abstract: Water contamination with nitrate (NO3−) (from fertilizers) and perchlorate (ClO4−) (from rocket fuel and explosives) is a widespread environmental problem. I employed the Membrane Biofilm Reactor (MBfR), a novel bioremediation technology, to treat NO3− and ClO4− in the presence of naturally occurring sulfate (SO42−). In the MBfR, bacteria reduce oxidized pollutants that act as electron acceptors, and they grow as a biofilm on the outer surface of gas-transfer membranes that deliver the electron donor (hydrogen gas, (H2). The overarching objective of my research was to achieve a comprehensive understanding of ecological interactions among key microbial members in the MBfR when treating polluted water with NO3− and ClO4− in the presence of SO42−. First, I characterized competition and co-existence between denitrifying bacteria (DB) and sulfate-reducing bacteria (SRB) when the loading of either the electron donor or electron acceptor was varied. Then, I assessed the microbial community structure of biofilms mostly populated by DB and SRB, linking structure with function based on the electron-donor bioavailability and electron-acceptor loading. Next, I introduced ClO4− as a second oxidized contaminant and discovered that SRB harm the performance of perchlorate-reducing bacteria (PRB) when the aim is complete ClO4− destruction from a highly contaminated groundwater. SRB competed too successfully for H2 and space in the biofilm, forcing the PRB to unfavorable zones in the biofilm. To better control SRB, I tested a two-stage MBfR for total ClO4− removal from a groundwater highly contaminated with ClO4−. I document successful remediation of ClO4− after controlling SO4 2− reduction by restricting electron-donor availability and increasing the acceptor loading to the second stage reactor. Finally, I evaluated the performance of a two-stage pilot MBfR treating water polluted with NO3− and ClO4−, and I provided a holistic understanding of the microbial community structure and diversity. In summary, the microbial community structure in the MBfR contributes to and can be used to explain/predict successful or failed water bioremediation. Based on this understanding, I developed means to manage the microbial community to achieve desired water-decontamination results. This research shows the benefits of looking "inside the box" for "improving the box". / Dissertation/Thesis / Ph.D. Sustainability 2014

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O Mycobacterium leprae, agente etiolÃgico da hansenÃase, nÃo à cultivÃvel em meio axÃnico. A quantificaÃÃo do bacilo realizada pelo exame baciloscÃpico e histopatolÃgico à Ãtil para a classificaÃÃo da hansenÃase na escolha e monitoramento do tratamento. No entanto, esta metodologia produz resultados de especificidade e sensibilidade limitada. A PCR em tempo real (qPCR) à um ensaio sensÃvel e especÃfico que permite a quantificaÃÃo a partir de diversas amostras e pode ser utilizada no diagnÃstico diferencial de muitos patÃgenos. Atà o momento, nenhum estudo avaliou a sensibilidade e especificidade da qPCR para o diagnÃstico da hansenÃase utilizando amostras de secreÃÃo nasal. Portanto, este estudo teve como objetivo detectar e quantificar o DNA de M. leprae por qPCR em amostras de secreÃÃo nasal e biÃpsias de pacientes com hansenÃase, correlacionando com a avaliaÃÃo clÃnica e o Ãndice baciloscÃpico. Foram analisadas 61 amostras de muco nasal e 19 amostras de biÃpsia de lesÃo de pele (pareadas) de casos confirmados de hansenÃase atendidos no Centro de ReferÃncia Nacional em Dermatologia SanitÃria Dona LibÃnia. Todas as amostras foram submetidas à extraÃÃo e amplificaÃÃo de DNA por nested PCR da regiÃo de 238 pb da sequÃncia RLEP2. Posteriormente as amostras foram submetidas à quantificaÃÃo da regiÃo 16S rRNA do genoma de M. leprae pela qPCR, cuja especificidade foi verificada atravÃs da curva de dissociaÃÃo (Tm=79,5ÂC). O mÃtodo foi suficientemente sensÃvel para detectar 20 fg de DNA de M. leprae, o equivalente a quatro bacilos. Na anÃlise da secreÃÃo nasal, o ensaio foi capaz de confirmar o diagnÃstico em 89,7% dos casos multibacilares (MB), e 73,3% dos casos paucibacilares (PB). A sensibilidade foi de 100% na analise de biÃpsias de pacientes MB. O nÃmero de bacilos detectados nas amostras de secreÃÃo nasal de pacientes com hansenÃase se manteve no intervalo de 1,39 x 103 a 8,02 x 105 bacilos, enquanto a detecÃÃo em biÃpsia de pacientes MB variou de 1,87 x103 a 1,50 x 106. A PCR em tempo real à mais sensÃvel do que a PCR convencional e pode ser utilizada como uma ferramenta complementar para o diagnÃstico clÃnico e histopatolÃgico da hansenÃase. / Mycobacterium leprae, the etiologic agent of leprosy, is not cultivable in axenic medium. The quantification of bacilli performed by skin bacilloscopy and histopathology is useful for the clinical classification of leprosy and treatment monitoring. However, this methodology yields low results regards to sensitivity and specificity. On the other way, the real-time quantitative PCR (qPCR) is a sensitive and specific assay that allows quantification of a varied of samples and also can be used for differential diagnosis of many pathogens. To this date, no studies have evaluated the sensitivity and specificity of qPCR for the diagnosis of leprosy from nasal secretion samples. Therefore, this study aimed at detecting and quantifying DNA from M. leprae by qPCR from nasal secretion and biopsy samples from leprosy cases, correlating with clinical and bacteriological index. We analyzed 61 samples of nasal secretion and 19 biopsy specimens of skin lesion (paired) from confirmed leprosy cases seen at the National Reference Center for Sanitary Dermatology Dona LibÃnia. All samples were subjected to DNA extraction followed by amplification by nested PCR of a region of 238 bp which targets a RLEP2 repetitive sequence. The samples were subjected to quantification of 16S rRNA region of the genome of M. leprae by qPCR, which specificity was verified by amplicon melting temperature (Tm = 79.5  C). The method was able to detect amounts over to 20 fg of M. leprae DNA, equivalent to four bacilli units. Nasal secretion assay was able to confirm the diagnosis in 89.7% of the multibacillary cases (MB) and 73.3% of the paucibacillary cases (PB). In addition, MB patient biopsies sensitivity was 100%. The number of bacilli detected in nasal secretion samples from leprosy patients ranged from 1.39 x 10 to 8.02 x 105 bacilli, while detection in MB patient biopsy ranged from 1,87 x 103 to 1,50 x 106. The real-time PCR is more sensitive than conventional PCR and can be used as a complementary tool for the clinical and histopathological diagnosis of leprosy.

Os herpesvírus bovinos tipo 1 (BoHV-1) e tipo 5 (BoHV-5) e o vírus da diarreia viral bovina (BVDV), são causas importantes de prejuízos sanitários e econômicos na bovinocultura. Estes agentes têm sido eventualmente detectados em búfalos (Bubalus bubalis) embora o papel desses vírus na biologia dessa espécie seja ainda desconhecido. A produção de búfalos vem se desenvolvendo expressivamente no Brasil, onde esses animais são mantidos frequentemente próximos ou associados à criação de bovinos. Como parte inicial de um projeto mais amplo sobre infecções por BoHV-1, BoHV-5, BuHV-1 e BVDV em búfalos, o presente estudo objetivou analisar a presença de anticorpos neutralizantes e genomas virais em amostras de soros bubalinos coletados em rebanhos do Estado do Rio Grande do Sul. Foram examinados 339 soros de animais maiores de 12 meses, de ambos os sexos, de diferentes raças e tipo de exploração. Os animais não eram vacinados para quaisquer vírus de interesse na pesquisa e se apresentavam sem alterações clinicamente aparentes na ocasião da coleta. Para a detecção de anticorpos neutralizantes, as amostras de soro foram examinadas através do teste de soroneutralização (SN) frente a diferentes amostras de herpesvírus: BoHV-1.1 (amostra LA) , BoHV-5b (amostra A663) e BuHV-1 (amostra b6). Cento e oito destas amostras foram examinadas adicionalmente frente ao BoHV-1.1 (amostra EVI123/98) e BoHV-1.2a (amostra SV265/96), para permitir uma avaliação da sensibilidade da SN utilizando como vírus de desafio todas as variantes disponíveis de BoHV-1 e BoHV-5. A sensibilidade da SN foi menor quando considerados os resultados obtidos com cada um dos vírus separadamente. Quando os resultados positivos frente a amostra LA, SV265/96 e A663 foram somados, a prova se apresentou mais sensível. Apesar disso, a SN não permitiu a identificação dos vírus que efetivamente haviam infectado os animais, devido ao elevado grau de reatividade cruzada. Para a verificação da circulação de pestivírus, foram realizados testes de SN utilizando a amostra “Oregon C24V” de BVDV-1 como vírus de desafio. Das 176 amostras analisadas, 19 (10,8 %) apresentaram anticorpos neutralizantes reagentes com o vírus utilizado. Paralelamente, para a detecção de genomas de pestivírus, foi desenvolvida uma reação em cadeia da polimerase quantitativa (qPCR) capaz de detectar os três tipos conhecidos de BVDV (1, 2 e 3) e otimizada para o exame das amostras de soros bubalinos. Cinco amostras (2,8 %) continham genomas virais, sugerindo a ocorrência de prováveis infecções persistentes nos animais, semelhante ao que ocorre em bovinos. Os resultados obtidos evidenciam a circulação de herpesvírus e pestivírus nas populações bubalinas analisadas. Mais estudos são necessários para definir quais os agentes efetivamente causadores de infecções nesta espécie animal. / Bovine herpesvirus type 1 (BoHV-1), 5 (BoHV-5) and bovine viral diarrhoea virus (BVDV) are major causes of sanitary and economic losses in cattle. These agents have been eventually detected in water buffaloes (Bubalus bubalis), although their role in host species biology is unknown. Buffalo production has had significant increase in Brazil, where these animals are often kept close to or in association with cattle. As part of a wider project on infections with BoHV-1, BoHV-5, BuHV-1 and BVDV in buffaloes, the present study aimed to examine the occurrence of neutralizing antibodies in buffaloes serum samples collected in farms from Rio Grande do Sul State. Three hundred and thirty nine serum samples were collected from animals with ages above 12 months, of both genders, of different races and under different management practices. The animals were not vaccinated for any virus studied in the present study and were all clinically healthy at the time of sample collection. For neutralizing antibodies detection, serum samples were tested in serum neutralization (SN) tests against different herpesviruses: BoHV-1.1 (strain LA), BoHV-5b (strain A663) e BuHV-1 (strain b6). One hundred and eight of theses samples were additionally examined with BoHV-1.1 (strain EVI123/98) and BoHV-1.2 (strain SV265/96), to compare SN sensitivity using all available variants of BoHV-1 and BoHV-5 as challenge viruses. The SN sensitivity was lower when results obtained with each virus were considered separately. Sensitivity was maximum when the positive results against strain LA, SV265/96 and A663 were added. Despite this, the SN did not allow the identification of the virus which had actually infected animals, in view of the high degree of antibody cross-reactivity. In order to assess the circulation of pestiviruses, SN tests were carried out using the sample "Oregon C24V" of BVDV-1 as challenge virus. Out of the 176 analyzed samples, 19 (10.8 %) presented neutralizing antibodies to pestivirus. At the same time, for the detection of genomes of pestiviruses, was developed a quantitative polymerase chain reaction (qPCR) able to detect the three known types of BVDV (1, 2 and 3) and optimized for examining bubaline sera. Five samples (2.8 %) contained viral genomes, suggesting the occurrence of persistent infections in animals, analogous to what occurs in cattle. The results obtained confirm circulation of herpesvírus and pestiviruses in examined buffalo populations. Further studies are needed to define which of these viruses effectively infect this animal species.

Made available in DSpace on 2014-08-20T13:32:46Z (GMT). No. of bitstreams: 1 dissertacao_railson_schreinert_dos_santos.pdf: 1331296 bytes, checksum: 2121f264ff3310f6f460595517aafdab (MD5) Previous issue date: 2012-07-27 / Rice (Oryza sativa L.) is one of the most important cereals in the world being cultivated in lowland ecosystems, which have a common characteristic: lack of drainage conditions. The lack of oxygen, due to submergence of seedlings, and iron toxicity are two of the major abiotic stresses which rice plants are subjected. Recently it was realized the importance of some ethylene responsive transcription factors (ERFs) in plant response to different stresses, especially to oxygen depletion. Considering it, two studies were made in order to evaluate the expression of ERFs under different stresses. In a first study seven genes were selected, based on previous results obtained from Genevestigator analysis, and had their transcriptional expression evaluated trough quantitative PCR (qPCR) in rice seedlings under oxygen depletion and iron overload. In the second study thirteen ERFs, which had no information available in the literature, also had their transcriptional levels evaluated in seedlings under conditions of hypoxia and anoxia through qPCR in a cultivar susceptible to flooding (Bonança) and a tolerant one (Nipponbare). As general conclusions it was once more emphasized the importance of ERF genes on plant response to environmental stresses. The specific function of each of these genes is yet to be studied. In silico analysis suggests the methylation of promoters as a possible way to regulate them. More studies about the function of these ERFs and about the role of methylation in plant stress response should be done. / O arroz (Oryza sativa L.) é um dos cereais mais importantes no mundo, sendo muito cultivado em ecossistemas de várzea, os quais apresentam uma característica comum: condições de deficiência de drenagem. A falta de oxigênio, devido à submergência das plântulas, e a toxidez por excesso de ferro são dois dos maiores estresses abióticos dentre os quais as plantas de arroz são submetidas. Recentemente percebeu-se a importância de alguns fatores de transcrição responsivos ao etileno (ERFs) na resposta vegetal à diferentes estresses, em especial à deficiência de oxigênio. Considerando-se o exposto efetuaram-se basicamente dois estudos visando avaliar a expressão de ERFs em diferentes estresses. Em um primeiro estudo sete genes foram selecionados a partir de resultados anteriores obtidos de análise no Genevestigator e estes tiveram sua expressão transcricional avaliada por PCR quantitativa (qPCR) em plântulas de arroz sob deficiência de oxigênio e excesso de ferro. No segundo estudo treze ERFs, os quais não apresentavam informações disponíveis na literatura, também tiveram seus níveis de expressão transcricional avaliados em plântulas sob condições de estresse por hipoxia e anoxia, também através de qPCR em uma cv. sensível à inundação (Bonança) e uma tolerante (Nipponbare). Como conclusões gerais se ressalta, novamente, a importância dos ERFs na resposta vegetal frente a estresses ambientais. A função específica de cada um destes genes ainda necessita ser estudada. Uma análise in silico nos promotores destes genes indica a metilação como possível forma de regulação transcricional. Mais estudos sobre a função destes ERFs e sobre o papel da metilação na resposta de arroz a estresses deverão ser feitas.

Submitted by Taiwany Oliveira (taiwanyrodriguess@gmail.com) on 2018-10-22T14:48:18Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Approved for entry into archive by Marcos Roberto Gomes (mrobertosg@gmail.com) on 2018-10-22T15:27:12Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-10-23T14:07:04Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) / Made available in DSpace on 2018-10-23T14:07:04Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) TESE DE DOUTORADO LIGIA FERNANDES ABDALLA.pdf: 4835970 bytes, checksum: 41de8abe90eea5b202c2800675761f12 (MD5) Previous issue date: 2018-09-26 / Zika virus (ZIKV) is an emergent arbovirus of the family Flaviviridae and the genus Flavivirus, which until 2007 was restricted to some cases of mild disease in Africa and Asia. In Brazil, it`s suspected that the entry of the virus occurred during the 2013 Confederations Cup and in the first half of 2015, there were already confirmed cases in states in all regions of the country. The Brazilian epidemic revealed that the usually mild and self-limiting infection could be related to neurological disorders. The objectives of this study were to clarify numerous questions regarding ZIKV infection, aiming to contribute to a better understanding of the mechanisms involved in the immunopathogenesis and course of the disease. As a result, the first report of atrial fibrillation in patients with Zika was described and could be considered an atypical manifestation during the virus infection; elevation of proinflammatory cytokines during ZIKV infection was observed; CXCL10 chemokine was identified as a potential biomarker for infection; saliva was characterized as the fluid of choice for the detection of Zika virus in the acute phase of the disease and a logistic regression model for the classification of cases of zika in relation to dengue was set up based on the clinical evaluation. / O Zika virus (ZIKV) é um arbovírus emergente da família Flaviviridae e do gênero Flavivirus, que até 2007 estava restrito a alguns casos de doença leve na África e na Ásia. No Brasil, suspeita-se que a entrada do vírus tenha se dado durante a Copa das Confederações de 2013 e no primeiro semestre de 2015, já havia casos confirmados em estados de todas as regiões do país. A epidemia brasileira revelou que a infecção geralmente leve e autolimitada poderia estar relacionada à distúrbios neurológicos. Os objetivos deste trabalho era esclarecer inúmeros questionamentos existentes em relação à infecção pelo ZIKV, visando contribuir com uma melhor compreensão dos mecanismos envolvidos na imunopatogênese e curso da doença. Como resultados, descreveu-se o primeiro relato de fibrilação atrial em pacientes com Zika, podendo ser considerado uma manifestação atípica durante a infecção pelo vírus; observou-se elevação de citocinas pró-inflamatórias durante a infecção ZIKV; identificou-se a quimiocina CXCL10 como um biomarcador potencial de infecção; caracterizou-se a saliva como fluído de escolha para o detecção do vírus Zika na fase aguda da doença e, montouse um modelo de regressão logística para a classificação de casos de zika em relação à dengue, com base na avaliação clínica.

L’éthyl tert-butyl éther (ETBE) et le méthyl tert-butyl éther (MTBE) sont des éthers carburants utilisés comme additifs dans les essences sans plomb. Du fait de leur utilisation massive, de nombreux cas de pollutions d’aquifères ont été répertoriés, en particulier pour le MTBE, et ces composés représentent donc un risque sanitaire potentiel. Des travaux récents ont permis de mettre en évidence différents micro-organismes capables de dégrader ces composés malgré leur faible biodégradabilité dans l'environnement. Néanmoins, une meilleure compréhension de l'écologie et de la régulation de ces capacités de dégradation permettrait une meilleure gestion de la bioremédiation de sites contaminés par l'ETBE ou le MTBE.L’objectif de la thèse, réalisée dans le cadre d'un projet ANR Blanc (MiOxyFun), est de mieux comprendre l'écologie des communautés microbiennes impliquées dans la dégradation de ces éthers et leur relation avec la régulation ainsi qu'avec les cinétiques de dégradation de ces composés par des membres spécifiques de ces communautés. Ainsi, à partir de différents échantillons environnementaux venant de sites pollués par l'ETBE ou le MTBE, des enrichissements ont pu être réalisés en laboratoire afin d'étudier leurs microflores. Ces enrichissements ont été étudiés notamment pour leurs cinétiques de dégradation, la composition de leurs communautés bactériennes, et pour l'isolement de souches bactériennes directement impliquées dans la dégradation de ces composés. L'étude des cinétiques de dégradation de l'ETBE ou du MTBE par différents enrichissements obtenus sur ETBE (cinq) et sur MTBE (six) a permis de montrer des profils de dégradation très différents. La dégradation était généralement lente et s'accompagnait d'un faible rendement en biomasse avec parfois accumulation transitoire de tert-butanol (TBA). Les capacités de dégradation d'autres composés des essences (BTEXs et n-alcanes) étaient aussi différentes d'un enrichissement à l'autre, le benzène, entre autres, étant dégradé par 10/11 enrichissements. Des techniques d'empreinte moléculaire (RISA, DGGE) ont permis de constater que les communautés bactériennes présentes dans les cinq enrichissements sur ETBE étaient différentes de celles sur les enrichissements sur MTBE. Les enrichissements sur ETBE ont fait spécifiquement l'objet d'une étude par analyse de banques de clones réalisées à partir des gènes codant l'ARNr 16S de ces enrichissements. Cette étude a montré la prédominance des Proteobacteria dans trois enrichissements, la prédominance des Acidobacteria dans un autre ainsi qu'une composition plus héterogène dans le cinquième. De plus, des Actinobacteria ont été détectées dans les 5 enrichissements.En parallèle, plusieurs souches possédant des capacités de dégradation ont été isolées des enrichissements: Rhodococcus sp. IFP 2040, IFP 2041, IFP 2042, IFP 2043 (dégradant l'ETBE jusqu'au TBA), une Betaproteobacteria IFP 2047 (dégradant l'ETBE), Bradyrhizobium sp. IFP 2049 (dégradant le TBA), Pseudonocardia sp. IFP 2050 (dégradant l'ETBE et le MTBE), Pseudoxanthomonas sp. IFP 2051 et une Proteobacteria IFP 2052 (dégradant le MTBE). Une étude par qPCR sur les gènes codant l'ARNr 16S a montré la prédominance de certaines souches isolées dans les enrichissements ETBE. Enfin, plusieurs gènes connus comme étant impliqués dans la dégradation des éthers carburants ont pu être mis en évidence dans les enrichissements et dans certaines des souches isolées. / ETBE and MTBE are fuel oxygenates added to unleaded gasoline to improve combustion. Due to their extensive use, numerous aquifers have been contaminated, particularly by MTBE. The use of ETBE and MTBE is considered to represent an environmental risk. Recent research has uncovered a range of microorganisms capable of degrading these compounds, even though their environmental half-lives are long. Improved understanding of the ecology and regulation of this degradative ability could improve the management of the ETBE and MTBE contaminated site remediation. The aim of this work, taking place in the framework of the ANR project MiOxyFun was to investigate the ecology of ETBE- and MTBE-degrading microbial communities and their relationship to the regulation and kinetics of ETBE- and MTBE-degradation by specific members of these communities. Several ETBE- and MTBE-degrading microbial communities were enriched in the laboratory from environmental samples from contaminated sites throughout the world. These enrichments were examined for their degradation kinetics, microbial community structure, and used to isolate specific community members actively degrading ETBE and/or MTBE. The ETBE or MTBE biodegradation kinetics of the five ETBE- and six MTBE- degrading enrichments demonstrated a diversity of biodegradation rates. Overall, biodegradation was generally slow and associated to a low biomass yield. Tert-butanol (TBA) was transiently produced in several cases. Biodegradation of other gasoline compounds (BTEXs and n-alkanes) was tested and varied among the enrichments studied. Benzene, however, was degraded in 10 out of the 11 enrichments. DNA fingerprinting techniques (RISA, DGGE) showed that the microflora present in the five ETBE enrichments were different from those of the MTBE enrichments. The ETBE enrichments were studied further by sequencing the 16S rRNA genes extracted, amplified and cloned from these enrichments. Proteobacteria dominated three of the ETBE enrichments, Acidobacteria in another one, and a more heterogeneous composition was found in the fifth ETBE enrichment. Actinobacteria were detected in all five enrichments. Several strains with ETBE or MTBE degradation capacities were isolated: Rhodococcus sp. IFP 2040, IFP 2041, IFP 2042, IFP 2043 (degrading ETBE to TBA),a Betaproteobacteria IFP 2047 (degrading ETBE), Bradyrhizobium sp. IFP 2047 (degrading TBA), Pseudonocardia sp. IFP 2050 (degrading ETBE and MTBE), Pseudoxanthomonas sp. IFP 2051 and a Proteobacteria IFP 2052 (degrading MTBE). Quantification of the 16S rRNA gene confirmed the relatively high number of these isolates in some of the ETBE enrichments. Several genes involved in ETBE and/or MTBE biodegradation were detected in some of the enrichments and in some of the isolated strains.

Interactions among microbial organisms often cannot be observed directly, but they can be inferred genetically using new molecular techniques. The analysis of secondary metabolite gene expression produced by co-cultured marine microbial species allows us to see how these organisms interact with one another when kept in the same environment. Co-cultures of three different strains of marine bacteria, P. aeruginosa PAO1, Roseobacter denitrificans OCH114, and Salinispora arenicola CNS-205 were grown in a laboratory setting, and using the Real-Time qPCR method gene expression levels of two different secondary metabolite producing genes from each organism was accessed across three time points. P. aeruginosa PAO1’s secondary metabolite genes RdhA and PhzH stayed repressed through all co-cultures and time points in this study, and Roseobacter denitrificans OCH-114’s secondary metabolite genes metallo-beta-lactamase and DMSP lyase were up-regulated after the 30 minute time point in the P. aeruginosa-R. denitrificans co-culture and at the 0 minute time point in the R. denitrificans-S. arenicola co-culture.

A variety of secondary metabolites with promising antimicrobial and anti-tumor properties have been identified in marine organisms. Sponges, in particular, have been the source of several of these, including discodermolide from Discodermia dissoluta. While metagenomic studies have been undertaken to identify genes involved in discodermolide production, presently, a transcriptomic approach has not been taken to characterize the metagenome of D. dissoluta. Samples of D. dissoluta were collected from a site in the Bahamas and screened for secondary metabolite production. Some specimens of D. dissoluta were positive for discodermolide while others were not. In order to determine which genes are differentially expressed between the two specimens, suppression subtractive hybridization (SSH) was performed utilizing a chemistry negative and chemistry positive morphotype as the “driver” and “tester” populations respectively. Here we demonstrate the efficacy of SSH through the identification of transcripts related to symbiosis and secondary metabolite production by metatranscriptomic and bioinformatics analyses of the resulting subtracted library as well as a 16S rRNA library. Additionally, we have confirmed differential gene expression of selected sequences utilizing quantitative polymerase chain reaction (qPCR) with SYBR Green chemistry to screen and characterize genes, some of which appear to be related to novel metabolism and unknown functions related to symbiosis within the complex sponge-microbial community.

Antimicrobial engineered nanomaterials (ENM) are increasingly incorporated into products despite limited understanding of the interactions between ENMs and bacteria that lead to toxic impacts. The hazard posed by increasing environmental release of antimicrobial ENMs is also poorly characterized. The overall objective of this thesis is to inform questions about the types of interactions that lead to an ENM inducing bacterial toxicity. Many antimicrobial ENMs are soluble, and the ion plays an important role in their toxicity. Some believe that, beyond release of ions, ENM toxicity is expected to derive from a nanoparticle (NP)-specific effect. This research compares bacterial responses to ENMs, their metal salts, and/or their transformed species within different experimental settings to improve our understanding of the interactions that enable ENM bacterial toxicity. The first objective is to characterize the potential hazard posed by pristine and transformed antimicrobial ENMs on microbial communities within a complex environmental system. One pair of ENMs (Ag0 and Ag2S) led to differential short-term impacts on surficial sediment microbial communities, while the other did not (CuO and CuS), showing that ENM transformation does not universally lead to distinct impacts. The metal ion (Cu2+) had a more profound microbial community impact than did any of the four ENMs. By 300 days the microbial community structure and composition re-converged, suggesting minimal long-term impacts of high pulse inputs of antimicrobial ENMs on microbial communities within complex environments. The second objective is to identify NP-specific effects of a common antimicrobial ENM on a model bacterium. Analysis of transcriptional responses identified NP-specific induction of a membrane stress responsive gene, providing evidence of a NP-specific effect. Otherwise, our results suggest that CuO NP toxicity triggers the same stress responses as does Cu2+, but at more moderate levels. Two ion treatments with the same total Cu input – one with pulse addition and one with gradual addition that was meant to better represent the slow dissolution of the CuO NP – led to temporally distinct responses. This calls for the use of more representative ion controls for comparison against soluble NP impacts in future nanotoxicity studies. The third objective is to investigate the potential use of CuO ENMs to reduce virulence and growth of an emerging bacterial pathogen. CuO NP exposure led to reduction in relative expression of three Staphylococcus aureus virulence factor genes, especially in methicillinresistant S. aureus (MRSA) clinical isolates. Growth was inhibited at high CuO NP concentrations for all four isolates, too. Comparison across all genes assayed showed isolatespecific transcriptional responses, but with NP- and ion-induced responses showing clear differences for each isolate, too. Altogether, this research contributes novel knowledge that will guide efforts to characterize potential hazard from release of ENMs into the environment and to apply ENMs for effective antibacterial treatment.

CADASIL is a neurodegenerative autosomal dominant hereditary disease with late onset. Main symptoms are migraines with aura, cerebral ischemic events, cognitive impairment and dementia. The disease is caused by a mutation in the NOTCH3 gene. The major mutation type changes the number of cysteine residues in the EGF-like repeats of the Notch3 protein. In Czech Republic, currently used methods for molecular genetic analysis of the CADASIL disease are Sanger sequencing and MLPA. But there are patients with CADASIL-like symptoms who were not confirmed by these methods. Therefore, the aim of this thesis was to implement transcript analysis by Sanger sequencing of cDNA PCR products and quantitative real-time PCR (qPCR) to analyze gross deletions and duplications to clarify the molecular genetic basis of the disease. By transcript analysis, the existence of the transcript variant X1 was experimentally confirmed in control samples. Moreover, the results from transcript analysis showed that non-typical missense mutation c.1725G>A (p.T575=) which does not directly change the number of cysteine residues, can cause the CADASIL disease via missplicing and subsequent causing deletion including cysteine residues. The other tested variants did not show any changes in the transcript level. The qPCR method did not...