thesis concerns the development of surface-enhanced infrared and Raman spectroscopic approaches: surface-enhanced infrared absorption spectroscopy (SEIRAS) combined with perfusion cell and surface-enhanced Raman spectroscopy (SERS) combined with electrochemistry. Within the first project different proteins were studied: Lactose Permease (LacY), complex I and IM30.The pKa of Glu325 in LacY WT and in different mutants carrying mutations in the proton translocation active center was determined. WT complex I was oxidized with different oxidizing agents and reduced with NADH. Corresponding redox-induced conformational changes were studied. The evidence was given that Mg2+ ions induce conformational changes in the protein IM30.Within the second project the spectroelectrochemical cell containing gold grid electrode was adopted for the studies of redox active proteins. This gold grid serves both as working electrode and as SERS active substrate. First Cyt c, Hb and Mb were used to validate the setup and then the approach was extended to study a membrane protein.

Orientadora: Janete Apparecida Desidério Sena / Banca: Manoel Victor Franco Lemos / Banca: Cristina Lacerda Soares Petrarolha Silva / Banca: Gislayne Fernandes Lemes Trindade Vilas-Bôas / Banca: Odair Aparecido Fernandes / Resumo: Aedes aegypti (Linnaeus), principal vetor da dengue no Brasil, tem sido combatido com o uso maciço de produtos químicos, contribuindo com o desenvolvimento de resistência e inviabilizando o controle do inseto. Bioinseticidas à base de Bacillus thuringiensis vêm apresentando resultados satisfatórios no controle de dípteros, devido à produção de proteínas bioinseticidas denominadas Cry (cristal), Cyt (citolíticas) e Chi (quitinase) e os efeitos sinergísticos existentes entre elas. O presente trabalho objetivou a seleção de isolados de B. thuringiensis, portadores de genes cry, cyt e chi com alta eficiência no controle de A. aegypti. Uma coleção de 1073 isolados de B. thuringiensis, provenientes de diversas regiões brasileiras, foi submetida à Reação em Cadeia da Polimerase (PCR), com base nos iniciadores específicos para os genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. Os isolados dípteros-específico foram avaliados quanto à estruturação genética e através de bioensaios seletivos e quantitativos, para determinação da CL50 e CL90. Apenas 45 (4,19%) apresentaram amplificação para os genes cry e cyt e destes, 25 (54,34%) eram quitinolíticos. Foram definidos 21 haplótipos entre os 45 isolados de B. thuringiensis, os quais foram submetidos aos bioensaios seletivos, indicando 13 isolados que causaram 100% de mortalidade às larvas de A. aegypti. Os bioensaios quantitativos e análise de agrupamento permitiram selecionar quatro isolados altamente tóxicos às larvas de A. aegypti. A identificação dos genes cry, cyt e chi de B. thuringiensis e análise dos efeitos sinergísticos entre as toxinas, associadas à análise da toxicidade a insetos vetores permitiram a seleção de isolados que poderão ser utilizados em formulações de novos bioinseticidas brasileiros, podendo contornar possíveis problemas de resistência. / Abstract: Aedes aegypti (Linnaeus), the main vector of dengue fever in Brazil, has been controlled with massive use of chemical products wich, contributed to the development of resistance decreasing the insect control efficacy. Bioinsecticides based on Bacillus thuringiensis is currently presenting satisfactory results on controlling dipterans, due to the production of Cry proteins (crystal proteins), Cyt (citolitic) and Chi (chitinase) with synergistic effects between them. The present work aimed to select B. thuringiensis isolates with cry, cyt and chi genes exhibiting high efficiency on A. aegypti larvae. A bacterial collection containing 1073 isolates of B. thuringiensis, obtained from different Brazilian locations, had their DNA isolated and submitted to PCR amplifications using specific primers for the genes cry4Aa, cry4Ba, cry10Aa, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa e chi. The dipterans-especific isolates were then evaluated for the genetic structure and through selective and latter to quantitative bioassays for the determination of LC50 and LC90. Forty five out of 1073 isolates (4.19%) presented amplicons for the cry and cyt genes and from these 25 (54.34%) were detected as chitinolitic. Twenty one haplotypes were identified among the 45 isolates of B. thuringiensis, which were submitted to the selective bioassays, indicating 13 isolates that caused 100% mortality of A. aegypti larvae. The quantitative bioassays and the statistical analysis have pointed four toxic isolates to A. aegypti larvae. The identification of the cry, cyt and chi genes of B. thuringiensis and the analysis of the synergistic effects among the toxins, along with the toxicity analysis towards the vector insects allowed the selection of a set of isolates that can be used for the formulation of new bioinsecticides from Brazilian origin, that would avoid or at least delay the appearance of resistance problems. / Doutor

Στην παρούσα διπλωματική εργασία γίνεται χρήση τριών διαφορετικών ειδών λεπτών υμενίων διοξειδίου του τιτανίου (TiO2) ως στερεό υπόστρωμα για την ακινητοποίηση πρωτεϊνών με σκοπό την ανάπτυξη και σύγκριση αμπερομετρικών βιοαισθητήρων με ευαισθησία στο υπεροξείδιο του υδρογόνου (H2O2). Το πρώτο είδος των λεπτών υμενίων TiO2 είναι από την εμπορική πάστα της Dyesol η οποία παρασκευάζεται με τη μέθοδο sol-gel, το δεύτερο είδος είναι από μία πάστα δικής μας παραγωγής με τη μέθοδο sol-gel και το τρίτο είδος είναι από μία πάστα που παρασκευάσαμε χρησιμοποιώντας την έτοιμη νανοδομημένη σκόνη TiO2, Degussa P – 25. Τα υμένια TiO2 που έχουν παρασκευαστεί από την εμπορική πάστα της Dyesol χρησιμοποιήθηκαν και για τη δημιουργία υβριδικού υποστρώματος με νανοσωματίδια Αργύρου το οποίο μπορεί να βρει εφαρμογή τόσο στη φωτοκατάλυση όσο και στην ανάπτυξη πιο ευαίσθητων αμπερομετρικών βιοαισθητήρων. Αρχικά περιγράφεται η λειτουργία των βιοαισθητήρων καθώς και οι σημαντικότεροι τύποι βιοαισθητήρων που έχουν κατασκευαστεί μέχρι σήμερα. Σημαντικό ρόλο στην επιτυχή κατασκευή ενός βιοαισθητήρα παίζει η επιλογή του υλικού που θα χρησιμοποιηθεί ως υπόστρωμα / ηλεκτρόδιο (στη συγκεκριμένη περίπτωση, υμένια TiO2) καθώς και ο τρόπος που ακινητοποιείται το βιομόριο πάνω σε αυτό, γι’ αυτό και έχει δοθεί έμφαση στην ανάλυση των παραπάνω πληροφοριών. Στη συνέχεια περιγράφεται η δομή και η φυσική λειτουργία της πρωτεΐνης, (κυτόχρωμα c), που χρησιμοποιήθηκε ως το βιομόριο επιλογής για την ανάπτυξη του βιοαισθητήρα. Αναλύονται οι κρυσταλλικές δομές του διοξειδίου του τιτανίου και οι βασικές φυσικοχημικές τους ιδιότητες. Επίσης γίνεται περιγραφή του φαινομένου της φωτοκατάλυσης, ενώ αναφέρονται τα πλεονεκτήματα και τα μειονεκτήματα του TiO2 για την εφαρμογή αυτή. Στο τέλος του πρώτου κεφαλαίου γίνεται αναφορά και στα νανοσωματίδια αργύρου καθώς και στους λόγους που βοηθούν στην αύξηση της φωτοκαταλυτικής απόδοσης του TiO2. Στη συνέχεια περιγράφονται οι πειραματικές διατάξεις που χρησιμοποιήθηκαν τόσο για τον χαρακτηρισμό των υμενίων TiO2 όσο και για την αναλυτική μελέτη της ακινητοποίησης του κυτοχρώματος c και των νανοσωματιδίων αργύρου πάνω σε αυτά. Περιγράφεται επίσης, η ηλεκτροχημική κυψελίδα 3 ηλεκτροδίων και οι τεχνικές της κυκλικής βολταμμετρίας και της φασματοηλεκτροχημείας που επιλέχθηκαν για τη μελέτη των ηλεκτροχημικών ιδιοτήτων των υμενίων TiO2 με ή χωρίς ακινητοποιημένη πρωτεΐνη, των υμενίων TiO2 με ακινητοποιημένα νανοσωματίδια αργύρου καθώς και για την ανάπτυξη των αμπερομετρικών βιοαισθητήρων με ευαισθησία στο H2O2. Τέλος, περιγράφεται η σύνθεση των τριών διαφορετικών παστών TiO2, η πειραματική διαδικασία εναπόθεσης των υμενίων του TiO2 σε υποστρώματα αγώγιμου υάλου και κατόπιν, ακολουθεί η ανάλυση των πειραματικών αποτελεσμάτων. / In this report 3 different types of TiO2 films were used as solid substrates for the immobilization of proteins in order to be used for the development and evaluation of amperometric biosensors for hydrogen peroxide (H2O2). The first type of thin TiO2 films were made using a Dyesol sol gel commercial TiO2 paste, the second type was produced from a TiO2 paste produced in our lab following a standard sol-gel procedure and the third type of films were produced from a paste prepared using Degusa P25 TiO2 powder. In addition, the thin TiO2 films produced from the Dyesol paste were modified with Ag nanoparticles in order to examine their electrochemical behavior which could lead to enhanced photcatalytic and/or biosensing performance. In the first chapter, a general decription of the different types of biosensors developed so far is presented and emphasis is given to their function and applications. In order to develop a successful biosensor, the choice of the material to be used as the solid substrate is very important as well as the type of the biomolecule used as the recognition element. The sensitivity and response of the biosensor is greatly enhanced by the method used to immobilize the biomolecule on the solid support in a stable and functional way. Therefore in this work both the material, TiO2 films, and the biomolecule of choice, Cytochrome-c, are presented in detail and in particular their physicochemical properties, their functions and applications. Moreover the different methods that have been used for the successful immobilization of biomolecules on solid surfaces are well documented. Furthermore, the photocatalytic properties of the TiO2 films are discussed and how they are enhanced by the deposition of silver nanoparticles on their surfaces that could also lead to the development of more sensitive and accurate amperometric biosensors. In the second chapter, the experimental techniques and procedures used for the characterization of the resulting TiO2 films and for the adsorption process of protein and/or silver nanoparticles on their surfaces are well presented. Furthermore, details are given of the electrochemical techniques (cyclic voltammetry and spectroelectrochemistry) used to evaluate the electrochemical performance of the resulting films with or without protein or silver nanoparticles. A detailed description of the 3 electrode electrochemical used to perform these experiments is also presented. Finally emphasis is given to the procedures used for the development of the electrochemical biosensors for H2O2. Finally, a description of the procedures used for the synthesis of the 3 different TiO2 pastes and of the method used for the production of thin TiO2 films on conducting glass is given followed by the presentation, analysis and discussion of the data collected.