Cette thèse traite le développement de dispositifs, basés sur la technologie "laboratoire sur puce"(LOC) qui visent à contrôler l'environnement des systèmes biologiques pour des applications macro et microbiologiques. En effet, les caractéristiques de la microfluidique permettent de manipuler l'environnement cellulaire à un niveau supérieur à celui du degré de contrôle atteignable avec les techniques ordinaires. Dans ce travail de thèse sera explorée la possibilité de profiter de ces fonctions afin de développer des outils de diagnostic peu coûteux et pourtant efficaces. En particulier, on rapporte le développement de systèmes microfluidiques permettant une perfusion des médias fluide et rapide, ainsi qu'une plateforme LOC capable de réaliser des PCRq hautement multiplexes. Au sujet des systèmes de perfusion, le but était d'obtenir une substitution du médium entourant les particules afin d'augmenter les capacités de séparation des modules de tri microfluidiques couplés. L'efficacité de notre approche a été validée par les hauts taux de séparation obtenus (>90%) avec l'utilisation de notre système de perfusion microfluidique couplé à une puce d'acoustophorèse. De plus, nous avons conçu et développé un système de thermalisation microfluidique capable d'opérer des changements de température en moins de 1s. Plus spécifiquement, cette plateforme exploite l'échange de chaleur entre un liquide de thermalisation qui circule dans une puce microfluidique et l'échantillon. Ces performances de thermalisation, et le rapport surface/volume élevé typique des appareils microfluidiques, ont permis d'effectuer 50 cycles de PCRq et l'analyse de courbe de fusion en moins de dix minutes. / The topic of this manuscript is the development of microdevices, based on "lab on chip" (LOC) technology, aimed to the environmental control and regulation of biological systems for macro and microbiological applications. Indeed, microfluidics possesses some inherent features which allow the manipulation of the environment at the cell and sub-cell level which are superior than the degree of control achievable with standard techniques. In this thesis work the possibility to leverage these features to develop inexpensive yet effective diagnostic tools is explored. In particular, we report the development of microfluidic systems which allow seamless and fast media perfusion and a novel LOC platform capable of performing highly multiplexed real-time PCR assays. Concerning the microfluidic perfusion systems, the aim was to achieve in-flow substitution of the particles' surrounding media in order to enhance the separation capabilities of the coupled microfluidic sorting modules. The effectiveness of our approach was validated by obtaining high separation purities (>90%) using our microfluidic perfusion system coupled with an acoustophoresis chip to discern two population of micro-sized beads. Moreover, we conceived and developed a microfluidic thermalisation system capable of sub-second temperature switches. Specifically, this platform relies on conductive heat exchange between a thermalisation liquid flowing inside a microfluidic chip and the biological sample. These thermalisation performances, and the high surface to volume ratio typical of microfluidic devices, allowed to perform 50 qPCR cycles and subsequent melting curve analysis in less than ten minutes.

Real-time or quantitative PCR (qPCR) is an innovative method used to determine the amount of amplifiable DNA in a biological sample. Typically, a fluorescent dye is introduced during thermal cycling, causing a change in fluorescent output as the double-stranded DNA (dsDNA) product accumulates. Both TaqMan® and Plexor HY System methods detect PCR inhibition through the monitoring of internal control sequences. Alternatively, SYBR®Green and Plexor detect inhibition through melt curve effects. Previous work using SYBR®Green intercalation has demonstrated that inhibitors can affect melt curves differently depending on their structure and mode of action. Inhibitors that bind DNA can cause melt curve shifts while those primarily affecting Taq polymerase do not. Unlike SYBR®Green, Plexor dyes are fluorescently linked to a modified base, 5'-methylisocytosine (iso-dC), adjacent to the 5' end of the dsDNA. This produces minimal interference in dsDNA structure making it an ideal procedure for measuring these effects. In this study, inhibition of qPCR was evaluated by observing the effects of various inhibitor concentrations and amplicon lengths on DNA amplification.

Taste-and-odor (T&O) compounds are frequently produced by cyanobacterial blooms in bodies of water. Geosmin, perhaps the most common T&O compound produced by these blooms, is not effectively removed by conventional water treatment processes and frequently causes the tap water to have an off flavor. Although geosmin is not harmful when ingested, it damages the consumers' confidence in the cleanliness of their water. There are treatment options for geosmin removal, but the most common methods are often not implemented until complaints are made by consumers.There has been an increasing amount of research on the use of polymerase chain reaction (PCR)-based methods that can detect the presence of the geosmin synthase gene which is responsible for the production of geosmin. If the geosmin synthase gene is found to be present in an emerging cyanobacterial bloom, water treatment facilities can prepare in advance to treat for geosmin. In this study, we developed a qPCR (quantitative polymerase chain reaction) assay that can detect the presence of the geosmin synthase gene in several species of cyanobacteria within the Anabaena genus. We tested our assay, as well as PCR assays designed by Giglio et al. (2008) and Suurnäkki et al. (2015) on extracted Anabaena flos-aquae DNA, biosynthesized Anabaena ucrainica DNA and DNA extracted from environmental samples of Deer Creek Reservoir, Strawberry Reservoir, and Utah Lake. It is important to note that the geosmin gene was not confirmed to be present in any of the environmental samples nor in the Anabaena flos-aquae DNA and our assay did not test positive on these samples. Our qPCR assay was very successful when used with the biosynthesized Anabaena ucrainica DNA. We used the results to estimate a DNA standard curve that can be used to estimate the starting concentration of the geosmin synthase gene. Because our assay was not successfully used with any extracted DNA, further testing and calibration may be necessary to produce a DNA standard curve that is representative of DNA that is extracted. Further calibration of the DNA standard curve was not done because there were no geosmin events during the course of the research.Development of PCR-based methods of detecting geosmin-producing cyanobacteria requires genetic sequencing information of the target-organisms. Thus, further development of PCR-based methods requires that the local geosmin-producers be identified and sequenced. Our assay as well as the assay designed by Moore (2019) can assist with the identification of these species by classifying their genus.

No description available.

The aim of this thesis is the study of the genetic substance of the important domestication sign pod dehiscence. Two types of pea were analyzed, with indehiscence pods JI92 (Pisum sativum subsp. sativum) and wild field pea with dehiscent pods JI64. (Pisum sativum subsp. elatius). By reciprocal crossbreeding of these two lines, were created recombinant inbred lines (RILs), of a total of 134 RILs lines were selected with 9 contrast lines. We utilized the massive parallel sequencing of the 3'ends of the cDNA, obtained by reverse transcription of mRNA isolated from the seam. Thanks to this method, 3 candidate genes were generated. Subsequently, we determined the expression of these three candidate genes for the using quantitative Real-Time PCR (RT-qPCR). Amplification curves and Ct values generated from the RT-qPCR were subsequently used to generate graphs to show the degree of expression of the candidate genes. The most suitable candidate was the Ps15 gene, which is present in LGIII in the Dpo1 region, and therefore could be responsible for pod dehiscence.

Maize (Zea mays) is globally considered as an important cereal crop, and a major staple food in developing countries such as Africa (WARD et al. 1999). In South Africa, maize is considered the most important grain crop as it is the main feed grain used for animals and a staple food for the population (FAO 2012). Maize can also be used for the production of maize-based ethanol, which can be used as a bio-fuel. In the USA, approximately 40% (11 million tonnes) of maize produced in 2012 was used for the production of bio-fuel (FAO 2012). Maize production in Africa was estimated to be less than two and on average 1.4 tons per hectare and remains below world average (FAO 2012). It was expected that South African crop production would decrease by approximately six percent during the 2012/2013 growing season as droughts during February and March 2013 in the North West and Free State provinces led to below-average maize yields in these production areas (FAO 2012; USDA 2013). Over the last few years maize production output has not been increasing together with the increasing population growth rate and thus puts pressure on commercial farmers to produce more maize for food security purposes and economical growth. The FAO states that agricultural production still needs to increase by up to 60% (80% in developing countries) within the next four years to be able to cope with an estimated global population growth of 39% by 2050. / Dissertation (MSc)--University of Pretoria, 2014. / National Research Foundation (NRF) / Plant Science / MSc / Unrestricted

No description available.

Bakgrund: För att barn inte ska behöva känna oro vid tumörterapi blir de nedsövda med anestetikum. Vid behandling av tumörer används joniserande strålning. Dock har tidigare studier funnit att exponering av joniserande strålning hos barn kan vara en bidragande faktor till kognitiva dysfunktioner. Man har också sett en synergistisk effekt mellan anestetikum och joniserande strålning. Syfte: Syftet med denna studie är att undersöka samverkanseffekten mellan olika anestetika och joniserande strålning. Metod: NMRI möss exponerades på postnatal dag 10. Totalt var det 6 exponeringsgrupper. Mössen exponerades antingen med ketamin (7,5 mg/kg) eller propofol (20 mg/kg). Individerna som exponerades för strålning (200 mGy) fick detta 1 timme efter administration av singeldos anestetikum. Provtagningen skedde 24 timmar efter exponering. Metoden qPCR användes för att undersöka uppregleringen och nedregleringen av gener. Resultat: En korrelation kunde observeras för generna Keap1 & Nrf2, BDNF & Grin2b och Jagged-1 & Hes5 genom att studera Spearmans korrelationskonstant. Inga signifikanta resultat erhölls för generna som studerades (p <0,05). Medelvärdena visade ingen signifikant förändring i relativ FC. Slutsats: Kombinationsbehandling med anestetikum + joniserande strålning visade inga större skillnader mellan exponeringsgrupperna i motsats till tidigare genomförda studier. Detta kan bero på tiden mellan exponering och provtagning. Det finns en korrelation mellan gener som har en teoretisk sammankoppling.

The objectives of this study were to determine: the effects of maternal melatonin (MEL) supplementation during late gestation on the histological and molecular regulation in the Longissimus dorsi (LM) muscle of fetal bovine offspring, composition and gene expression of fetal perirenal (PR) adipose tissue, and LM gene expression in postnatal offspring at birth and d 195 of age. Maternal supplementation of MEL during late gestation resulted in no difference in calf fetal body weight or birth weight. However, at d 195 of age, calves from MEL treated dams had an average body weight increase of 20 kg. Fetal LM weight and length tended to be increased in calves from MEL treated dams. Fetal gene expression of calves from MEL treated dams resulted in: increased LM adenosine monophosphate-activated protein kinase-α (AMPK) and decreased PR adiponectin (ADIPOQ), CCAAT enhancer binding protein alpha (CEBPA), proliferator activated receptor gamma (PPARg), and stearoyl-CoA desaturase (SCD). The improved metabolic status of LM coupled with the decrease in adipogenic gene expression, could result in calves from MEL treated dams having improved lean muscle accretion and reduced overall adiposity during postnatal development.

Background: Cyclosporine is an immunosuppressive agent used to treat immune-mediated and inflammatory diseases in dogs. We have developed a pharmacodynamic (PD) assay that measures interleukin-2 (IL-2) produced by activated T cells to measure the immunosuppressive effects of cyclosporine. Hypothesis/objectives: Our retrospective study extracted data from samples submitted to our laboratory to obtain descriptive statistics, to determine whether assay results predicted treatment effectiveness, and to determine whether cyclosporine formulation or breed affected PD responses. Animals: 1,110 samples were analyzed over 4 years. Methods: Extracted data was analyzed to determine whether there was a relationship between assay results and clinical control, and whether either formulation or breed affected results. Results: We found no relationship between assay results and control of signs, and found that breed did not affect results. At comparable doses, proprietary modified cyclosporine was more immunosuppressive than proprietary non-modified cyclosporine, and both proprietary and generic modified formulations had similar efficacy.