Heat pasteurization is an effective water treatment to address the emerging plant pathogen issue associated with increased water recycling practices in the ornamental horticulture industry. The current protocol that recommends treating water at 95"C for 30 s, however, faces two major challenges: its energy cost and environmental footprint. We hypothesized that temperature required to inactivate major pathogens in re-circulated water may be substantially lowered from 95"C with extended exposure time. The goal of this study was to test this hypothesis and make this water decontamination technology economically more attractive while reducing its environmental impact. Specific objectives were to (1) examine the effect of water temperature on the survival of Phytophthora and bacterial species, two major groups of plant pathogens in water recycling systems, and (2) elucidate the underlying biological mechanisms by which plant pathogens are killed at those temperatures. Lab assays were performed to determine the survival of zoospores and chlamydospores of P. nicotianae, and oospores of P. pini as well as seven bacterial species after heat treatments at given periods of time. Greenhouse experiments were conducted to determine the applicability of the lab assay data to the real world using annual vinca (Catharanthus roseus) and P. nicotianae as a model system. The results of these studies indicated that the water temperature required to eliminate Phytophthora and bacterial species can be lowered to 48"C from 95"C if treatment time extends to 24 h. Two major steps were taken to elucidate the underlying biological mechanisms. Firstly, a scheme based on the DNA fingerprint and sequence analysis was developed for characterizing bacterial species in irrigation water, after comparing two typing strategies, three sample concentration methods, and evaluating conditions in denaturing gradient gel electrophoresis (DGGE) profiling. Bacterial species detected by culture-dependent and -independent strategies were rather different. The greater bacterial diversity was detected when water samples were concentrated by using both methods than centrifugation or filtration alone. As for DGGE profiling, 40 to 60% denaturant concentrations at 70 V for 16 h revealed the highest bacterial diversity. Secondly, water samples were taken from an irrigation reservoir in a local nursery and analyzed for bacterial diversity following heat treatments at 42 and 48"C. After these heat treatments "-proteobacteria, "-proteobacteria, and Firmicutes became dominant which presents a substantial shift of bacterial community structure compared to those in the control water at 25"C. Among the dominant in treated water were Bacillus, Pseudomonas, Paenibacillus, Brevibacillus, and Lysobacter species, which may have potential biocontrol activities against plant pathogens. This study provided the scientific basis for developing a more energy-efficient and environmentally sound heat pasteurization protocol for water decontamination. / Ph. D.

In this project rRNA maturation was investigated with the help of mathematical models of processing pathways from pre-rRNA to mature rRNA species. Previously described models were transferred from Excel to Mathematica. Additionally, two Mathematica based software applications were created, which help to analyse metabolic [3H]-Uracil labelling of pre-rRNA species. The first program, M Fit helps to visualize dependencies in the pre-rRNA processing. The other program S Fit tries to find a best fit of the model response to labelling time course data, hence optimizing parameter values. To validate the model anything that has an influence on the co-transcriptional cleavage is of interest, as these would have distinct effects on the 20S pre-rRNA labelling curve. A list of proteins which might play a role in A2 cleavage of the 35S was compiled and Rat1 was selected as the first candidate to investigate. All prerRNA species except the 35S pre-rRNA consist of two populations. One set created by nascent transcript cleavage (35S gets cleaved during transcription process) and one set created by released transcript cleavage (if a fully transcribed 35S pre-rRNA was released). These two species are not usually distinguishable on gels. However, with the help of the models the two different populations can be differentiated. This allows useful predictions to be made about [3H]-Uracil labelling courses in cases of high or low co-transcriptional cleavage. Experimental data for Rat1 depletion strains indeed showed an inhibition of co-transcriptional cleavage with a curve pattern as predicted by the models. Loss of another ribosome synthesis factor Srp40 was predicted to inhibit cotranscriptional pre-rRNA methylation. Of particular interest here was the effect on the 20S as this species supposed to be mostly methylated co-transcriptionally. Labelling with [3H] methionine the 20S curve for the Srp40 deletion mutant should have an earlier onset as compared to 20S curve from the corresponding wild type strain. A higher tritium response was shown for srp40Δ as compared to wild type; this might proof loss of co-transcriptional methylation.

In fishes, elopomorphs have a leptocephalous stage in the life cycle. Elopomorpha includes tenpounders (Elops), tarpons (Megalops), bonefishes (Albula), spine eels (Notacanthus), apodes and gulper eels. They are highly diversified in morphology and habitat utilization, Their monophyly is based on sharing of this larval stage. However, not all researches on these group accept the idea that this stage is of relationship. If this is true, the concept of elopomorpha must be re-evaluated. In attempt to elucidate their phylogenetic relationships, mtDNA 12S rRNA sequences were analyzed and data suggest that: (1)Elopomorpha is a monophyletic group. In other word, leptochphalous stage is a valid phylogenetic indictor, and it is not the result of convergency to environment; (2)Elops and Megalops share a common ancestor, and is a primitive group for Elopomorpha; (3)Megalops is the primitive lineage of Elopomorpha; (4)Albula and Notacanthus share a common ancestor, and they are the sister group of Anguilliformes; (5)Anguilliformes is a monophyletic group; (6)Muraenidae is a primitive group of Anguilliformes; (7)A high speciation rate might have taken place in Apodes within a short period of time; (8)Synaphobranchidae is a monophyletic group and it is the primitive group of Congroidei. (10) Synaphobranchid species have short interspecific genetic distance among species.

AN ABSTRACT OF THE DISSERTATION OF MANISHA DEOGHARIA, for the Doctor of Philosophy degree in Molecular Biology, Microbiology and Biochemistry presented on May 16, 2018, at Southern Illinois University, Carbondale TITLE: PSEUDOURIDINE MODIFICATIONS IN HUMAN tRNAs AND ARCHAEAL rRNAs MAJOR PROFESSOR: DR. RAMESH GUPTA RNAs undergo several post-transcriptional modifications inside the cell. The most abundant modification found in RNA is pseudouridine. Pseudouridine is present in all major classes of RNA. The classical TΨC sequence of tRNA reflects T (ribothymidine or 5-methyluridine) at position 54 in most Bacteria and Eukarya, and Ψ and C at positions 55 and 56, respectively, in nearly all tRNAs. TrmA and TruB homologs produce T54 and Ψ55, respectively, in Bacteria and Eukarya. However, archaeal tRNAs commonly have Ψ54 (or m1Ψ54) instead of T54, and Pus10 produces both Ψ54 and Ψ55 in these tRNAs. The pus10 gene is present in nearly all Archaea and most eukaryotes, but not in Bacteria and yeast. This coincides with the presence of Ψ54 in archaeal tRNAs and certain tRNAs (for Gln, Trp, Pro Thr, etc.) of animals, and its absence in the tRNAs of Bacteria and yeast. tRNAs for Trp and Pro that function as primers for replication of retroviruses also contain Ψ54. We found that Pus10 is the Ψ54 synthase in eukaryotes. The Ψ54 activity is specific for certain tRNAs, and it requires a conserved Am1AAU sequence at positions 57-60 of the tRNA for its maximum activity. Recombinant Pus10 can also form Ψ54 in select tRNAs and presence of m1A at position 58 is necessary for its maximum activity. Humans have two paralogs of TruB, TruB1, and TruB2 which are predicted to be the Ψ55 synthases for cytoplasmic and mitochondrial tRNAs, respectively. We found that recombinant human Pus10 can also modify Ψ55 of tRNAs in vitro. This Ψ55 activity of human Pus10 is not selective for specific tRNAs. Another pseudouridine synthase, Cbf5, which functions in guide dependent manner, is necessary for Ψ production in 23S rRNA of H. volcanii. Cbf5 is the catalytic component of the box H/ACA ribonucleoprotein complex that brings about these modifications. It consists of a guide RNA and three core proteins Nop10, Gar1, and L7Ae along with Cbf5. We found that Nop10 is necessary for Ψ production in 23S rRNA.

No description available.

A conversão de áreas de florestas da Amazônia em áreas agrícolas e pastoris desregula processos relacionados ao estoque de carbono, sendo considerada depois da queima de combustíveis fósseis a atividade que mais contribui com a emissão de gases do efeito estufa, dentre os quais se encontra o metano. A produção de metano é intermediada pelas arquéias metanogênicas, que atuam na decomposição anaeróbia da matéria orgânica. Portanto, para compreender as alterações do fluxo desse gás no ecossistema amazônico, é necessário que as comunidades microbianas envolvidas nesse processo sejam estudadas. Dessa forma, o presente estudo teve por objetivo monitorar e caracterizar comunidades de arquéias metanogênicas, por análises de enriquecimento destas comunidades, em amostras de solo provenientes de floresta primária, floresta secundária e pastagem da região amazônica. As amostras de solo foram colocadas em meio enriquecido com a adição de acetato, metanol ou H2:CO2, separadamente, para estimular os metabolismos aceticlástico, metilotrófico e hidrogenotrófico. O monitoramento desses cultivos foi realizado por análises de emissão de metano por cromatografia gasosa, quantificação do gene mcrA pela técnica de PCR quantitativo (qPCR) e caracterização da comunidade metanogênica por meio de microscopia e sequenciamento da região V4-V5 do gene 16S rRNA. Analisando a emissão de metano entre os três tipos de fontes de carbono para as três amostras de solo, os enriquecimentos com metanol apresentaram uma produção maior de metano em relação as amostras com o acetato e muito superior aos cultivos com atmosfera de H2:CO2. A maior média de produção de metano ocorreu nos enriquecimentos com metanol, indicando que a via metilotrófica embora considerada alternativa, pode ser importante na produção de metano no solo amazônico. Por meio da técnica de qPCR foi possível quantificar o gene mcrA das amostras de pastagens logo no tempo inicial da incubação, o que não foi possível para as amostras florestais. No tempo final, o número de cópias desse gene foi similar para os três perfis de solo. Foi possível observar pela caracterização fenotípica dos enriquecimentos agregados de células característicos do gênero Methanosarcina, gênero que foi identificado posteriormente pelo sequenciamento do gene 16S rRNA, além de células em formatos de bastonetes e cocos. Os resultados do sequenciamento permitiram identificar 7 grupos distintos de arqueias metanogênicas, afiliados aos filos Euryarchaeota e Bathyarchaeota. Nas amostras iniciais de pastagens foram identificadas sequências que se afiliaram a todos esses grupos, enquanto as amostras florestais apresentaram sequencias afiliadas apenas ao gênero Methanosarcina. A composição final da comunidade das amostras de pastagens foi similar a inicial, porém mais abundante. Os enriquecimentos de amostras de solo de floresta primária e secundária apresentaram uma composição distinta, devido ao enriquecimento de grupos que não foram identificados no início da incubação. Os resultados obtidos mostraram que embora as arqueias metanogênicas estejam em baixa abundância nos solos florestais, podem ser enriquecidos quando submetidos a condições favoráveis, atingindo produção de metano e alcançando composição similar as amostras de pastagens. / Amazonian forest conversion into agricultural and livestock areas disrupts processes related to carbon stock, being considered, after the fossil fuels burning, the activity contributes most to greenhouse gases emission, of which is methane. Methane production is mediated by methanogenic archaea, acting in organic matter anaerobic decomposition. Therefore, to understand the changes in the flow of this gas in the Amazonian ecosystem, it is necessary to study microbial communities involved in this process. This study aims to monitor and characterize methanogenic archaeal communities by population enrichment from soil samples collected in primary, secondary and pasture of the Amazon region. Soil samples were placed into an enriched medium and received separately acetate, methanol, and H2:CO2 to stimulate the three metabolism types: acetoclastic, methylotrophic and hydrogenotrophic. Monitoring was performed by methane emission analysis by gas chromatography, mcrA quantification by the quantitative PCR and the community characterization was performed by microscopy and sequencing of the V4-V5 region of the 16S rRNA gene. Analyzing the methane emission by the three types of carbon sources in the three soil samples, methanol enrichments presented a higher methane yield than the acetate samples and much larger than cultures with H2:CO2. These results indicate that methylotrophic pathway, although considered as an alternative, may be important in methane production in the Amazonian soil. Was possible to quantify the mcrA gene by qPCR from pasture samples at the initial incubation time, which was not possible for forest samples. In incubation final time, copies number of this gene was similar for the three soil profiles. The phenotypic characterization of enrichments revealed aggregated cells, characteristic of the genus Methanosarcina, later identified by 16S rRNA sequencing. The cells in rod-shaped and cocci formats were also observed. Was identify by sequencing 7 different methanogenic archaeas groups affiliated with Euryarchaeota and Bathyarchaeota phylum. In the initial pasture samples, were identified sequences affiliated with all these groups, while forest samples presented sequences affiliated with only a Methanosarcina genus. Pasture samples showed a final community composition similar to initial, however more abundant. Soil samples enrichment from primary and secondary forest presented a distinct composition due to groups enrichment that was not identified at incubation beginning. These results showed that although methanogenic archaeas are in low abundance in forest soils, they can be enriched when submitted to favorable conditions, archive methane production and reaching similar composition pasture samples.

A conversão de áreas de florestas da Amazônia em áreas agrícolas e pastoris desregula processos relacionados ao estoque de carbono, sendo considerada depois da queima de combustíveis fósseis a atividade que mais contribui com a emissão de gases do efeito estufa, dentre os quais se encontra o metano. A produção de metano é intermediada pelas arquéias metanogênicas, que atuam na decomposição anaeróbia da matéria orgânica. Portanto, para compreender as alterações do fluxo desse gás no ecossistema amazônico, é necessário que as comunidades microbianas envolvidas nesse processo sejam estudadas. Dessa forma, o presente estudo teve por objetivo monitorar e caracterizar comunidades de arquéias metanogênicas, por análises de enriquecimento destas comunidades, em amostras de solo provenientes de floresta primária, floresta secundária e pastagem da região amazônica. As amostras de solo foram colocadas em meio enriquecido com a adição de acetato, metanol ou H2:CO2, separadamente, para estimular os metabolismos aceticlástico, metilotrófico e hidrogenotrófico. O monitoramento desses cultivos foi realizado por análises de emissão de metano por cromatografia gasosa, quantificação do gene mcrA pela técnica de PCR quantitativo (qPCR) e caracterização da comunidade metanogênica por meio de microscopia e sequenciamento da região V4-V5 do gene 16S rRNA. Analisando a emissão de metano entre os três tipos de fontes de carbono para as três amostras de solo, os enriquecimentos com metanol apresentaram uma produção maior de metano em relação as amostras com o acetato e muito superior aos cultivos com atmosfera de H2:CO2. A maior média de produção de metano ocorreu nos enriquecimentos com metanol, indicando que a via metilotrófica embora considerada alternativa, pode ser importante na produção de metano no solo amazônico. Por meio da técnica de qPCR foi possível quantificar o gene mcrA das amostras de pastagens logo no tempo inicial da incubação, o que não foi possível para as amostras florestais. No tempo final, o número de cópias desse gene foi similar para os três perfis de solo. Foi possível observar pela caracterização fenotípica dos enriquecimentos agregados de células característicos do gênero Methanosarcina, gênero que foi identificado posteriormente pelo sequenciamento do gene 16S rRNA, além de células em formatos de bastonetes e cocos. Os resultados do sequenciamento permitiram identificar 7 grupos distintos de arqueias metanogênicas, afiliados aos filos Euryarchaeota e Bathyarchaeota. Nas amostras iniciais de pastagens foram identificadas sequências que se afiliaram a todos esses grupos, enquanto as amostras florestais apresentaram sequencias afiliadas apenas ao gênero Methanosarcina. A composição final da comunidade das amostras de pastagens foi similar a inicial, porém mais abundante. Os enriquecimentos de amostras de solo de floresta primária e secundária apresentaram uma composição distinta, devido ao enriquecimento de grupos que não foram identificados no início da incubação. Os resultados obtidos mostraram que embora as arqueias metanogênicas estejam em baixa abundância nos solos florestais, podem ser enriquecidos quando submetidos a condições favoráveis, atingindo produção de metano e alcançando composição similar as amostras de pastagens. / Amazonian forest conversion into agricultural and livestock areas disrupts processes related to carbon stock, being considered, after the fossil fuels burning, the activity contributes most to greenhouse gases emission, of which is methane. Methane production is mediated by methanogenic archaea, acting in organic matter anaerobic decomposition. Therefore, to understand the changes in the flow of this gas in the Amazonian ecosystem, it is necessary to study microbial communities involved in this process. This study aims to monitor and characterize methanogenic archaeal communities by population enrichment from soil samples collected in primary, secondary and pasture of the Amazon region. Soil samples were placed into an enriched medium and received separately acetate, methanol, and H2:CO2 to stimulate the three metabolism types: acetoclastic, methylotrophic and hydrogenotrophic. Monitoring was performed by methane emission analysis by gas chromatography, mcrA quantification by the quantitative PCR and the community characterization was performed by microscopy and sequencing of the V4-V5 region of the 16S rRNA gene. Analyzing the methane emission by the three types of carbon sources in the three soil samples, methanol enrichments presented a higher methane yield than the acetate samples and much larger than cultures with H2:CO2. These results indicate that methylotrophic pathway, although considered as an alternative, may be important in methane production in the Amazonian soil. Was possible to quantify the mcrA gene by qPCR from pasture samples at the initial incubation time, which was not possible for forest samples. In incubation final time, copies number of this gene was similar for the three soil profiles. The phenotypic characterization of enrichments revealed aggregated cells, characteristic of the genus Methanosarcina, later identified by 16S rRNA sequencing. The cells in rod-shaped and cocci formats were also observed. Was identify by sequencing 7 different methanogenic archaeas groups affiliated with Euryarchaeota and Bathyarchaeota phylum. In the initial pasture samples, were identified sequences affiliated with all these groups, while forest samples presented sequences affiliated with only a Methanosarcina genus. Pasture samples showed a final community composition similar to initial, however more abundant. Soil samples enrichment from primary and secondary forest presented a distinct composition due to groups enrichment that was not identified at incubation beginning. These results showed that although methanogenic archaeas are in low abundance in forest soils, they can be enriched when submitted to favorable conditions, archive methane production and reaching similar composition pasture samples.

Thousands of parrots all over the world suffer from illnesses and medical complications that can result from interactions between their immune system and bacteria in their digestive tract. The aim of this master's thesis is to understand the link between symptoms of these medical issues, the composition of blood and gastrointestinal microbiota in parrots. Using the hematological methods, 198 blood samples representing 53 parrot species were analyzed. The composition of microbiome was defined by combination of a molecular approach using bacterial 16S rRNA gene sequencing in 132 fecal samples, 12 intestine samples, 228 cloacal swabs and 236 beak swabs representing in total 61 parrot species and a diagnostic approach by psittacine fecal Gram's stain method. Significant association of hematological parameters with individual, environmental and clinical factors was observed, as well as its considerable interspecific variability. Absolute heterophile and lymphocyte counts have been shown more useful for infectious and autoimmune disease monitoring than H/L ratio. Relative numbers of basophiles were the best indicator for behavioral disorders. In relation to hematological parameters, the effect of the bacterial family Flavobacteriaceae, as part of the oral microbiota, and the bacteria Escherichia or...

Translation initiation factor IF1 is a small, essential and ubiquitous protein factor encoded by a single infA gene in bacteria. Although several important functions have been attributed to IF1, the precise reason for its indispensability is yet to be defined. It is known that IF1 binds to the ribosomal A-site during initiation, where it primarily contacts ribosomal RNA (rRNA) and induces large scale conformational changes in the small ribosomal subunit. To shed more light on the function of IF1 and its interaction with the ribosome, we have employed a genetic approach to elucidate structure-function interactions between IF1 and rRNA. A selection has been used to isolate second site suppressor mutations in rRNA that restore the growth of a cold sensitive mutant IF1 with an arginine to leucine substitution in position 69 (R69L).  This yielded two classes of suppressors – one class that mapped to the processing stem of 23S rRNA – a transient structure important for proper maturation of 23S rRNA; and the other class to the functional sequence of 16S rRNA. Suppressor mutations in the processing stem of 23S rRNA were shown to disrupt efficient processing of 23S rRNA. In addition, we report that at least one of the manifestations of cold sensitivity associated with the mutant IF1 is at the level of ribosomal subunit association. These results led to a model whereby the cold sensitive R69L mutant IF1 results in aberrant ribosomal subunit association properties, while the 23S processing stem mutations indirectly suppress this effect by decreasing the pool of mature 50S subunits available for association.  Spontaneous suppressor mutations in 16S rRNA were diverse in position and phenotypic properties, but all mutations affected ribosomal subunit association, in most cases by directly decreasing the affinity of the 30S for 50S subunits. Site directed mutagenesis of select positions in 16S rRNA yielded additional suppressor mutations that were localized to the mRNA and streptomycin binding sites on the small ribosomal subunit. We suggest that the 16S rRNA suppressors occur in positions that affect the conformational dynamics brought about by IF1. Taken together, this work indicates that the major function of IF1 is the modulation of ribosomal subunit association brought about through conformational changes of the 30S subunit. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Made available in DSpace on 2014-06-12T17:35:11Z (GMT). No. of bitstreams: 2 arquivo4409_1.pdf: 676822 bytes, checksum: 4a1153b3912f6483c79f20c422b118f1 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2002 / Oligonucleotídeos foram construídos com base na sequência primária do gene codificando o rRNA de Plasmodium para amplificar DNA de P. falciparum, P. vivax, P. malariae e P. ovale, de maneira gênero-específica. Três sistemas de PCR foram utilizados: PCR simples, hemi-nested PCR convencional e hemi-nested PCR em um único tubo, desenvolvidos em nosso laboratório. Na PCR simples, composta de 30 ciclos, foram utilizados os oligonucleotídeos GJ1 e HR842 (20 pmol/50&#956;l), já testados por nosso grupo. Na hemi-nested PCR convencional utilizou-se três oligonucleotídeos (GJ1, PGFO3 e HR842), em duas reações sequenciais, sendo o PGFO3 construído durante o desenvolvimento do presente trabalho, visando a detecção do gênero Plasmodium. O par GJ1 e HR842 foram utilizados como oligonucleotídeos externos na primeira reação, e o PGFO3 como interno, ancorado ao HR842 na segunda reação. A hemi-nested PCR em um único tubo consistiu em 60 ciclos (92ºC, 30s; 58ºC, 30s e 72ºC, 45s), e concentrações limitantes de oligonucleotídeos externos (4 pmols/50&#956;l) participavam da PCR sem competição com os oligonucleotídeos internos durante os primeiros 15 ciclos da reação e 40 pmol/50&#956;l de primers internos (imobilizados na face interna da tampa do microtubo) foram introduzidos na PCR no 16º ciclo. As concentrações dos outros componentes da reação foram as mesmas utilizadas nas reações convencionais de PCR. Observou-se que a quantidade mínima de DNA genômico detectada pela PCR simples, hemi-nested PCR e hemi-nested PCR em um único tubo foi de 10 pg; 0,01 pg e 0,1 pg, respectivamente. Apesar da hemi-nested PCR em um único tubo ter sido menos sensível que a heminested PCR convencional, é muito mais simples e conveniente, já que o risco de contaminação cruzada é bem menor. Esses sistemas moleculares de diagnóstico podem ser usados em situações quando se requer uma alta sensibilidade e especificidade, tais como na avaliação da eficiência de quimioterapia, detecção precoce de infecção e prevenção de transmissão a partir de pacientes hipoparasitêmicos