Evaluation of candidate factors for the post-transcriptional regulation of microRNA expression

RNA Silencing ist ein kürzlich entdeckter, natürlich vorkommender Mechanismus zur Regulation von Genexpression in zahlreichen Organismen. Die Schlüsselfunktion des RNA Silencing wird von kleinen RNA-Molekülen namens microRNAs (miRNAs) getragen, die als post-transkriptionelle Regulatoren auf mRNAs einwirken, um Translationsinhibition oder Degradierung zu induzieren. Die Entstehung von miRNAs wurde in der Vergangenheit intensiv erforscht, weshalb zahlreiche beteiligte Proteinfaktoren bereits identifiziert werden konnten. Die Regulation der miRNA-Biogenese ist allerdings noch ein Forschungsfeld mit vielen offenen Fragen. Jüngste Untersuchungen zeigen, dass die Vorstufe von miR-138 in Mausgeweben differentiell exprimiert wird, während die reife miRNA lediglich in Nervengewebe exprimiert wird. Es konnte auch gezeigt werden, dass diese Beobachtung auf einen unbekannten Proteinfaktor zurückzuführen ist, der die Prozessierung von der Vorstufe zur reifen Form blockiert. Einige Kandidaten konnten durch chromatographische Methoden identifiziert werden, wobei das Heterodimer SRP9/14 am vielversprechendsten war. In der vorliegenden Studie wurde SRP9/14 als potentieller Inhibitor für die miRNA-Biogenese mittels zweier Methoden evaluiert: einerseits wurde das Heterodimer als rekombinantes Protein exprimiert und auf seine Funktionalität als Inhibitor von miR-138 in in vitro Assays getestet. Andererseits wurde die Expression von SRP9/14 in HeLa-Zellen durch siRNAs abgeschaltet und die Zellextrakte funktionell auf Vorhandensein des Inhibitors getestet. Beide Methoden zeigten, dass es sich bei SRP9/14 nicht um den gesuchten Inhibitor handelt.RNA silencing is a recently discovered naturally occurring mechanism to regulate gene expression in a wide range of organisms. Key players of RNA silencing are small RNA molecules termed microRNAs (miRNAs), which act as post-transcriptional regulators on mRNAs to induce inhibition of translation or degradation. The process of how miRNAs are generated has been investigated intensely in the last years and many protein factors involved have been identified. However, regulation of miRNA biogenesis is still a field of research with many open questions. Recent studies have indicated that miR-138 displays differential expression of its precursor form in mouse tissues, while the mature miRNA is expressed solely in neural tissues. This phenomenon was shown to be due to an unknown protein factor inhibiting processing of the precursor to the mature miRNA. Several candidate proteins have been identified by chromatography methods of which the heterodimer SRP9/14 was the most promising. In this thesis, evaluation of SRP9/14 as a putative inhibitor of miRNA biogenesis was performed using two approaches: first, the heterodimer was expressed as recombinant protein and tested for its ability to inhibit processing of pre-miR-138 in in vitro assays. Second, SRP9/14 was knocked-down in HeLa cells using siRNAs followed by testing depleted cell extracts inhibitory activity using in vitro processing assays. Both approaches demonstrated that SRP9/14 does not inhibit pre-miR-138 maturation and has to be excluded from the list of putative inhibitors

Establishment of a Cre/loxP recombination system for N-terminal epitope tagging of genes in Tetrahymena

<p>Abstract</p> <p>Background</p> <p>Epitope tagging is a powerful strategy to study the function of proteins. Although tools for C-terminal protein tagging in the ciliated protozoan <it>Tetrahymena thermophila </it>have been developed, N-terminal protein tagging in this organism is still technically demanding.</p> <p>Results</p> <p>In this study, we have established a Cre/loxP recombination system in <it>Tetrahymena </it>and have applied this system for the N-terminal epitope tagging of <it>Tetrahymena </it>genes. Cre can be expressed in <it>Tetrahymena </it>and localizes to the macronucleus where it induces precise recombination at two loxP sequences in direct orientation in the <it>Tetrahymena </it>macronuclear chromosome. This Cre/loxP recombination can be used to remove a loxP-flanked drug-resistance marker from an N-terminal tagging construct after it is integrated into the macronucleus.</p> <p>Conclusions</p> <p>The system established in this study allows us to express an N-terminal epitope tagged gene from its own endogenous promoter in <it>Tetrahymena</it>.</p

The Burden of Invasive Bacterial Infections in Pemba, Zanzibar

BACKGROUND: We conducted a surveillance study to determine the leading causes of bloodstream infection in febrile patients seeking treatment at three district hospitals in Pemba Island, Zanzibar, Tanzania, an area with low malaria transmission. METHODS: All patients above two months of age presenting to hospital with fever were screened, and blood was collected for microbiologic culture and malaria testing. Bacterial sepsis and malaria crude incidence rates were calculated for a one-year period and were adjusted for study participation and diagnostic sensitivity of blood culture. RESULTS: Blood culture was performed on 2,209 patients. Among them, 166 (8%) samples yielded bacterial growth; 87 (4%) were considered as likely contaminants; and 79 (4%) as pathogenic bacteria. The most frequent pathogenic bacteria isolated were Salmonella Typhi (n = 46; 58%), followed by Streptococcus pneumoniae (n = 12; 15%). The crude bacteremia rate was 6/100,000 but when adjusted for potentially missed cases the rate may be as high as 163/100,000. Crude and adjusted rates for S. Typhi infections and malaria were 4 and 110/100,000 and 4 and 47/100,000, respectively. Twenty three (51%), 22 (49%) and 22 (49%) of the S. Typhi isolates were found to be resistant toward ampicillin, chloramphenicol and cotrimoxazole, respectively. Multidrug resistance (MDR) against the three antimicrobials was detected in 42% of the isolates. CONCLUSIONS: In the presence of very low malaria incidence we found high rates of S. Typhi and S. pneumoniae infections on Pemba Island, Zanzibar. Preventive measures such as vaccination could reduce the febrile disease burden

Cost of Illness Due to Typhoid Fever in Pemba, Zanzibar, East Africa

The aim of this study was to estimate the economic burden of typhoid fever in Pemba, Zanzibar, East Africa. This study was an incidence-based cost-of-illness analysis from a societal perspective. It covered new episodes of blood culture-confirmed typhoid fever in patients presenting at the outpatient or inpatient departments of three district hospitals between May 2010 and December 2010. Cost of illness was the sum of direct costs and costs for productivity loss. Direct costs covered treatment, travel, and meals. Productivity costs were loss of income by patients and caregivers. The analysis included 17 episodes. The mean age of the patients, was 23 years (range=5-65, median=22). Thirty-five percent were inpatients, with a mean of 4.75 days of hospital stay (range=3-7, median=4.50). The mean cost for treatment alone during hospital care was US$21.97 at 2010 prices (US$ 1=1,430.50 Tanzanian Shilling\u2500TSH). The average societal cost was US$154.47 per typhoid episode. The major expenditure was productivity cost due to lost wages of US$ 128.02 (83%). Our results contribute to the further economic evaluation of typhoid fever vaccination in Zanzibar and other sub-Saharan African countries

Role of malondialdehyde epitopes in sterile inflammation

Im Kontext einer entzündlichen Immunantwort tritt auch erhöhter oxidativer Stress auf, der zur Fragmentierung von Lipiden sowie zur Bildung von sogenannten oxidationsspezifischen Epitopen wie Malondialdehyd-(MDA)-Epitopen führen kann. Diese Epitope wurden nicht nur in zahlreichen entzündlichen Erkrankungen wie bei der Alzheimer-Krankheit, akuter Lungenschädigung, Multipler Sklerose und altersabhängiger Makuladegeneration festgestellt, sondern auch in ernährungsbedingten Krankheiten wie nicht-alkoholischen Fettleber- und Herz-Kreislauf-Erkrankungen. Obwohl mehrere Hypothesen im Hinblick auf den Zusammenhang zwischen ernährungsbedingten Veränderungen wie beispielsweise Hyperlipidämie und den darauffolgenden entzündlichen Reaktionen existieren, herrscht bis heute keine Klarheit über die zugrunde liegenden Mechanismen. Während in vitro Untersuchungen bereits zeigen konnten, dass MDA-Epitope eine pro-inflammatorische Wirkung auf Zellen des angeborenen Immunsystems haben und dass wichtige Plasmaproteine wie natürliche Antikörper vom Isotyp IgM oder Komplementfaktor H eine bedeutende Rolle bei der Neutralisierung von MDA-Epitopen spielen, sind die funktionalen Konsequenzen einer solchen Interaktion weiterhin unbekannt. In meinem Dissertationsprojekt möchte ich MDA-vermittelte Entzündungsantworten im Detail charakterisieren, deren Signalwege und Rezeptoren auf Immunzellen identifizieren, und die Rolle von MDA-Epitopen als Gefahr-assoziiertes molekulares Muster, besser bekannt unter dem Begriff„"damage-associated molecular pattern“, im Rahmen von Entzündungen untersuchen. Zusammenfassend konnte ich anhand von RNA-Sequenzierungsanalysen zahlreiche Prozesse identifizieren, die sowohl charakteristisch für Signalwege des angeborenen Immunsystems als auch für oxidativen Stress sind, und die im Lebergewebe von LDLR-KO-Mäusen bei cholesterinhaltiger Diät verstärkt aktiviert werden. Des Weiteren konnte ich zeigen, dass MDA-Epitope in entzündetem Lebergewebe insbesondere auf apoptotischen Zellen auftreten und sowohl Zytokinsekretion als auch Infiltration von Leukozyten stimulieren. Für eine MDA-induzierte Zytokinsekretion in vitro waren die Scavenger-Rezeptoren CD36 und MSR-1 erforderlich. Außerdem konnten endogen gebildete MDA-Epitope in vivo durch intravenöse Gabe eines spezifischen MDA-Antikörpers neutralisiert werden, was eine verringerte Leberentzündung in LDLR-KO-Mäusen bei cholesterinhaltiger Diät zur Folge hatte. Abschließend habe ich experimentelle Daten für ein besseres Verständnis des Zusammenhangs zwischen einer cholesterinhaltigen Diät und der darauffolgenden Entzündung in der Leber geliefert, was einen potentiellen, neuen Anknüpfungspunkt für Therapien darstellt könnte.Increased oxidative stress during inflammatory responses leads to the generation of damaged lipids and proteins with oxidation-specific epitopes such as malondialdehyde (MDA) epitopes. These lipid peroxidation-derived moieties have been associated with many inflammatory diseases including Alzheimers disease, acute lung injury, multiple sclerosis and age-related macular degeneration. Importantly, malondialdehyde-epitope production is also observed in diet-induced inflammatory diseases such as nonalcoholic fatty liver disease and cardiovascular disorders. There have been many hypotheses trying to link the diet-induced changes such as hyperlipidemia with the subsequent inflammatory events but a clear insight into the underlying mechanism has remained elusive. Recently, natural IgM antibodies as well as complement factor H have been identified as major malondialdehyde-binding proteins in plasma. Additionally, malondialdehyde epitopes were demonstrated to be pro-inflammatory in vitro but the functional responses they induce in cells of innate immunity are not well understood. In my thesis project, I wanted to characterize the malondialdehyde-induced pro-inflammatory responses in more detail, identify signalling pathways and associated receptors on immune cells that sense the presence of malon- dialdehyde epitopes, and elucidate the functional role of malondialdehyde epitopes as danger-associated molecular pattern with the capacity to elicit sterile inflammation. Together, I could identify innate immunity and oxidative stress processes as major response pathways in livers of Ldlr / mice that were fed a Western-type diet using RNA sequenc- ing and in silico functional analyses of transcriptome data. Furthermore, I showed that malondialdehyde epitopes are detectable in hepatic inflammation predominantly on dying cells and stimulate cytokine secretion as well as leukocyte recruitment in vitro and in vivo. Malondialdehyde-induced cytokine secretion in vitro was dependent on the presence of the scavenger receptors CD36 and MSR1. Moreover, in vivo neutralization of endogenously generated malondialdehyde epitopes by intravenous injection of a specific malondialdehyde antibody resulted in decreased hepatic inflammation in Ldlr/ mice on a Western-type diet. In conclusion, I have provided evidence for a better understanding of the link between a lipid-rich diet and liver inflammation and suggested a putative novel point for therapeutic intervention.submitted by Mag. Clara Jana Lui BuschZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität, Dissertation, 2017OeB

Isolation and Long-term Cultivation of Mouse Alveolar Macrophages

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages

Long-term culture-expanded alveolar macrophages restore their full epigenetic identity after transfer in vivo

International audienceAlveolar macrophages (AMs) are lung tissue-resident macrophages that can be expanded in culture, but it is unknown to what extent culture affects their in vivo identity. Here we show that mouse long-term ex vivo expanded AMs (exAMs) maintained a core AM gene expression program, but showed culture adaptations related to adhesion, metabolism and proliferation. Upon transplantation into the lung, exAMs reacquired full transcriptional and epigenetic AM identity, even after several months in culture and could self-maintain long-term in the alveolar niche. Changes in open chromatin regions observed in culture were fully reversible in transplanted exAMs and resulted in a gene expression profile indistinguishable from resident AMs. Our results indicate that long-term proliferation of AMs in culture did not compromise cellular identity in vivo. The robustness of exAM identity provides new opportunities for mechanistic analysis and highlights the therapeutic potential of exAMs