Brazil is the largest citrus producer in the world and has a large global citrus market share. However, several diseases affect the crop, being postbloom fruit drop (PFD) one of them. PFD has gained importance in São Paulo State for the displacement of citrus areas to regions with weather conditions more favorable for this disease. The accurate identification of the causal agent of the PFD has been performed and it was renamed as Colletotrichum abscissum. The origin of the initial inoculum is still an enigma for PFD epidemics and the hypotheses that the initial inoculum could be present in propagation material have been discussed but it has never been demonstrated. The objective of this work was to detect and quantify Colletotrichum abscissum from citrus leaves of budwood increase block and citrus nursery plants by qPCR. Four commercial citrus farms from São Paulo State, Brazil with budwood increase block and citrus nursery plants of Pera and Valencia sweet orange varieties were used for this work. C. abscissum was detected in budwood increase block and in nursery plant in both varieties (Valencia and Pera) at the four farms sampled. Out of 122 budwood increase block samples, 89 (73%) were positive for C. absicissum. From nursery plants, out of 175 samples, 129 (73%) were detected with the pathogen. The majority of the positive samples of budwood increase blocks and nursery plants contained 10 to 200 and 10 to 400 conidia of C. absicissum, respectively. With the methods used was not possible to isolate the fungus from vegetative material. This finding suggests a new long distances dispersion type of C. abscissum in the cycle of postbloom fruit drop by propagation material. Confirmation of C. abscissum in budwood increase block and nursery plants would lead to update regulations for the production of certified citrus nursery trees and searching for new control strategies of the pathogen. / O Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.

Five separate field experiments were conducted across different environmental locations in Texas for the purpose of quantifying concentrations of different phytochemical groups (ascorbic acid, capsaicinoids, and flavonoids) within various pepper species, as well as, to identify the most optimum environment to promote expression of the aforementioned phytochemical. Depending on the particular experiment, quantitative measurements were then used in more detail in one of three ways: for identification of the most superior individuals and optimum environmental locations to express elevated concentrations of a particular phytochemical (first three experiments), to calculate heritability and % heterosis estimates for various fruit characteristics and phytochemical levels (fourth experiment), or for use in a specific biotechnology technique to potentially identify a molecular marker linked to elevated levels of ascorbic acid (AA) and flavonoids (quercetin and luteolin) (fifth experiment). In the first experiment, significant differences in fruit weight, capsaicin, and dihydrocapsaicin (DHC) were revealed in fruit tissue of five Habanero (Capsicum chinense) hybrids in comparison to a popular, commercial check (Kukulkan F1) after being grown across three different environmental locations (College Station, Uvalde, and Weslaco). Fruit grown at the Weslaco location was found to be larger and contained more capsaicin and DHC than those produced in Uvalde or College Station. While flavonoid contents were variable and low in all genotypes and locations, a few hybrids showed some potential for use in future crossing schemes to compete against the commercial check. Our results further suggested that variation in phytochemicals in fruit tissue of Habanero genotypes can be exploited by selecting in an appropriate environment. In the second experiment, analysis of four jalapeño hybrids in comparison to three commercial checks (Dragon, Ixtapa, and J1845), as well as, two cayenne hybrids in comparison to one commercial check (Mesilla) were compared after growing in three different locations (Amarillo, College Station, and Uvalde). Results demonstrated that the College Station location contributed to production of fruit containing higher concentrations of both AA and flavonoids, while those grown in Amarillo produced fruit with higher capsaicinoids. As expected, cayenne samples contained more AA and flavonoids than jalapeño samples. In comparison to the commercial jalapeño checks, all jalapeño hybrids generally expressed less capsaicinoids. With respect to AA and flavonoid concentrations, a few jalapeño hybrids proved to have some potential to compete against their respective commercial cultivars. For the cayenne market, one genotype revealed its potential use in mild markets and for farmers interested in a new hybrid expressing both appreciable levels of phytochemicals, as well as, more visually aesthetic attributes. Statistical analyses from the third experiment found all the F-values for each characteristic to be significant except the Location x Genotype (L x G) component for fruit wall thickness. For this experiment, a total of 21 different C. annuum (jalapeño, Serrano, and cayenne) genotypes were evaluated after growing in two diverse environmental locations (Uvalde and Weslaco). In general, peppers grown in Weslaco produced fruit with higher concentrations of AA and capsaicinoids, with a few exceptions, while fruit grown at Uvalde generally was larger in size and contained higher concentrations of flavonoids. In comparison to their respective commercial checks (jalapeño – Dragon, Tormenta; Serrano – Halcon, Magnum45; cayenne – Mesilla), a few hybrids were arguably more desirable for their respective markets with respect to different fruit measurements, AA, capsaicinoid, and flavonoid concentrations. This evidence further suggested the potential benefit this material could have for growers interested in replacing current material in the industry and to more successfully provide consumers with a healthier ingredient to better sustain their lives. In the fourth experiment, results confirmed our hypotheses that paprika type material has higher AA and flavonoid concentrations than Serrano peppers, while the opposite is possible for capsaicinoid expression. For this experiment, our goal was to identify the relative ease of incorporating different traits of interest into an improved specimen evaluating a combination of 29 F1 paprika and Serrano pepper (C. annuum) hybrids along with 19 of their respective parents. We were also able to identify how productive certain crossing combinations were at expressing a significant amount of positive heterosis for different characteristics of interest. From our correlation analyses, we were able to identify that a significant association can exist between fruit weight and fruit diameter, as well as when other characteristics were evaluated. In all, our results were able to reveal how effective certain combinations of parent material are towards production of offspring with improved traits expressing appreciable fruit characteristics and elevated phytochemical concentrations. Finally, the quantitative measurements produced in our F2 molecular marker experiment found significant amounts of variation for both flavonoids (quercetin and luteolin) and AA expression. It was evident that incorporation of the necessary genes of interest present within fruit tissue across this F2 family for these different phytochemicals were passed from ‘Ca377’ (P1) to several of these offspring. Three candidate primers were eventually identified for their potential polymorphic expression. However, after statistically analyzing the results, only one primer was identified as expressing a significant association (although the value was still relatively low) of variability with respect to luteolin. We also identified a significant association between quercetin and luteolin, quercetin and total flavonoids (quercetin+luteolin), as well as, luteolin and total flavonoids (quercetin+luteolin) in fruit tissues of different pepper material. From our results, we were able to arguably conclude that an environmental component may serve a more essential role in activating the necessary physiological processes to produce specific secondary metabolites. Although our RAPD technique was fairly straightforward and useful, some may argue that a more reliable technique would have been better. Therefore, we postulate that success may be possible in the near future if a different molecular marker technique is used.

Neosporosis is a parasite disease caused by Neospora caninum protozoan and characterized as a major cause of abortion in cattle. Eventually is possible birth of animals with neurological signs, stillbirths or birth of clinically normal calves, but chronically infected. Although there are many researches related to bovine neosporosis, there are still doubts about the cycle and the pathogenesis of N. caninum infection, so that is suitable for certain control and prophylaxis strategies to disease. Gerbils are considered highly sensitive of the parasite replication. Thus, this study aimed to verify the occurrence of infection with N. caninum by oral inoculation of tachyzoites in gerbils (M. unguiculatus). Seventeen animals were orally inoculated with 4x105 N. caninum tachyzoites. The detection frequency of total DNA samples of N. caninum showed that the protozoan infectivity occurs via this route of inoculation, noting that 81.8 % of the inoculated animal parasite DNA was detected. In relation to tissues, it can be observed that the DNA parasite was found in all organs evaluated (heart, lungs, kidneys, liver, spleen, brain), by variable frequency, demonstrating the wide distribution of this parasite in animals tissues. / Neosporose é uma enfermidade de origem parasitária, causada pelo protozoário Neospora caninum e caracteriza-se como uma das principais causas de aborto em bovinos. Eventualmente, é possível ocorrer o nascimento de animais com sinais neurológicos, natimortos, ou ainda, o nascimento de bezerros clinicamente normais, porém cronicamente infectados. Embora existam inúmeras pesquisas relacionadas à neosporose bovina, ainda existem imprecisões quanto ao ciclo e patogenia da infecção por N. caninum, para que possam ser determinadas estratégias adequadas ao controle e profilaxia da doença. Os gerbils (Meriones unguiculatus) são considerados altamente sensíveis a replicação do protozoário. Assim, este estudo teve como principal objetivo verificar a ocorrência de infecção pelo N. caninum através da inoculação oral de taquizoítos em gerbils. Foram inoculados 17 animais pela via oral, com 4x105 taquizoítos de N. caninum. A frequência de detecção total de DNA de N. caninum das amostras demonstrou que ocorre infectividade pelo protozoário através desta via de inoculação, observando que em 81,8% dos animais inoculados foi detectado DNA do parasita. Em relação aos tecidos, pode-se observar que o DNA do protozoário foi encontrado em todos os órgãos avaliados (coração, pulmões, rins, fígado, baço, cérebro), com frequências variáveis, demonstrando assim a ampla distribuição deste protozoário em diferentes tecidos dos animais.

A interação entre bactérias e plantas resulta na ocorrência de vários processos biológicos no ambiente e pode ser regulada por diferentes fatores. Considerando que um recíproco reconhecimento ocorre entre as espécies de bactérias e plantas, alterações nos fatores bióticos e abióticos interferem diretamente nesta interação levando a modificações na composição das comunidades bacterianas associadas às plantas. No presente trabalho foram avaliados diferentes fatores que estão diretamente relacionados a este assunto. Atualmente, a aplicação de técnicas de microbiologia molecular permite acessar alterações causadas nestas comunidades de maneira independente do cultivo bacteriano. Desta maneira, foi demonstrado que na rizosfera de plantas de tabaco, os diferentes estágios de desenvolvimento são os principais determinantes da composição da comunidade bacteriana em detrimento do genótipo das plantas, sejam estas transgênicas ou convencionais. Utilizando plantas transgênicas e convencionais de eucalipto de diferentes genótipos, foi verificado que diferentes plantas não transgênicas podem apresentar comunidades bacterianas mais distintas do que quando os clones não transgênicos são comparados com os transgênicos. No entanto, efeitos específicos podem ocorrer na interação bactéria-planta, como o observado pela inibição da população de Methylobacterium spp. em plantas transgênicas de eucalipto TR-15. Avaliando o efeito da inoculação de bactérias endofíticas na composição de comunidades bacterianas associadas à plantas de batata de diferentes cultivares, os resultados mostram que a inoculação de Pseudomonas putida altera a comunidade bacteriana de maneira similar a alteração da variedade da planta. Os demais isolados avaliados, classificados como Paenibacillus sp. e M. mesophilicum, causam menores alterações na composição das comunidades bacterianas. Adicionalmente foi demonstrado que a colonização das plantas de batata pelo endófito P. putida resulta em pequena alteração no perfil metabólico da planta hospedeira. Por fim, buscando melhores métodos de isolamento da comunidade bacteriana da rizosfera de batata, os resultados mostraram que a mimetização do ambiente pode resultar em melhor acesso da diversidade bacteriana por meio de isolamento e cultivo das espécies componentes desta comunidade. / The plant-bacteria interactions result in the occurrence of biological process in the environment and might be regulated by different factors. Considering that a reciprocal recognition occur amongst bacteria and plants species, shifts in biotic and abiotic factors interfere directly on these interactions, leading to modifications in the composition of bacterial communities to plants associated. In the present work different factors related to this subject were evaluated. Nowadays, the applications of techniques of molecular microbiology allow assessing the shifts caused on these communities by a culture independent approach. On this way, it was demonstrated that in rhizosphere of tobacco plants, different stages of plants development are main determinants of bacterial community composition rather than the plants genotypes, transgenic or not. Using plants transgenic or not, carrying different genotypes, it was verified that different non-transgenic plants could harbor bacterial communities more distinct than those observed in association with transgenic plants. However, specific effects could be observed like the inhibition of Methylobacterium spp. population in eucalyptus transgenic plants TR-15. Considering the effect of endophytic bacteria inoculation in the composition of bacterial communities associated to different cultivars of potato plants, the results show that inoculation of Pseudomonas putida causes similar shifts in the bacterial community similarly to those observed when different cultivars are considered. Other evaluated strains, classified as Paenibacillus sp. and M. mesophilicum, caused minor alterations in the composition of bacterial communities. In addition, it was demonstrated that plant colonization by endophytic P. putida results in small effects on the metabolic profile of host plant. At least, aiming better methodologies for bacteria isolation from potato rhizosphere, the results show that mimicking the natural environment could result in a better assessment of bacterial diversity by isolation of species present in this community.

This study was conducted to measure the reduction in polycyclic aromatic hydrocarbons (PAHs) content in oil sludge by co-composting the sludge with pig, cow, horse and poultry manures under laboratory conditions. Four kilograms of soil spiked with 800g of oil sludge was co-composted differently with each manure in a ratio of 2:1 (w/w) spiked soil: manure and wood-chips in a ratio of 2:1 (w/v) spiked soil: wood-chips. Control was set up similar as the one above but without manure. Mixtures were incubated for 10 months at room temperature. Compost piles were turned weekly and moisture level was maintained at between 50% and 70%. Moisture level, pH, temperature, CO2 evolution and oxygen consumption were measured monthly and the ash content at the end of experimentation. Bacteria capable of utilizing PAHs were isolated, purified and characterized by molecular techniques using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), amplification of the 16S rDNA gene using the specific primers (16S-P1 PCR and 16S-P2 PCR) and the amplicons were sequenced. Extent of reduction of PAHs was measured using automated soxhlet extractor with Dichloromethane as the extraction solvent coupled with gas chromatography/mass spectrometry (GC/MS). Temperature did not exceed 27.5OC in all compost heaps, pH ranged from 5.5 to 7.8 and CO2 evolution was highest in poultry manure at 18.78μg/dwt/day. Microbial growth and activities were enhanced. Bacteria identified were Bacillus, Arthrobacter and Staphylococcus species. Results from PAH measurements showed reduction between 77 and 99%. The results from the control experiments may be because it was invaded by fungi. Co-composting of spiked soils with animal manures enhanced the reduction in PAHs. Interestingly, all bacteria isolated and identified in this study were present in all treatments, including the control. / Environmental Sciences / M.Sc. (Environmental Sciences)

Thesis (MSc)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Non-native species are recognized as a major component of global environmental change. Their ecological impacts are numerous and include the alteration of whole ecosystem processes as well as the loss of native biodiversity. As such, understanding the processes that drive the invasion of non-native species is essential for the control and management thereof. Numerous research approaches have been used to provide insight on the history and ecology of non-native species invasions. However, recent approaches employing molecular techniques have greatly helped in solving taxonomic issues associated with some of these species; identifying sources of invasions; and shedding light on colonization dynamics. Lantana camara, a globally invasive and highly variable species complex, is one of the most notorious plant invaders in South Africa. The species has been associated with negative impacts in agricultural areas, decreased invertebrate diversity, livestock mortality, and where it occurs along riparian areas- decreased water quality and obstruction to accessing water sources. This project aimed to review L. camara invasions in South Africa and to unravel patterns of spread in L. camara along the Sabie-Sand catchment in South Africa’s flagship protected area, the Kruger National Park, using a molecular approach. The findings of the first part of the study highlight that L. camara has successfully spread across South Africa with only four known introduction events, and this can be attributed to the species’ broad ecological tolerance, its use in the horticultural industry, and a variety of dispersal vectors (birds, humans and rivers). Furthermore, although sale of the species is prohibited in the country, it will continue to spread naturally and also has the potential to expand its distribution under changing climate scenarios. The second part of the study highlights that spread along the Sabie-Sand catchment is primarily river-driven and that the Sand tributary is the invasion source in the system. Because the Sand tributary originates outside the Kruger National Park, and only a small portion is under park management, the implications for spread are important. / AFRIKAANSE OPSOMMING: Nie-inheemse spesies word erken as 'n belangrike komponent van die globale omgewing verandering. Hulle ekologiese impak is talle en sluit in die verandering van ekosisteem prosesse asook die verlies van inheemse biodiversiteit. As sodanig, die begrip van die prosesse wat lei tot die inval van 'n nie-inheemse spesies is noodsaaklik vir die beheer en bestuur daarvan. Talle navorsingsbenaderings is gebruik om insig te gee oor die geskiedenis en ekologie van nie-inheemse spesies invalle. Onlangse benaderings soos die gebruik van molekulêre tegnieke, help in die oplossing van taksonomiese kwessies wat verband hou met 'n paar van hierdie spesies,in die identifisering van bronne van invalle, en om lig te werp op die kolonisasie dinamika. Lantana camara, 'n wêreldwye indringende en spesie kompleks, is een van die mees berugte plantindringer in Suid-Afrika. Die spesie is geassosieer met negatiewe gevolge in landbou gebiede, afgeneem ongewerwelde diversiteit, vee mortaliteit, en waar dit voorkom saam oewer gebiede - afgeneem kwaliteit van die water en obstruksie tot waterbronne. Hierdie projek is daarop gemik om om L. camara invalle in Suid-Afrika te hersien en patrone van verspreiding te ontrafel in L. camara langs die Sabie-Sand-opvanggebied in Suid-Afrika se vlagskip beskermde gebied, die Kruger Nasionale Park, met behulp van 'n molekulêre benadering. Die bevindinge van die eerste deel van die studie wys dat L. camara het suksesvol versprei oor die hele Suid-Afrika met slegs vier bekende inleiding gebeure, en dit kan toegeskryf word aan die spesie se breë ekologiese verdraagsaamheid, die gebruik daarvan in die hortologie bedryf, en 'n verskeidenheid van die verspreiding vektore (voëls, mens en riviere). Verder, hoewel die verkoop van die spesie in die land verbied word, sal dit natuurlik voortgaan om te versprei en het ook die potensiaal om uit te brui onder veranderende klimaat scenario's. Die tweede deel van die studie wys dat versprei langs die Sabie-Sandopvanggebied hoofsaaklik rivier-gedrewe is en dat die Sand sytak die inval bron is. Omdat die Sand sytak buite die Kruger Nasionale Park ontstaan, en slegs 'n klein gedeelte onder park bestuur is, is die implikasies vir verspreiding belangrik. / Financial support from the DST-NRF Centre of Excellence for Invasion Biology and the Working for Water (WfW) Programme through their collaborative project on “Research for Integrated Management of Invasive Alien Species” and Stellenbosch University’s Subcommittee B Young Researcher's Fund

O presente trabalho esteve inserido em um projeto mais amplo de cooperação internacional intitulado Conservation and Sustainable Management of Below-Ground Biodiversity CSMBGBD/ BiosBrasil, implementado pela United Nations Environment Programmer (UNEP) na bacia do Alto Solimões, Amazônia Ocidental, estado do Amazonas. Esta região é território remanescente de povos indígenas e permanece conservada, sendo um importante hotspot de biodiversidade. Ainda assim, as áreas de estudo foram caracterizadas por paisagens antropizadas com diferentes sistemas de usos da terra. Amostras de solos foram coletadas em um período de elevado índice pluviométrico, nos anos de 2008 e 2009, em áreas caracterizadas por floresta primária tropical, cultivos semiperenes de mandioca manejados por prática agrícola de corte-equeima, pastagens implantadas nos anos de 1970 e áreas florestais em estádios avançados de regeneração (>10 anos de abandono). As amostras foram analisadas pelas técnicas de DGGE, ARISA, clonagem e sequenciamento a fim de obter uma caracterização das estruturas de comunidades de Archaea, Bacteria e microfungos e da composição e diversidade de um grupo funcional de Archaea envolvido no processo de oxidação de amônia nos ambientes do solo. Os resultados permitiram concluir que o uso da terra tem um grande efeito sobre as estruturas de comunidades de Archaea, Bacteria e microfungos presentes no solo e sugerem que longo período de abandono das áreas é necessário para cumprir com a resiliência dos ecossistemas amazônicos no contexto de recomposição da paisagem. Adicionalmente, os dados revelaram que a riqueza e a diversidade de comunidades de Archaea oxidadoras de amônia foram capazes de refletir sensivelmente as alterações percebidas nos ambientes do solo em decorrência do desmatamento de áreas de floresta primária e uso subsequente com cultivo agrícola tradicional e pastagem na região do Alto Solimões, Amazônia Ocidental. / The present study was part of a wider project of international cooperation entitled Conservation and Sustainable Management of Below-Ground Biodiversity CSMBGBD/ BiosBrasil, established by the United Nations Environment Programmer (UNEP) at the basin of Alto Solimões, Western Amazon, Amazonas State. This region is a remanescent territory of indigenous people and remains conserved, being considered an important hotspot of biodiversity. Even though, the studied areas were characterized by mosaic landscapes under different land use systems. Soil samples were collected in a period of high pluviometric index in the years 2008 and 2009 in areas characterized by tropical rainforest, semi permanent manioc cultivation under agricultural management of slash-and-burn, pasture established in the 1970s and forested areas at a higher stage of regeneration (>10 years abandoned). The samples were analyzed by DGGE, ARISA techniques, cloning and sequencing in order to obtain a characterization of the community structure of Archaea, Bacteria and microfungi and the composition and diversity of an archaeal functional group involved in the process of ammonia oxidation in the soil environments. The results allowed to conclude that land use has a great effect on the community structure of Archaea, Bacteria and microfungi present in the soil and suggest that long period of abandon of the areas is necessary to accomplish with the resilience of Amazonian ecosystems in the context of landscape recomposition. Additionally, the data revealed that richness and community diversity of ammonia oxidizing Archaea were able to sensitively reflect the changes observed in the soil environment due to deforestation of rainforest areas and subsequent use with traditional agriculture cultivation and pasture in the region of Alto Solimões, Western Amazon.

Vias redox são importantes reguladores da homeostase e sinalização celular, mas o entendimento dos mecanismos desses processos é incompleto. Tiol-proteínas como a dissulfeto isomerase proteica (PDI) podem ser moduladores dessas vias. A PDI(PDIA1) é o protótipo da família das PDIs, cuja função canônica é o enovelamento redox de proteínas no retículo endoplasmático. Além disso, PDI exerce regulação de NADPH oxidases, as principais fontes de oxidantes celulares, e é necessária para ativação de RhoGTPases, organização do citoesqueleto e migração de células vasculares. No estudo de mecanismos pelos quais a PDI regula RhoGTPases, mostramos, em redes computacionais e em experimentos de co-imunoprecitação, associação entre PDIA1 e o regulador de RhoGTPases RhoGDIalfa. Além disso, identificamos forte proximidade entre os genes codificando estas proteínas. Neste estudo, caracterizamos o perfil e implicações desta sintenia gênica.A análise bioinformática pelos programs Ensembl, NCBI e UCSC evidencia um padrão de sintenia entre diferentes isoformas destas duas famílias: PDIA1 (P4HB), PDIA2 (PDIP) e PDIA8 (Erp27) são vizinhos, respectivamente, a RhoGDIbeta, RhoGDIy e RHOGDIalfa, com correspondentes regiões intergênicas de 7.1, 2.9 e 0.14 kb em distintos cromossomos em H. sapiens. O padrão dessa sintenia foi fortemente conservado emC. elegans, alguns peixes e uniformemente em anfíbios, répteis, aves e mamíferos. Leveduras expressam no mesmo cromossomo , porém em locais distantes (i.emacrossintenia) ortólogos da PDIA1 e RhoGDI?, mas não expressam outras PDIs e RhoGDIssintênicasnos eucariotos complexos. No entanto, sintenia entre PDI e RhoGDI foi também observada na planta A. thaliana, sem evidência de um ancestral comum. Os pares sintênicos associam-se a blocos vizinhos conservados, porém diversos para cada par, enquanto cada bloco contem um gene codificando um distinto regulador da PP1 (fosfatase proteica-1). Análise filogenética mostrou topologia semelhante entre as duas famílias.Análise dos dados do estudo ENCODE e predição pelo Softberry identificou sítios de ligação a fatores de transcrição comuns entre os distintos pares, cuja ontologia indicou principalmente desenvolvimento, processos metabólicos e resposta imune. O estudo de possíveis implicações funcionais dessa sintenia mostrou que manipulações da expressão proteica de PDIA1 não promovem mudança consistente na expressão proteica de RhoGDIalfa, tanto in vitro (silenciamento da PDI por siRNA e superexpressão por vetor lentiviral induzível) como in vivo (camundongo transgênico com superexpressão constitutiva da PDIA1). No entanto, as mudanças da expressãogênica de ambos os genes na camada íntima de artérias carótidas de camundongo durante remodelamento induzido por fluxo foram fortemente correlacionadas. Experimentos de coimunoprecipitação e co-localização à microscopia confocal sugeriram interação física entre PDIA1 e RhoGDIAalfa. Deste modo, estes dados mostram um intrigante padrão de conservação evolutiva da proximidade gênica entre PDIs e RhoGDIs, não usual em eucariotos. Genes sintênicos frequentemente codificam proteínas que tendem a interagir física e/ou funcionalmente. Com efeito, nosso dados sugerem co-regulação e interação física entre PDIA1 e RhoGDIAalfa, corroborando a convergência entre essas proteínas como possível mecanismo envolvido na regulação redox do citoesqueleto pela PDIA1 / Redox pathways are important regulators of homeostasis and cell signaling, but the understanding of the mechanisms of these processes is incomplete. Thiol proteins such as protein disulfide isomerase (PDI) can be modulators of these pathways. PDI (PDIA1) is the prototype of the family of PDIs whose canonical function is a redox protein folding in the endoplasmic reticulum. In addition, PDI exerts regulatory NADPH oxidase, the main sources of cellular oxidant, and is required for activation RhoGTPases, cytoskeletal organization and migration of vascular cells. In the study of mechanisms by which regulates PDI RhoGTPases, we showed in computer networks and co-imunoprecitation experiments association between PDIA1 and the regulator of RhoGTPases, RhoGDI?. In addition, we identified strong proximity of the genes encoding these proteins. In this study, we characterize the profile and implications of this synteny. .A bioinformatic analysis by programs Ensembl, NCBI and UCSC shows a pattern of synteny between different isoforms of these two families: PDIA1 (P4HB), PDIA2 (PDIP) and PDIA8 (Erp27) are neighbors , respectively RhoGDIalfa, and RhoGDIy RHOGDIbeta with corresponding intergenic regions 7.1, 2.9 and 0:14 kb in different chromosomes of H. sapiens. The pattern of this synteny was strongly maintained in C. elegans, some fish and evenly amphibians, reptiles, birds and mammals. Yeasts express on the same chromosome, but in distant places (i.e macrosintenia) orthologs of PDIA1 and RhoGDI?, but do not express other syntenics PDIs and RhoGDIs in complex eukaryotes. However, synteny between PDI and RhoGDI was also observed in the plant A. thaliana, no evidence of a common ancestor. The syntenic pairs are associated with the stored neighboring blocks, but different for each pair, while each block contains a gene encoding a regulator of distinct PP1 (protein phosphatase-1). Phylogenetic analysis showed similar topology between the two famílias. The identified binding sites common transcription factors between different pairs, which mainly indicated ontology development, metabolic and immune response. The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDI, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). The study of possible functional implications of synteny showed that manipulations of PDIA1 protein expression do not promote consistent change in protein expression RhoGDIalfa, both in vitro (silencing of PDI by siRNA and overexpression of inducible lentiviral vector) and in vivo (transgenic mice overexpressing constitutive of PDIA1). However, changes of gene expression of both genes in the intima of mouse carotid arteries during remodeling induced by flow were strongly correlated. Immunoprecipitation experiments and co-location to confocal microscopy suggested physical interaction between PDIA1 and RhoGDIAalfa. Thus, these data show an intriguing pattern of evolutionary conservation of gene proximity between POIs and RhoGDIs not common in eukaryotes. sintênicos genes often encode proteins that tend to interact physically and / or functionally. Indeed, our data suggest co-regulation and physical interaction between PDIA1 and RhoGDIAalfa, supporting the convergence of these proteins as a possible mechanism involved in redox regulation of cytoskeleton by PDIA1

Tecidos vegetais, incluindo as sementes, são habitados por microrganismos denominados endofíticos, cuja interação com a planta pode conferir características vantajosas ao hospedeiro. Sabe-se que o crescimento de plantas é influenciado por fatores como a síntese de ácido-indolacético (AIA), solubilização de fosfato, fixação de nitrogênio e controle de fungos fitopatogênicos. Antes da comercialização, as condições de armazenamento de sementes de soja podem restringir o desenvolvimento de microrganismos devido à baixa temperatura e umidade. Esse fato leva ao interesse de exploração de microrganismos endofíticos resistentes a essas condições. O estudo e a caracterização dessas comunidades são de grande interesse agronômico e biotecnológico, sendo possível sua aplicação em sementes, introduzindo no campo plantas com superior potencial de produção. Com os objetivos de comparar a comunidade bacteriana endofítica de sementes de soja geneticamente modificadas e convencionais; e de isolar e caracterizar essas comunidades, sementes de 12 cultivares de soja foram amostradas, de onde 3504 isolados bacterianos foram obtidos. Os isolados foram agrupados morfologicamente de acordo com a coloração e taxa de crescimento das colônias, sendo representantes de cada grupo morfológico (no total 176 isolados) agrupados pela técnica de ARDRA (Análise de Restrição de DNA Ribossomal Amplificado). Um total de 12 ribotipos foi observado compondo a comunidade cultivada de bactérias endofíticas de sementes de soja. Representantes destes ribotipos tiveram seus genes 16S rDNA parcialmente sequenciados, identificando os integrantes desta comunidade como: Acinetobacter sp., Bacillus sp., Brevibacterium sp., Chryseobacterium sp., Citrobacter sp., Curtobacterium sp., Enterobacter sp., Methylobacterium sp., Microbacterium sp., Micromonospora sp., Pantoea sp., Paenibacillus sp., Pseudomonas sp., Ochrobactrum sp., Streptomyces sp. e Tsukamurella sp. A comunidade bacteriana endofítica de sementes de soja provenientes de plantas genticamente modificadas apresentou uma diversidade maior comparada à comunidade bacteriana de plantas convencionais. Em relação ao potencial biotecnológico desta comunidade, os resultados demonstraram que os isolados foram capazes de controlar o crescimento de fungos fitopatogênicos por antagonismo (18%), sintetizar AIA (100%), solubilizar fosfato (39%) e fixar nitrogênio (18%). Os isolados com os melhores resultados nas análises in vitro foram inoculados em sementes de soja e avaliados em casa de vegetação quanto à habilidade de promover o crescimento das plantas. As plantas apresentaram diferentes respostas à inoculação das bactérias. A maior parte dos tratamentos mostrou influência negativa das bactéria nas plantas, enquanto que um isolado de Enterobacter sp. aumentou a massa da matéria seca de raiz. Mesmo não diferindo estatisticamente, alguns isolados mostraram tendência de aumento e outros de diminuição de biomassa da planta. / Plant tissues, including seeds, are inhabited by microorganisms called endophytes, whose interaction with the plant can offer advantages to the host. It is known that plants growth promotion is induced by indole acetic acid (IAA) synthesis, phosphate solubilization and nitrogen fixation, among others. The control of phytopathogenic fungi is also related to a good plant development. Before commercialization, the seed storage conditions can restrict the development of microorganism, due to the low temperature and humidity. This fact leads to the interest of exploring resistant microorganisms to those conditions. The study and the characterization of these communities are of great agronomic and biotechnological interest, being possible its application onto seeds, introducing in the field plants with a greater production potential. In this context, the aim of this study was to isolate and identify the endophytic bacteria community in soybean seeds and study the capacity of these isolates to promote growth in the host plant, including: phosphate solubilization, nitrogen fixation, IAA synthesize and antagonism against phytopathogenic fungi. From seeds of 12 cultivars, 3504 bacteria were isolated. The isolates were morphologically grouped according to the coloration and growth rate of the colony. Representatives of each morph group, totalizing 176, were analyzed using the Amplified Ribosomal Restriction Analysis (ARDRA) technique. A total of 12 ARDRA ribotypes were observed in the cultivable endophytic community of soybean seeds. Representatives of each ribotype had their 16S rDNA gene partially sequenced, allowing to the identification of the members of this community as: Acinetobacter sp., Bacillus sp., Brevibacterium sp., Chryseobacterium sp., Citrobacter sp., Curtobacterium sp., Enterobacter sp., Methylobacterium sp., Microbacterium sp., Micromonospora sp., Pantoea sp., Paenibacillus sp., Pseudomonas sp., Ochrobactrum sp., Streptomyces sp. and Tsukamurella sp. The endophytic bacterial community of soybean seeds from genetically modified plants showed a greater diversity compared to the bacterial community of conventional seeds. In relation to the biotechnological potential of the community, the outcomes demonstrate that the isolates were able to antagonist phytopathogenic fungi (18%), synthesize IAA (100%), solubilize phosphate (39%) and fix nitrogen (18%). The isolates with best in vitro outcomes were inoculated onto seeds and tested in greenhouse for their ability to promote growth in soybean. The plants answered differently to the inoculation of each bacterial isolate. The major part of the treatments demonstrated a negative influence of bacteria onto plants, while one Enterobacter sp. isolate increased the dry mass weight of roots. Even not differing statistically, some isolates showed a tendency to increase, meanwhile others to decrease the biomass of the plant.

Les mucilages pélagiques marins (MPM) sont des phénomènes mondiaux sporadiques et, dans certaines régions, chroniques dans les zones côtières. Ces agrégats gélatineux, enrichis en matière organique et en microorganismes, forment des écosystèmes marins autonomes transitoires allant de 0,5 cm à plusieurs kilomètres de long. Ils correspondent à des étapes évolutives de la neige marine non-sédimentée maintenue dans la zone photique. Durant les dernières décennies une intensification des apparitions des MPM ont été recensées. Ainsi, les MPM sont devenus un sujet de préoccupation pour les populations qui exploitent les ressources côtières et dans le contexte du fonctionnement global de l'écosystème. Malgré l’intérêt scientifique grandissant au cours des dernières décennies au sujet de ces phénomènes, notamment en mer Adriatique, les causes de formation, la dynamique et le rôle respectif des microorganismes dans de tels systèmes restent énigmatiques. La plupart des études réalisées sur les MPM étaient axées uniquement sur les communautés microbiennes eucaryotes par l’utilisation de techniques microscopiques. Or de nos jours, les méthodes moléculaires permettent de se concentrer également sur l'ensemble de la communauté, y compris les procaryotes. Afin d'approfondir notre compréhension de ces phénomènes nous avons étudié un type MPM qui est apparu de manière récurrente et abondante au cours de la dernière décennie dans le sud du Golfe de Gascogne (France), le «Liga». Dans un premier temps, nous avons étudié la formation de ce MPM au travers d’une année complète en suivant la dynamique des communautés archées, bactériennes et eucaryotes par la technique de T-RFLP, technique d’empreinte moléculaire ciblant la petite sous-unité du gène codant pour ARNr. Cette approche a révélé que les communautés microbiennes du Liga étaient différentes des communautés microbiennes marines pour les trois domaines du vivant, et que ces deux communautés étaient gouvernées par des paramètres environnementaux dont la variation était saisonnière. Les archées n’ayant pas pu être détectés dans le Liga, nous nous sommes focalisés sur la structure des communautés bactériennes et eucaryotes au moyen de technologies de séquençage à haut débit. Cette méthode a révélé que le Liga était principalement composé d'espèces marines, même si ces communautés étaient significativement différentes des espèces marines. Dans le Liga, les communautés eucaryotes étaient principalement composées de dinoflagellés, de zooplancton et de cnidaires. Les communautés bactériennes étaient principalement composées d’Alphaproteobacteria et de Gammaproteobacteria. La diversité fonctionnelle du Liga fraîchement formé a été également étudiée pendant les saisons d'apparition de ce phénomène, au printemps et à l'automne. Nous avons ainsi mis en évidence que les communautés microbiennes du Liga avaient potentiellement moins de capacités de résistance au stress et que ces communautés étaient potentiellement plus virulentes que les communautés microbiennes marines. / Marine pelagic mucilage (MPM) is worldwide phenomena occurring sporadically and, in certain regions, episodically in coastal areas. These gelatinous aggregates, enriched in organic matter and microorganisms, form autonomous transitory marine ecosystems ranging from 0.5 cm to several kilometers. They correspond to evolving stages originating from the non-settling early marine snow maintained in the photic zone. During the last decades intensification of MPM events have been noticed. They became a matter of concern both for populations exploiting coastal resources and in the context of global ecosystem functioning. Although increased scientific attention has been paid during last decades to these phenomena in specific areas such the Adriatic Sea, the causes of appearance, the dynamics, and the respective role of microorganisms in such systems remain enigmatic. Most of the studies performed on MPM focused on eukaryotic microbial communities using microscopic techniques. However molecular methods allow nowadays focusing also on the whole community including the prokaryotic part. In order to deeper our understanding of these phenomena we studied a MPM that occurred recurrently and more frequently during the last decade in the south of the Bay of Biscay (France), the “Liga”. In a first step we investigated the formation of this MPM through a complete year by following the dynamics of archaeal, bacterial and eukaryotic communities using T-RFLP fingerprinting targeting the small subunit of rRNA genes. This approach revealed that Liga’s microbial communities where different from marine microbial communities for the three domains of life and that both marine mucilage and marine communities were linked with seasonal patterns. As archaea were not found in the Liga, we focused on bacterial and eukaryotic communities’ structures through high throughput sequencing. The molecular composition revealed that the Liga was mainly composed of marine species although these communities were significantly different from marine species. In the Liga, eukaryotic communities were mainly composed of dinoflagellates, zooplankton and cnidarians species and bacterial communities were mainly composed of Alphaproteobacteria and Gammaproteobacteria. Functional diversity of freshly-formed Liga was targeted during its seasons of apparition, in spring and in autumn. We highlighted that Liga’s microbial communities had less potential capabilities to resist to stress conditions and that these communities were potentially more virulent than marine microbial communities.