An Evaluation Design to Assess Participant Satisfaction With The Project Lead The Way Teacher Training Program

Plan B Paper for M.A. in Comparative and International Development Educatio

The role and importance of gene polymorphisms in the development of atherosclerosis

The development of atherosclerosis is a multifactorial process. The purpose of the study was to examine three genetic polymorphisms playing a role in the metabolic processes underlying the disease. We compared the data of 348 atherosclerotic non-diabetic patients with 260 atherosclerotic diabetic patients and 384 healthy controls. We analyzed the prevalence of myocardial infarction and stroke in three different groups of patients carrying different polymorphisms. It was proved that if the mutant TT eNOS Glu298ASP variant is present, a significantly higher number of myocardial infarctions can be observed than in patients carrying heterozygote GT or normal GG genotype. We proved that in the case of MTHFR 677CT heterozygote variants, the occurrence of myocardial infarction is significantly higher and the difference is also significant in case of the 677TT homozygote variant. It was verified that among patients with the mutant TNF-α AA genotype the occurrence of cardiovascular events was significantly higher. Screening the genetically high risk groups on the long run should be considered as an early detection opportunity that may give better chances for prevention and treatment. Understanding the inflammatory mechanisms of the atherosclerosis may give new therapeutical targets to pharmacologists

Development of a Site-Directed Integration Plasmid for Heterologous Gene Expression in <i>Mycoplasma gallisepticum</i>

<div><p>Deciphering the molecular basis of the interactions between the parasite <i>Mycoplasma gallisepticum</i> and its avian hosts suffers from the lack of genetic tools available for the pathogen. In the absence of well established methods for targeted disruption of relevant <i>M. gallisepticum</i> genes, we started to develop suicide vectors and equipped them with a short fragment of <i>M. gallisepticum</i> origin or replication (<i>oriC</i>_MG). We failed to create a disruption vector, although by adding a further short fragment of the <i>M. gallisepticum tufB</i> upstream region we created a “Trojan horse” plasmid. This is fully integrated into the genomic DNA of <i>M. gallisepticum</i>, always at the same site, <i>oriC</i>_MG, and is able to carry and express any gene of interest in the genetic background of <i>M. gallisepticum</i>. Successful expression of a heterologous gene was shown with the <i>lacZ</i> gene of <i>E. coli</i>. When used for gene complementation or expression of hybrid genes in <i>M. gallisepticum</i>, a site-specific combined integration/expression vector constitutes an improvement on randomly integrating transposons, which might have unexpected effects on the expression of chromosomal genes. </p> </div

Schematical illustration of <i>lacZ</i> expression/integration vectors.

<p>The <i>lacZ</i> gene (black arrow) was subcloned with (p5TlacZ+) or without its own SD sequence (SD_lac, black circle) (p5TufPOlacZ) downstream of a putative <i>tuf</i>PO cassette consisting of the first 4 codons of <i>tufB</i> (<i>tufB</i>`, open square), its SD sequence (SD_tuf , open circle), and its assumed promoter <i>tuf</i>PO (open triangle). For control reasons, plasmids were created where the SD_lac was inserted into <i>tufB</i>´ (p5TSDlacZ), or the <i>tufB</i> frame was terminated by creating a frame shift (p5TlacZdis), or the SD_lac – <i>lacZ</i> unit was subcloned in reverse orientation to <i>tuf</i>PO (p5lacZ-). For control reasons, the SD_lac – <i>lacZ</i> unit was also subcloned into transposon Tn<i>4001</i>mod behind the P_out promoter [20] (pISMlacZ+) and in opposite direction (pISMlacZ-). LacZ activities of transformed <i>E. coli</i> or <i>M. gallisepticum</i> were analyzed in 2 (<i>E. coli</i>) or 4 (<i>M. gallisepticum</i>) independent assays and are given as Miller units [16]; however, they should not be compared to each other as different methods for standardization of <i>E. coli</i> and <i>M. gallisepticum</i> samples had to be used. </p

Integration of plasmid pINT into the <i>M. gallisepticum</i> genome.

<p>(<b>A</b>) Schematic representation of the origin of replication of <i>M. gallisepticum</i>. The region between <i>dnaN</i> and <i>soj</i> (parA) was annotated by Papazisi et al. to contain the origin of replication [22]. It contains DnaA boxes (black ovoids), short repeats (grey triangles), and AT-rich regions (dashed boxes). A 420-bp fragment (black bar) upstream of the <i>dnaN</i> gene enabled plasmid pINT to become integrative. Coordinates are given according to GenBank entry NC004829. (<b>B</b>) Genetic elements of plasmid pINT. ColE1, <i>E</i>. <i>coli</i> origin of replication; <i>lac</i>PO, promoter of <i>E</i>. <i>coli</i> lactose operon; <i>oriC</i>_MG, 420-bp fragment of <i>M</i>. <i>gallisepticum </i><i>oriC</i> region; <i>tetM</i>, tetracycline resistance gene; <i>tet</i>PO, promoter of <i>tetM</i>; (<b>C</b>) Southern blot analysis of <i>M. gallisepticum</i> RCL1 transformants harbouring pINT. Hybridization of an <i>oriC</i> probe to <i>Pvu</i>II DNA fragments of 5.6 and 6.1 kb shows the full-length integration of the 4.3-kb plasmid pINT (P, plasmid linearized by <i>Pvu</i>II) into the genomic 7.3-kb <i>Pvu</i>II fragment (MG, untransformed RCL1) which encompasses the genomic origin of replication. Ten out of 50 randomly selected Tet^R transformants are shown. M, DIG-labeled DNA Molecular Weight Marker II (Roche).</p

Map of the putative <i>M. gallisepticum</i><i>tufB</i> promoter sequence.

<p>Depicted is the genomic sequence between the tRNA^Trp open reading frame (MGA_trna24; locus tags according to GenBank entry NC004829) and the housekeeping gene <i>tufB</i> which encodes the EF-Tu protein (MGA_1033). Arrows indicate start and end sequences of open reading frames which are additionally indicated by italicized letters, and putative transcriptional elements (-35 box, -10 box, Shine-Dalgarno sequence [SD]) are highlighted by underlined bold letters. </p