Made available in DSpace on 2016-12-23T13:56:14Z (GMT). No. of bitstreams: 1 Cynara Oliveira Possamai.pdf: 3245812 bytes, checksum: 7de0caeadb98d478a083db43020f22f8 (MD5) Previous issue date: 2012-08-28 / O gênero Acanthamoeba compreende protozoários anfizóicos que estão presentes nos mais diversos ambientes, podendo causar no homem doenças graves, como a ceratite amebiana e a encefalite amebiana granulomatosa. Os fatores envolvidos na patogenicidade de Acanthamoeba não são inteiramente conhecidos, por isso, alguns marcadores vêm sendo investigados na tentativa de identificar linhagens capazes de causar infecção. O objetivo deste trabalho foi investigar a ocorrência de Acanthamoeba em amostras clínicas e ambientais, bem como caracterizar os isolados obtidos por parâmetros morfológicos, genotipagem e avaliação do potencial patogênico. Foram coletadas amostras de raspado de córnea de pacientes com suspeita de ceratite amebiana e amostras ambientais provenientes de poeira, solo, piscina, água potável, água de inundação e água do mar da região metropolitana de Vitória-ES. Todas as amostras foram cultivadas em meio ágar soja. Além da cultura, as amostras de raspado de córnea também foram coletadas em salina de Page e submetidas a uma reação de semi-nested PCR. Culturas positivas para Acanthamoeba, identificadas com base na morfologia dos cistos e trofozoítos, foram selecionadas, clonadas e classificadas nos grupos morfológicos I, II ou III. A genotipagem dos isolados foi realizada a partir do sequenciamento parcial do gene 18S rDNA e o potencial patogênico das culturas clonadas foi avaliado por meio de ensaios de termotolerância e osmotolerância. Foram cultivadas em ágar 90 amostras ambientais, 16 de raspado de córnea e nove de lentes de contato (LC). Dessas, 38 (33 ambientais, quatro clínicas e uma de LC) foram positivas para Acanthamoeba, sendo obtidos 28 clones (24 ambientais, três clínicos e um de LC). Dentre eles, 26 apresentaram características morfológicas do grupo II, um do grupo I (solo) e um clone (água potável) não foi classificado de acordo com os parâmetros morfológicos de classificação. Quatro casos de ceratite amebiana foram confirmados somente por diagnóstico molecular. Todos os isolados clínicos, o de LC e a maioria dos isolados ambientais sequenciados foram classificados como pertencentes ao genótipo T4. Dentre os isolados ambientais, dois foram agrupados no genótipo T11 (piscina) e um no T1 (poeira). Todos os isolados clonados submetidos aos ensaios de termotolerância apresentaram crescimento a 28ºC e a 37ºC. Em contrapartida, nenhum isolado cresceu a 42ºC. Nos testes de osmotolerância, todos os isolados se desenvolveram a 0,1M e a 0,5M de manitol e a maioria deles cresceu à concentração de 1,0M. Os resultados deste estudo pioneiro no Espírito Santo confirmam a predominância do grupo morfológico II e do genótipo T4 em isolados clínicos e ambientais de Acanthamoeba e relata pela primeira vez no Brasil o isolamento de Acanthamoeba pertencente ao genótipo T1. Este trabalho demonstra também a presença de isolados potencialmente patogênicos no ambiente, inclusive em amostras de água de inundação e de água do mar, o que pode representar um fator de risco para o desenvolvimento de infecções causadas por Acanthamoeba. Além disso, a metodologia desenvolvida neste estudo poderá ser utilizada para um diagnóstico mais rápido, sensível e específico de ceratite por Acanthamoeba / The genus Acanthamoeba comprises amphizoic protozoa with a wide environment distribution. They can cause serious diseases in humans, such as amoebic keratitis and granulomatous amoebic encephalitis. Thus, the factors involved in the pathogenicity of Acanthamoeba are being investigated as major interests to identify strains able to cause infection. The aim of this study was to investigate the occurrence of Acanthamoeba both in clinical and environmental samples, characterize the isolates by morphological parameters, genotyping and also evaluate the pathogenic potential. Clinical samples were collected from patients with a suspicious diagnosis of amoebic keratitis throughout corneal scrapings. Environmental samples were collected from dust, soil, swimming pool, tap, sea, and flood waters in Vitoria metropolitan regions, Espirito Santo State, Brazil. All samples were cultured on soy agar. Samples from corneal scrapings were also collected in Page saline and subjected to a reaction of semi-nested PCR. Positive cultures for Acanthamoeba, previously identified based on the cysts and trophozoites morphology, were selected, cloned and classified into morphological groups I, II or III. Genotyping of isolates was performed by partially sequencing the 18S rDNA gene while the pathogenic potential of cloned cultures was assessed by thermo and osmotolerance assays. From all samples cultured on agar, 90 were from environmental sources, 16 from corneal scrapings and nine from contact lenses (CL). Of these, 38 (33 from environmental samples, four from clinical samples and one from CL sample) were positive for Acanthamoeba. Among the 38 positive isolates, 28 were successfully cloned (24 from environmental isolates, three from clinical isolates and one from CL isolate). Twenty six cloned samples showed morphological characteristics of group II, one of group I (soil) and one (tap water) could not be classified according to morphological parameters. Four cases of amoebic keratitis could only be confirmed by molecular diagnosis. All clinical, CL and most of the environmental isolates sequenced were classified as T4 genotype. Among the environmental isolates, two were grouped in genotype T11 (pool) and one in T1 (dust). All cloned isolates subjected to the thermotolerance assay grew at 28 ºC and 37 ºC. The same result was observed in osmotolerance tests at 0.1M and 0.5M mannitol. Neverthless, while most of the cloned isolates were able to grow at 1.0M mannitol, none of the isolates were able to grow at 42 ºC. The present study confirms the predominance of morphological group II and genotype T4 in clinical and environmental isolates of Acanthamoeba in Espirito Santo State and first time reports the T1 genotype isolation of Acanthamoeba in Brazil. This work also demonstrates the presence of potentially pathogenic isolates at the environment, including samples of flood and sea waters, which may represent a risk factor for the development of infections caused by Acanthamoeba. Furthermore, the methodology used in this study could be used for a fast, sensitive and specific diagnosis of Acanthamoeba keratitis

Made available in DSpace on 2016-06-02T20:21:35Z (GMT). No. of bitstreams: 1 6077.pdf: 10672804 bytes, checksum: 8a7ba5e1820e96664e3cbb7f872f6f3e (MD5) Previous issue date: 2013-10-28 / Financiadora de Estudos e Projetos / Over the recent years, the incidence of human fungal infections has shown a significant increase. Aspergillus fumigatus is a filamentous fungus opportunistic pathogen responsible for many human respiratory diseases, including invasive pulmonary aspergillosis, which is the most serious form of infection . Studies show that A. fumigatus virulence has a multifactorial process associated with its structure, capacity for growth, adaptation to stress conditions, evasion mechanisms of the immune system and ability to cause harm to the host. CWI (via cell wall integrity ) is a signaling cascade activated in yeast cells under conditions of cell wall stress and plays a role in the adaptation of various fungal pathogens in the human host . In many fungi , CWI is triggered by activation of protein kinase C ( PKC ) and that this pathway is associated with the transcription of genes related to maintaining the integrity of the cell wall and its redevelopment. In this work, a mutant Gly579Arg (G579R) was constructed by transformation mediated by inserting a gene replacement cassette comprising a G2044C transversion located in the cysteine-rich domain controller C1B pkcA of A. fumigatus. From the phenotypic analysis of the mutant strain was observed in the involvement of pkcAG579R CWI since the mutant showed high sensitivity to agents such as CR (congo red) and CFW (calcofluor white) . Furthermore, pkcA is also involved in tolerance to oxidative stress caused by paraquat and menadione. Additionally it was found to increase the sensitivity of the mutant pkcAG579R temperature variations as well as the inhibitor of Hsp90 radicicol. Como CWI is related to the transcriptional activation of biosynthetic genes and rugged cell wall (such as glucan synthase, glucanosil chitin synthases and transferases) the abundance of major genes coding for these enzymes was analyzed by RT-PCR in real time. Based on the tests can be α -1 ,3 glucan synthase ( agsA-C ) dependent signaling mediated PkcA for correct expression. Furthermore, genes such as β-1,3 glucan synthase (fksA) glucanosyltransferase (gelA-C) and some chitin synthases (chsB-E-C) appear not to be dependent function and CWI PkcA . These data demonstrated the role of pkcA signaling cascade in the maintenance of cell wall and thermotolerance in A. fumigatus. This work was the first in which a systematic analysis of gene pkcA was conducted in the human opportunistic fungal pathogen A. fumigatus. / Ao longo dos últimos anos, a ocorrência de infecções fúngicas humanas vem apresentando um aumento expressivo. Aspergillus fumigatus é um fungo filamentoso patógeno oportunista responsável por diversas doenças respiratórias humanas, incluindo aspergilose pulmonar invasiva, que é a forma de infecção mais grave. Estudos demonstram que o A. fumigatus possui um processo de virulência multifatorial associado a sua estrutura, capacidade de crescimento, adaptação em condições de estresse, mecânismos de evasão do sistema imune e capacidade de causar danos ao hospedeiro. A CWI (via de integridade da parede celular) é uma cascata de sinalização ativada nas células fúngicas sob condições de estresse de parede celular e desempenha um papel na adaptação de vários fungos patogênicos no hospedeiro humano. Em muitos fungos, CWI é desencadeada através da ativação da proteína quinase C (PKC) sendo que esta via está associada à transcrição de genes relacionados com a manutenção da integridade da parede celular e sua remodelação. Neste trabalho o mutante Gly579Arg (G579R) foi construído através da transformação mediada pela inserção de um cassete de substituição gênica que compreende uma transversão G2044C localizado no domínio regulador rico em cisteína C1B da pkcA de A. fumigatus. A partir da análise fenotípica desse mutante foi possível observar o envolvimento de pkcAG579R na CWI uma vez que a linhagem mutante mostrou alta sensibilidade a agentes como o CR (congo red) e CFW (calcofluor white). Além disso, pkcA está envolvido também na tolerância ao estresse oxidativo causado por menadiona e paraquat. Adicionalmente verificou-se o aumento da sensibilidade da linhagem mutante pkcAG579R à variações de temperatura bem como ao inibidor de Hsp90, radicicol. Como a CWI está relacionada à ativação transcricional de genes de biossíntese e reforço de parede celular (como por exemplo glucanas sintases, quitinas sintases e glucanosil transferases), a abundância dos principais genes que codificam essas enzimas foi analisada através de RTPCR em tempo real. Baseado nos testes pode-se verificar que as α-1,3 glucana sintase (agsA-C) dependem da sinalização mediada por PkcA para sua expressão. Por outro lado, genes como a β-1,3 glucana sintase (fksA), glucanosiltransferases (gelA-C) e algumas quitinas sintases (chsB-C-E) parecem não ser dependente da CWI e da função de PkcA. Esses dados demostraram parte do papel de pkcA na cascata de sinalização da manutenção da parede celular e termotolerância em A. fumigatus. Este trabalho foi o primeiro no qual uma análise sistemática do gene pkcA foi conduzida no fungo patógeno oportunista humano A. fumigatus.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T14:16:38Z No. of bitstreams: 2 Dissertação - Elen Regozino Muniz - 2015.pdf: 5452848 bytes, checksum: 01e704bb99491be79de20d410d521a0f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T14:18:52Z (GMT) No. of bitstreams: 2 Dissertação - Elen Regozino Muniz - 2015.pdf: 5452848 bytes, checksum: 01e704bb99491be79de20d410d521a0f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-13T14:18:52Z (GMT). No. of bitstreams: 2 Dissertação - Elen Regozino Muniz - 2015.pdf: 5452848 bytes, checksum: 01e704bb99491be79de20d410d521a0f (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-04-01 / Rhipicephalus microplus, known as tick of the cattle, cause great economic losses each year in Brazil. Entomopathogenic fungi stand out as an alternative to the exclusive control and indiscriminate use of the chemical. However, ambient temperature above the threshold considered optimal for these organisms can interfere with your biology, and compromise their development and effectiveness. In the present study evaluated biological parameters seven strains of Metarhizium spp. (IP1, IP34, IP46, IP60, IP119, IP125 and IP146) originated from the Central Brazil, incubated at 32 ± 0.5°C or 27 ± 1°C. In addition, the virulence was assessed for each strain of R. microplus engorged females. IP119, Metarhizium anisopliae s.s., was selected among the other isolates for virulence tests against R. microplus submitted with stressful temperature condition (32 ± 0.5°C) or low relative humidity (RH = 75%) to present considerable heat tolerance and virulence in the screening tests. Tests with conidia formulated in oil-water emulsion (liquid vaseline 5%), carboxymethylcellulose gel or emulgel 5% were conducted to evaluate the conidial thermotolerance when exposed to 45°C for 4 hours or 6 hours, and to test the efficacy of formulated to control engorged female R. microplus the stressful conditions of temperature (32°C) and low humidity (RH = 75%). It was concluded that a constant temperature of 32°C or exposure for a few hours at 45°C is stressful condition for the isolates. The conidia IP119 when bathed in liquid vaseline jelly were more tolerant of exposure to 45°C for 4 hours. IP119 was shown to be virulent for engorged female R. microplus, being the oil-water emulsion 5% of the most effective formulation. / Rhipicephalus microplus, conhecido como carrapato-dos-bovinos, causa grandes percas econômicas por ano no Brasil. Fungos entomopatogênicos se destacam como controle alternativo ao uso exclusivo e indiscriminado do químico. Contudo, a temperatura ambiente acima do limiar considerado ótimo para estes organismos pode interferir na sua biologia, e comprometer seu desenvolvimento e eficácia. No presente estudo foram avaliados parâmetros biológicos de sete linhagens de Metarhizium spp. (IP1, IP34, IP46, IP60, IP119, IP125 e IP146), originados do cerrado do Centro-Oeste brasileiro, incubados a 32±0,5°C ou 27±1°C. Além disso, foi avaliada a virulência de cada linhagem para fêmeas ingurgitadas de R. microplus. Metarhizium anisopliae s.s., IP119, foi selecionado dentre os demais isolados para os testes de virulência contra R. microplus submetidos a com condição de temperatura estressante (32±0,5°C) ou em baixa umidade relativa (UR=75%) por apresentar notável tolerância ao calor e virulência nos testes de triagem. Testes com os conídios formulados em emulsão óleo-água (vaselina líquida 5%), gel carboximetilcelulose ou emulgel 5% foram realizados para avaliar a termotolerância dos conídios quando expostos a 45°C por 4 ou 6 horas, e testar a eficácia dos formulados para o controle de fêmeas ingurgitadas de R. microplus nas condições estressantes de temperatura (32°C) e umidade baixa (UR=75%). Concluiu-se que a constante temperatura de 32°C ou a exposição por poucas horas a 45°C foi condição estressante para os isolados estudados. Os conídios de M. anisopliae s.s. (IP119) quando banhados em emulsão óleo-água 5% foram mais tolerantes em exposição a 45°C por 4 horas. M. anisopliae s.s. (IP119) mostrou ser virulento para fêmeas ingurgitadas de R. microplus, tendo sido a emulsão óleo-água 5% a formulação mais eficaz.

O fungo entomopatogênico Isaria fumosorosea (Ascomycota: Hypocreales: Cordycipitaceae) é encontrado comumente nos solos e infectando diversas espécies de artrópodes. Bioprodutos à base de I. fumosorosea são utilizados principalmente na América do Norte e Europa para o controle de mosca-branca, pulgões e trips. Entretanto, no Brasil ainda não existe nenhum produto registrado à base deste patógeno. O objetivo deste trabalho foi estabelecer parâmetros para a seleção de isolados promissores para o desenvolvimento de um biopesticida à base de I. fumosorosea. Os isolados foram avaliados em três etapas sequênciais: esporulação em meio de cultura artificial, fermentação em grão de arroz e tolerância à radiação ultravioleta B e temperatura alta. A partir de uma coleção de 172 isolados de I. fumosorosea provenientes do solo e insetos de diferentes biomas do Brasil, foram escolhidos 72 isolados por apresentar visualmente maior esporulação das colônias em SDYA (Sabouraud-dextrose-extrato de levedura-ágar). Posteriomente, esses isolados foram utilizados para a fermentação sólida em garrafas de vidro contendo 50 gramas de arroz parboilizado. Destes, 14 isolados foram avaliados quanto à produção de conídios em sacos de polipropileno contendo arroz parboilizado e quanto aos efeitos da exposição à radiação ultravioleta B (UV-B) por 0, 2, 4, 6 e 8 horas e temperatura de 45°C por 0, 30, 60 e 90 minutos. Os isolados ESALQ-4556 e ESALQ-4778 apresentaram os maiores rendimentos de conídios em garrafas de vidro (4,65 e 4,40 ×109 conídios/g arroz seco) e em sacos de polipropileno (3,96 e 3,25 ×109 conídios/g arroz seco). Os isolados ESALQ-1296 e ESALQ- 3415 apresentaram maior tolerância aos efeitos da radiação ultravioleta-B e aquecimento. Para a elaboração de uma formulação do tipo dispersão oleosa foi ajustado o ponto de secagem dos conídios para incrementar o tempo de prateleira e avaliadas diferentes misturas do adjuvante KBRAdj e óleos vegetais. Foram utilizados conídios do isolado ESALQ-1296 produzidos em arroz com atividade de água (aw) de 0,527 e conídios secos com aw= 0,410; 0,248 e 0,182 adicionados ao adjuvante KBRAdj puro. A sobrevivência dos conídios mais secos foi maior do que de conídios úmidos. O adjuvante promoveu proteção aos conídios umidos, apresentando viabilidades significativamente maiores do que conídios umidos sem adjuvante em todos os períodos de armazenamento. Conídios com aw= 0,182 sem adjuvante apresentaram as maiores viabilidades (70,3%) após 60 dias de armazenamento. Para desenvolver uma formulação do tipo dispersão oleosa foram testadas misturas de 25% de óleo de soja ou canola + 75% do adjuvante KBRAdj com ou sem sílica gel, em conídios úmidos (aw=0,546 e UR= 13,18%) ou secos (aw=0,175 e UR= 4,42%). A adição de 25% de óleo vegetal ou sílica gel na formulação não contribuiu para o incremento do período de prateleira da formulação. A viabilidade dos conídios nas diferentes formulações após 90 dias de armazenamento variou de 42,3 ± 0,03% a 56,9 ± 0,01%. Estudos futuros deverão ser conduzidos para incrementar o período de prateleira e avaliar a virulência destas formulações para o controle de pragas, especialmente mosca-branca e psilídeo-dos-citros. / The entomopathogenic fungus Isaria fumosorosea (Ascomycota: Hypocreales: Cordycipitaceae) is commonly found in soil and infecting several species of arthropods. I. fumosorosea based bioproducts are mainly used in North America and Europe for the control of whitefly, aphids and thrips. However, in Brazil there is still no registered product based on this pathogen. The objective of this study was to establish parameters for the selection of promising isolates for developing I. fumosorosea based biopesticides. The isolates were evaluated in three sequential steps: sporulation on artificial medium, sporulation on rice grains, tolerance to ultraviolet-B radiation and high temperature. From a collection of 172 I. fumosorosea isolates from soil and insects of different Brazilian biomes, 72 isolates were chosen based on visual observations of higher sporulation of colonies in SDYA (Sabouraud-dextrose- yeast extract-agar). Later, these isolates were used for the solid fermentation in glass bottles containing 50 g of parboiled rice. Of these, 14 strains were evaluated for conidia production in polypropylene bags containing parboiled rice and the effects of exposure to ultraviolet-B (UV-B) for 0, 2, 4, 6 and 8 hours and 45°C for 0, 30, 60 and 90 minutes. The ESALQ-4556 and ESALQ-4778 isolates showed the highest spore yields in glass bottles (4.65 and 4.40 × 109 spores/g dry rice) and in polypropylene bags (3.96 and 3.25 × 109 spores/g dry rice). The ESALQ-1296 and ESALQ-3415 isolates showed a higher tolerance to the UV-B radiation and heating. For the development of oil dispersion formulations, the drying point of the conidia was adjusted to increase the shelf-life in different mixtures of the KBRAdj adjuvant and vegetable oils. Conidia produced on rice from the isolate ESALQ-1296 without drying with water activity (aw) = 0.527 and dried conidia with aw of 0.410; 0.248 and 0.182 added to the pure adjuvant. Survival of the dryer conidia was greater than humid conidia. The adjuvant provided protection to humid conidia, with significantly higher viability than conidia without drying and without adjuvant at all storage periods. Conidia with aw = 0.182 without adjuvant showed the highest viability (70.3%) after 60 days of storage. To develop an oil dispersion type formulation it were tested mixtures with 25% of soy or canola oil + 75% of KBRAdj adjuvant with or without silica gel using humid (aw = 0.546 and RH = 13.18%) or dried conidia (aw = 0.175 and RH = 4.42%). Addition of 25% of vegetable oil or silica gel in the formulation did not contribute to the increase of the shelf-life. The conidia viability in the different formulations after 90 days of storage ranged from 42.3% ± 0.03% to 56.9 ± 0.01. Future studies must be conducted to increase the shelf- life and to assess the virulence of these formulations for pest control, particularly whitefly and the citrus psyllid.

Cette recherche a été focalisée sur l’étude de la variabilité génétique et des mécanismes associés à une adaptation d’A. bisporus à la culture dans des conditions subtropicales. Elle a été subdivisée en trois parties : 1- identification de la diversité de l’aptitude à fructifier à haute température (FHT+) dans un échantillon de souches sauvages collectées dans plusieurs localités de pays d’Amérique du Nord et d’Europe. Dans cet ensemble nous avons inclus des représentants des trois variétés d’Agaricus à savoir: les variétés bisporus, burnettii et eurotetrasporus; 2- la caractérisation d’un gène candidat de thermo-tolérance dans des souches FHT+ et FHT- et l’identification de son implication dans le caractère FHT+; 3- l’étude du déterminisme génétique du caractère FHT+. Toutes les souches de la variété burnettii sont capables de produire des champignons avec de forts rendements à haute température. Cette aptitude est un élément de leur adaptation aux conditions climatiques rencontrée par la population originale. Seulement quelques souches d’A. bisporus var. bisporus ont révélés un bon potentiel pour fructifier à 25°C, mais sans relation avec les conditions climatiques de leur zone d’origine. Le gène étudié n’était que légèrement impliqué dans la réaction au stress thermique. Nous l’avons renommé aap1 car nous avons montré qu’il appartient à une nouvelle sous-famille des homologues du gène YAP1 de levure. Il n’est pas un contributeur dominant pour la thermo-tolérance d’A. bisporus, mais la protéine qu’il code peut être impliquée comme facteur de transcription de résistance générale au stress. En fait le caractère FHT+ est un caractère quantitatif sous contrôle polygénique. Il peut être hérité d’un parent de la variété burnetti dans une descendance inter variétale. Au cours de ce travail, des souches d’A. bisporus possédant des potentiels intéressants pour la culture en conditions subtropicales ont été identifiées et les bases génétiques pour un programme de création variétale pour introduire ces caractères dans des souches déjà cultivées ont été identifiées. / This work focused mainly on studying genetic variability andmechanisms associated with an adaptation of Agaricus bisporus for cultivation undersubtropical conditions of Mexico. The research was divided into three parts: 1-Identification of the diversity for fructification at a high temperature (FHT+) in a pool ofwild strains gathered in numerous locations of North America and Europe andrepresentative of the three varieties known in the species: bisporus, burnettii y eurotetrasporus; 2- characterization of a candidate gene of thermo-tolerance in FHT+ and FHT- strains and identification of its involvement in the FHT+ trait; 3- studies on the genetic determinism of FHT+ trait. All the strains of A. bisporus var burnettii produced mature sporophores at high temperatures with high yields. This ability is a part of the adaptation to the climatic conditions faced by the original population. For the A. bisporus var bisporus only some strains expressed the FHT+ trait and very few produced significant yield at high temperatures. There was no correlation with the geographical origin of the strains. The candidate gene studied was only weakly involved in the FHT+ trait. This study allowed a better characterization of one of the genes. We renamed it aap1 because it belongs to a new sub-family of counterparts of the gene YAP1 of yeast. It is not a dominant contributor to the thermo-tolerance of A. bisporus, but the protein that it encodes can be involved as a factor in transcription of general resistance to stress. Actually FHT+ was shown to be a quantitative trait under polygenic control, and it can be inherited from the A. bisporus var. burnettii in an intervarietal progeny. Strains of A. bisporus with interesting potential for cultivation under subtropical conditions and genetic bases of breeding programs for introducing this potential in already cultivated strains had been identified.

L'adaptation des plantes face au stress thermique est cruciale pour leur survie et implique des réponses spécifiques telles que l’induction de protéines chaperonnes et la production d'espèces réactives de l'oxygène (ROS). Les glutaredoxines (GRX), une famille de protéines thiol anti- oxydantes, jouent un rôle important dans la régulation redox et la réponse au stress oxydatif. Mes études se concentrent sur GRXS17, une protéine multi-domaine à cœur fer-soufre. Malgré un phénotype de développement sévère du mutant grxs17 à des températures normales et plus élevées, peu est connu sur les fonctions biochimiques et les rôles intracellulaires précis de GRXS17. J’ai montré au cours de ma thèse que GRXS17 fonctionne comme une chaperonnedépendante de l’oxydation de la cellule. Elle présente à la fois une activité de type foldase mais également holdase. L'exposition aux stress oxydatif et thermique provoque le passage d'une forme dimérique à des complexes à poids moléculaires élevés ce qui est consistant avec une activité holdase. J’ai également montré que GRXS17 est requis pour la tolérance à des hausses de température de manière dépendante de ses cystéines catalytiques. Des approches de transcriptomique, métabolomique et protéomique montrent une réponse au stress thermique retardée dans le mutant grxs17, des défauts dans l’accumulation de certains métabolites clés, et ont permis d'identifier de potentiels nouveaux interactants de GRXS17 dans des conditions de stress thermique. Ces éléments soutiennent la fonction chaperonne de GRXS17 dans desconditions normales et de stress thermiques. / Adaption of plants to heat stress is crucial for their survival and involves dedicated response such as chaperones proteins induction and production of reactive oxygen species (ROS). Glutaredoxins (GRX), a family of thiol redox proteins, play an important role in redox regulation and response to oxidative stress. The focus of our studies is on GRXS17 which is a multi-subunit iron-sulfur glutaredoxin. Despite the severe developmental phenotype of the grxs17 mutant at normal and elevated temperatures, relatively little is known about the biochemical functions and precise intracellular roles of GRXS17. I show during my thesis that GRXS17 function as a foldase and holdase redox-dependent chaperone. Oxidative and heat stress exposure cause a shift from a dimeric form to high MW complexes which is concordant with a holdase activity. I show that GRXS17 is required for the tolerance to moderated heatstress in a Cys-dependent manner. Transcriptomic, metabolomic and proteomic approaches show heat stress response delayed in grxs17, key-metabolites defects and allowed to identifynew potential GRXS17-interactor under heat stress conditions, supporting a potential protecting function of GRXS17 against stress damage.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-09-05T13:57:56Z No. of bitstreams: 1 arquivototal.pdf: 1065428 bytes, checksum: 87d41d3e8d39921b88e283f449f37caa (MD5) / Made available in DSpace on 2017-09-05T13:57:56Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1065428 bytes, checksum: 87d41d3e8d39921b88e283f449f37caa (MD5) Previous issue date: 2016-03-21 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Salmonella enterica subespecie enterica is a pathogen frequently involved in outbreaks worldwide. Serovars S. Enteritidis e S. Typhimurium belonging to paghe type 4 (PT4) are prevalent among the etiological agentes of salmonellosis outbreaks involving chicken meat. The aim of this study was to assess changes in thermotolerance and capability to survive to gastrointestinal conditions of Salmonella Enteritidis PT4 and S. Typhimurium PT4 incorporated in chicken breast following exposure to abiotic stresses (cold, acid and osmotic) imposed during food processing. The effects of stressing conditions imposed by Origanum vulgare L. essential oil (oregano), a candidate substance for use as antimicrobial in foods, was also assessed. The exposure to cold stress (5°C for 5 h) did not result in increased tolerance to acid stress (lactic acid at 5 μL/g and 2.5 μL/g) in all tested strains. Cells of S. Typhimurium and S. Enteritidis previously exposed to acid stress showed higher tolerance to osmotic stress (NaCl at 75 mg/g or 37.5 mg/g,) compared to non-acid-exposed cells. The exposure to osmotic stress without previous exposure to acid stress caused a salt-concentration dependent decrease in S. Enteritidis or S. Typhimurium. Exposure to oregano essential oil at subinhibitory concentrations (1.25 μL/g and 0.625 μL/g) decreased the acid and omostic tolerance in both S. Enteritidis and S. Typhimurium. The subsequent exposure to cold, acid and osmotic stress conditions increased the thermotolerance in all test strains. Under simulated gastrointestinal conditions, the cells that survived the subsequent stress exposure (persisters) showed higher tolerance to acidic conditions (approximately 1.2 log CFU/g) compared with control cells. Persisters cells of S. Enteritidis and S. Typhimurium showed higher survival rates at the end of the in vitro digestion. These results show that subsequent exposure to stress conditions increase the thermotolerance and enhanced the survival under gastrointestinal condition in S. Enteritidis PT4 and S. Typhimurium PT4, which may contribute to the persistence of this pathogen within food matrix and host. / Salmonella enterica subespécie enterica é um patógeno frequentemente envolvido em surtos alimentares em todo o mundo. Os sorovares S. Enteritidis e S. Typhimurium pertencentes ao fagotipo 4 (PT4) são prevalentes entre os agentes etiológicos de surtos de salmonelose envolvendo carne de frango. O objetivo do presente estudo foi avaliar mudanças na termotolerância e a capacidade de sobreviver em condições gastrointestinais simuladas de Salmonella Enteritidis PT4 e S. Typhimurium PT4 inoculadas em peito de frango após a exposição subsequente a estresses abióticos (frio, ácido e osmótico) impostos durante o processamento de alimentos. Os efeitos da exposição ao óleo essencial de Origanum vulgare L. (orégano), uma substância proposta para uso como antimicrobiano em alimentos também foram avaliados. A exposição ao estresse frio (5 °C durante 5 h) não aumentou a viabilidade das células na exposição subsequente ao estresse ácido (ácido láctico - 5 μL/g e 2,5 μL/g) nas cepas testadas. Células de S. Enteritidis ou S. Typhimurium previamente expostas ao estresse ácido mostraram maior tolerância ao estresse osmótico (NaCl - 75 mg/g e 37,5 mg/g) em comparação com células não expostas ao ácido. A exposição ao estresse osmótico sem prévia exposição ao estresse ácido causou um decréscimo concentração-dependente em ambas as cepas testadas. A exposição ao óleo essencial de orégano em concentrações subletais (1,25 μL/g e 0,625 μL/g) diminuiu a tolerância ao estresse ácido e osmótico em S. Enteritidis e S. Typhimurium. A exposição subsequente aos estresses frio, ácido e osmótico em todas as concentrações testadas aumentou a termotolerância nas cepas testadas. Em condições gastrointestinais simuladas, as células sobreviventes dos tratamentos de subsequente exposição ao estresse (células persistentes) apresentaram maior tolerância às condições ácidas em comparação às células controles. Células persistentes de S. Enteritidis e S. Typhimurium apresentaram taxas de sobrevivência maiores (aproximadamente 1,2 log UFC/g) que as células controle no final da digestão in vitro. Estes resultados mostram que a exposição subsequente a condições de estresse aumenta a termotolerância e a sobrevivência em condições gastrointestinais em S. Enteritidis PT4 e S. Typhimurium PT4, o que pode contribuir para a persistência deste patógeno na matriz alimentar e no hospedeiro.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T12:47:20Z No. of bitstreams: 2 Tese - Cíntia das Chagas Bernardo - 2016.pdf: 2168975 bytes, checksum: f20144021bf112df62693342cfba7dae (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-08-25T12:47:45Z (GMT) No. of bitstreams: 2 Tese - Cíntia das Chagas Bernardo - 2016.pdf: 2168975 bytes, checksum: f20144021bf112df62693342cfba7dae (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-08-25T12:47:45Z (GMT). No. of bitstreams: 2 Tese - Cíntia das Chagas Bernardo - 2016.pdf: 2168975 bytes, checksum: f20144021bf112df62693342cfba7dae (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-07-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The current study compared the virulence of conidia and blastospores of Metarhizium anisopliae s.l. (IP 363), Metarhizium robertsii (IP 146) and Beauveria bassiana s.l. (IP 361 and GC 307) against Rhipicephalus microplus, and the tolerance to heat and UV-B radiation; in addition, it evaluated the development of conidia and blastospores of the isolates IP 146 and IP 361 on the tick cuticle by scanning electron microscopy (SEM). Larvae and engorged females of R. microplus were treated (larvae: 106, 107 or 108 propagules/mL, females: 107 propagules/mL) by immersion in conidia or blastospores suspensions of tested isolates. The higher percentage of larval mortality was obtained in the group treated with conidia of IP 361, which had lower LC50. In bioassays with engorged females, IP 146 and IP 361 blastospores, provided tick percent control superior to 90%, while conidia of the same isolates, promoted 70.97% and 63.29% of tick control, respectively. Ticks were treated topically with 50 μL of fungal suspension (107 propagules/mL) and incubated at different times from 0 to 96 hours to analyze its development on the engorged females cuticle; after each incubation time, the females were fixed and analyzed by SEM. Blastospores of both isolates have started development 4 hours after treatment, demonstrating rapid development and suggesting penetration by the tick natural openings; at 4 hours incubation, indicative of penetration of IP 361 blastospores through the cuticle was observed, but no signs of penetration was observed with IP 146 blastospores at any time evaluated. Furthermore, fungal suspensions (103 propagules/mL) were exposed to heat (45 °C) for several time periods, then inoculated in Petri plates with BDAY medium plus chloramphenicol (0.055% v/v), and incubated for 7 days at 27 °C and RH ≥ 80%. ARSEF 324 conidia (79.1%) were more tolerant to heat than conidia of IP 363 (55.5%), IP 146 (1.5%), GC 307 (0%), and IP 361 (0% ) at 2 hours exposure, as well as blastospores after 60 minutes exposure, demonstrating mean percent CFU of 100%, 12.3%, 30.7%, 55% and 0%, respectively. Fungal suspensions (103 propagules/mL) were also inoculated on BDAY in Petri plates, and exposed to UV-B radiation; after treatment, plates were incubated for 7 days at 27 °C and RH ≥ 80%. No difference in mean relative percent CFU between conidia and blastospores was observed. Suitable adjuvants which aim at protecting fungal propagules against stressful abiotic factors are required for conidia and blastospores; however, the selection of isolates with marked natural tolerance to heat and UV-B radiation may increase performance of bioproducts. Accordingly, it is suggested that blastospores are promising fungal propagules for biological control of ticks, since they were virulent against R. microplus; in addition, the rapid development of blastospores on the tick cuticle indicates they may be exposed shortly to harmful environmental abiotic factors. / O presente estudo comparou conídios e blastosporos de Metarhizium anisopliae s.l. (IP 363), Metarhizium robertsii (IP 146) e Beauveria bassiana s.l. (IP 361 e CG 307) quanto a virulência para Rhipicephalus microplus, e quanto a tolerância ao calor e a radiação UV-B; foi avaliado ainda o desenvolvimento de conídios e blastosporos dos isolados IP 146 e IP 361 na cutícula de carrapatos por meio da microscopia eletrônica de varredura (MEV). Larvas e fêmeas ingurgitadas de R. microplus foram tratadas (larvas: 106, 107 ou 108 propágulos/mL; fêmeas: 107 propágulos/mL) por imersão, com conídios e blastosporos dos isolados avaliados. Maior percentual de mortalidade foi obtido nos grupos de larvas tratadas com conídios de IP 361, o qual obteve menor CL50. No bioensaio com fêmeas, blastosporos de IP 146 e IP 361 proporcionaram percentual de controle superior a 90%, enquanto que conídios dos mesmos isolados promoveram mortalidade de 70,97% e 63,29%, respectivamente. Para análise do desenvolvimento dos propágulos na cutícula de fêmeas ingurgitadas, essas foram tratadas topicamente com 50 µL de suspensão fúngica (107 propágulos/mL), incubadas em diferentes tempos de 0 a 96 horas; após cada tempo de incubação as fêmeas foram processadas e analisadas por MEV. Blastosporos de ambos isolados iniciaram seu desenvolvimento já após 4 horas de tratamento, demonstrando rápido desenvolvimento e sugerindo penetração por aberturas naturais do carrapato; com 4 horas de incubação foi possível ver a penetração de blastosporos de IP 361 através da cutícula, situação não evidenciada pelo isolado IP 146 em nenhum tempo avaliado. Nos testes de tolerância ao calor, o isolado de Metarhizium acridum (ARSEF 324) foi inserido como isolado padrão, por ser conhecida a sua tolerância. Suspensões fúngicas (103 propágulos/mL) foram expostas ao calor (45°C) por diferentes tempos, em seguida inoculadas em placas de Petri com meio BDAY acrescido de cloranfenicol (0,055% v/v) e incubadas por 7 dias a 27°C, e UR ≥ 80%. Conídios de ARSEF 324 (79,1%) demonstraram ser mais tolerantes ao calor do que conídios de IP 363 (55,5%), IP 146 (1,5%), CG 307 (0%) e IP 361 (0%) no tempo de 2 horas de exposição, assim como blastosporos no tempo de 60 minutos, demonstrando percentual relativo médio de 100%, 12,3%, 30,7%, 55% e 0%, respectivamente. Nos testes de exposição à UV-B, suspensões fúngicas (103 propágulos/mL) foram inoculadas em placas de Petri contendo BDAY e expostas a diferentes doses de radiação; após tratamento, as placas foram incubadas por 7 dias a 27°C, e UR ≥ 80%. Não houve diferença do percentual relativo médio de conídios e blastosporos expostos a mesma dose de radiação. Adjuvantes adequados que visem proteger os propágulos fúngicos contra fatores abióticos estressantes são requeridos para formulações de conídios e blastosporos; no entanto, a seleção de isolados naturalmente mais tolerantes ao calor e a radiação UV-B podem favorecer o desenvolvimento de bioprodutos. Nesse sentido, acredita-se que blastosporos sejam promissores para o biocontrole de carrapatos, já que estes se demonstraram virulentos para R. microplus, além de apresentarem rápido desenvolvimento sobre a cutícula desse artrópode, o que pode indicar menor tempo de exposição desses propágulos a fatores abióticos limitantes no ambiente.

Grapevine (Vitis spp.) is one of the most widely cultivated perennial fruit crops in the world and its economic relevance is mainly related to wine production. In recent years, the increased frequency of extreme phenomena such as heat waves has been acknowledged as one of the most significant climate variables negatively affecting grape yield and berry composition, with consequences also on wine quality. Thus, studying the physiological, metabolic and genetic factors that are involved in grapevine response to high temperatures is essential to improve the knowledge of mechanisms underlying thermotolerance, aiming to support plant breeding innovation and the development of new management strategies in viticulture. In this work, a segregating population obtained from the crossing of ‘Rhine Riesling’ and ‘Cabernet Sauvignon’ was studied in the field with a multidisciplinary approach. The progeny (around 120 genotypes) was evaluated for phenological traits affected by changing temperatures, in particular bud burst, flowering and véraison, while physiological response to heat stress was assessed in various hot summer days by measuring chlorophyll fluorescence kinetics and stomatal conductance. Measures were collected in the early morning as control and in the afternoon during hot hours. Phenotypic data were then used in combination with a high-density linkage map (average distance between adjacent markers 0.78 cM), previously developed using genotypic information from 139 individuals, to perform QTL analysis. Based on physiological responses to high temperatures, selected individuals showing contrasting behaviour, together with parental lines, were further studied in controlled conditions. In the field, in fact, plants may be subjected to combined stresses and changes in environmental conditions may heavily influence plants response. With the experiment in controlled condition, on the other hand, plants were stressed at higher temperatures, compared to the ones registered in the field, by maintaining all the other sources of variability constant. In the growth chamber plants were studied for their physiological response to heat stress by using the same approach adopted in the field. To better understand mechanisms involved in grapevine adaptation to heat stress conditions, individuals with contrasting behaviour were studied also for their metabolome modifications, both in the field and in controlled conditions. Volatile organic compounds (VOCs) were investigated with an untargeted approach applying conventional methods of analysis. Accumulation of VOCs in grapevine leaves was analysed using gas chromatography coupled with mass spectrometry (GC-MS) after a pre-concentration with a solid-phase micro-extraction (SPME) approach. On the other hand, VOCs emission during stress was investigated in controlled conditions thanks to the use of the Closed-Loop Stripping Analysis (CLSA) which allows the collection of VOCs directly emitted by plants. Analysis was then performed with GC-MS. Metabolic alterations of non-volatile compounds were examined with an untargeted analysis using high-performance liquid chromatography coupled with a high-resolution mass spectrometer equipped with an electrospray soft ionization (HPLC-HR-ESI-MS). In this work a metabolomic workflow was developed, starting from sample collection and extraction to sample analysis and data interpretation. The analytical method developed allowed the preliminary evaluation of leaf metabolome alterations due to stress factors. In fact, the use of a weak cation-exchange mixed mode column, in combination with a data dependent acquisition mode, allowed a first wide screening of both primary and secondary metabolites resulting in a good compromise for metabolic fingerprinting. QTL analysis on the segregating population allowed the identification of several QTLs, related to both phenological and physiological traits, with the discovery of interesting putative candidate genes for grapevine resilience to changing temperatures. This is the first time that a similar approach has been applied to a perennial fruit crop by analysing chlorophyll fluorescence and leaf transpiration traits related to heat stress. On the other hand, the multidisciplinary approach allowed the fine characterization of Rhine Riesling and Cabernet Sauvignon response to high temperatures, both in controlled and field conditions, a tentative classification of ‘tolerant’ and ‘susceptible’ progeny individuals and the identification of metabolic pathways altered during heat stress in the susceptible plants. Together with the implementation of a novel metabolomic workflow based on HPLC-HR-ESI-MS, this work represents a novelty in studies on grapevine response to changing temperatures, as it considered not only the berry metabolism but the resilience of the plants itself, paving the way for future studies on thermotolerance.

Ophioderma longicauda (Bruzelius, 1805) est un complexe d’espèces cryptiques incluant six lignées mitochondriales (L1-L6), dont certaines (L2-L3-L4) incubent leur descendance, alors que d'autres se reproduisent probablement via des larves lécithotrophes. Afin de définir les limites d’espèces dans le complexe O. longicauda, le statut reproductif des lignées L1 et L3 a été étudié. L’analyse morphologique et génétique a montré qu’il s’agissait d’espèces biologiques différentes, avec notamment différentes périodes de reproduction. De plus, l’analyse par DAPC de 31 marqueurs génétiques a montré que le complexe O. longicauda était constitué de six groupes génétiques distincts. Deuxièmement, l’influence des traits d’histoire de vie sur la connectivité et la diversité génétique a été étudiée. Pour ce faire, 10 marqueurs ont été séquencés pour six populations sympatriques des lignées L1 et L3 en Grèce. La structure génétique était très marquée pour l’espèce incubante L3, tandis que l’espèce dispersante L1 n’a pas montré de structure génétique à cette échelle. L’analyse de la diversité génétique pour ces 10 marqueurs a montré que celle des dispersantes était trois à quatre fois plus élevée que celle des incubantes. De plus, l’analyse de la diversité génétique dans les transcriptomes des L1 et L3 a montré qu’elle était 1.5 à 2 fois plus élevée chez les dispersantes que chez les incubantes. Finalement, deux canaux ioniques impliqués dans la mobilité des spermatozoïdes ont montré une évolution sous sélection positive. Ces résultats suggèrent que la compétition des spermatozoïdes pourrait être un mécanisme d’isolement pré-zygotique chez Ophioderma longicauda. / Ophioderma longicauda (Bruzelius, 1805) is a cryptic species complex including six mitochondrial lineages (L1-L6), of which three (L2-L3-L4) brood their juveniles, whereas the other lineages most likely reproduce using lecithotrophic larvae. In order to define the species limits in the O. longicauda complex, the reproductive status of lineages L1 and L3 was studied. The morphological and genetic study showed that they were different biological species, with notably different reproductive periods. In addition, the analysis of 31 genetic markers using DAPC showed that the O. longicauda complex included six distinct genetic groups. Secondly, the influence of life-history traits on connectivity and genetic diversity was studied. To do so, 10 markers were sequenced for six sympatric populations of lineages L1 and L3 from Greece. The genetic structure was high for the brooding species, whereas the broadcasting species did not display any genetic structure at that scale. The analysis of genetic diversity for these 10 markers showed that diversity was three to four times higher in broadcasters than in brooders. In addition, the analysis of genetic diversity in the L1 and L3 transcriptomes showed that diversity was 1.5 to 2 times higher in broadcasters than in brooders. Finally, two ion channels involved in sperm motility showed an evolution under positive selection. These results suggest that sperm competition might be a mechanism of pre-zygotic isolation in Ophioderma longicauda.