Investigations of Coxsackie-adenovirus-receptor (CAR) mRNA expression of rats during organ development and in cultured rat cardiomyocytes - Importance of CAR expression for adenovirus-mediated gene transfer into rat cardiomyocytes

Titelblatt, Inhaltsverzeichnis, Lebenslauf 1\. Einleitung 2\. Literaturübersicht 3\. Material und Methoden 4\. Ergebnisse Teil 1 4\. Ergebnisse Teil 2 4\. Ergebnisse Teil 3 5\. Diskussion 6\. Zusammenfassung 7\. Summary 8\. Literaturverzeichnis 9\. AnhangZiel der Untersuchungen war es zum einen, mittels kompetitiver RT-PCR die mRNA-Expression von rCAR1 und rCAR2 in verschiedenen Organen während der peripartalen Phase sowie bei juvenilen und adulten Ratten zu untersuchen. Um weitere Informationen über die Regulation von CAR im kardialen System zu erhalten, sollte die rCAR1ý und rCAR2-mRNA-Expression in neonatalen Rattenkardiomyozytenkulturen in Abhängigkeit von der Zelldichte und der Kulturdauer untersucht werden. Weiterhin erfolgten mit Hilfe eines hCAR- exprimierenden adenoviralen Vektors Untersuchungen zur Bedeutung der CAR- Expression für den adenoviralen Gentransfer in Rattenkardiomyozytenkulturen. Es zeigte sich, dass die mRNA beider rCAR-Isoformen (rCAR1 und rCAR2) in allen untersuchten Organen exprimiert wird, die rCAR2-mRNA-Expression jedoch nur etwa ein Zehntel der rCAR1-mRNA-Expression betrug. Unabhängig von der Expressionshöhe zeigten beide Isoformen in allen untersuchten Organen, mit Ausnahme des Gehirns, einen tendenziell vergleichbaren Expressionsverlauf während der Entwicklung. Die für das Gehirn ermittelten unterschiedlichen Expressionsverläufe zwischen rCAR1- und rCAR2-mRNA deuten darauf hin, dass rCAR2 während der frühembryonalen Gehirnentwicklung von Bedeutung sein könnte. Weiterführende Untersuchungen in dieser Entwicklungsphase könnten dies konkretisieren. Der mittels kompetitiver RT-PCR ermittelte Verlauf der mRNA-Expression von rCAR1 und rCAR2 während der Organentwicklung zeigte vor allem im Gehirn und in der Skelettmuskulatur, aber auch im Herzen und in der Niere während der Embryonalentwicklung eine höhere CAR-mRNA-Expression, die mit zunehmendem Alter im Zuge der Organreifung abnahm. Dieser Rückgang war im Gehirn und in der Skelettmuskulatur besonders drastisch. Dagegen konnte in der Lunge und der Leber keine derartige Beziehung gefunden werden. Untersuchungen an neonatalen Rattenkardiomyozyten demonstrierten, dass die rCAR-mRNA-Expression in Kulturen mit einer geringen Zelldichte 24 h nach der Isolierung deutlich höher war als in Kulturen mit einer höheren Zelldichte (rCAR1 bis 9,1 mal höher; rCAR2 bis 2,1 mal höher) und die Expression mit zunehmender Zelldichte annähernd linear abnahm. Eine Verlängerung der Kulturdauer über 48 und 72 Stunden hatte keinen weiteren Einfluß auf die CAR1 -mRNA-Expression. Die im Gehirn, in der Skelettmuskulatur, im Herzen und bedingt in der Niere während der embryonalen und neonatalen Entwicklung sowie in Rattenkardiomyozyten mit geringer Zelldichte und entsprechend geringen Zellkontakten ermittelte höhere rCAR-mRNA-Expression ist ein wichtiger Hinweis dafür, dass CAR eine Bedeutung bei der Organentwicklung hat, in deren Rahmen CAR wahrscheinlich als Zelladhäsionsmolekül fungiert und am Aufbau von Zellkontakten (ýPfadfinderý-Funktion) beteiligt ist. Die unter Verwendung eines hCAR-exprimierenden Vektors durchgeführten Untersuchungen an neonatalen Rattenkardiomyozytenkulturen konnten deutlich machen, dass die CAR-Expression auch in Kardiomyozyten die Effizienz der AdV- Aufnahme und somit die Gentransfereffizienz entscheidend beeinflusst. So führte die Expression von rekombinantem hCAR auf der Zelloberfläche sowohl zu einer erhöhten AdV-Bindung als auch zu einer erhöhten AdV-Aufnahme und Transgenexpression.It was the aim of this study to investigate the rCAR1- and rCAR2-mRNA expression in different organs during peripartal organ development of rats and compare it with the rCAR1- and rCAR2-mRNA expression detected in juvenil and adult rats. In order to get inside into regulation of CAR in the cardial system rCAR1- and rCAR2-mRNA was investigated in neonatal rat cardiomyocytes dependend on cell density and culture time. Finally, impact of CAR for adenoviral gene transfer into cardiomyocytes was investigated by expression of hCAR in cultured neonatal rat cardiomyocytes. The rCAR-isoforms-mRNAs were expressed in all investigated organs. However rCAR2-mRNA expression reached only approximately 10 % of expression of rCAR1-mRNA. However, exclusively in brain, changing of rCAR1- and rCAR2-mRNA expression during organ development was similar. Relative high expression of rCAR2-mRNA in the brain in the early prenatal development indicate that rCAR2 may have a special importance this course of brain development. Additionally investigation should be help to examine this. Especially in brain, skeletal muscle, heart and kidney a stronger rCAR-mRNA expression was detectable during embyonal and peripartal development compare to expression found in juvenil and adult rats as measured by competitive RT- PCR. The decrease was most prominent in brain and skeletal muscle. No significant changes in rCAR1- and rCAR2-mRNA expression were found in the liver and the lung. Investigation of cultured neonatal rat cardiomyocytes demonstrated that rCAR- mRNA was significant higher expressed if cells were seeded at a low density (rCAR1 up to 9.1 fold; rCAR2 up to 2.1 fold) compare to cells seeded at a high density if analyzed 24 hrs after isolation of the cells. Decrease of rCAR-mRNA expression was almost linear. Increasing of culture time for 48 hrs and 72 hrs did not result in further changing rCAR1-mRNA expression. Comparatively high rCAR-mRNA expression in brain, skeletal muscle, heart and kidney during embryonal and neonatal organ development and in cultured neonatal rat cardiomyocytes seeded at low density (result in few cell-cell contacts), indicate that CAR may have importance in organ development, possibly act as a cell adheasion molecule and may be involved in pathfinding for cell-cell contact formation in several organs. Investigation of cultured neonatal rat cardiomyocytes using a hCAR expressing AdV demonstrate that CAR is involved in adenovector attachment and influences over this way the gene transfer efficiency into cardiomyocytes. So it was shown that hCAR expressed on the surface of cardiomyocytes increased adenovector attachment and uptake as well as transgene expression severalfold

Risk-based life cycle mim strategy for coastal structures - Effect of pre-existing damages on failure probability

A risk-based strategy for monitoring, inspection and maintenance (MIM) is described as a key component of an overall framework for life-cycle engineering and management. Its application for coastal structures is exemplarily outlined for sea/estuary dikes and harbor quay walls. The necessity of the incorporation of pre-existing damages into this strategy is exemplarily illustrated for quay walls, showing how this will affect the failure probability in comparison to a quay wall without any pre-existing damages.German Federal State Lower Saxon

Development of a biodegradable microstent for minimally invasive treatment of Fallopian tube occlusions

Obstructions of the Fallopian tube represent one of the most common reasons for an unfulfilled desire to have children. Microstent technology opens up new therapeutic possibilities to restore the natural lumen of the Fallopian tube within a single treatment. Within the current work we developed a self-expandable biodegradable microstent for gynecological applications. Based on a novel microstent design, prototypes were manufactured from poly-L-lactide tubing by means of fs-laser cutting. Microstent prototypes were characterized morphologically by means of scanning electron microscopy and biaxial laser scanning. As manufactured, a microstents outside diameter of about 2.3 mm and a strut thickness/width of about 114 µm/103 µm was measured. Mechanical characterization of microstents included bending as well as crimping and release behavior. After crimping to a minimum diameter of 0.8 mm and consecutive release, a microstent recovery to a diameter of 1.8 mm was found. Therefore, proof-of-concept for the self-expandable microstent could be successfully provided. © 2020 by Walter de Gruyter Berlin/Boston 2020

The microphthalmia-associated transcription factor p.E318K mutation does not play a major role in sporadic renal cell tumors from caucasian patients

OBJECTIVE The transcription factor MITF (microphthalmia-associated transcription factor) is known to induce expression of hypoxia-inducible factor (HIF1-α), which is involved in renal carcinogenesis. The MITF p.E318K mutation leads to deficient SUMOylation of MITF, resulting in enhanced activation of its target genes. A case-control study on melanoma patients who coincidentally were affected by renal cell carcinoma (RCC) has revealed an elevated risk for mutation carriers to be affected by one or both of these malignancies, suggesting a possible role for MITF p.E318K in renal carcinogenesis. The same study described an MITF mutation frequency of 1.5% in a small cohort of sporadic RCC, but comprehensive data on sporadic renal cell tumors are missing. We therefore tested a large cohort of sporadic renal tumors for MITF p.E318K mutation status. METHODS Genomic DNA was extracted from 426 formalin-fixed, paraffin-embedded sporadic renal tumors that had been graded according to the 2004 WHO classification of renal tumors and staged according to the 2002 TNM classification. The tumor cohort was enriched with papillary and chromophobe RCC, and also contained benign oncocytomas. DNA was tested for MITF p.E318K by pyrosequencing. RESULTS Of 403 analyzable tumors, 402 renal tumors were wild-type ones, and only 1 case showed the MITF p.E318K mutation. This tumor was a clear-cell RCC (pT3b N0 M0 G3 according to the TNM classification 2002). The affected patient was male, 61 years old, and had no known coexisting malignancies. CONCLUSION The MITF p.E318K mutation does not appear to play a major role in sporadic RCC carcinogenesis, but is possibly restricted to a rare subpopulation of inherited RCC

Whole Genome Association Mapping of Plant Height in Winter Wheat (<i>Triticum aestivum</i> L.)

<div><p>The genetic architecture of plant height was investigated in a set of 358 recent European winter wheat varieties plus 14 spring wheat varieties based on field data in eight environments. Genotyping of diagnostic markers revealed the <i>Rht-D1b</i> mutant allele in 58% of the investigated varieties, while the <i>Rht-B1b</i> mutant was only present in 7% of the varieties. <i>Rht-D1</i> was significantly associated with plant height by using a mixed linear model and employing a kinship matrix to correct for population stratification. Further genotyping data included 732 microsatellite markers, resulting in 770 loci, of which 635 markers were placed on the ITMI map plus a set of 7769 mapped SNP markers genotyped with the 90 k iSELECT chip. When Bonferroni correction was applied, a total of 153 significant marker-trait associations (MTAs) were observed for plant height and the SSR markers (−log₁₀ (P-value) ≥4.82) and 280 (−log₁₀ (P-value) ≥5.89) for the SNPs. Linear regression between the most effective markers and the BLUEs for plant height indicated additive effects for the MTAs of different chromosomal regions. Analysis of syntenic regions in the rice genome revealed closely linked rice genes related to gibberellin acid (GA) metabolism and perception, i.e. GA20 and GA2 oxidases orthologous to wheat chromosomes 1A, 2A, 3A, 3B, 5B, 5D and 7B, <i>ent</i>-kaurenoic acid oxidase orthologous to wheat chromosome 7A, <i>ent</i>-kaurene synthase on wheat chromosome 2B, as well as GA-receptors like DELLA genes orthologous to wheat chromosomes 4B, 4D and 7A and genes of the GID family orthologous to chromosomes 2B and 5B. The data indicated that besides the widely used GA-insensitive dwarfing genes <i>Rht-B1</i> and <i>Rht-D1</i> there is a wide spectrum of loci available that could be used for modulating plant height in variety development.</p></div

Development of a biodegradable microstent for minimally invasive treatment of Fallopian tube occlusions

Obstructions of the Fallopian tube represent one of the most common reasons for an unfulfilled desire to have children. Microstent technology opens up new therapeutic possibilities to restore the natural lumen of the Fallopian tube within a single treatment. Within the current work we developed a self-expandable biodegradable microstent for gynecological applications. Based on a novel microstent design, prototypes were manufactured from poly-L-lactide tubing by means of fs-laser cutting. Microstent prototypes were characterized morphologically by means of scanning electron microscopy and biaxial laser scanning. As manufactured, a microstents outside diameter of about 2.3 mm and a strut thickness / width of about 114 μm / 103 μm was measured. Mechanical characterization of microstents included bending as well as crimping and release behavior. After crimping to a minimum diameter of 0.8 mm and consecutive release, a microstent recovery to a diameter of 1.8 mm was found. Therefore, proof-of-concept for the self-expandable microstent could be successfully provided

Linear regressions of the most PH reducing (“best”) and the most PH promoting (“worst”) SNP-alleles with PH-BLUEs.

<p>Linear regression resulted in a relationship between PH-BLUEs and the 20, 10 or 5 “best” or “worst” SNP-alleles from different loci in 372 varieties.</p

Boxplots for plant height.

<p>Boxplots for eight field environments are depicted in green, the untreated control in a nursery is shown in red. Asterisks mark outlier varieties.</p

Manhattan plots of SSR or SNP marker alleles associated with plant height.

<p>The plot presents significant MTAs at a threshold of –log₁₀ (P-value) ≥4.0 for BLUEs sorted according to their chromosomal location. The red line indicates the threshold for Bonferroni correction, –log₁₀ (P-value) ≥4.82 (SSR) and ≥5.89 (SNP).</p