Production and improvement of virosomes for the use as vectors transfering transgenes into bovine sperm cells

Deckblatt-Impressum Zitat Inhalt Abkürzungen Einführung Zielsetzung Material und Methoden Ergebnisse Diskussion Zusammenfassung Summary Anhang Literaturverzeichnis Publikationshinweis Danksagung SelbständigkeitserklärungDas Ziel vieler Studien ist die Generierung transgener Nutztiere. Für den Transfer der Fremd-DNA in die Eizelle wird hauptsächlich die Mikroinjektion angewendet. So gewonnene Embryonen werden in Leihmütter transferiert, jedoch entwickeln sich beim Rind nur aus ca. 1% der Embryonen ein lebendes, gesundes Kalb. Um die geringe Effizienz zu steigern, wurden andere Techniken entwickelt, unter anderem der Spermien-vermittelte Gentransfer (SMGT). Hierbei handelt es sich um eine Technik, bei der im einfachsten Fall durch Inkubation der Spermien mit DNA-haltigen Medien diese die fremde DNA bei der Befruchtung in die Eizellen tragen. Vorteile dieser Methode sind der geringe apparative Aufwand und die Tatsache, dass Gewinnung und Aufbewahrung der Spermien von Nutztieren gut etabliert sind. Der Transfer von DNA in Zellen ist auf unterschiedlichen Wegen möglich, unter anderem durch Verwendung von Viren. Hierbei wird das natürliche Potenzial der Viren ausgenützt, ihr Genom in Zellen einzuschleusen und sich dort zu replizieren. In der vorliegenden Arbeit wird über die Herstellung von fusogenen Vesikeln berichtet, die in ihrer künstlichen Lipidhülle das Oberflächenprotein des Influenza Virus, Influenzahämagglutinin, tragen. Um die Virosomen zu optimieren, wurden verschiedene Schritte während des Rekonstitutionsprotokolls variiert und die Virosomen im Hinblick auf Lipid-, Protein- und DNA-Gehalt bewertet, sowie die Fusion der Virosomen mit kryokonservierten Bullenspermien und die Gentransferrate beurteilt. Es konnte festgestellt werden, dass in den so hergestellten Virosomen nahezu ausschließlich Influenzahämagglutinin enthalten ist, dass der Hauptteil der Lipide in den Virosomen wiedergefunden werden kann und dass eine Zugabe von 6 mg Cholesterol auf 25,15 mg Gesamtlipid notwendig ist für die Fusion der Virosomen mit den Spermien. Werden Virosomen für verschiedene Zelltypen präpariert, scheint es notwendig zu sein, den Cholesterolgehalt der exogenen Lipide speziell auf die Zellart abzustimmen. Des Weiteren konnte festgestellt werden, dass die so hergestellten Virosomen in der Lage sind, unabhängig vom Individuum, dem Reifestadium der Spermien oder der Spezies des Donorss mit Spermien zu fusionieren. Die Gentransferrate variiert zwischen 1,5 und 302 Plasmiden pro Spermienzelle. Zusammenfassend ist zu sagen, dass Influenzavirosomen eine Alternative bei der Generierung landwirtschaftlicher Nutztiere sein könnten.Generating transgenic livestock is the aim of many studies. To transfer foreign DNA into oocytes usually pronuclear microinjection is applied. Embryos extracted by this technique are transferred into foster mothers, but only 1% of transferred zygotes results in the birth of living, healthy calves. To improve the efficiency new methods have been developed, among others sperm mediated gene transfer (SMGT). By this technique sperm cells, incubated with DNA containing media, carry the transgene into the oocyte during fertilization. Several methods have been published to transfer foreign genes into living cells. One method is to utilize the natural potential of viruses to transfer and replicate their genetic material into cells. Here experiments are reported that were designed to exploit the fusion potential of reconstituted influenza virus envelopes (virosomes) for the transfer of foreign DNA into bovine spermatozoa. A protocol was developed for the preparation of virosomes bearing influenza X-31 hemagglutinin inserted into artificial lipid bilayers. To optimize virosomes for the sperm mediated transfer different steps during the reconstitution protocol were varied. Criteria for improving the virosomes were beside lipid, protein and plasmid content of virosomes also fusion and gene transfer efficiency to sperm cells. Results of this study indicated that the protein inserted into the lipid bilayers consists mainly of influenza hemagglutinin. From the added lipids the major amount could be found in the virosomes. Interestingly adding about 6 mg cholesterol to a total amount of lipids from 25.15 mg seems to be necessary to promote fusion with cryopreserved bull spermatozoa. But the optimal lipid mixture seems to depend on the target cell. Additionally virosomes are able to fuse to sperm cells independent from individuum, developmental stage or species. Gene transfer to sperm cells varies from 1.5 to 302 plasmids per sperm cell. Taken together this makes virosomes a promising tool for transfering foreign DNA into sperm cells of farm animals

Mineralocorticoid receptors in non‐alcoholic fatty liver disease

Liver diseases are the fourth common death in Europe responsible for about 2 million death per year worldwide. Among the known detrimental causes for liver dysfunction are virus infections, intoxications and obesity. The mineralocorticoid receptor (MR) is a ligand‐dependent transcription factor activated by aldosterone or glucocorticoids but also by pathological milieu factors. Canonical actions of the MR take place in epithelial cells of kidney, colon and sweat glands and contribute to sodium reabsorption, potassium secretion and extracellular volume homeostasis. The non‐canonical functions can be initiated by inflammation or an altered micro‐milieu leading to fibrosis, hypertrophy and remodelling in various tissues. This narrative review summarizes the evidence regarding the role of MR in portal hypertension, non‐alcoholic fatty liver disease, liver fibrosis and cirrhosis, demonstrating that inhibition of the MR in vivo seems to be beneficial for liver function and not just for volume regulation. Unfortunately, the underlying molecular mechanisms are still not completely understood

Warming the early Earth - CO2 reconsidered

Despite a fainter Sun, the surface of the early Earth was mostly ice-free. Proposed solutions to this so-called "faint young Sun problem" have usually involved higher amounts of greenhouse gases than present in the modern-day atmosphere. However, geological evidence seemed to indicate that the atmospheric CO2 concentrations during the Archaean and Proterozoic were far too low to keep the surface from freezing. With a radiative-convective model including new, updated thermal absorption coefficients, we found that the amount of CO2 necessary to obtain 273 K at the surface is reduced up to an order of magnitude compared to previous studies. For the late Archaean and early Proterozoic period of the Earth, we calculate that CO2 partial pressures of only about 2.9 mb are required to keep its surface from freezing which is compatible with the amount inferred from sediment studies. This conclusion was not significantly changed when we varied model parameters such as relative humidity or surface albedo, obtaining CO2 partial pressures for the late Archaean between 1.5 and 5.5 mb. Thus, the contradiction between sediment data and model results disappears for the late Archaean and early Proterozoic.Comment: 53 pages, 4 tables, 11 figures, published in Planetary and Space Scienc

Contemporary predictors of death and sustained ventricular tachycardia in patients with repaired tetralogy of Fallot enrolled in the INDICATOR cohort

Objective: Patients with repaired tetralogy of Fallot (TOF) experience increased rates of mortality and morbidity in adulthood. This study was designed to identify risk factors for death and ventricular tachycardia (VT) in a large contemporary cohort of patients with repaired TOF. Methods: Subjects with repaired TOF from four large congenital heart centres in the USA, Canada and Europe were enrolled. Clinical, ECG, exercise, cardiac magnetic resonance (CMR) and outcome data were analysed. Results: Of the 873 patients (median age 24.4 years), 32 (3.7%) reached the primary outcome (28 deaths, 4 sustained VT; median age at outcome 38 years; median time from CMR to outcome 1.9 years). Cox proportional-hazards regression identified RV mass-to-volume ratio ≥0.3 g/mL (HR, 5.04; 95% CI 2.3 to 11.0; p<0.001), LV EF z score<−2.0 (HR, 3.34; 95% CI 1.59 to 7.01; p=0.001), and history of atrial tachyarrhythmia (HR, 3.65; 95% CI 1.75 to 7.62; p=0.001) as outcome predictors. RV dysfunction was predictive of the outcome similar to LV dysfunction. In subgroup analysis of 315 subjects with echocardiographic assessment of RV systolic pressure, higher pressure (HR 1.39; 95% CI 1.19 to 1.62; p<0.001) was associated with death and sustained VT independent of RV hypertrophy and LV dysfunction. Conclusions: RV hypertrophy, ventricular dysfunction and atrial tachyarrhythmias are predictive of death and sustained VT in adults with repaired TOF. These findings may inform risk stratification and the design of future therapeutic trials

Mind the gap: A process model for diagnosing barriers to key account management implementation

Today, many firms develop and implement key account management (KAM) programs to manage the relationships with strategically important customers. The implementation of KAM programs requires the configuration of special activities, actors, and resources dedicated to key accounts, which poses major challenges for managerial practice. Firms often underestimate the fundamental organizational change required for a successful implementation of KAM. The objective of this article is to advance extant knowledge on KAM by developing a framework that outlines essential processes to assess and diagnose barriers to KAM implementation. In our article, we integrate extant knowledge on KAM organization and enactment, and we propose a four-step process model that links the concepts of embeddedness, differentiation, integration, and alignment. In addition, we illustrate our model in a case study analysis with a large-scale European industrial company. The findings of our study allow us to derive avenues for further research on KAM implementation as well as implications for management practice

Familial hypercholesterolaemia in children and adolescents from 48 countries: a cross-sectional study

Background: Approximately 450 000 children are born with familial hypercholesterolaemia worldwide every year, yet only 2·1% of adults with familial hypercholesterolaemia were diagnosed before age 18 years via current diagnostic approaches, which are derived from observations in adults. We aimed to characterise children and adolescents with heterozygous familial hypercholesterolaemia (HeFH) and understand current approaches to the identification and management of familial hypercholesterolaemia to inform future public health strategies. Methods: For this cross-sectional study, we assessed children and adolescents younger than 18 years with a clinical or genetic diagnosis of HeFH at the time of entry into the Familial Hypercholesterolaemia Studies Collaboration (FHSC) registry between Oct 1, 2015, and Jan 31, 2021. Data in the registry were collected from 55 regional or national registries in 48 countries. Diagnoses relying on self-reported history of familial hypercholesterolaemia and suspected secondary hypercholesterolaemia were excluded from the registry; people with untreated LDL cholesterol (LDL-C) of at least 13·0 mmol/L were excluded from this study. Data were assessed overall and by WHO region, World Bank country income status, age, diagnostic criteria, and index-case status. The main outcome of this study was to assess current identification and management of children and adolescents with familial hypercholesterolaemia. Findings: Of 63 093 individuals in the FHSC registry, 11 848 (18·8%) were children or adolescents younger than 18 years with HeFH and were included in this study; 5756 (50·2%) of 11 476 included individuals were female and 5720 (49·8%) were male. Sex data were missing for 372 (3·1%) of 11 848 individuals. Median age at registry entry was 9·6 years (IQR 5·8-13·2). 10 099 (89·9%) of 11 235 included individuals had a final genetically confirmed diagnosis of familial hypercholesterolaemia and 1136 (10·1%) had a clinical diagnosis. Genetically confirmed diagnosis data or clinical diagnosis data were missing for 613 (5·2%) of 11 848 individuals. Genetic diagnosis was more common in children and adolescents from high-income countries (9427 [92·4%] of 10 202) than in children and adolescents from non-high-income countries (199 [48·0%] of 415). 3414 (31·6%) of 10 804 children or adolescents were index cases. Familial-hypercholesterolaemia-related physical signs, cardiovascular risk factors, and cardiovascular disease were uncommon, but were more common in non-high-income countries. 7557 (72·4%) of 10 428 included children or adolescents were not taking lipid-lowering medication (LLM) and had a median LDL-C of 5·00 mmol/L (IQR 4·05-6·08). Compared with genetic diagnosis, the use of unadapted clinical criteria intended for use in adults and reliant on more extreme phenotypes could result in 50-75% of children and adolescents with familial hypercholesterolaemia not being identified. Interpretation: Clinical characteristics observed in adults with familial hypercholesterolaemia are uncommon in children and adolescents with familial hypercholesterolaemia, hence detection in this age group relies on measurement of LDL-C and genetic confirmation. Where genetic testing is unavailable, increased availability and use of LDL-C measurements in the first few years of life could help reduce the current gap between prevalence and detection, enabling increased use of combination LLM to reach recommended LDL-C targets early in life

Consensus guidelines for the use and interpretation of angiogenesis assays

The formation of new blood vessels, or angiogenesis, is a complex process that plays important roles in growth and development, tissue and organ regeneration, as well as numerous pathological conditions. Angiogenesis undergoes multiple discrete steps that can be individually evaluated and quantified by a large number of bioassays. These independent assessments hold advantages but also have limitations. This article describes in vivo, ex vivo, and in vitro bioassays that are available for the evaluation of angiogenesis and highlights critical aspects that are relevant for their execution and proper interpretation. As such, this collaborative work is the first edition of consensus guidelines on angiogenesis bioassays to serve for current and future reference

Das Körperbild von Jugendlichen im Zusammenhang mit Aggression

Barbara Maria SchreierAlpen-Adria-Universität Klagenfurt, Diplomarbeit, 2016(VLID)241395

Social behaviours in Neolamprologus pulcher

362 observations of 8 social behaviours of the cooperatively breeding cichlid fish Neolamprologus pulcher bred in a paternal half-sibling breeding design. Includes behaviours, covariates that explained some variation in behaviours and random effects for quantitative genetic analyses