Florações de cianobactérias tóxicas em corpos dágua doce usados como fonte para o consumo humano, recreação e irrigação são freqüentes nos dias de hoje devido à eutrofização destes ambientes. O monitoramento de linhagens tóxicas é importante para a prevenção dos efeitos adversos causados por suas toxinas na saúde de humanos e animais. Métodos rápidos e sensíveis para a detecção precoce das cianobactérias tóxicas em estações de tratamento de água e em programas de monitoramento de mananciais são de fundamental interesse para a prevenção desses efeitos. Atualmente, contagem de células, identificação de cianobactérias por microscopia óptica e análises químicas ou imunológicas das toxinas são usadas nos monitoramentos. Em anos recentes, métodos moleculares estão sendo desenvolvidos e propostos para o diagnóstico rápido e sensível da presença de cianobactérias tóxicas em diversos ambientes. Este estudo teve por objetivo implementar uma metodologia capaz de acessar e quantificar as cianobactérias do gênero Microcystis na Praia dos Namorados, no reservatório de Salto Grande, Americana, SP, local cujo uso recreacional é bastante intenso e florações de cianobactérias são freqüentemente observadas. Simultaneamente, buscou-se detectar o potencial de produção de microcistinas desses organismos. A técnica de PCR Quantitativa em Tempo Real (qPCR) foi empregada com essa finalidade e dois conjuntos de oligonucleotídeos LUX foram desenvolvidos tendo como alvo dois genes distintos. Seqüências de cpcBA-IGS do operon da ficocianina (PC) foram obtidas para sete linhagens de cianobactérias brasileiras, as quais foram alinhadas com outras seqüências existentes em banco de dados público, e permitiram o desenho dos iniciadores de qPCR QPCF/QPCR, capazes de amplificar fragmentos de 144 pb. Da mesma forma, outras 11 sequências inéditas foram obtidas para uma região do domínio da N-methyltransferase do gene da sintetase de microcistinas (mcyA) e permitiram o desenvolvimento dos iniciadores QmcyANMTF/ QmcyA-NMTR, capazes de amplificar fragmentos de 154 pb. Ambos os conjuntos foram marcados uma única vez com os fluoróforos FAM (QPCF/QPCR) ou JOE (QmcyA-NMTF/QmcyA-NMTR). Curvas de calibração para ambos os genes foram estabelecidas com regressão linear simples usando os valores de Ct (número de ciclos da qPCR em que a fluorescência atinge um limiar fixo pré-determinado) e concentrações conhecidas de DNA gênomico (em número de células equivalente) da Microcystis sp. NPLJ-4 (produtora de microcistina). As diluições utilizadas para o estabelecimento dessas curvas variaram de 1:10 a 1:10-5 e as concentrações de DNA foram correlacionadas com o respectivo número de células na amostra, obtido pela técnica de Utermöhl em laboratório certificado, como metodologia independente. Para PC e mcyA, as equações de regressão foram y = 43,977 - 1,8097Ln(x) (R2 = 0,99, p<0,05) e y = 42,932 - 1,8449Ln(x) (R2 = 0,99, p<0,05), respectivamente, sendo y = Ct com limiar de fluorescência fixo avaliado em 0,03 e x = quantidade de DNA na amostra em concentrações conhecidas (dada como log do número de células equivalente). Para ambos os genes analisados, o limite de detecção foi de 100 células por reação. Eficiências de 79 e 76% foram alcançadas nas amplificações para PC e mcyA, respectivamente. Posteriormente, essa metodologia foi aplicada a duas outras linhagens isoladas de Microcystis e em amostras ambientais de água, que foram coletadas sempre no mesmo ponto (Praia dos Namorados), em quatro períodos distintos (dez 06, abr 07, set 07 e nov 07). Genótipos PC (em número de células mL-1) foram quantificados pela técnica de qPCR em todas as amostras analisadas. Porém, genótipos mcy puderam ser determinados apenas em M. aeruginosa NPJB1 (0,5%) e na amostra referente à primeira coleta (2,7%). Os resultados de número de células em todas as análises feitas usando a técnica de Utermöhl foram superiores aos obtidos pela qPCR. A comparação entre as médias dos valores de quantificações gerados por Utermöhl e qPCR (PC) mostrou diferença significativa (Teste t de Student, p <0,05). Nas amostras ambientais, com exceção da primeira coleta, a presença de outros gêneros de cianobactérias cocóides, como Radiocystis e Sphaerocavum, foi observada, e suas distinções não foram possíveis nas observações microscópicas após a disrupção das colônias. Estudos adicionais realizados com a região cpcBA-IGS desses outros gêneros mostraram 96% de identidade com Microcystis, e seqüências IGS bastante similares. Assim, existe a possibilidade de que os iniciadores desenhados neste estudo estejam também amplificando esses dois gêneros de cianobactérias. Em adição, a contagem em microscópio pode ter superestimado os resultados, enquanto que a técnica de qPCR mostrou baixa eficiência de amplificação. O ensaio imunológico ELISA (Enzyme-Linked Immunosorbent Assay) identificou a produção de microcistinas dos tipos LR, RR ou YR em todas as amostras com concentrações maiores que 0,1 g L-1, com exceção da M. aeruginosa NPCD1 (não produtora de microcistinas). Os resultados obtidos neste estudo indicam que o número de células de Microcystis, estimado pela técnica de qPCR pode ser usado para o monitoramento ambiental na Praia dos Namorados, em atendimento à Portaria MS 518. Além disso, demonstram que é possível inferir a proporção de genótipos mcyA a partir da determinação de genótipos PC. Contudo, a validação dessa técnica requer maiores estudos, incluindo a utilização de mais gêneros de cianobactérias e análises de outros reservatórios brasileiros, para melhor avaliar sua confiabilidade para uso no monitoramento ambiental. Para fins de monitoramento em larga escala, a técnica de qPCR mostrou-se mais viável economicamente em relação à contagem de células por microscopia, devido a sua maior capacidade de processamento (18 amostras dia-1) / Toxic cyanobacterial blooms in freshwater bodies used as a source for human consumption, recreation and irrigation are frequent nowadays due to the eutrofication of these environments. The monitoring of toxic strains is important for the prevention of the side effects caused by their toxins in human and animal health. Rapid and sensitive methods for early detection of toxic cyanobacteria in water treatment stations and aquatic monitoring programs are essential for the prevention of these effects. Currently, cells counting, cyanobacterial identification using optical microscopy and chemical or immunological analyses of the toxins are applied for monitoring purposes. In recent years, molecular approaches are being developed and proposed for rapid and sensitive diagnosis of the presence of toxic cyanobacteria in several environments.This study aimed to implement a methodology capable of access and quantify Microcystis cyanobacterial genus at the Praia dos Namorados, in the Salto Grande reservoir, Americana, SP, where recreation is very intense and cyanobacterial blooms are frequently observed. Simultaneously, it was searched to detect the potential for microcystins production by these organisms. The Real Time Quantitative PCR (qPCR) technique was employed for these purpose and two sets of LUX primers were developed to target two distinct genes. Sequences of cpcBA-IGS of the phycocyanin operon (PC) were obtained for seven Brazilian cyanobacterial strains, which were aligned with other existing public database sequences, and allowed to design the qPCR QPCF/QPCR primers, able to amplify fragments of 144 bp. In the same way, 11 novel sequences were obtained from a region of the N-methyltransferase domain of the microcystin sinthetase gene (mcyA) and allowed the development of QmcyANMTF/ QmcyA-NMTR primers, able to amplify fragments of 154 bp. Both primer sets were labeled only once with FAM (QPCF/QPCR) or JOE (QmcyA-NMTF/QmcyA-NMTR) fluorophors. Standard curves for both genes were established with simple linear regression using the Ct values (the PCR cycle numbers at which the fluorescence reaches a predetermined threshold level) and known Microcystis sp. NPLJ-4 (microcystin producer) genomic DNA concentrations (in cell number equivalents). The dilutions used for the establishment of these curves ranged from 1:10 to 1:10-5 and the DNA concentrations were correlated with the respective cell numbers of the sample, obtained by Utermöhl technique in a certified laboratory, as an independent methodology. For PC and mcyA, the regression equations were y = 43.977 - 1.8097Ln(x) (R2 = 0.99, p<0,05) and y= 42.932 - 1.8449Ln(x) (R2 = 0.99, p<0,05), respectively, where y is the Ct at the set fluorescence threshold level (0.03) and x is the amount of known DNA concentrations in the sample (given as log cell number equivalents). For both analyzed genes, the detection limit was 100 cells per reaction. Efficiencies of 79 and 76% were achieved for PC and mcyA amplifications, respectively. Subsequently, this methodology was applied to two other isolated Microcystis strains and to environmental water samples, which were collected always in the same location (Praia dos Namorados), in four different periods (Dez 06, Apr 07, Set 07 and Nov 07). PC genotypes (in cell numbers mL-1) were quantified by the qPCR technique in all analyzed samples. However, mcy genotypes could be determined only in M. aeruginosa NPJB1 (0.5%) and in the first collected sample (2.7%). The results of cell numbers in all the analyses performed using Utermöhl technique were superior then those obtained by qPCR. The comparison between the average quantification values generated by Utermöhl and qPCR (PC) showed significant difference (Test t of Student, p<0,05). In the environmental samples, except for the first one collected, the presence of other cocoid cyanobacterial genera, such as Radiocystis and Sphaerocavum, was observed, and their distinctions were not possible by microscope observation after colonies disruption. Further studies carried out with the cpcBA-IGS region of these other genera showed 96% of identity with Microcystis, and very similar IGS sequences. Then, there is a possibility that the primers designed in this study are also amplifying these two cyanobacterial genera. In addition, microscopic cells counting may have overestimated the results, whereas qPCR technique showed low efficiency of amplification. The ELISA immunological assay (\"Enzyme-Linked Immunosorbent Assay\") identified the LR, RR or YR microcystins types in all samples analysed with concentrations higher than 0.1 mg L-1, with exception of M. aeruginosa NPCD1 (no microcystin producer). The results obtained in this study suggest that Microcystis cell numbers estimated by qPCR technique can be used for the environmental monitoring of the Praia dos Namorados, in attendance to the MS 518 regulation. Furthermore, they demonstrate that it is possible to infer the mcyA genotypes proportion from the PC genotypes determination. However, further studies are required for the validation of this technique, including the use of more cyanobacterial genera and other Brazilian reservoirs analyses, to better evaluate the reliability for its use in the environmental monitoring. For large scale monitoring, the qPCR technique is more economically viable when compared to the microscopic cells counting, due to its large processing capacity (18 samples day-1)

A síntese de selenocisteína e sua incorporação co-traducional em selenoproteínas como resposta a um códon UGA em fase requerem uma complexa maquinaria molecular. Em eucariotos, foram identificados componentes que participam da reação de formação de selenocisteína: Seril-tRNA sintetase (SerRS), O-fosfoseril-tRNA quinase (PSTK), SECIS Binding Protein 2 SBP2, um fator de elongação específico para Sec (EFSec), selenofosfato sintetase 1 (SPS1) e selenofosfato sintetase 2 (SPS2), SEPSECS, proteína ligante de RNA SECp43, proteína ribossomal L30, um tRNA de inserção de selenocisteína (tRNASec, SELC) e uma sequência específica no RNA mensageiro (elemento SECIS). O primeiro passo da incorporação de selenocisteína em proteínas é realizado pela SerRS, que aminoacila o tRNA com serina através da ativação da serina por Mg+2 e ATP, levando a formação de um intermediário ligado a enzima (Ser-AMP). Posteriormente, ocorre a mudança do radical Ser do intermediário Ser-AMP para o tRNASec, e subsequentemente, a conversão enzimática de Ser-tRNASec para Sec-tRNASec. Através de análises in sílico nosso grupo identificou componentes da maquinaria de inserção de selenocisteína em espécies de Kinetoplastida. Foram identificados homólogos de tRNASec e as enzimas TbSerRS, TbSPS2, TbPSTK, TbSepSecS e TbEFSec. Nosso principal alvo é o estudo estrutural da SerRS de Trypanosoma brucei nativa e em complexo com o tRNASec e com as isoformas do tRNASer. Uma nova metodologia no processo de purificação desta enzima foi desenvolvida e, através das técnicas de cromatografia de exclusão molecular, espalhamento de luz dinâmico e ultracentrifugação analítica conseguimos determinar o estado oligomérico da TbSerRS. O resultado de dímeros em solução corroborou com dados reportados na literatura, além de verificarmos por meio de estudos de cinética enzimática que a enzima encontra-se ativa sob as condições utilizadas. A técnica de ultracentrifugação analítica de sedimentação em equilíbrio também nos permitiu verificar a formação do complexo SerRS-tRNA, mas não nos possibilitou definir a estequiometria deste complexo. Estudos estruturais da enzima nativa e em interação com os tRNAs SELC e com as isoformas do tRNASer, L-serina, um análogo não hidrolisável de AMP, MgCl2, e com porções menores dos tRNAs foram realizados por meio da cristalografia por difração de raios X. Através dessa técnica, dezessete conjunto de dados foram coletados, processados e estão em fase de refinamento. Algumas análises estruturais possibilitaram confirmar a presença de duas moléculas de glicerol em cada monômero na região do sítio ativo para a estrutura da TbSerRS nativa e uma molécula de dAMP para o complexo TbSerRS-dAMP. / The synthesis of selenocysteine and its co-translational incorporation in selenoproteins in response to a UGA codon in frame require complex molecular machinery. In eukaryotes, components that participate in the reaction of selenocysteine formation were identified: SeryltRNA synthetase (SerRS), O-phosphoseryl-tRNA kinase (PSTK), SECIS Binding Protein 2 - SBP2, a selenocysteine-specific elongation factor (EFSec), selenophosphate synthetase 1 (SPS1) and selenophosphate synthetase 2 (SPS2), SEPSECS, SECp43 RNA binding protein, ribosomal protein L30, selenocysteine tRNA (tRNASec, SELC), and a specific sequence in the messenger RNA (SECIS element). The first step for selenocysteine incorporating is performed by SerRS that aminoacylates the tRNA with serine through serine activation by Mg2+ and ATP leading to the formation of an intermediate linked to the enzyme (Ser-AMP). Subsequently, the change of the Ser radical to tRNASec takes place followed by the enzymatic conversion of Ser-tRNASec to Sec-tRNASec. Through in silico analysis our group has identified components of the selenocysteine insertion machinery in species of Kinetoplastida. Homologues of tRNASec and the enzymes TbSerRS, TbSPS2, TbPSTK, TbSepSecS and TbEFSec were identified. Our main target is the structural study of the native SerRS from Trypanosoma brucei and SerRS in complex with the tRNASec and the tRNASer isoforms. A new methodology in the purification process of this enzyme has been developed, and through molecular exclusion chromatography, dynamic light scattering and analytical ultracentrifugation techniques we were able to determine the oligomeric state of TbSerRS. The result of dimers in solution corroborated with the data reported in the literature. Moreover, we were able to verify through studies of enzyme kinetics that the enzyme is active. The sedimentation equilibrium analytical ultracentrifugation technique also demonstrated the formation of the SerRS-tRNA complex, however, it did not allow the definition of the complex stoichiometry. Structural studies of the native enzyme and its interaction with SELC, tRNASer isoforms, L-serine, a non-hydrolyzable AMP analog, MgCl2, and smaller portions of tRNAs were performed by X-ray diffraction crystallography. Through this technique, seventeen data sets were collected, processed, and are being submitted to refinement processes. Initial structural analysis allowed the confirmation of the presence of two glycerol molecules in each monomer in the active site region in the native structure of TbSerRS and one dAMP molecule in the TbSerRS-dAMP complex.

Consumption of endophyte-infected tall fescue results in a syndrome of negatively altered physiological systems, collectively known as fescue toxicosis. Another challenge to endophyte-infected tall fescue -based beef cattle operations is that the soils often are selenium (Se) poor, necessitating the need to provide supplemental Se. To test the general hypothesis that different forms of supplemental Se would ameliorate the negative effects of fescue toxicosis, predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing an endophyte-infected tall fescue pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as sodium selenite (ISe), SELPLEX (OSe), or an 1:1 blend of ISe and OSe (MIX). Steers were commonly weaned and depleted of Se for 98 d. Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised and subjected to summer-long common grazing of an endophyte-infected tall fescue pasture (0.51 ppm ergot alkaloids: ergovaline plus ergovalinine; 10.1 ha). Selenium treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The first project objective was to determine the effect of forms of supplemental Se on whole blood Se, serum prolactin, liver glutamine synthetase (GS) activity, carcass parameters, and growth performance (Experiment 1). In Experiment 1, whole blood Se increased for all treatments from day 0 to 22 and then did not change. Across periods, MIX and OSe steers had greater whole blood Se than ISe steer. Compared to ISe steers, MIX and OSe steers had more serum prolactin. Liver GS mRNA, protein content, and activity were greater in MIX and OSe steers than ISe steers. However, the ADG and carcass parameters were not affected by Se treatments. The second project objective was to determine the effect of forms of supplemental Se on serum clinical parameters of Experiment 1 steers (Experiment 2). In Experiment 2, across periods, MIX steers had more serum albumin than OSe, and ISe steers, respectively. Serum alkaline phosphatase (ALP) activity was greater in MIX and OSe steers. In addition, blood urea nitrogen (BUN), serum sodium, phosphorus, and magnesium concentration were affected by Se treatments. Partial correlation analysis revealed that serum albumin, BUN, and ALP activity were correlated with whole blood Se concentration. The third project objective was to evaluate the hepatic transcriptome profiles of Experiment 1 steers using microarray and targeted RT-PCR analyses (Experiment 3). In Experiment 3, bioinformatic analysis of microarray data indicated that hepatic glutamate/glutamine, proline, arginine, and citrulline metabolism was affected by different forms of supplemental Se. The mRNA expression of critical proteins involved in glutamate/glutamine (GLS2, GLUD1, GLUL), proline (PYCR1, ALDH18A1), and urea (ARG1, ARG2, OAT, NAGS, OTC, ORNT1) metabolism were differentially expressed by Se treatments. Collectively, we conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes 1) increased whole blood Se content, an indicator of greater whole-body Se assimilation; 2) increased serum prolactin, albumin, and ALP, the reduction of which are hallmarks of fescue toxicosis; and 3) altered hepatic nitrogen metabolism, as indicated by changes in key enzymes of glutamate/glutamine, proline, and urea metabolism. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.

As nitrogen (N) is a critical nutrient for plant growth, the development of synthetic N fertilisers dramatically changed agricultural production in the twentieth century. Improvement in N use efficiency (NUE) has been a focus of breeding for grain crop species, since protein is an important component of the harvested product. The study of NUE in sugarcane has lagged behind grain crops, mainly because N is not a component of sucrose, the primary product of the traditional sugarcane industry. Recently, improvement in NUE has become a focus of sugarcane breeding, due largely to environmental concerns regarding pollution from high N fertilisation, and the increasing cost of N fertilisers. This thesis aimed to gain an initial understanding of the genetic basis for variability in NUE in sugarcane. This was achieved through: (i) the screening of 168 sugarcane genotypes under limiting and non-limiting N supply in two glasshouse experiments; (ii) the mapping of marker-trait associations (MTA) for biomass and physiological traits under limiting and non-limiting N supply in a sugarcane mapping population; (iii) the analysis of expression of candidate genes encoding enzymes involved in the central processes of N assimilation and remobilisation in plants; and (iv) the mapping of candidate genes in a sugarcane genetic map. Genetic variation was identified for growth traits as well as physiological traits including %N, internal NUE (iNUE, g dry weight g-1 N) and leaf glutamine synthetase (GS) activity in a sugarcane mapping population. These traits were also analysed for linkage with genetic markers. Genetic variation in the screened genotypes was higher under limiting N supply, a finding that was reflected by the fact that marker-trait associations (MTA) for increases in iNUE were not identified under non-limiting N supply in the commercial parent of the mapping population. Contrary to findings in grain crop species, there was no link between GS activity and other traits, either through phenotypic correlations or co-location of MTA. The expression of candidate genes encoding GS, nitrate reductase (NR) and alanine amintotransferase (AlaAT) was quantified with Sequenom™ MassARRAY technology. Plants were grown under growth-limiting N supply, non-limiting N supply, or a N-pulse treatment, which consisted of growth-limiting N supply followed by non-limiting N supply 24 hours prior to sampling. Two genes, scAlaAT.d and scGS1.a, encoding AlaAT and GS respectively, were identified as non-responsive to changes in N supply, whereas scAlaAT.a, scGS1.b and scGS1.c had significantly (p<0.05) increased expression under a N-pulse, indicating an important role for these genes in the response of sugarcane to a sudden increase in N availability. The location of candidate genes associated with variation in NUE in a sugarcane genetic map were sought through restriction fragment length polymorphism (RFLP) markers. Twenty-two probes were screened, of which two generated single-dose markers, allowing the mapping of a single allele of scAspAT, encoding aspartate aminotransferase, and two alleles of scGS2, encoding plastidic GS. Because of the economic and environmental consequences of inefficient N fertiliser application, the development of sugarcane cultivars with improved NUE is essential. Since variation for NUE exists, especially in unimproved sugarcane varieties, this may be achieved through traditional breeding methods by screening existing breeding populations under limiting N supply. Additionally, an improved understanding of the genetic basis of variation for NUE in sugarcane should be pursued by further analysis of candidate gene response to changing N availability by screening widely varying cane species for differences in gene expression, enzyme activity and metabolite profiles. The further addition of candidate gene locations to sugarcane genetic maps will aid both future marker-assisted selection in breeding, and a fundamental understanding of genetic control of NUE variation. Through the development of sugarcane cultivars with improved NUE and an enhanced knowledge of the genetic control underpinning sugarcane N physiology, concerns regarding high N fertiliser applications may be mitigated and sustainability ensured.

Reductive acetogenesis via the acetyl-CoA pathway is a hydrogenotrophic pathway that has the potential to reduce methanogenesis from ruminant livestock. However our understanding of the organisms capable of this transformation (acetogens) is hindered by a lack of specific molecular tools for this group. In the present thesis, a PCR primer set specific for a wide range of acetogens was developed, targeting the acetyl-CoA synthase (ACS) gene which is unique to the acetyl-CoA pathway. ACS was found to be useful marker for potential acetogens and ACS sequences could be used to infer family-level phylogeny for many acetogens. ACS gene specific primers were used in combination with existing molecular tools targeting the gene encoding formyltetrahydrofolate synthetase (FTHFS, present in the acetyl-CoA pathway but not unique to it) and 16S rRNA genes, as well as cultivation techniques, to investigate acetogen diversity in the rumen and two analogous gut systems where microbial hydrogenotrophy differs: the forestomach of a native Australian marsupial, the tammar wallaby Macropus eugenii; and the developing rumen of young lambs. Novel potential acetogens present naturally in the rumen of pasture fed and grain fed cattle affiliated with the Ruminococcaceae/Blautia group and distantly with the Lachnospiraceae. A large diversity of potential acetogens with functional genes affiliating broadly between the Lachnospiraceae and Clostridiaceae though without a close sequence from a cultured relative were also detected. Rumen acetogen enrichment cultures revealed the presence of a known acetogen, Eubacterium limosum, in grain fed cattle, as well as novel acetogens affiliating with the Lachnospiraceae and Ruminococcaceae/Blautia group. The novel potential acetogen population detected in this study may represent an important hydrogenotrophic group in the rumen that we understand very little about and that requires further investigation. The tammar wallaby, which exhibits foregut fermentation analogous to that of the rumen but resulting in lower methane emissions, housed a different acetogen population to that of the bovine rumen (LIBSHUFF, p <0.0001) though novel potential acetogens in the tammar wallaby forestomach affiliated broadly in the same family groups (Blautia group, Lachnospiraceae and between Lachnospiraceae and Clostridiaceae without a close cultured isolate). Acetogen enrichment cultures from the tammar wallaby forestomach facilitated isolation of a novel acetogen, which was closely related to potent reductive acetogens from kangaroos. The differences between the acetogen population of the tammar wallaby forestomach and the bovine rumen may be a factor in explaining lower methane emissions and methanogen numbers in tammar wallabies relative to ruminants. Using a gnotobiotically reared lamb model, the unique acetogen population present in the developing rumen was identified and it’s response to methanogen colonisation examined. The acetogen E. limosum and potential acetogen Ruminococcus obeum were identified as well as a small diversity of novel potential acetogens affiliating with the Blautia group and the Lachnospiraceae. A small but diverse population of naturally resident methanogens were also identified in gnotobiotically reared lambs that had been isolated at 17 hours of age. After inoculation with Methanobrevibacter sp. 87.7, methanogen numbers in gnotobiotically reared lambs significantly increased but acetogen diversity was not altered, indicating that this population is resilient to methanogen colonisation to some degree. The potential acetogen population in gnotobiotically reared lambs was significantly different (LIBSHUFF, p < 0.0001) to that in conventionally reared sheep, which indicates that factors other than methanogen establishment alone, probably relating to other microbes and associated hydrogen concentrations in the rumen, affect acetogens during rumen development.

Two cellular innate responses, the dsRNA protein kinase (PKR) pathway and the 2'-5' oligoadenylate synthetase (OAS)/RNase L pathway, are activated by dsRNAs produced by viruses and reduce translation of host and viral mRNAs. PKR activation results in eIF2a phosphorylation. As a consequence of eIF2a phosphorylation, stress granules (SGs) are formed by the aggregation of stalled SG proteins with pre-initiation complexes and mRNA. West Nile virus (WNV) infections do not induce eIF2a phosphorylation despite upregulation of PKR mRNA and protein suggesting an active suppression of PKR activation. Assessment of the mechanism of suppression of PKR activation in WNV-infected cells indicated that WNV infections do not induce PKR phosphorylation so that active suppression is not required. In contrast to infections with "natural" strains of WNV, infections with the chimeric W956 infectious clone (IC) virus efficiently induce SGs in infected cells. After two serial passages, the IC virus generated a mutant (IC-P) that does not induce SGs efficiently but does induce the formation of NS3 granules that persist throughout the infection. This mutant was characterized. 2'-5' oligoadenylate synthetases (OAS) are activated by viral dsRNA to produce 2-5A oligos that activate RNase L to digest viral and cellular RNAs. Resistance to flavivirus-induced disease in mice is conferred by the full-length 2'-5' oligoadenylate synthetase 1b (Oas1b) protein. Oas1b is an inactive synthetase that is able to suppress the in vitro synthetase activity of the active synthetase Oas1a. The ability of Oas1b to inhibit Oas1a synthetase activity in vivo and to form a heteromeric complex with Oas1a was investigated. Oas1b suppressed 2-5A production in vivo. Oas1a and Oas1b overexpressed in mammalian cells co-immunoprecipitated indicating the formation of heteromeric complexes by these proteins. Unlike mice, humans encode a single OAS1 gene that generates alternatively spliced transcripts encoding different isoforms. Synthetase activity has previously been reported for only three of the isoforms. The in vitro synthetase activity of additional OAS1 isoforms was analyzed. All tested isoforms synthesized higher order 2-5A oligos. However, p44A only produced 2-5A dimers which inhibit RNase L.

Soils in many agricultural areas have high pH, resulting in low availability of zinc and iron. Plants grown on such soils suffer from either Zn or Fe deficiency or both. The efficient plant genotypes grown normally in calcerous soils were found to evolve some strategies to acquire the iron which is in insoluble form. Iron efficient graminaceous monocots release iron chelating substances, mugineic acid family phytosiderophores (MAs), in response to iron deficiency stress. Several researchers have suggested that phytosiderophores also can play role in grass Zn nutrition and thus it may be possible that it is the uptake mechanism for Zn efficiency. Several possible genes that take role in phytosiderophore synthesis or found to be induced under iron deficient conditions were identified in several organisms but not on wheat. In this study, the efficient barley cultivar Tokak-157, efficient wheat cultivar Kira&ccedil / -66 and relatively less efficient wheat cultivar BDMM-19 were grown in normal growth conditions for 1 week and transfered to zinc deficient, iron deficient and both zinc and iron deficient nutrient solutions. After growing 1 week on these conditions, plants grown on both zinc and iron deficient nutrient solutions were retransfered to zinc and iron sufficient conditions. Degenerate primers were designed for the conserved regions of previously identified genes that take role in phytosiderophore synthesis or induced under iron deficient conditions and RT-PCRs were performed. The complete open reading frame of IDI-1(Iron deficiency induced-1) gene and the putative gene fragment for SAM-s (S-adenosylmethionine synthetase) were identified.

Carbamoyl phosphate synthetase (CPS) from E. coli catalyzes the formation of carbamoyl phosphate, an intermediate in the biosynthesis of pyrimidine nucleotides and arginine, from glutamine, bicarbonate and two molecules of MgATP. This reaction is catalyzed by three separate active sites that are separated in space by ~100 Å. The transfer of ammonia and carbamate through the two intramolecular tunnels was investigated by molecular dynamics simulations and experimental characterization of mutations within. The presence of an unstable reaction intermediate, carboxyphosphate, was established. A method for studying the synchronization of the two active sites on the large subunit of CPS was developed. The potential of mean force (PMF) calculations along the ammonia and carbamate transfer pathways indicate a low free-energy path for the translocation of ammonia. The highest barrier for ammonia is 7.2 kcal/mol which corresponds to a narrow turning gate surrounded by the side chains of Cys-232, Ala-251, and Ala-314 in the large subunit. A blockage in the passageway was introduced by the triple mutant C232V/A251V/A314V, which was unable to synthesize carbamoyl phosphate. The release of phosphate is necessary for the injection of carbamate into the carbamate tunnel. Two mutants, A23F and G575F, were designed to block the migration of carbamate through carbamate tunnel. The mutants retained only 1.7 percent and 3.8 percent of the catalytic activity for the synthesis of carbamoyl phosphate relative to the wild-type CPS, respectively. Formate can be utilized by CPS in the absence of bicarbonate to form formyl phosphate. This intermediate was observed by 31P, 13C, and 1H NMR. For the three NMR methods a peak corresponding to formyl phosphate was observed at 2.15 ppm (31P) , 162.4 ppm (13C), and 8.39 and 7.94 ppm (1H). The rate of formation of formyl phosphate is 0.025 ± 0.005 s-1. Formamide was not detected in the presence of an ammonia source. Fluorescence anisotropy measurements on the C551A/S171C and C551A/S717C mutants provided insight into a possible mechanism of synchronization between the two active sites on the large subunit. The biggest fluorescence anisotropy change was observed at the N-terminal domain in the presence of AMPPNP and ATP.

Malaria is a treatable communicable disease yet remains a common cause of death and disease especially among pregnant women and children. Most of malaria's worldwide burden disproportionately lies in Southeast Asia and Sub-Saharan Africa. Western medicine's 100+ year history of combating Plasmodium falciparum has taught us that the global population of malaria parasites has a unique and dangerous ability to rapidly evolve and spread drug resistance. Recently it was documented that resistance to the first-line antimalarial artemisinin may be developing in Southeast Asia.

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.