Several genera of bacteria residing in the sea surface microlayer and in the near-surface layer of the ocean have been found to be involved in the production and decay of surfactants. Under low wind speed conditions, surfactants can suppress short gravity capillary waves at the sea surface and form natural sea slicks. These features can be observed with both airborne and satellite-based synthetic aperture radar (SAR). Using a new microlayer sampling method, a series of experiments have been conducted in the Straits of Florida and the Gulf of Mexico in 2013 to establish a connection between the presence of surfactant-associated bacteria in the upper layer of the ocean and sea slicks. In a number of cases, sampling coincided with TerraSAR-X and RADARSAT-2 satellite overpasses to obtain SAR images of each study site. Samples collected from slick and non slick conditions have been analyzed using real time PCR techniques to determine Bacillus relative abundance in each area sampled. Previous work has shown that the sea surface microlayer plays a role in air-sea gas exchange, sea surface temperature, climate-active aerosol production, biochemical cycling, as well as the dampening of ocean capillary waves. Determining the effect of surfactant-associated bacteria on the state of the sea surface may help provide a more complete global picture of biophysical processes at the air-sea interface and uptake of greenhouse gases by the ocean.

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.

Syfilis är en sexuellt överförbar infektion som orsakas av spiroketen Treponema pallidum subspecies pallidum. Laboratoriediagnostik utgörs i första hand av serologiska test tillsammans med kliniska fynd men på grund av olika faktorer är dessa inte helt pålitliga. Ett flertal olika PCR-metoder utvecklats som påvisar patogenen med hjälp av T-pallidum-specifika gener. En väl studerad gen med hög specificitet är polA-genen som anses vara en mycket robust och känslig metod och lämpar sig väl för att påvisa T. pallidum i kliniska material. Syftet med projektet är att utveckla och optimera en kvalitativ realtids-PCR i singelplex format för verifiering av T. pallidum, för diagnostisering av syfilis. I detta projekt har en realtids-PCR riktad mot polA för detektion av T. pallidum utvecklats. Metodens prestanda utvärderades med en kommersiellt framställd DNA-kontroll med avseende på sensitivitet, specificitet detektions-gräns. När samtliga moment verifierats och fastställts testades metoden på kliniskt material från sår. Den T. pallidum-specifika PCR-analysen visade att det inte förekom korsreaktivitet mot andra agens som förväntas finnas i sår från patient med misstänkt syfilis. Metoden uppvisade en känslighet vid spädning 1:100 (cirka 13 kopior/µl) och en precision på 36,3±0,8 Ct. Det kliniska provet visade på förekomst av T. pallidum dock är känsligheten något sämre än existerande referensmetod. Metoden kan användas i rutindiagnostik av T. pallidum dock bör utfallet från extraktion av DNA från sår studeras ytterligare för att eventuellt öka utbytet och detektera lägre koncentrationer i kliniskt material. / Syphilis is a sexually transmitted infection caused by the spirochete Treponema pallidum subsp. pallidum. Laboratory diagnostics consist primarily of serological tests together with clinical findings for different reasons these methods are not reliable. A variety of PCR methods has been developed that target the pathogen using T. pallidum-specific genes. A well-studied gene with high specificity is the polA gene which is considered very robust and sensitive method and well suited for detection of T. pallidum in clinical materials. The aim of the project is to develop and optimize a qualitative real-time PCR in single-plex format for the verification of T. pallidum, for the diagnosis of syphilis. A real-time PCR targeting polA was developed for detection of T. pallidum. An evaluation of the method's performance was done with a commercially produced DNA control with regards to sensitivity, specificity detection limit and precision. When all test where verified and established, the method was tested on clinical material from ulcers. The results of the T. pallidum-specific PCR-assay showed that there was no cross-reactivity to other agents that are expected to be in ulcers from patients with suspected syphilis. The method showed a sensitivity at dilution 1:100 (about 13 copies/μl) and a precision of 36.3 ± 0.8 Ct.The clinical specimen showed presence of T. pallidum, however, the sensitivity was not as good than the existing reference method. The method can be used in routine diagnostics of T. pallidum, however, the outcome of extraction of DNA from ulcers should be further studied in order to increase the yield and detect lower concentrations in clinical material.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that are necessary in memory and cognition. They are pentameric and consist of α and β subunits. They are most commonly heteromeric but, can sometimes be homomeric. nAChRs are activated by many ligands including nicotine (exogenous) and acetylcholine (endogenous).nAChRs are located on hippocampal interneurons. The interneurons, although sparse, control the synchronous firing of the pyramidal cells. However, the hippocampal interneuron structure and function is quite diverse and not fully characterized. Therefore, we sought to quantify nAChR subunit mRNA levels using real-time PCR of CA1 hippocampal interneurons.Surprisingly we found that the α3 and β2 mRNA subunits were the highest expressed and highest co-expressed subunits. Additionally, the α4 mRNA subunit was the lowest expressed of the subunits detected. The α4 subunit is one of the most pharmacologically targeted nAChR subunits and is found throughout the rest of the brain at much higher levels than the α3 mRNA subunit. Upon PCR analysis two subpopulations of the α3 and β2 subunits emerged: those that contained 3X more α3 than β2 and those that contained 3X more β2 than α3. Therefore, we hypothesized that two likely α3β2 nAChR stoichiometries are present in hippocampal interneurons. We differentiated their kinetic properties using electrophysiology.Additionally, like the α4 subunit, the α7 subunit is highly targeted in cognitive therapeutics. Since, the α7 subunit is the most characterized nAChR subunit, there are current efforts to develop allosteric modulators of the α7 subunit. The α7 subunit is found at moderate levels within hippocampal interneurons and remains a valid target. Current treatment options for Alzheimer's disease, and other dementias are limited and only mildly effective. Therefore, we sought to characterize the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2) on α7.Furthermore, there are no current methods to distinguish the α7 from the α7β2 nAChRs during whole cell electrophysiological recordings. Therefore, we also characterized the PAM-2 effect on α7β2 nAChRs. Our results highlight at least 2 ways PAM-2 can be used to differentiate α7 from the α7β2 during whole-cell recordings.

We were developing detection methods for three food allergens of plant origin. We used real-time PCR for soy detection in food oriented on gene lec, that is coding lektine specific for soy. On this target sequence were oriented PCR system with primers Le2F and Le2R and TaqMan probe Le2P. Detection limit (2,75 pg), practical detection limit (0,02 %), inclusivity and exclusivity were determined. Whole system were quantified. Real-time PCR for pistachio detection were based on primers and probe for gene COR. Detection limit (3,5 pg), practical detection limit (0,002 %), inclusivity and exclusivity were determined. For almond detection we were not succeed system, that fulfil all qualitative parametres.

In der vorliegenden Dissertation sollte der Krankheitsverlauf einer KHV-Infektion untersucht werden sowie der Einfluss den Haltungsbedingungen von Nutzkarpfen auf dessen Krankheitsverlauf (KHV-Disease; KHVD) haben. Zusätzlich sollte untersucht werden, ob Plötzen (Rutilus rutilus), Schleien (Tinca tinca) und Karauschen (Carassius carassius) als potentielle Carrierfische, ebenso als empfängliche Spezies eine Rolle spielen können.

(anglický jazyk) The most common senile dementia, Alzheimer disease (AD), is characterized by a decline of memory and high cognitive functions. Typical post-mortem brain lesions are extracellular amyloid deposits, intracellular neurofibrilary tangles and ruined cholinergic and other neurotransmitters systems. Connection between damaged central cholinergic system and beta-amyloid accumulation remains obscure. We examined parietal cortex of young adult (7- month-old) female APPswe/PS1dE9 double transgenic mice which develope beta-amyloid fragments at high rate. Cholinergic synapses of these mice demonstrate functional presynaptic (stimulated acetylcholine release) as well as postsynaptic (muscarinic receptor-induced G- protein activation) deficits and reduction of cholinergic markers. The mRNA levels of choline acetyltransferase, vesicular acetylcholine transporter and M1 to M4 subtypes of muscarinic receptors were determined in transgenic and littermate controls using qPCR. Obtained experimental data does not show any changes in measured mRNA levels. These observations indicate that reduction of cholinergic synaptic markers and function is due to posttranscriptional events.

The work is focused on the observation of the effect of selenium nanoparticles [Se] on seedlings of head cabbage, whose seeds were inoculated with Xanthomonas campestris pv campestris (Pammel) Dowson. The paper summarizes protocols of nanoparticle synthesis using various methods. A special part was devoted to methods of synthesis of selenium [Se] nanoparticles. Methods of conventional synthesis and methods of biological synthesis have been described. When using a preparation containing nanoparticles, the effect on bacteria inoculated on seed was clearly seen. Increasing concentration of the product visibly appeared to be stronger. The use of SelenBact appears to be very promising when applied to seed contaminated with bacteria Xanthomonas campestris pv campestris (Pammel) Dowson.

Master’s thesis DNA markers for bacterial infection diagnosis engages in matters of pathogenic bacteria that cause urinary tract infections and in matters of diagnostic methods for this bacteria with focus on the usage of DNA probes. The aim of this thesis was to find, design and test suitable DNA probes by using quantitative Real-Time PCR. Probes were designed based on specific bacterial sequences of 16S rRNA gene fragment. Afterwards, testing and optimisation of Real-Time PCR was conducted. Successful optimisation was managed for two probes out of twelve – one for S. aureus, second for P. aeruginosa. These results represent the basis for the follow-up experimental work to ensure improvement and reproducibility of the experiments.

Quantification of human DNA in forensic samples is an important step during STR profiling because the STR genotyping is sensitive to the quantity of DNA used in the PCR reaction. This study focuses on the importance of quantification in the entire process of genetic analysis. Two real time PCR platforms (Roche LightCycler480 System and ABI 7900 RT PCR) were used to compare two commercial kits in terms of DNA quantification. It was found out that accuracy of absolute quantification values in commercial quantification kits is strongly dependent on the construction of calibration curve. Especially low template DNA samples were used to assess whether QuantifilerTM or Plexor® HY System can determinate a minimum quantification value (cut off value) below which STR profiles would consistently fail to be detected. The usage of Plexor® HY System enabled to determine the cut off quantification value more exactly probably due to different molecular background and chemistry used in this kit. Reliability and other issues connected with cut off value are discussed. In order to better understand the relationship between the quantity of DNA and the number of detectable loci series the dilution experiment with standard DNA007 was done. Quantitative and qualitative consequences of input DNA amount in evaluation of...