No description available.

Wing polyphenism in insects is a type of phenotypic plasticity where environmental factors trigger the development of a set of discrete wing morphologies. In the water strider Gerris buenoi, photoperiods are the main environmental cue that trigger wing morph determination. The genetic mechanisms connecting environmental cues and the determination of wing morph in G. buenoi are not clear. However, recent experimental work suggests that engagement of the Hippo pathway via ecdysone signalling is a promising model for further investigation. In this study, a reverse-transcription quantitative PCR (RT-qPCR) protocol was developed, aimed at elucidating this potential transduction pathway by quantifying gene expression of Fat, Dachsous, Yorkie, EcR, E75 and E74. This was done using melt curve analysis, gel electrophoresis, sequencing of RT-qPCR products and qPCR standard curves. Additionally, wing morph distribution in extreme and intermediate photoperiods were examined. Wing morph proportions were significantly different between adults emerging in the intermediate photoperiods 15.30:8.30 and 15:9 (hours light : hours dark). An effect of sex was observed, with a higher probability of males becoming long-winged compared to females. This has likely evolved as a result of a dispersal-reproduction trade-off. Taken together, this study provided insight for future investigations of periodically induced wing morph determination and its genetic mechanisms in G. buenoi that will contribute to the understanding of phenotypic plasticity.

No description available.

This term project deals with the problem of scheduling real-time tasks in overload conditions and techniques for lowering power consumption. Each of these parts features mechanisms and reasons for their using. There are also described specific algorithms, that are implemented, in operating system uC/OS-II, and compared in next phase of master's thesis.

Die Therapie der HIV Infektion besteht aus Kombination mehrerer antiretroviraler Substanzen und birgt ein erhöhtes Risiko an Arzneimittelwechselwirkungen. Das bekannte Problem der Virusresistenz kann zudem durch Enzyminduktion begünstigt werden. Das Ziel der vorliegenden Arbeit lag in Untersuchungen zu Einflüssen der Virustatika auf die Expression von Cytochrom P450 Enzymen: 1A1, 1B1, 3A4 sowie der P-Glykoproteins (MDR1) an immortalisierten Zellsystemen. Die Protease Inhibitoren Indinavir, Nelfinavir, Ritonavir und Saquinavir induzierten die Regulation der mRNA Expression über den Aryl-Kohlenwasserstoff-Rezeptor (AhR) und den Pregnan-X-Rezeptor (PXR) dosisabhängig und signifikant. Die Nukleosidischen Reverse Transkriptase Inhibitoren Zalcitabin, Zidovudin und Lamivudin sowie der Nicht-Nukleosidische Inhibitor Nevirapin zeigten induktive Eigenschaften nur für die AhR Zielgene CYP1A1 und CYP1B1. Amprenavir und Efavirenz aktivierten die PXR-Regulation. Die möglichen Auswirkungen der Induktion der untersuchten Gene wurden ausführlich diskutiert. Die molekularen Grundlagen der interindividuell variierenden Aktivität von CYP3A wurden in einer Probandenstudie untersucht. Es wurden die mRNA Expression in den Leukozyten, die Aktivität des Enzyms und einige bekannte Polymorphismen unter Einwirkung von Rifampicin untersucht und diskutiert. / The therapy of HIV infection requires a combination of several antiretroviral substances accompanying risk factors for drug-drug interactions. Moreover, virus resistance can be promoted by enzyme induction caused by antiretroviral drugs. The aim of the study was to investigate the influences of antiretroviral substances on the expression of cytochrome P450 enzymes: 1A1, 1B1, 3A4 and p-glycoprotein (MDR1) using immortalized cell systems. The protease inhibitors indinavir, nelfinavir, ritonavir and saquinavir induced significantly the regulation of mRNA expression through the aryl hydrocarbon receptor (AhR) and the pregnane-x-receptor (PXR) in a concentration-dependent manner. The nucleoside reverse transcriptase inhibitors zalcitabine, zidovudine and lamivudine and the non-nucleoside reverse transcriptase inhibitor nevirapine showed inductive properties only for the AhR target genes CYP1A1 and CYP1B1.Amprenavir and efavirenz activated the PXR target genes. Potentially effects of the described induction are discussed. In a second part of the work, the molecular mechanisms of the individual varying activity of the CYP3A enzyme were investigated applying an in vivo study. CYP3A4 mRNA expression and rifampicin mediated induction in leucocytes were correlated with systemic enzyme activity under induction and known polymorphisms.

Nicotine exerts a therapeutic effect in ulcerative colitis (UC) but the mechanism underlying this effect, is not clear. However, this effect may imply that nicotine has some, as yet to be discovered, effect on the immune system. The aim of the work described in this thesis was to characterise the nicotinic acetylcholine receptors (nAChRs) on human peripheral blood lymphocytes in term of receptor subtype. To achieve this, a combination of radioligand binding assays, pharmacological and molecular biological techniques were used. The data obtained from the binding studies suggested that the presence of one binding site for (-)- nicotine on human peripheral blood lymphocytes with a Kd 15 ± 5.759 nM (1.5 ± 5.759 x 10-8 M) and Bmax 2253 ± 409 sites/cell. The competition studies showed that ligands competing with [3H]-(-)-nicotine were (-)-nicotine, epibatidine and α-bungarotoxin, while others ligands for nAChRs displaced radiolabelled nicotine in insignificant quantities. Thus, radioligand-binding experiments suggest that the binding site for nicotine on human peripheral blood lymphocytes is a nAChR containing α7 and possibly α4 or/and b2 containing nAChR subunits. No evidence was obtained to suggest the presence of a non-cholinergic nicotine receptor. Furthermore, considerable subject to subject variation in the specific binding of radiolabelled nicotine was observed. Because of this only tentative conclusions could be drawn from radioligand binding data. Polymerase chain reaction (RT-PCR) was then used to demonstrate mRNA for the subunits of nAChRs suggested by radioligand binding studies. Data obtained show that the human peripheral blood lymphocytes tested, expressed mRNAs for α4, α5, α7, β2 neuronal nAChRs subunits and β1 muscle nAChR subunit. Expression of the α5 mRNA subunit of nAChR was observed in the lymphocytes in each sample of lymphocytes tested. In contrast, the expression pattern of mRNAs for α4, α7, β1, and β2 mRNAs subunits of nAChRs, varied between individuals. Finally, Western blot analysis was used to confirm that mRNA expression resulted in the expression of protein for nAChR subunits in human peripheral lymphocytes using monoclonal antibodies against α4, α5, α7, and β2 nAChR subunits, which had been detected by RT-PCR. The results obtained from the Western blot analysis show that protein for α4, α5, and α7 nAChR subunits was expressed in most, but not all of the human peripheral blood lymphocyte samples tested and some of the bands obtained were faint. In contrast, protein for the β2 nAChR subunit was observed in a few samples tested and the bands were faint. From the results obtained in this study, it is possible to conclude that human peripheral blood lymphocytes may contain nAChRs with subunit compositions of α4β2, α4β2α5, and/or α7. However, further studies are necessary to show whether or not the single binding site for nicotine demonstrated by radioligand binding experiments is due to one or all of these nAChRs. Thus, the findings of the present study suggest the presence of nAChR on human peripheral blood lymphocytes. Nicotine and its effect may occur through these non- neuronal nAChRs mechanisms. Such a mechanism of action could account for the beneficial of nicotine in ulcerative colitis. Furthermore, a compound that acts on these receptors, but not on nAChRs found on other cells may have therapeutic utility in the treatment of inflammation.

Le Cancer du Col Utérin (CCU) chez la femme est provoqué par le virus oncogénique VPH. La métastase lymphatique ganglionnaire est un facteur pronostique majeur pour l’évolution de ce cancer et sa présence influence la décision thérapeutique. En général, l’envahissement ganglionnaire est diagnostiqué par histologie, mais cette méthode est laborieuse et parfois prise en défaut pour détecter les micrométastases et les cellules cancéreuses isolées et pour donner des résultats rapides en per opératoire. L’outil moléculaire que nous désirons développer pour combler cette lacune est basé sur une analyse d’ARN des gènes du VPH exprimés par les cellules du CCU. Ceci sera fait par transcription réverse de l’ARN cellulaire couplé à une réaction quantitative en chaine par polymérase en temps réel (RT-qPCR). Cette technique devrait nous permettre une détection et une évaluation rapide des micrométastases pour aider à déterminer immédiatement un pronostic fiable et la thérapie associée. C’est un test précis, sensible et rapide pour détecter un envahissement ganglionnaire dans le CCU visant à améliorer la gestion thérapeutique. Le projet est basé sur trois objectifs. En premier lieu, valider les marqueurs moléculaires E6 et E7 de VPH16 et 18 à partir des échantillons frais et des échantillons fixés dans des blocs de paraffine. En deuxième lieu, déterminer la fiabilité et la sensibilité des marqueurs pour la détection des macrométastases, des micrométastases et les cellules tumorales isolées en utilisant la technique de RT-qPCR. En troisième lieu et parallèlement au travail présenté dans ce mémoire, il est nécessaire de constituer une base de données des patientes qui ont le virus VPH16 et 18 intégré dans leur génome, qui ont été traitées et dont nous connaissons déjà le diagnostic final afin de valider la méthode (biobanque). Nous avons réussi à extraire de l’ARNm de haute qualité à partir d’échantillons complexes, à détecter les gènes E6 et E7 de VPH16 et 18 en RT-qPCR, et à déterminer précisément la limite de détection de E6 et E7 dans les échantillons frais qui est une proportion de 0,008% de cellules cancéreuses. Dans les échantillons fixés dans la paraffine, cette limite est de 0,02% et 0,05% pour E6-E7-VPH16 et E6-E7-VPH18 respectivement. Ceci comparativement à une limite de détection histologique de 1% qui est déterminée par immunohistochimie de CK19. Enfin, notre protocole est validé pour VPH18 dans les ganglions lymphatiques du CCU. / The presence of lymph nodes metastasis in uterine cervical carcinoma influences therapeutic management and patient survival. The gold standard for metastasis detection is histology. However, histology lacks sensitivity to detect micrometastasis or isolated cancer cells and is not an efficient method for immediate diagnosis during surgery. The molecular tool that we want to develop to fill this gap is based on an analysis of expressed RNA transcripts derived from the HPV genome in cells of uterine cervical carcinoma (UCC). This will be done by reverse transcription of cellular RNA coupled to a quantitative polymerase chain reaction in real-time (RT-qPCR). This technique could allow detection and rapid assessment of micrometastasis to help determine prognosis and an immediate reliable combination therapy. The proposed technique would be a specific test, sensitive and rapid to detect lymph node involvement in the UCC to improve therapy management. Our objective is to constitute a patient bank containing genetic and clinical information. This genetic information will be used to test and improve new molecular markers for UCC metastasis. These markers will be validated using comparisons to traditional histological results and evaluated for their capacity to detect lymph nodes micrometastasis. Ultimately, we wish to develop a reliable molecular diagnosis method useful during surgery and improve our knowledge about the clinical evolution of metastatic UCC. Currently, we are able to extract high quality mRNA from formalin-fixed cells mounted in paraffin blocks and to detect E6 and E7 from HPV16 and HPV18 using RT-qPCR. We have specifically determined the detection limit of E6 and E7, which is 0.008% in the fresh samples and 0.02% and 0.05% for HPV16-E6-E7 and HPV18- E6-E7 respectively in the samples fixed in paraffin blocks. Comparatively, the histological detection limit was determined to be around 1% using immunohistochemistry for CK19 expression. Finally, our protocol has been validated for HPV18 in UCC patient lymph nodes

<p>Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated.</p><p><i>Nostoc punctiforme</i> PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by <i>hupSL</i>. The transcription of <i>hupSL</i> was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in <i>N. punctiforme</i>, the <i>hypFCDEAB</i> operon. These genes were found to be situated upstream of <i>hupSL</i> in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon.</p><p>The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of <i>N. punctiforme</i> was constructed, where <i>hupL</i> was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the <i>hupL</i>^- mutant and in wild type <i>N. punctiforme</i> was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup^- mutant of the related strain <i>Nostoc</i> PCC 7120 were also studied. </p><p>This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.</p>

Antibiotics are biologically active and are globally used in humans and animal medicine for treatment and in sub-therapeutic amounts as growth promoters in animal husbandry, aquaculture and agriculture. After excretion, inappropriate disposal and discharge from drug production facilities they enter into water bodies either as intact drugs, metabolites or transformed products. In water environments they promote development of antibiotic resistance genes (ARGs) which could serve as a reservoir and be horizontally transferred to human-associated bacteria and thus contribute to AR proliferation. Measurement of antibiotics has been revolutionized with the usage of solid phase extraction (SPE) for enrichment followed by Liquid chromatography mass spectrometry (LC-MS). On-line SPE coupled to LC-MS/MS has the advantages of high sample throughput, low sample preparation time and minimal solvent utilization.  Constructed wetlands (CWs) are potential alternatives to conventional treatment plants to remove organic pollutants. A study at Plönninge, Halmstad was performed to assess the impact of bacterial community pattern and development of resistance in spiked (n=4) and control (n=4). CWs were spiked with antibiotics at environmentally relevant concentrations continuously for 25 days. Shannon Index (H’) were used to determine the bacterial diversity and real-time PCR detected and quantified antibiotic resistance genes (ARGs) sulI, tetA, tetB, erm, dfrA1, qnrS and vanB and class 1 integrons intI1. No significant differences in bacterial compositions or in ARGs or integron concentrations could be discerned between exposed and control wetlands. A study conducted in Northern Pakistan showed that the antibiotic levels in most studied rivers were comparable to surface water measurements in unpolluted sites in Europe and the US. However, high levels of antibiotics were detected in the river in close vicinity of the 10 million city Lahore, e.g. 4600 ng L−1 sulfamethoxazole. Highest detected levels were at one of the drug formulation facilities, with measured levels up to 49000 ng L−1 of sulfamethoxazole for example. The highest levels of ARGs detected, sul1 and dfrA1, were directly associated with the antibiotics detected at the highest concentrations, sulfamethoxazole and trimethoprim. In the study in UK, sewage epidemiology surveillance is used to measure the oseltamivir carboxylate (OC), metabolite of oseltamivir (parent drug) in twenty four time proportional hourly influent samples from two WWTPs and then back-calculations were made to assess the compliance of drug.  Predicted users of oseltamivir, based on measured OC in waste water, ranged from 3-4 and 120-154 people for the two WWTP catchments, respectively, which are consistent with the projected use from national antiviral allocation statistics, 3-8 and 108-270, respectively. Scenario analysis suggests compliance was likely between 45-60% in the study regions.

Molecular hydrogen is a potential energy carrier for the future. Nitrogen-fixing cyanobacteria are a group of photosynthetic microorganisms with the inherent ability to produce molecular hydrogen via the enzyme complex nitrogenase. This hydrogen is not released, however, but is recaptured by the bacteria using an uptake hydrogenase. In this thesis, genes involved in cyanobacterial hydrogen metabolism were examined, and the possibility of employing genetically modified cyanobacteria for hydrogen production was investigated. Nostoc punctiforme PCC 73102 (ATCC 29133) is a nitrogen-fixing filamentous cyanobacterium containing an uptake hydrogenase encoded by hupSL. The transcription of hupSL was characterised, and putative regulatory elements in the region upstream of the transcription start site were identified. One of these, a binding motif for the global nitrogen regulator NtcA, was further investigated by mobility shift assays, and it was found that the motif is functional in binding NtcA. Also, a set of genes involved in maturation of hydrogenases was identified in N. punctiforme, the hypFCDEAB operon. These genes were found to be situated upstream of hupSL in the opposite direction, and they were preceded by a previously unknown open reading frame, that was found to be transcribed as part of the same operon. The potential for hydrogen production by filamentous cyanobacteria was investigated by studying mutant strains lacking an uptake hydrogenase. A mutant strain of N. punctiforme was constructed, where hupL was inactivated. It was found that cultures of this strain evolve hydrogen during nitrogen fixation. Gas exchange in the hupL- mutant and in wild type N. punctiforme was measured using a mass spectrometer, and conditions under which hydrogen production from the nitrogenase could be increased at the expense of nitrogen fixation were identified. Growth and hydrogen production in continuous cultures of a Hup- mutant of the related strain Nostoc PCC 7120 were also studied. This thesis advances the knowledge about cyanobacterial hydrogen metabolism and opens possibilities for further development of a process for hydrogen production using filamentous cyanobacteria.