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<p>Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials.</p><p>Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.</p>

Crown gall disease is an agricultural problem caused by the soil-borne bacterium, Agrobacterium tumefaciens. A. tumefaciens oncogenes cause transformed plant cells to overproduce the hormones, auxin and cytokinin. High hormone levels cause unorganized plant cell growth resulting in a gall. Control of crown gall disease is difficult because after plant cell transformation has occurred, the bacterium is no longer required for the disease to progress. Apple trees engineered to express double-stranded RNA of two A. tumefaciens oncogenes, ipt and iaaM, silenced the expression of the wild-type oncogenes and prevented crown gall disease. Only the iaaM oncogene was targeted for posttranscriptional gene silencing (PTGS) as measured by biological assays and by quantitative reverse-transcription polymerase chain reaction (q-RTPCR) on transgenic tissue. However, if the translation initiation sequence of the iaaM construction was eliminated, gall formation was not prevented, indicating that translatable RNA initiates silencing whereas untranslatable RNA does not. Other data indicate that the Arabidopsis thaliana micro-RNA pathway gene is involved in A. tumefaciens-mediated tumorigenesis. A. thaliana plants with a mutation in HEN1, a gene required for micro-RNA maturation, demonstrated a tenfold reduction in tumorigenesis upon A. tumefaciens infection compared to wild-type. The same plant line showed no difference in T-DNA transfer and nuclear uptake. / Graduation date: 2006

Port-Orford-cedar (Chamaecyparis lawsoniana) is a forest tree native to a small area of Oregon and California. A root disease caused by Phytophthora lateralis causes widespread mortality of Port-Orford-cedar. This dissertation examines three important elements of the Port-Orford-cedar P. lateralis pathosystem related to breeding for disease resistance: use of resistant rootstocks to maintain genotypes of Port-Orford-cedar for breeding; the heritability and genetic basis of disease resistance; and variability in virulence and DNA fingerprint among a sample of P. lateralis isolates. Port-Orford-cedar was reciprocally grafted to western redcedar (Thuja plicata), incense cedar (Calocedrus decurrens), and Alaska yellow-cedar (Chamaecyparis nootkatensis). Port-Orford-cedar scion graft success was moderate with western redcedar and incense cedar, but extreme overgrowth of the rootstock by the scion indicated incompatibility. Xylem union was good, but phloem union was incomplete or lacking. Nearly all Port-Orford-cedar rootstocks and seedlings exposed to P. lateralis died of root disease. Four percent of the Alaska yellow-cedar exposed also died, confirming this tree as a host for P. lateralis. Resistance of Port-Orford-cedar to P. lateralis is rare. A small number of trees have been identified exhibiting resistance. A number of families were tested to determine the genetic basis for resistance. Estimates of narrow-sense and family mean heritability of resistance, as exhibited by restriction of lesion length after inoculation, were determined. Both narrow-sense and family mean heritabilities were between 0.61 and 0.98 in most tests. Between 21% and 32% of the variance was due to differences among families. Thirteen isolates of P. lateralis were collected from three hosts throughout the geographic range of the fungus. Variation in growth rate on artificial media at three temperatures, virulence when used to inoculate Port-Orford-cedar, and DNA fingerprint were compared. There were significant differences in growth rate among isolates at 24C, but fewer differences at lower temperatures and on a rich medium. One isolate produced significantly shorter lesions in three different inoculation tests. Isolates differed at only two of 189 bands produced by Inter Simple Sequence Repeat (ISSR) DNA primers, indicating very little genetic variation among isolates. / Graduation date: 2001

In den letzten 20 Jahren hat sich der Maiszünsler (<i>Ostrinia nubilalis HÜBNER</i>), aus der Schmetterlingsfamilie der Pyralidae oder Zünsler, zum bedeutendsten tierischen Schädling des Maises (<i>Zea mays</i>) entwickelt. Eine Möglichkeit den Befall des Maiszünslers abzuwenden, bietet der Anbau von <i>Bacillus thuringiensis</i>-Mais (Bt-Mais). Mit Hilfe der Gentechnik wurden Gene des Bakteriums <i>Bacillus thuringiensis</i> übertragen, die einen für Fraßinsekten giftigen Wirkstoff bilden, wodurch die Pflanzen während der kompletten Vegetation vor den Larven des Maiszünslers geschützt sind.<br><br> Ziel des vorliegenden Projektes war es, in einer 3-jährigen Studie die Auswirkungen des großflächigen Anbaus von Bt-Mais auf die ökologische Situation und den Handlungsrahmen des integrierten Pflanzenschutzes komplex zu untersuchen. Dazu wurden in Betrieben im Oderbruch, das als permanentes Befallsgebiet des Maiszünslers gilt, in den Jahren 2002 bis 2004 jährlich zwei Felder mit jeweils einer Bt-Sorte und einer konventionellen Sorte angelegt. Zusätzlich wurden biologische und chemische Maiszünsler-Bekämpfungsvarianten geprüft.<br><br> Durch verschiedene Methoden wie Bonituren, Ganzpflanzenernten, Bodenfallenfänge und Beobachtungen des Wahlverhaltens von (Flug-)insekten konnten Aussagen zum Vorkommen von Insekten und Spinnentieren getroffen werden, wobei hierfür Daten aus Untersuchungen der Jahre 2000 und 2001 im Oderbruch ergänzend herangezogen werden konnten. Durch Ertragsmessungen, Energie- und Qualitätsermittlungen, sowie Fusarium- und Mykotoxinanalysen konnte der Anbau von Bt-Mais als neue Alternative zur Bekämpfung des Maiszünslers bewertet werden.<br><br> Bezüglich des Auftretens von Insekten und Spinnentieren wurden im Mittel der fünfjährigen Datenerhebung beim Vergleich der Bt-Sorte zur konventionellen Sorte, mit Ausnahme der fast 100 %igen Bekämpfung des Maiszünslers, keine signifikanten Unterschiede festgestellt. Hierfür wurde ein besonderes Augenmerk auf Thripse, Wanzen, Blattläuse und deren Fraßfeinde, sowie mittels Bodenfallenfängen auf Laufkäfer und Spinnen gerichtet.<br><br> Die erwarteten ökonomischen Vorteile wie etwa Ertragsplus oder bessere Nährstoff- und Energiegehalte durch geringeren Schaden beim Anbau von Bt-Mais als Silomais blieben in den Untersuchungsjahren aus. Allerdings zeigten Fusarium- und Mykotoxinanalysen eine geringere Belastung des Bt-Maises, was möglicherweise auf den geringeren Schaden zurückzuführen ist, da beschädigte Pflanzen für Fusarium und Mykotoxine anfälliger sind.<br><br> Desweiteren konnten erste methodische Ansätze für ein auf EU-Ebene gefordertes, den Anbau von Bt-Mais begleitendes Monitoring, erarbeitet werden. So konnten Vorschläge für geeignete Methoden, deren Umfang sowie des Zeitpunktes der Durchführungen gemacht werden. / In the last 20 years the European corn borer (<i>Ostrinia nubilalis</i>, <i>Pyralidae</i>) has become the most important pest in maize (<i>Zea mays</i>). One of a couple of possibilities to reduce the infestation by the European corn borer is the cultivation of <i>Bacillus thuringiensis</i> maize (Bt maize). Genetic engineering transmitted genes from <i>Bacillus thuringiensis</i>, which produce a substance that is toxic to feeding insects and thus protect plants against the larvae of the European corn borer during the whole vegetation.<br><br> The present project is a 3-year study to identify the effects of Bt maize growing on the ecological situation and the possibilities of integrated plant protection. From 2002 to 2004, two fields in the Oderbruch region, where Ostrinia nubilalis occurs, were each planted with Bt maize and a conventional maize variety every year. Furthermore, a biological and a chemical strategy against the European corn borer were verified.<br><br> Different methods like counts, harvest of whole plants, pitfall traps and observation the landing behaviour of flying insects were used to determine the abundance of insects and spiders. Furthermore, we could use additional data from studies obtained in the Oderbruch region in 2000 and 2001. The determination of yield, quality and energy content of the crops as well as of the degree of Fusarium infection and contamination by mycotoxins led to the conclusion that the cultivation of Bt maize is a new alternative strategy to control the European corn borer.<br><br> The average occurrence of insects and spiders did not differ significantly between Bt maize and the conventional variety in the 5 years of data recording. The only exception is the almost total control of the European corn borer. Attention was especially paid to thrips, bugs, aphids and their feeding enemies and using ground traps to ground beetles and spiders.<br><br> The expected economic benefits like increased yield or nutrient and energy content of the crop as a result of a minimized damage to silage Bt maize were not achieved in the years under investigation. However, the analysis of Fusarium and mycotoxins indicated a lower exposure of Bt maize, which may result from a lower damage caused by Ostrinia nubilalis, and damaged plants are more susceptible to Fusarium and mycotoxins.<br><br> Furthermore, we developed a first methodological approach for the monitoring procedure of Bt maize growing required by the EU. We have made proposals on appropriate methods, their extent as well as the optimum time of their application.

Regulation of gene expression occupies a central role in the control of the flow of genetic information from genes to proteins. Regulatory events on multiple levels ensure that the majority of the genes are expressed under controlled circumstances to yield temporally controlled, cell and tissue-specific expression patterns. The combined set of expressed RNA transcripts constitutes the transcriptome of a cell, and can be analysed on a large-scale using both sequencing and microarray-based methods. The objective of this work has been to develop tools for analysis of the transcriptomes (methods), and to gain new insights into several aspects of the stem cell transcriptome (applications). During recent years expectations of stem cells as a resource for treatment of various disorders have emerged. The successful use of endogenously stimulated or ex vivo expanded stem cells in the clinic requires an understanding of mechanisms controlling their proliferation and self-renewal. This thesis describes the development of tools that facilitate analysis of minute amounts of stem cells, including RNA amplification methods and generation of a cDNA array enriched for genes expressed in neural stem cells. The results demonstrate that the proposed amplification method faithfully preserves the transcript expression pattern. An analysis of the feasibility of a neurosphere assay (in vitro model system for study of neural stem cells) clearly shows that the culturing induces changes that need to be taken into account in design of future comparative studies. An expressed sequence tag analysis of neural stem cells and their in vivo microenvironment is also presented, providing an unbiased large-scale screening of the neural stem cell transcriptome. In addition, molecular mechanisms underlying the control of stem cell self-renewal are investigated. One study identifies the proto-oncogene Trp53 (p53) as a negative regulator of neural stem cell self-renewal, while a second study identifies genes involved in the maintenance of the hematopoietic stem cell phenotype. To facilitate future analysis of neural stem cells, all microarray data generated is publicly available through the ArrayExpress microarray data repository, and the expressed sequence tag data is available through the GenBank. / QC 20100927

<p>Disruption of coordinated impulse propagation in the heart as a result of fibrosis or myocardial infarction can create an asynchronous substrate with poor conduction and impaired contractility. This can ultimately lead to cardiac failure and make the heart more vulnerable to life-threatening arrhythmias and sudden cardiac death. The transplantation of exogenous cells into the diseased myocardium, "cardiac cell therapy," has been proposed as a treatment option to improve compromised cardiac function. Clinical trials of stem cell-based cardiac therapy have shown promising results, but also raised concerns about our inability to predict or control the fate of implanted cells and the electrical consequences of their interactions with host cardiomyocytes. Alternatively, genetically engineered somatic cells could be implanted to selectively and safely modify the cardiac electrical substrate, but their unexcitable nature makes them incapable of electrically repairing large conduction defects. The objective of this thesis was thus to develop a methodology to generate actively conducting excitable cells from an unexcitable somatic cell source and to demonstrate their utility for studies of basic electrophysiology and cardiac cell therapy.</p><p>First, based on the principles of cardiac action potential propagation, we applied genetic engineering techniques to convert human unexcitable cells (HEK-293) into an autonomous source of excitable and conducting cells by the stable forced expression of only three genes encoding an inward rectifier potassium (Kir2.1), a fast sodium (Na_v1.5), and a gap junction (Cx43) channel. Systematic pharmacological and electrical pacing studies in these cells revealed the individual contributions of each expressed channel to action potential shape and propagation speed. Conduction slowing and instability of induced arrhythmic activity was shown to be governed by specific mechanisms of I_Na inhibition by TTX, lidocaine, or flecainide. Furthermore, expression of the Na_v1.5 A1924T mutant sodium channel or Ca_v3.3 T-type calcium channel was utilized to study the specific roles of these channels in action potential conduction and demonstrate that genetic modifications of the engineered excitable cells in this platform allow quantitative correlations between single-cell patch clamp data and tissue-level function.</p><p>We further performed proof-of-concept experiments to show that networks of biosynthetic excitable cells can successfully repair large conduction defects within primary excitable tissue cultures. Specifically, genetically engineered excitable cells supported active action potential propagation between neonatal rat ventricular myocytes (NRVMs) separated by at least 2.5 cm in 2-dimensional and 1.3 cm in 3-dimensional cocultures. Using elastic films with micropatterned zig-zag NRVM networks that mimicked the tortuous conduction patterns observed in cardiac fibrosis, we showed that electrical resynchronization of cardiomyocyte activation by application of engineered excitable cells improved transverse conduction by 370% and increased cardiac twitch force amplitude by 64%. This demonstrated that despite being noncontractile, engineered excitable cells could potentially improve both the electrical and mechanical function of diseased myocardial tissue. </p><p>Lastly, we investigated how activation and repolarization gradients at the interface between cardiomyocytes and other excitable cells influence the vulnerability to conduction block. Microscopic optical mapping of action potential propagation was used to quantify dispersion of repolarization (DOR) in micropatterned heterocellular strands in which either well-coupled or poorly-coupled engineered excitable cells with a short action potential duration (APD), seamlessly interfaced with NRVMs that had a significantly longer APD. The resulting electrical gradients originating from the underlying heterogeneity in intercellular coupling and APD dispersion were further manipulated by the application of barium chloride (BaCl2) to selectively prolong APD in the engineered cells. We measured how the parameters of DOR affected the vulnerable time window (VW) of conduction block and found a strong linear correlation between the size of the repolarization gradient and VW. Reduction of DOR by BaCl2 significantly reduced VW and showed that VW correlated directly with dispersion height but not width. Conversely, at larger DOR, VW was inversely correlated with the dispersion width but independent of the dispersion height. In addition, despite their similar APDs, poorly-coupled excitable cells were found to significantly increase the maximum repolarization gradient and VW compared to well-coupled excitable cells, but only at larger DOR.</p><p>In summary, this thesis presents the novel concept of genetically engineering membrane excitability and impulse conduction in previously unexcitable somatic cells. This biosynthetic excitable cell platform is expected to enable studies of ion channel function in a reproducible tissue-level setting, promote the integration of theoretical and experimental studies of action potential propagation, and stimulate the development of novel gene and cell-based therapies for myocardial infarction and cardiac arrhythmias.</p> / Dissertation

Analysis of genetic relatedness is of great importance in forensic casework such as immigration and identification cases. The conventional methods for relationship testing are not sufficient in the most complicated cases, because more genetic markers are required to obtain results with satisfactory statistical security. This study demonstrates that microarrays, which can be used to genotype thousands of single nucleotide polymorphisms (SNPs), could be a promising solution to this problem. The microarray technique used in this study performed very well on blood samples and also worked well in combination with whole genome amplification, but did not generate any results when used on severely degraded materials. Markers suitable for relatedness analysis were selected from the microarray and were successfully tested on families with known genetic relations. Although a maximum of 64 autosomal markers were used, there is a great potential of selecting the hundreds or thousands of markers that may be required in some cases of relatedness investigation.

Vaginitis is a vaginal infection which affects many women all over the world. The disorder is characterized by an infection of the vaginal area which can cause problems like abnormal vaginal discharge, itching and redness. The two most common causes of vaginitis are bacterial vaginosis and Candida vaginitis. The prevalence of bacterial vaginosis in Sweden is around 10-20 % and approximately 75 % of all women will once in their lifetime suffer from vaginal yeast infection. The clinical symptoms of vaginal infections are not specific and the diagnosis methods of bacterial vaginosis and Candida vaginitis are subjective and depended on the acuity of the clinician. Due to the lack of standardized and objective diagnostic tools, misdiagnosis and consequently incorrect treatment may occur. As vaginal infections and symptoms impact greatly of women´s quality of life and vaginitis have been associated with serious public health consequences, it is essential to diagnose and treat the conditions correctly. Hence, there is a great need of better methods of diagnosing these conditions. The aim of this master thesis was to develop quantitative species-specific real-time PCR assays to use in diagnosing the two most common causes of vaginitis i.e. bacterial vaginosis and Candida vaginitis. Potential markers for bacterial vaginosis (Atopobium vaginae, BVAB2, Gardnerella vaginalis, Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus iners, Megasphaera type 1, Megasphaera type 2, Mobiluncus curtisii, Mobiluncus mulieris and Leptotrichia/Sneathia species) and Candida vaginitis (Candida albicans, Candida glabrata, Candida parapsilosis and Candida tropicalis) were chosen. Primers and probes were designed and tested on reference strains and vaginal samples. Single- and multiplex PCR reactions were successfully optimized with the designed oligonucleotides. Furthermore, standard curves with excellent linearity were created and covered more than five orders of magnitude. These developed quantitative species-specific real-time PCR assays will, in a prospective medical validation, quantify 300 vaginal samples from women visiting the RFSU Clinic in Stockholm.

Fenugreek (Trigonella foenum-graecum L.) is a medicinal plant with potential applications in the natural health product industry. In a multi-environmental setting, 10 genotypes were tested across 14 growing environments (using a Randomized Complete Block Design), representing irrigated and rainfed growing conditions in southern Alberta, Canada over two cropping years (2006 and 2007). The objectives of this study were (1) to determine seed yield, plus content and productivity of selected bioactive compounds (galactomannan, diosgenin and 4-hydroxyisoleucine), (2) to assess the impact of growing environment on these variables and (3) to identify promising genotypes for breeding and industrial use. Using principal component and cluster analyses, the study provides insight on the relative influence of growing environments and genes on the biochemical and agronomical traits as well as identifies genotypes based on performance and stability. These are useful as parental materials in cultivar development for the Canadian natural health product industry. / xiii, 154 leaves : ill. (some col.) ; 29 cm