Die Breite der Wirkungen von Aldosteron auf Nierenzellen wurde lange Zeit unterschätzt. Inzwischen zeigte sich ein nicht unerheblicher Anteil des Hyperaldosteronismus an arterieller Hypertonie und ebenso mehren sich die Hinweise auf damit assoziierter erhöhter Inzidenz für maligne Entartung von Nierengewebe. In dieser Arbeit wurde der Effekt von Hyperaldosteronismus auf Nierenzellen von Ratten in vivo untersucht. Mittels real time quantitative PCR wurden die relative Expressionsveränderungen der mRNA von validierten Nierenschädigungsmarkern im Hyperaldosteronismusmodell kontrolliert beobachtet und statistisch ausgewertet. Anders als im analog durchgeführten Vorversuch mit DOCA an der Stelle von Aldosteron, ließ sich größtenteils kein über der natürlichen Streuung der Daten liegender, signifikanter Effekt der Nierenschädigung durch überhöhte Aldosteronspiegel nachweisen. Hierfür kommen vielfältige Gründe in Frage. Neben der technischen Variabilität, der Beschaffenheit der internen Kontrolle, potentiell vorhandenen Inhibitoren und der Qualität der mRNA, konnten eine Reihe von weiteren Gründen als Ursache für die Diskrepanz zu den Ergebnissen der mit DOCA behandelten Tiere ausgeschlossen werden. Neben der theoretischen Möglichkeit inter-methodischer Differenzen und sich daraus ergebender Variationen, sowie der noch weiter zu untersuchenden Rolle des Glukokortikoidrezeptors durch dessen variable gleichzeitige Aktivierung, ist die Interpretation im Sinne eines zu gering ausgeprägten Schädigungseffektes durch den Hyperaldosteronismus für den gewählten Stichprobenumfang naheliegend. Hiermit stimmt auch die Tatsache überein, dass der Effekt der Behandlung mit Aldosteron im Vergleich zur Behandlung mit DOCA von vorne herein deutlich geringer ausfallend erwartet wurde. / The broad spectrum of effects of aldosterone on renal cells has been underestimated for a long time. Meanwhile it has been shown that hyperaldosteronism has a considerable share of all cases of arterial hypertension, and the indications for an associated higher incidence of malignant transforming of kidney tissue are also increasing. The subject of this study was to investigate the effect of Hyperaldosteronism on kidney cells in rats. By means of real-time quantitative PCR, the change in the relative expression of mRNA of validated kidney cell damage markers in the hyperaldosteronism model were monitored and statistically evaluated under controlled conditions. In contrast to the previous pre-test with DOCA instead of aldosterone, a significant effect of renal impairment due to excessive aldosterone levels could not be detected. Numerous reasons are conceivable for that. In addition to the technical variability, the nature of the internal control, potentially present inhibitors and the quality of the mRNA, a number of further reasons could be excluded as a cause of the discrepancy with the results of the animals treated with DOCA. Besides the theoretical possibility of inter-methodical differences and resulting variations, as well as the role of the glucocorticoid receptor, which is still to be investigated, the closest interpretation is a damage effect too small to be detected by the given sample size. This is also in agreement with the fact that the effect of the treatment with aldosterone compared with the treatment with DOCA was expected to be significantly lower from the outset.

Im Rahmen dieser Arbeit wurden Fibroblasten und Keratinozyten, welche in vitro auf unterschiedlichen Scaffolds sowohl gemeinsam als auch in Monokulturen gezüchtet wurden, mittels Real-time PCR auf ihre Genausschüttung untersucht, um festzustellen wie sich die Unterlage auf die Genausschüttung auswirkt. Hierzu wurden die Proben sowohl auf die Genexpressionsmarker für die Basallamina Kollagen IV, Laminin 1 und 5 als auch auf die Genexpressionsmarker für die frühe Differenzierung Keratin K13 und K14 untersucht. Als Referenzgen wurde β-Actin ausgewählt, da dieses Gen in den Vorversuchen mit zwei weiteren Referenzgenen die stabilste Expression gezeigt hatte. Die Genexpressionsanalyse zeigte, dass nur in den Kokulturen von Keratinozyten und Fibroblasten eine ausgewogene Genexpression stattfindet, da sich die Zellen darin beeinflussen und regulieren. / Fibroblasts and keratinocytes were cultured in vitro on different scaffolds in monocultures and cocultures and examined by RT-qPCR for gene expression. Gene expression analysis was made for genes coding for basement Membrane collagen IV, laminin 1 and 5 and for early differentiation keratin K13 and K14. β-Actin was used as reference gene, because it showed in preliminary tests with two other reference genes most stable expression. Gene expression analysis showed only in cocultures of fibroblasts and keratinocytes balanced gene expression, because the two cell types affect and regulate each other.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

With advances in molecular technology, the study of human ageing has turned to DNA for answers as to how humans age. Due to the size of the human genome and the longevity of humans, organisms with smaller genomes and shorter lifespans have frequently been the center of research studies in ageing. Studies of Drosophila melanogaster, Caenorhabditis elegans, yeast, and mice have uncovered specific genes that up and down regulate with age and stress. Research has yet to produce, however, results from an organism known for its longevity. Amblyomma americanum is an excellent candidate for this, as it can survive for years unfed. Two groups of 75 unfed adult A. americanum were monitored in a control environment of 85% relative humidity and an experimental environment designed to induce physiological stress at 75% relative humidity. Five ticks were tested for transcript abundance of five candidate ageing genes initially and at the 25th, 75th, and 95th percent mortality. These tests provided evidence that Amblyomma americanum undergoes changes in gene expression with age on a genetic level.

Aging, characterized by a time-dependent functional decline, eventually results in the death of an organism. Unfortunately, this complex biological phenomenon is poorly understood. In order to dissect the molecular changes associated with aging, the identification and molecular characterization of the genes that regulate this universal process is absolutely necessary. The expectation is that the isolated genes potentially have human homologues and can be experimentally analyzed in Drosophila melanogaster in order to determine basic function. In an attempt to find candidate genes that may influence aging, the enhancer trap technique was utilized to identify age-related regulatory elements. The genomic regions surrounding the insertion site of the enhancer trap lines have the potential to be regulated by the characterized enhancer. A previous screen determined the temporal pattern of 180 enhancers trap lines, known as DJ lines. Many of these lines demonstrated an expression pattern that was associated with age. Several of the genes within the nearby genomic regions of six sequenced DJ lines, DJ695, DJ710, DJ849, DJ767, DJ761 and DJ694, were chosen for transcript quantification. Prior to gene quantification, reverse transcription, an essential step in the experimental procedure, was assessed for the error it incorporated into quantification. Specifically, an exogenous molecule was used to ensure that unsuccessful reverse transcription reactions had the potential to be identified and, soon after, discarded. This was achieved through the use of a spike RNA molecule, Luciferase. Luciferase was shown to be a diagnostic tool that can be used in determining reverse transcription efficiency. Eight genes were chosen from the aforementioned DJ lines and quantitative PCR revealed that the natural regulation of some genes were comparable to the, previously obtained, expression pattern of the enhancer trap line. Although the expression of other genes did not correlate to that of the enhancer trap lines, all genes exhibited expression patterns that were age-associated. The known functions of these candidate genes and the relevant homologues are discussed. These findings validate the use of the enhancer trap technique in the identification of candidate genes involved in the aging process. / Thesis (Master, Biology) -- Queen's University, 2009-02-09 10:59:16.871

Zugl.: München, Univ., Diss., 2008

München, Universiẗat, Diss., 2008.

Zugl.: Braunschweig, Techn. Univ., Diss., 2007

Zugl.: Bonn, Univ., Diss., 2009

Resumo: A severidade dos sintomas provocados pelo Huanglongbing (HLB) ou Greening, a rápida progressão na incidência de plantas afetadas nos pomares e o fato de esta doença afetar indistintamente todas as variedades comerciais de citros, contribuíram para este estudo que visou conhecer o padrão de colonização da bactéria Candidatus Liberibacter americanus (Lam) e Candidatus Liberibacter asiaticus (Las) em plantas cítricas. O PCR convencional é o teste atualmente utilizado na diagnose. Apesar de ser uma técnica reconhecidamente sensível, para o caso do HLB tem sido apenas confirmatório, ou seja, somente permite detecção da presença da bactéria em amostras de folhas sintomáticas. Surgiram aprimoramentos da técnica de PCR, como é o caso do Nested PCR e o PCR quantitativo (qPCR), demonstrado neste trabalho ser o método empregado mais sensível que o PCR convencional. Assim, através deste estudo pode-se concluir que Las e Lam se concentraram prioritariamente nas partes com sintomas de HLB das plantas de campo, naturalmente inoculadas e infectadas por liberibacter, se diferenciando na média do número estimado de cópias de liberibacter por grama de folha de plantas afetadas, sendo Las com 5,63 contra 5,01 em plantas afetadas por Lam (título bacteriano). A proporção de amostras positivas para Lam foram de 21 (19%) contra 12 (11,2%) amostras positivas para Las. A maior parte das amostras foi negativa para ambas as liberibacters (96 - Las e 90 - Lam). O pequeno acréscimo nos resultados positivos obtido pelo método de qPCR, aliado ao seu alto custo, não justifica a substituição do PCR convencional por este método na diagnose laboratorial do HLB, ficando restrito somente a pesquisa / Abstract: The degree of severity of the symptoms developed by Huanglongbing (HLB) or Greening, the fast incidence of diseased plants at different orchards and the fact that this phytopathogen affects various commercial citrus varieties have contributed to the proposition of the present work, that aimed to know the colonization pattern developed by the bacteria Candidatus Liberibacter americanus (LAM) and Candidatus Liberibacter asiaticus (LAS) on citrus plants. The conventional PCR is the current used assay to detect HLB. Besides being a sensitive and specific technique it is currently seen and a molecular technique that confirms the already symptomatic leaves of diseased plants. Improvements of the PCR technique named the Nested PCR and the quantitative PCR is described in this work as the most sensitive to detect the phytopathogen prior to the development of the symptoms disease. So, based on the results obtained in this work it was possible to conclude that LAS and LAM concentrate showed a tendency to appear mostly on parts of the plants exhibiting symptoms and that are already infected by these bacteria, showing difference when mean number of copies of liberibacter per g of leaf of infected plants, with LAS value of 5.63 as compared to 5.01 for LAM on plants with LAM detectable symptoms (high bacterial titer). The ratio of LAM positive samples was 21 (19%) against 12 (11.2%) positive for LAS. The majority of the samples were detected as negative for both bacteria (96% for LAS and 90% for LAM). The small increase on the positive results obtained when qPCR was used and considering its present high analysis cost, this type of PCR is not seen as adequate to diagnose LAM or LAS / Orientador: Manoel Victor Franco Lemos / Coorientador: Silvio Aparecido Lopes / Banca: Nelson Arno Wulff / Banca: Janete Apparecida Desiderio Sena / Mestre