Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway.

No description available.

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization.

Polycyclic aromatic hydrocarbons, produced by anthropological and natural activities, are hazardous through formation of oxidative radicals and DNA adducts. Growth of Mycobacterium sp. strain KMS, isolated from a contaminated soil, on the model hydrocarbon pyrene induced specific proteins. My work extends the study of isolate KMS to the gene level to understand the pathways and regulation of pyrene utilization. Genes encoding pyrene-induced proteins were clustered on a 72 kb section on the KMS chromosome but some also were duplicated on plasmids. Skewed GC content and presence of integrase and transposase genes suggested horizontal transfer of pyrene-degrading gene islands that also were found with high conservation in five other pyrene-degrading Mycobacterium isolates. Transcript analysis found both plasmid and chromosomal genes were induced by pyrene. These processes may enhance the survival of KMS in hydrocarbon-contaminated soils when other carbon sources are limited. KMS also grew on benzoate, confirming the functionality of an operon containing genes distinct from those in other benzoate-degrading bacteria. Growth on benzoate but not on pyrene induced a gene, benA, encoding a benzoate dioxygenase α-subunit, but not the pyrene-induced nidA encoding a pyrene dioxygenase α-subunit; the differential induction correlated with differences in promoter sequences. Diauxic growth occurred when pyrene cultures were amended with benzoate or acetate, succinate, or fructose, and paralleled delayed expression of nidA. Single phase growth and normal expression of benA was observed for benzoate single and mixed cultures. The nidA promoters had potential cAMP-CRP binding sites, suggesting that cAMP could be involved in carbon repression of pyrene metabolism. Growth on benzoate and pyrene requires gluconeogenesis. Intermediary metabolism in isolate KMS involves expression from genes encoding a novel malate:quinone oxidoreductase and glyoxylate shunt enzymes. Generation of C3 structures involves transcription of genes encoding malic enzyme, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate synthase. Carbon source modified the transcription patterns for these genes. My findings are the first to show duplication of pyrene-degrading genes on the chromosome and plasmids in Mycobacterium isolates and expression from a unique benzoate-degrading operon. I clarified the routes for intermediary metabolism leading to gluconeogenesis and established a potential role for cAMP-mediated catabolite repression of pyrene utilization.

It has been found in previous studies that there is a correlation between the placenta regulatory genes and the weight gain of the mother, Body Mass Index (BMI) as well as the birthweight of the fetus. When the mother gains weight / is overweight, this will affect the gene expression in the placenta, and in turn this triggers the weight gain of the fetus. The aim of the study was to investigate the correlation between the lipoprotein lipase gene and the mother's BMI, weight gain and the child's birth weight by extracting RNA from the placentas and analysing its quality and concentration. cDNA was generated from RNA using reverse transcription and gene expression was amplified using real-time PCR. The data from real-time PCR was used in the comparative Ct-method to calculate a 2˄(-ΔΔCt)-value which represents the RNA-level of the LPL-gene. Lastly this value was analysed by using the two-statistic methods, Pearson's rank correlation and Spearman's correlation, which showed that the value of the correlation coefficient for all the variables was close to the value of zero. The closer the value is to zero, the weaker the association becomes between the different variables. The correlation was 0.045, 0.112 and 0.044 for the child's birth weight, mother's BMI respective weight gain. The results from this study shows that there is no correlation between LPL and the mother's weight gain, BMI, or the child's birth weight.

Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.

Prokaryotic cells such as Escherichia coli use glucose as their preferred carbon source. In the absence of glucose, these cells resort to other sources to generate glucose and this process of de novo synthesis of glucose is termed gluconeogenesis. Phosphoenolpyruvate carboxykinase (Pck) is one of the three enzymes important in regulating gluconeogenesis. It converts oxaloacetic acid (OAA) from the Krebs cycle to phosphoenolpyruvate (PEP), a glycolytic intermediate. The Pck structural gene (pckA) is regulated by catabolite repression. There is a 100-fold induction of pckA-lacZ fusions at the onset of stationary phase concurrent with induction of glycogen synthesis. Mutants affecting the expression of pckA were analysed to shed some light on the mechanism of its genetic regulation.<p>Spontaneous mutants isolated with Pck- (lack of PEP carboxykinase activity) and Suc- (inability to utilise succinate as carbon source) phenotypes were previously characterised as atpG mutants defective in the ã subunit of ATP synthase.<p>In this work we find by reverse transcriptase and real time quantitative PCR that levels of pckA mRNA are normal in the atpG mutants and that the defects in expression of pckA are therefore likely at the level of translation, protein assembly and/or protein degradation. As expected, ATP synthase activity and proton pumping in inside-out membrane vesicles were defective in these atpG mutants. It is likely that one of these defects is affecting regulation or expression of the pckA gene. It was observed that atpG mutants were defective in calcium-dependent transformation although they could be made competent for electroporation. The atpG mutants were also defective for growth of P1 bacteriophage although they could serve as recipients for P1-dependent generalised transduction. These latter phenotypes are also likely due to defects in energy metabolism.

No description available.

Die Telomere sind spezialisierte DNA-Protein-Komplexe, die sich an den Enden der Chromosomen der eukaryotischen Zellen befinden. Die Telomerase ist ein Ribonukleoprotein, welches für die vollständige Replikation der Telomere bei den meisten Eukaryoten verantwortlich ist. Die katalytische Untereinheit des Enzyms (hTERT beim Menschen) besitzt Reverse-Transkriptase-Aktivität, und nutzt eine integrierte RNA (hTR beim Menschen) als Template, um Telomer-Wiederholungssequenzen an den Enden der Chromosomen zu synthetisieren. Die Telomerase ist in den meisten normalen humanen somatischen Zellen unterdrückt. In den meisten Krebszellen jedoch, stellt die Reaktivierung der Telomerase zur Beibehaltung der Telomerlänge eine Voraussetzung für deren unbegrenztes Wachstumspotential dar. Im Rahmen dieser Arbeit sollte ein neuer, einfacher und selektiver Assay für den Nachweis der humanen Telomerase entwickelt werden. In dem neuen Assay sollten die beiden Kernkomponenten des Enzyms, die Protein-Untereinheit und die RNA, die Targets sein. Der Test ist in seiner Grundstruktur wie folgt aufgebaut : 1. Immobilisierung der Telomerase über die hTERT an eine Festphase, beschichtet mit Phosphorothioat-modifizierten (PS) Oligonukleotiden oder Heparin. Zusammen mit der Telomerase werden bei diesem Schritt die Heparin-bindenden Proteine, die in der Probe enthalten sind, an die Festphase gebunden. 2. Spezifischer Nachweis der hTR. Zur Detektion der hTR wird ein Oligonukleotid-Ligations-Assay (OLA) oder eine Reverse-Transkriptase-PCR (RT-PCR) eingesetzt. In der optimierten Endversion wurde zur Immobilisierung des Enzyms eine Festphase, beschichtet mit PS-Oligonukleotiden, verwendet. Die hTR wurde mittels RT-PCR nachgewiesen. Mit dem neuen Assay wurden erfolgreich 75 Tumorzellen detektiert. / Telomeres are specialized DNA-Protein structures located at the ends of linear eukaryotic chromosomes. Telomerase is a ribonucleoprotein, which is responsible for the complete replication of the telomeres in most eukaryotes. The catalytic reverse transcriptase protein subunit (hTERT in humans) of the nucleoprotein uses an integral RNA (hTR in humans) as a template for the addition of telomeric repeat sequences to the ends of chromosomes. Telomerase is repressed in most normal human somatic cells, while the reactivation of telomerase to maintain telomere length is necessary for the unlimited growth potential of most human cancer cells. The aim of this work was the development of a new, simple and selective assay for the detection of human telomerase. The targets of the new assay were the two core subunits of the enzyme : hTERT and hTR. The test comprises two principal steps : 1. Immobilization of the telomerase via the hTERT subunit on a solid phase, coated with heparin or phosphorothioate-modified (PS) oligonucleotides. In this step telomerase is bound together with the heparin-binding proteins of the analysed sample to the surface. 2. Specific detection of the hTR. For the detection of the hTR an oligonucleotide ligation assay (OLA) or a reverse transcriptase PCR (RT-PCR) was used. In the optimized final version of the assay a PS-coated solid phase was used for the immobilization of the enzyme. Reverse transcriptase PCR was applied for detection of the hTR. 75 tumor cells were successfully detected with the new assay.

Organic nitrates, such as nitroglycerine (NTG), are widely used for their potent vasodilator capacity in the management of coronary artery disease and heart failure. Unfortunately, their beneficial effect is rapidly lost due to the development of nitrate tolerance, which is translated by an impaired vasorelaxation to NTG and an increased oxidative stress production. Although the mechanisms of the development of nitrate tolerance are still not fully elucidated, much interest has been focused in treating nitrate-receiving patients together with other drugs in order to overcome the development of nitrate tolerance. The Nitric Oxide generating enzyme, eNOS, and the superoxide anion generating enzyme, NAD(P)H oxidase, have been suggested to play a role in the development of nitrate tolerance. The aim of this study was to analyse the underlying mechanism by which ramipril, an ACE inhibitor and rosuvastatin, a new molecule of the statin class, are able to protect against the development of nitrate tolerance in the aortas isolated from rats, wild-type (wt) and eNOS-/- mice. <p>These results show that ramipril as well as rosuvastatin are able to protect against the development of nitrate tolerance in the wt and eNOS-/- mice aortas suggesting that eNOS is not necessary for their protective effect. The aortas from nitrate tolerant rats and mice showed a significant increase in the NAD(P)H oxidase activation compared to the aortas from the control and from the co-treated ramipril+NTG or rosuvastatin+NTG animals. In line with these findings were the results obtained by RT-PCR analysis: the mRNA expression of the different subunits of the NAD(P)H oxidase, such as gp91phox, p22phox, were significantly decreased after rosuvastatin or ramipril treatment in wt and eNOS-/- mice aortas. Apocynin, the NAD(P)H oxidase inhibitor was also able to inhibit the development of nitrate tolerance in the rat and mouse aortas. <p>In conclusion, these results suggest that rosuvastatin and ramipril are able to protect against the development of nitrate tolerance by counteracting the nitrate-induced oxidative stress. The mechanism of protection involves a direct interaction with the NAD(P)H oxidase pathway and seems to be completely independent of the eNOS pathway. <p> / Doctorat en sciences pharmaceutiques / info:eu-repo/semantics/nonPublished