O Brasil é o maior produtor mundial de feijão com produção média anual de 3,5 milhões de toneladas, entretanto, um dos maiores problemas enfrentados por essa cultura é o déficit hídrico, que leva a uma redução considerável em seu rendimento. Dessa forma, a identificação de genes que controlam os mecanismos de defesa e adaptação do feijoeiro à falta de água durante o seu desenvolvimento é de grande utilidade. E, como a tolerância à seca é um caráter multigênico, genótipos com diferentes graus de tolerância apresentam expressão gênica diferencial, com ativação e/ou repressão de determinados genes. Diante disso, esse trabalho teve como objetivos: (i) identificar genes diferencialmente expressos em dois genótipos de feijoeiro comum, sendo um tolerante (BAT 477) e o outro suscetível (IACCarioca 80SH) à seca; (ii) verificar a expressão gênica espacial (em raízes, caules e folhas) e temporal (cinco níveis crescentes de déficit hídrico - 24 h, 48 h, 72 h, 96 h e 120 h de exposição ao estresse) por meio de RT-qPCR; (iii) predizer a função dos genes identificados, com base nos genes ortólogos de Arabidopsis thaliana, usando dados públicos de microarranjo disponíveis na plataforma Genevestigator. Para atingir esses objetivos, foi conduzido um experimento em casa-de-vegetação, sendo induzido o déficit hídrico, por meio da suspensão da irrigação, quando as plantas atingiram o estádio fenológico R5, mantendo sob suprimento hídrico adequado as plantas-controle. Foi então utilizada a técnica de cDNA-AFLP para isolar os transcritos diferencialmente expressos entre os dois genótipos, sob seca, a qual aliada ao sequenciamento possibilitou a identificação e anotação de 45 transcritos, sendo 21 exclusivamente expressos no genótipo tolerante e 24 no genótipo suscetível. Dentre os transcritos identificados no genótipo tolerante, podem ser listados pelo menos 11, com potencial de serem usados para transformação genética (chlorophyll A-B binding protein, HSP40, HSP70, glycosyl hydrolase, serine/threonine protein kinase, trehalose-6-phosphate synthase, E3 ubiquitin ligase, fructose biphosphate aldolase, mediator complex subunit 13, aquaporin nodulin MTN-3-related e TCP transcription factor), e no genótipo suscetível, podem ser listados nove (coatomer protein complex, monoamine-oxidase A repressor R1, synaptobrevin, haloacid dehalogenase-like hydrolase, ADP-ribosylation factor, mTERF, serine protease S1C HtrA-related, legume lectin e SWI/SNF-related chromatin binding). Na análise de expressão gênica espacial e temporal, os transcritos mais promissores para uso em trabalhos futuros de transformação genética foram: aquaporin nodulin MTN3, E3 ubiquitin ligase, serine/threonine protein kinase, glycosyl hydrolase e HSP 70 protein, uma vez que tiveram uma expressão bastante pronunciada no genótipo tolerante. Através das análises in silico, baseadas nos genes ortólogos de A. thaliana, foram descobertos processos e vias metabólicas que podem estar envolvidos na resposta do feijoeiro comum ao déficit hídrico. Além disso, foram identificados genes associados com a tolerância à seca, corroborando os dados experimentais. Sendo assim, os resultados obtidos nesse trabalho fornecem o entendimento necessário ao desenvolvimento de ferramentas moleculares (marcadores para genes diferencialmente regulados) para serem utilizados em programas de melhoramento, bem como para informação genética básica na anotação funcional do genoma do feijoeiro e também para utilização desses genes candidatos em trabalhos de transformação genética para obtenção de plantas mais tolerantes à seca. / Brazil is the largest producer of common beans, with average annual production of 3.5 million tonnes; however, one of the biggest problems faced by this crop is drought, which leads to a considerable reduction in their yield. Thus, the identification of genes that control the defense mechanisms and adaptation of common bean to drought during its development is very useful. Drought tolerance is a multigenic character, so genotypes with different degrees of water deficit tolerance exhibit differential gene expression, with activation and/or repression of certain genes. Therefore, this study aimed to: (i) identify differentially expressed genes in two common bean genotypes, one tolerant (BAT 477) and other susceptible (IAC-Carioca 80SH) to drought, (ii) verify the spatial (root, stem and leaves) and temporal (five increasing levels of water deficit - 24 h, 48 h, 72 h, 96 h and 120 h of stress exposition) gene expression by RTqPCR (iii) predict the genes function, based on Arabidopsis thaliana orthologous genes, using available data in the public microarray platform Genevestigator. To achieve these objectives, an experiment was conducted in a green house, being induced water deficit, by withholding water when the plants reached growth stage R5, maintaining adequate water supply under the control plants. To isolate transcripts differentially expressed between the two genotypes under drought was used the cDNA-AFLP technique, which coupled with sequencing enabled the identification and annotation of 45 transcripts, 21 exclusively expressed in the tolerant genotype and 24 in the susceptible one. Among the transcripts identified in the tolerant genotype, may be listed at least 11, with potential to be used in genetic transformation (chlorophyll A-B binding protein, HSP40, HSP70, glycosyl hydrolase, serine/threonine protein kinase, trehalose-6-phosphate synthase, E3 ubiquitin ligase, fructose biphosphate aldolase, mediator complex subunit 13, aquaporin nodulin MTN-3-related and TCP transcription factor), and in the susceptible genotype, can be listed nine (coatomer protein complex, monoamine-oxidase A repressor R1, synaptobrevin, haloacid dehalogenase-like hydrolase, ADP-ribosylation factor, mTERF, serine protease S1C HtrA-related, legume lectin and SWI/SNF-related chromatin binding). In the spatial and temporal gene expression analysis, the transcripts that stood out for use in genetic transformation future studies were: aquaporin nodulin MTN3, E3 ubiquitin ligase, serine/threonine protein kinase, glycosyl hydrolase and HSP 70 protein, since it had an expression quite pronounced in the tolerant genotype. Through the in silico analysis, based on orthologous genes of A. thaliana, was discovered processes and metabolic pathways that may be involved in the common bean response to drought. In addition, we identified genes associated with drought tolerance, corroborating the experimental data. Thus, the present results provide the necessary understanding to develop molecular tools (markers for differentially regulated genes) to be used in breeding programs, as well as basic genetic information in the common bean functional genome annotation and also to use these candidate genes for genetic transformation to obtain drought-tolerant plants.

O ciclo estral das cadelas difere das demais espécies domésticas. Estudos demonstraram que o aumento da concentração plasmática de P4 durante a fase luteínica das cadelas pode levar a alterações metabólicas como a resistência insulínica, acarretando complicações como Diabetes mellitus. A glicose é uma molécula transportada, na maioria das células, por proteínas transportadoras. O processo de instalação da resistência insulínica é caracterizado por alterações teciduais da expressão de algumas proteínas transportadoras de glicose, como o GLUT4. Atualmente 13 isoformas de proteínas transportadoras já foram seqüenciadas (GLUT1 ao GLUT13). O GLUT4 está presente nos músculos e no tecido adiposo, principalmente. Para avaliar se a expressão do GLUT 4 está presente nas células luteínicas e se esta expressão relaciona-se à produção de P4 e E2, 28 cadelas foram divididas em 7 grupos de acordo com os dias após a ovulação -p.o. (de 10 à 70 dias, n = 4 por grupo). Os ovários foram dissecados e congelados em nitrogênio líquido, o RNA extraído e o cDNA confeccionado e submetido ao PCR em tempo real. O gene GAPDH foi utilizado como controle endógeno para padronização da expressão do gene alvo. Foi coletado sangue para dosagem da glicemia, insulinemia, progesterona e estradiol. Para avaliar a regulação positiva do GLUT4 avaliamos também a expressão do mRNA do HIF-1α, destas mesmas cadelas. A expressão do GLUT4 apresentou tendência a aumento de expressão aos 20 dias (p. o.), quando comparado aos 10, 30 e 40 dias, pico de expressão aos 50 dias (p.o.), e então apresentou tendência a queda aos 60 e 70 dias p.o. Já a expressão do HIF-1α manteve-se muito semelhante através dos dias, tendendo a queda aos 10 e aos 40 dias pós ovulação, quando comparado com os demais grupos. Os resultados de dosagem de P4 e E2 variaram dentro do esperado para o diestro e não apresentaram correlação com a expressão de GLUT 4; a glicemia e insulina, aqui expressas através do índice HOMA (insulina x glicose % 22,5), apresentou pico aos 40 dias. Sabe-se que quanto mais alto o índice HOMA, menos este animal é sensível à insulina, ou seja, mais resistente à ela. Observou-se que o índice HOMA apresentou-se mais alto aos 40 dias, associado aos menores valores de expressão do GLUT4. Por outro lado, obtivemos o pico de expressão de GLUT4 aos 50 dias, quando o índice HOMA apresentou valores baixos. Sugere-se que a queda da P4 associada à elevação do estradiol plasmático possa influenciar o índice HOMA. Pode-se concluir que a expressão do GLUT4 no corpo lúteo de cadelas segue o padrão observado para tecidos sensíveis à insulina, nos quais existe uma maior expressão durante a fase de maior sensibilidade à insulina e diminuição drástica em fase de pré ou já instalada resistência insulínica. / The canine estral cycle differs from other domestic species. Some studies demonstrated that the increase of the plasmatic concentration of progesterone during canine luteinic phase can lead to metabolic alterations, such as insulinic resistance and may cause complications such as Diabetes mellitus. Glucose is a molecule that is transported in most cells by transporting proteins. The process of installation of the insulinic resistance is characterized by tissue alterations of the expression of some glucose transporting proteins, as GLUT4. Currenly, 13 isoforms of transporting proteins were sequenced (GLUT1 to GLUT13). GLUT4 is present mainly in muscle and fat tissue. In order to assess if GLUT4 expression is present in luteal cells, and if this expression is related to P4 and E2 production, 28 bitches were divided into 7 groups, in accordance with the days after the ovulation -p.o. (from 10 to 70 days, n=4 for group). The ovaries were dissected and frozen in liquid nitrogen. The RNA was extracted and the cDNA was made and submitted to real time PCR. GAPDH gene was used as endogenous conntrol to standardization of target gene expression. Blood was collected to glycemia, insulinemia, P4, and E2β dosage. To assess the positive regulation of GLUT4, we also assessed HIF-1α mRNA expression of the same bitches. GLUT4 expression showed a tendency to increase the expression on the twentieth day (p.o.), when compared to the 10th, 30th, and 40th days, expression top on the 50 th day (p.o.) , and then, it showed a tendency to foll on the 60th and 70th days p.o. HIF-1α expression was very similar over the days, tending to fall on the 10th and 40th days post ovulation, when compared to other groups. P4 and E2β dosage results varied according to thr expectations in diestrus and have not shown correlation with GLUT4 expression; glycemia and insulin, here expressed by HOMA index (insulin x glucose % 22,5) showed crest (highest point) on the 40 th day. It is knows that the higher the HOMA index, the less sensitive this animal is to insulin, it is, more resistant to it. It was observed that HOMA index was higher on the 40th day, associated to small values of the GLUT4 expression. Otherwise, the got the top of GLUT4 expression on the 50th day, when HOMA index showed low values. It has been suggested that P4 fall associated to the plasmatic E2 increase may influence HOMA index. We may conclude that GLUT4 expression into the corpus luteum of bitches follows the standard observed in insulin-sensitive tissues, in which there is a higher expression over the phase of higher sensitiveness to insulin and remarkable decrease in pre or even installed insulinic resistance.

A diarréia neonatal é o principal problema sanitário que acomete os bezerros nas primeiras semanas de vida, causando grandes prejuízos devido à morbidade, mortalidade, custos com tratamento e atraso no desenvolvimento. Os rotavírus são os mais importantes agentes virais causadores de gastroenterites em crianças e diarreia em diferentes espécies animais. Além do seu impacto econômico na produção animal, os bovinos podem atuar como reservatórios da diversidade genética e antigênica para humanos. Assim, o diagnóstico deste agente é fundamental para o desenvolvimento de medidas profiláticas mais especificas visando o seu controle. O objetivo deste trabalho foi desenvolver um método de PCR em Tempo Real para a detecção de rotavírus bovinos em sistema Taqman® tendo como alvo, a proteína não-estrutural 5 (NSP5). Para isso, 113 amostras de fezes bovinas foram coletadas de reabanhos do estado de São Paulo, e previamente testadas por PCR convencional. Para a padronização da PCR em Tempo Real, a estirpe padrão foi clonada, gerando um plasmídeo com 3x1010 cópias/reação. Como controle exógeno foi utilizado β-actina. O limite de detecção foi determinado por diluições seriadas na base 10, detectando até 6x101 cópias/µl. A curva padrão da PCR em Temo Real para a detecção do segmento codificador da proteína NSP5 teve como resultados, uma eficiência de 100,47%; com slope igual a -3,18 e R2 de 1,0. De um total de 113 amostras testadas pela PCR convencional 63 delas (55,7%) foram consideradas positivas para rotavírus. Dessas mesmas amostras testadas, 5 não amplificaram para β-actina e não foram incluídas nas análises posteriores. Para a PCR em Tempo Real o limite de detecção foi considerado o valor de 6x101 cópias/reação, sendo definido o ponto de corte o ciclo (Ct) de número 36 para o teste com a amostra viral a partir do DNA ligado em vetor plasmidial. Considerando-se o ponto de corte de 60 (6x101) cópias/reação, das 108 amostras testadas, 63 (58,3%) foram consideradas positivas ao teste. O valor de concordância obtido através do teste Kappa, a um intervalo de confiança de 95%, a partir dos resultados gerados entre os testes de PCR convencional e PCR em foi de 0.279 (baixa concordância). Os resultados obtidos nesse estudo demonstrou que o teste foi eficiente podendo ser utilizado para um diagnóstico rápido e eficiente do rotavirus do grupo A, aumentando assim o repertório dos testes já estabelecidos / Neonatal diarrhea is the main health problem affecting the calves in the first weeks of life, causing major losses due to morbidity, mortality, treatment costs and delayed development. Rotaviruses are the most important causative agents of viral gastroenteritis in children and in different animal species. In addition to its economic impact on livestock, cattle can act as reservoirs for genetic and antigenic diversity for human samples. Therefore, the diagnosis of this agent is critical to the development of more specific preventive measures for their control. The objective of this work is to develop a method of PCR for rotavirus detection in cattle using TaqMan® system targeting the 5 nonstructural protein (NSP5). For this, 113 samples of cattle feces were collected from farms of São Paulo, and previously tested by convencional PCR. To PCR standardization, the standard sample was cloned, generating plasmid 3x1010 copies/reaction. The β-actin was usede as exogenous control. The limit of detection was determined by serial dilutions, to detect 6 x101 copies/µl. The standard curve of PCR to encoding segment detection NSP5 protein had as a result, an efficiency of 108.5%; with slope equal to -3.18 and R2 equal 1.. From a total of 113 samples tested by conventional PCR 63 (55.7%) were positive for rotavirus. From these samples 5 not amplifyed for β-actin gene and were not included in subsequent analyzes. For detection limit of Real-Time PCR was considered the amount of 6x101 copies / reaction, the cut-off being defined cycle (Ct) number 36 to the test with a viral sample from the DNA ligated into plasmid vector. Considering the cut-off 60 (6x101) copies/reaction of the 108 samples tested, 63 (58.3%) were positive to the test. The correlation value obtained by the Kappa test, at a 95% confidence interval, based on results generated between the conventional and PCR testing PCR was 0.279 (low agreement). The results of this study demonstrated that the probe can be efficiently used for a fast and efficient diagnosis of rotavirus of group A, thereby enhancing the repertoire of the established tests

The bacterium Vibrio vulnificus is an autochthonous inhabitant of estuarine waters and also found in shellfish such as oysters. It is a human pathogen of importance in the seafood industry, and can also infect recreational water users. Currently, recognized methods of detection rely upon isolation of pure cultures which requires at least 24 hours. To reduce the time needed for identification of the pathogen and simultaneously ascertain the virulence potential of the strains present, real-time PCR assays and sample processing procedures were developed (Chapter 1). These assays discriminate between type A (environmental, generally lower virulence) and type B (clinical, higher virulence) isolates. The genetic relationships between environmental V. vulnificus strains isolated from permitted and prohibited shellfish harvesting areas was determined using BOX-PCR genomic fingerprinting coupled with sequence analysis of three proposed virulence markers: (1) the virulence correlated gene (vcg), (2) 16S rRNA type and (3) presence/absence of the vulnibactin gene (viuB) (Chapter 2). The real-time PCR assays were able to detect the presence of seeded V. vulnificus in environmental water at a concentration of 160 cells 100·ml-1. In seeded oyster homogenates, the assays were able to detect a minimum of 10³ cells and 10² cells per reaction of type A and type B respectively. The phylogenetic analysis separated the majority of type A/ vcgE strains isolated from permitted shellfish harvesting areas from those isolated from prohibited harvesting areas. The genomic (BOX-PCR) fingerprints of type A and type AB isolates were more similar to one another than to type B isolates. Only one type A/ vcgE isolate contained the viuB gene; however, eight type B/ vcgC isolates had that gene. No obvious grouping was discerned between type B/ vcgC isolates from permitted versus prohibited shellfish harvesting areas or between those possessing the viuB gene versus those lacking viuB. These data provide insight into the ecology and correlation between population biology and general water quality, as gauged by the classification of the shellfish growing area waters. The 16S typing assays can be used for routine rapid typing to aid in risk assessment and reduce infection frequency through consumption of contaminated seafood.

Seafood substitution is a worldwide problem due to factors such as limited monitoring coupled with complex supply chains. Red snapper (Lutjanus campechanus) is a highly valued and overfished species that is commonly substituted with other fish, such as tilapia, rockfish, and other snapper species. DNA barcoding is typically used by regulatory agencies to detect seafood substitution; however, it is expensive and time-consuming. A rapid, real-time PCR assay targeting red snapper was developed previously for use in fisheries management; however, it has not been tested for its ability to detect red snapper species substitution. The objective of this study was to assess the ability of the real-time PCR assay to identify red snapper fillets and differentiate red snapper from common substitute fish species in combination with DNA barcoding. A total of 21 fresh/frozen fillets labeled as “red snapper” were tested with real-time PCR, along with 57 fresh/frozen fillets representing 15 of the most common categories of fish mislabeled as red snapper. All samples were tested with DNA barcoding to confirm the identity of fish species. Real-time PCR parameters were optimized to reduce background signals associated with cross-reactivity. Overall, real-time PCR identified 4 samples as red snapper: 3 were authenticated as red snapper with DNA barcoding and 1 was identified as mahi-mahi. Overall, 40% of all samples and 91% of “red snapper” samples were considered mislabeled according to DNA barcoding. Red snapper was substituted with other snapper species (e.g., Lutjanus malabaricus, Lutjanus peru, Ocyurus chrysurus, and Rhomboplites aurorubens), rockfish (Sebastes flavidus and Sebastes brevispinis), sea bream (Pagrus major/Pagrus auratus), and mahi-mahi (Coryphaena hippurus). The real-time PCR assay tested in this study can serve as a rapid screening test for the detection of mislabeled species, which can then be confirmed with sequencing techniques. This species identification technique has the potential to be used by regulatory agencies to rapidly determine the authenticity of red snapper on-site.

The emergence of MNAzymes (Multi-component nucleic acid enzymes) provides a new approach for detection of target analytes in various applications. In this thesis, three novel MNAzyme-based methodologies were developed to expand the range of the applications of MNAzymes. MNAzymes can be coupled with DNA or RNA ligands called aptamers to generate an apta-MNAzyme system, which can be used for the detection of non-nucleic target analytes such as small molecules and proteins. Direct detection using apta-MNAzyme system is performed in a format, which was isothermal, fluorescent, rapid, and requires no protein enzymes. Apta-MNAzymes can be coupled with a signal amplification cascade to increase the sensitivity of the reaction. Another MNAzyme-based methodology termed truncated MNAzyme arm system was developed to discriminate the presence of a single base mismatch of two closely related sequences. The system employs a partzyme with a truncated sensor arm and a stabiliser oligonucleotide that binds adjacently to the truncated sensor arm to stabilise the active MNAzyme structure. Truncated MNAzyme real-time PCR system is capable of discriminating the presence of a single base mismatch in a target DNA with high specificity and sensitivity (down to approximately 10 gene copies). The generic nature of the system enables simultaneous detection of three SNP targets in a multiplex format. MNAzymes was also investigated with various strategies to discriminate DNA sequences that are either methylated or unmethylated. In this thesis, bisulphite-treated DNA samples present in as low as 0.032 % of methylated DNA in a background of unmethylated DNA were discriminated using MNAzyme real-time methylation specific PCR (MSP) system. Furthermore, the presence of 5-methylcytosines in a target sequence increases the melting temperature of the duplex DNA. This was exploited further to directly discriminate DNA methylation status of target sequences using the truncated MNAzyme arm system without the need for bisulphite modification. Findings in this thesis have broadened the scope of MNAzymes as versatile tools for many possible applications and flexible alternative to the current technologies.

Wheat is the most economically important crop forming one quarter of Australian farm production. The wheat industry is severely affected by diseases, with fungal pathogens causing the most important economic losses in Australia. The application of fungicides and chemicals can control crop diseases to a certain extent, however, it is expensive and public concern for the environment has led to alternative methods of disease control to be sought, including the use of microorganisms as biological control agents. Microorganisms are abundant in the soil adjacent to plant roots (rhizosphere) and within healthy plant tissue (endophytic) and a proportion possess plant growth promotion and disease resistance properties. Actinobacteria are gram-positive, filamentous bacteria capable of secondary metabolite production such as antibiotics and antifungal compounds. A number of the biologically active endophytes belonging to the Actinobacteria phylum were isolated in our laboratory. A number of these isolates were capable of suppressing the wheat fungal pathogens Rhizoctonia solani, Pythium sp. and Gaeumannomyces graminis var. tritici, both in vitro and in planta indicating the potential for the actinobacteria to be used as biocontrol agents. The aim of this research was to investigate the molecular mechanisms underlying this plant-microbe interaction. The indigenous microbial populations present in the rhizosphere and endophytic environment are critical to plant health and disruptions of these populations are detrimental. The culture-independent technique Terminal Restriction Fragment Length Polymorphism (T-RFLP) was used to characterise the endophytic actinobacteria population of wheat roots under different conditions. Soils which support a higher number of indigenous microorganisms result in wheat roots with higher endophytic actinobacterial diversity and level of colonisation. Sequencing of 16S rRNA gene clones, obtained using the same actinobacteria-biased PCR primers that were used in the T-RFLP analysis, confirmed the presence of the actinobacterial diversity, and identified a number of Mycobacterium and Streptomyces species. It was found that the endophytic actinobacterial population of the wheat plants contained a higher diversity of endophytic actinobacteria than reported previously, and that this diversity varied significantly among different field soils. The endophytic actinobacteria have previously been shown to protect wheat from disease and enhance growth when coated onto the seed before sowing. As the endophytes isolated were recognised as potential biocontrol agents, the impact on the indigenous endophytic microbial population was investigated. Utilising the T-RFLP technique it was established that the use of a commercial microbial inoculant, containing a large number of soil bacterial and fungal strains applied to the soil, disrupts the indigenous endophyte population present in the wheat roots. The hypothesis is that non-indigenous microbes proliferate and dominate in the soil preventing a number of endophytic-competent actinobacterial genera from access to the seed and ultimately endophytic colonisation of the wheat roots. This dramatically reduces diversity of endophytes and level of colonisation. In contrast the use of a single endophytic actinobacteria endophyte inoculant results in a 3-fold increase in colonisation by the added inoculant, but does not significantly affect this indigenous population. Colonisation of healthy plant tissues with fungal endophytes has been shown to improve the competitive fitness with enhanced tolerance to abiotic and biotic stress and improved resistance to pathogens and herbivores. In this study the fungal endophyte population of wheat plants grown in four different soils was analysed using partial sequencing of 18S rRNA gene sequences. Sequence anlaysis of clones revealed a diverse range of fungal endophytes. In this diverse range of fungal endophytes a number sequences were highly similar to those of previously known fungal phytopathogens. A number of sequences detected were similar to fungal species previously identified in soil or plant material but not as endophytes. The remaining sequences were similar to fungal species without a known relationship with plants. Plants have developed an inducible mechanism of defence against pathogens. In addition to local responses plants have developed a mechanism to protect uninfected tissue through a signal that spreads systemically inducing changes in gene expression. In the model plant Arabidopsis thaliana activation of the Systemic Acquired Resistance (SAR) pathway and the Jasmonate (JA)/Ethylene (ET) pathway is characterised by the production of pathogenesis-related (PR) and antimicrobial proteins resulting in systemic pathogen resistance. Endophytic actinobacteria, isolated from healthy wheat roots in our laboratory, have been shown to enhance disease resistance to multiple pathogens in wheat when coated onto the seed before sowing. Real Time RT-PCR was used to determine if key genes in the SAR and JA/ET pathways were induced in response to inoculation with endophytic actinobacteria. Inoculation of wild-type Arabidopsis thaliana with selected strains of endophytic actinobacteria was able to �prime� the defence pathways by inducing low level expression of SAR and JA/ET genes. Upon pathogen infection the defence-genes are strongly up-regulated and the endophyte coated plants had significantly higher expression of these genes compared to un-inoculated plants. Resistance to the bacterial pathogen Erwinia carotovora subsp. carotovora was mediated by the JA/ET pathway whereas the fungal pathogen Fusarium oxysporum triggered primarily the SAR pathway. Further analysis of the endophytic actinobacteria-mediated resistance was performed using the Streptomyces sp. EN27 and Arabidopsis defence-compromised mutants. It was found that resistance to E. carotovora subsp. carotovora mediated by Streptomyces sp. EN27 occurred via a NPR1-independent pathway and required salicylic acid whereas the jasmonic acid and ethylene signalling molecules were not essential. In contrast resistance to F. oxysporum mediated by Streptomyces sp. EN27 occurred via a NPR1-dependent pathway but also required salicylic acid and was JA- and ET-independent. This research demonstrated that inoculating wheat with endophytic actinobacteria does not disrupt the indigenous endophytic population and may be inducing systemic resistance by activating defence pathways which lead to the expression of antimicrobial genes and resistance to a broad range of pathogens.

<p>Aromatase is the enzyme that catalysis the conversion of androgens into estrogens. It´s a member of P450 cytochrome family and is encoded by the CYP19-gene. The enzyme aromatase has an important role in regulating physiological and behavioral sexual mechanisms. This includes for instance activation, motivation and maintenance of the reproductive behaviors. The sexual behavior is affected by a complex series of events that requires the connection of endogenous hormonal and neurochemical changes with social interactions, especially between the opposite sexes. The aim of the present study was to examine how social interactions effect the aromatase expression and activity in the guppy brain. Guppy males were introduced into four different social conditions: Isolated, all male conditions, heterospecific (with zebrafish females) and conspecific female guppies. The focal males were kept under these conditions for two respectively four days. The sexual behavior, of each of the focal males was recorded daily during 10 minutes. The males with the guppy females showed, in contrast to the males in the other groups, a high frequency of reproductive behaviors. The brains of the focal males were collected and the brain aromatase activity was measured using tritiated water assay. I have also tried to analyze the gene-expression of aromatase with RT-PCR. However I was unable to analyze the results with the RT-PCR, because of possible primer-dimerization. Due to the limited time schedule, we were not able to solve the problem. ANOVA performed on the aromatase activity, revealed no significant difference between the different treatment groups. The variance was highest in the zebrafish category and lowest in the isolated males. There was no significant correlation between the mean number of reproductive behaviors and the aromatase activity in males that were together with guppy females. The results do not support the hypothesis that social interactions can affect the brain aromatase activity in guppy males.</p>

<p>Chlamydia and gonorrhea are major causes of sexually transmitted infections (STI) in adolescents worldwide. The infections are caused by <em>Chlamydia trachomatis</em> or <em>Neisseria gonorrhoeae, </em>bacteria with clinical manifestations such as urethritis, prostatitis and epididymitis among men, and urethritis, cervicitis and upper genital tract infection (i.e. pelvic inflammatory disease) among women. However, in many cases of genital tract infection, the etiology remains uncertain. In light of this, <em>Mycoplasma genitalium</em> was somewhat accidentally isolated in 1980 after prolonged incubation of urogenital specimens from men with non-gonococcal urethritis. Following the initial isolation in 1980, repeated attempts have been made to recover the extremely fastidious organism from clinical samples by culture techniques, but isolates have been rare and difficult to obtain. With the development of PCR methods in the early 1990s, detection of <em>M. genitalium</em> infection became more feasible.</p><p>The aim in paper <strong>I</strong> was to compare three different PCR assays (conventional and real-time 16S rRNA gene PCR as well as real-time <em>Mycoplasma genitalium</em> adhesin protein (MgPa) gene PCR) for detection of <em>M. genitalium</em>. The study also determined the prevalence of <em>M. genitalium</em>. Clinical specimens collected from STI attendees, 381 men and 298 women, were used to determine the prevalence of <em>M. genitalium</em> and 213 of these specimens were used in the PCR comparative study. The prevalence of <em>M. genitalium</em> infection in men and women was 27/381 (7.1 %) and 23/298 (7.7 %) respectively. In the PCR comparative study, <em>M. genitalium </em>DNA were detected in 61/76 (80.3 %) of true-positive specimen by conventional 16S rRNA gene PCR, in 52/76 (68.4 %) by real-time 16S rRNA gene PCR and in 74/76 (97.4 %) by real-time MgPa gene PCR. Hence, real-time MgPa gene PCR is well suited for clinical diagnosis of <em>M. genitalium</em> in urogenital specimens from men and women.</p><p>The aim in paper <strong>II</strong> was to determine whether a patients’ endocervical swab specimen can be transported in first void urine (FVU) as combined specimens in detection of <em>Mycoplasma genitalium </em>by real-time PCR. The study also compared two different DNA extraction methods (manual Chelex DNA extraction and automated BioRobot M48 DNA extraction) for observation of possible PCR inhibition. Clinical specimens collected from 329 women attending a STI clinic were used in the study. A total of 100 endocervical swab specimens transported in FVU was used in the PCR inhibition analysis. <em>M. genitalium</em> was detected in 25/329 (7.6 %) women. Endocervical swab specimens transported in FVU demonstrate higher sensitivity compared to both FVU alone and specimens transported in 2-SP medium detecting 24/25 (96 %), 22/25 (88 %) and 17/25 (68 %) of <em>M. genitalium</em> positive women, respectively. Automated BioRobot M48 DNA extraction was shown to be superior to manual Chelex extraction leaving no PCR inhibition and slightly higher DNA yield and/or better sensitivity. The results from these two studies are important knowledge in establishing the future diagnostic level of this STI in our county and also nationally.</p>

<p> </p><p>Genotyping can be used to link genetic variation among individuals to certain diseases or conditions. Some known disorders and states that are dependent on single nucleotide polymorphism (SNPs) are lactose intolerance, venous thrombosis, hereditary hemochromatosis and the difference in sensibility among people to metabolise drugs.</p><p>In this project a new kit, TaqManÒ Sample-to-SNP KitÔ <strong>for extraction of DNA and preparation of the extract for genotyping with real-time PCR and allelic discrimination, was evaluated. QIAamp® DNA Blood Biorobot® MDx Kit was used as the reference method.</strong></p><p>The purpose of the comparison was to find a method that makes DNA extraction from blood samples cheaper and faster, but with the same reliability as the reference procedure.</p><p>The results of the evaluation showed a complete agreement of the genotype results between the methods tested, which means that the new method was as reliable as the reference method. The costs of reagents and material would be reduced with 52% if the new method is adopted, that alone would result in a cost reduction of 144 000SEK a year with a sample volume of 650 samples/month. The time for DNA extraction would also be reduced with the new procedure.</p><p> </p>