Development of the experimental system based on Cre/loxP recombination for polyomavirus mutant production.

Myší polyomavirus je významným zástupcem čeledi Polyomaviridae s potenciálním využitím v genové terapii a imunoterapii. Práce s virovými mutantami je důležitou součástí jeho studia, ale současné metody jejich produkce jsou pracné a poskytují nízký výtěžek. Tato práce se zabývá vývojem nového experimentálního sytému, který umožňuje pomocí Cre/loxP rekombinace generovat intaktní polyomavirový genom z rekombinantního plazmidu in vivo. Během rekombinace dochází k nevyhnutelnému vložení jednoho loxP místa do vzniknuvšího virového genomu. Byly připraveny dvě varianty produkčních plazmidů, které vytvářejí virový genom divokého typu s loxP místem vloženým mezi poly(A) signální sekvence časných a pozdních genů, nebo v intronové oblasti časných genů. Inzerce loxP místa mezi poly(A) signální sekvence měla dramatický dopad na expresi virových genů a vedla k úplné ztrátě infektivity viru. Naopak, substituce loxP místa v intronové oblasti časných genů produkci infekčního viru umožnila. Pro zajištění exprese Cre rekombinázy jsem také vytvořil stabilně transfekované buněčné linie, které mohou přípravu viru zjednodušit. Tato práce ukazuje, že nově navržený systém poskytuje uspokojivý výtěžek viru, řeší omezení vyskytující se u běžně používaných metod a může být využit pro produkci málo infekčních virových mutant. Powered by...Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologieFaculty of SciencePřírodovědecká fakult

Remnants of an ancient deltaretrovirus in the genomes of horseshoe aats (Rhinolophidae)

Endogenous retrovirus (ERV) sequences provide a rich source of information about the long-term interactions between retroviruses and their hosts. However, most ERVs are derived from a subset of retrovirus groups, while ERVs derived from certain other groups remain extremely rare. In particular, only a single ERV sequence has been identified that shows evidence of being related to an ancient , despite the large number of vertebrate genome sequences now available. In this report, we identify a second example of an ERV sequence putatively derived from a past deltaretroviral infection, in the genomes of several species of horseshoe bats (Rhinolophidae). This sequence represents a fragment of viral genome derived from a single integration. The time of the integration was estimated to be 11-19 million years ago. This finding, together with the previously identified endogenous in long-fingered bats (Miniopteridae), suggest a close association of bats with ancient deltaretroviruses

DNA hypomethylation and aberrant expression of the human endogenous retrovirus ERVWE1/syncytin-1 in seminomas

Additional file 3: Figure S1. Expression analysis of ERVWE1 and ERVFRDE1 loci normalized to the TBP gene. Expression from the ERVWE1 (A, B) and ERVFRDE1 (C, D) loci was analyzed by qRT-PCR in the panel of tumor samples. Both the full-length RNA (A, C) and spliced mRNA (B, D) forms were quantified. All the data were normalized to the expression of TBP. Each sample is represented by a dot and was measured as a technical triplicate. In each column, median with interquartile range is depicted. Significance was assigned as follows: **** for P-values <0.0001, *** for P-values <0.001, ** for P-values <0.01, * for P-values <0.05

Hemodynamics in Ruptured Intracranial Aneurysms

Incidental detection of unruptured intracranial aneurysms (UIA) has increased in the recent years. There is a need in the clinical community to identify those that are prone to rupture and would require preventive treatment. Hemodynamics in cerebral blood vessels plays a key role in the lifetime cycle of intracranial aneurysms (IA). Understanding their initiation, growth, and rupture or stabilization may identify those hemodynamic features that lead to aneurysm instability and rupture. Modeling hemodynamics using computational fluid dynamics (CFD) could aid in understanding the processes in the development of IA. The neurosurgical approach during operation of IA allows direct visualization of the aneurysm sac and its sampling in many cases. Detailed analysis of the quality of the aneurysm wall under the microscope, together with histological assessment of the aneurysm wall and CFD modeling, can help in building complex knowledge on the relationship between the biology of the wall and hemodynamics. Detailed CFD analysis of the rupture point can further strengthen the association between hemodynamics and rupture. In this chapter we summarize current knowledge on CFD and intracranial aneurysms

High aspect ratio nanomaterial-induced macrophage polarization is mediated by changes in miRNA levels

IntroductionInhalation of nanomaterials may induce inflammation in the lung which if left unresolved can manifest in pulmonary fibrosis. In these processes, alveolar macrophages have an essential role and timely modulation of the macrophage phenotype is imperative in the onset and resolution of inflammatory responses. This study aimed to investigate, the immunomodulating properties of two industrially relevant high aspect ratio nanomaterials, namely nanocellulose and multiwalled carbon nanotubes (MWCNT), in an alveolar macrophage model. MethodsMH-S alveolar macrophages were exposed at air-liquid interface to cellulose nanocrystals (CNC), cellulose nanofibers (CNF) and two MWCNT (NM-400 and NM-401). Following exposure, changes in macrophage polarization markers and secretion of inflammatory cytokines were analyzed. Furthermore, the potential contribution of epigenetic regulation in nanomaterial-induced macrophage polarization was investigated by assessing changes in epigenetic regulatory enzymes, miRNAs, and rRNA modifications.ResultsOur data illustrate that the investigated nanomaterials trigger phenotypic changes in alveolar macrophages, where CNF exposure leads to enhanced M1 phenotype and MWCNT promotes M2 phenotype. Furthermore, MWCNT exposure induced more prominent epigenetic regulatory events with changes in the expression of histone modification and DNA methylation enzymes as well as in miRNA transcript levels. MWCNT-enhanced changes in the macrophage phenotype were correlated with prominent downregulation of the histone methyltransferases Kmt2a and Smyd5 and histone deacetylases Hdac4, Hdac9 and Sirt1 indicating that both histone methylation and acetylation events may be critical in the Th2 responses to MWCNT. Furthermore, MWCNT as well as CNF exposure led to altered miRNA levels, where miR-155-5p, miR-16-1-3p, miR-25-3p, and miR-27a-5p were significantly regulated by both materials. PANTHER pathway analysis of the identified miRNA targets showed that both materials affected growth factor (PDGF, EGF and FGF), Ras/MAPKs, CCKR, GnRH-R, integrin, and endothelin signaling pathways. These pathways are important in inflammation or in the activation, polarization, migration, and regulation of phagocytic capacity of macrophages. In addition, pathways involved in interleukin, WNT and TGFB signaling were highly enriched following MWCNT exposure.ConclusionTogether, these data support the importance of macrophage phenotypic changes in the onset and resolution of inflammation and identify epigenetic patterns in macrophages which may be critical in nanomaterial-induced inflammation and fibrosis

Restoration of the former fast in Hodíškov

The subject of the bachelor thesis is a proposal for the restoration of the former fast house with its farm part in Hodíškov. The project is based on the subject BGA032 – Architectural Studio 4 - Restoration of Monuments. The first mention of the fast house comes from reports in the town registers from 1462 and 1483. However, in these years the inn was already standing. The origins of the tavern are not clearly known. The actual establishment of the fast is assumed to have taken place around 1417, when a significant event took place in the village, namely the settlement of a dispute at Hodíškov between the abbot of Žďár, Jan, and Čeněk of Lipý, the lord of Nové Město. Between 1813 and 1945, the Linsbauer family owned the Hodíškov fast, which contributed to the elevation of the village and the region. They adapted the farmhouse and the yard to the present-day appearance of a chateau. In the period 1850-1866 the former building was rebuilt and adapted to its present form, together with the addition of an outbuilding. At the same time, the building represents several functions, where 1PP serves as a pub, 1NP as a social space with a clubhouse, kitchen and sanitary facilities. In 2NP there are starter flats and the attic space is not used at the same time. The utility area serves as a garage for farm vehicles. The deck space is also unused here. The municipality has an idea of using the building for starter flats. However, this solution is contrary to the planning documentation. The new use of the building will be in line with the planning documentation, will bring the building back to life and will contribute to the development of the village. It is an investment project, with the function of a hotel resort with temporary housing, wellness, restaurant facilities and a traditional pub for the residents of the village. In addition to the historic building, the project envisages a new building in the farmyard, which will unify the visual character of the farmyard. The aim of the bachelor thesis is to modify the object so that its full potential is used. At the same time, the historical form of the building and the genius loci of the whole plot should be preserved. The building will be treated as a historically valuable and listed building