Mechanism of Genomic Instability in Cells Infected with the High-Risk Human Papillomaviruses

In HPV–related cancers, the “high-risk” human papillomaviruses (HPVs) are frequently found integrated into the cellular genome. The integrated subgenomic HPV fragments express viral oncoproteins and carry an origin of DNA replication that is capable of initiating bidirectional DNA re-replication in the presence of HPV replication proteins E1 and E2, which ultimately leads to rearrangements within the locus of the integrated viral DNA. The current study indicates that the E1- and E2-dependent DNA replication from the integrated HPV origin follows the “onion skin”–type replication mode and generates a heterogeneous population of replication intermediates. These include linear, branched, open circular, and supercoiled plasmids, as identified by two-dimensional neutral-neutral gel-electrophoresis. We used immunofluorescence analysis to show that the DNA repair/recombination centers are assembled at the sites of the integrated HPV replication. These centers recruit viral and cellular replication proteins, the MRE complex, Ku70/80, ATM, Chk2, and, to some extent, ATRIP and Chk1 (S317). In addition, the synthesis of histone γH2AX, which is a hallmark of DNA double strand breaks, is induced, and Chk2 is activated by phosphorylation in the HPV–replicating cells. These changes suggest that the integrated HPV replication intermediates are processed by the activated cellular DNA repair/recombination machinery, which results in cross-chromosomal translocations as detected by metaphase FISH. We also confirmed that the replicating HPV episomes that expressed the physiological levels of viral replication proteins could induce genomic instability in the cells with integrated HPV. We conclude that the HPV replication origin within the host chromosome is one of the key factors that triggers the development of HPV–associated cancers. It could be used as a starting point for the “onion skin”–type of DNA replication whenever the HPV plasmid exists in the same cell, which endangers the host genomic integrity during the initial integration and after the de novo infection

A bovine papillomavirus-1 based vector restores the function of the low-density lipoprotein receptor in the receptor-deficient CHO-ldlA7 cell line

BACKGROUND: The rationale of using bovine papillomavirus-1 (BPV-1) derived vectors in gene therapy protocols lies in their episomal maintenance at intermediate to high copy number, and stable, high-level expression of the gene products. We constructed the BPV-1 based vector harbouring the human low-density lipoprotein receptor (LDLR) gene cDNA and tested its ability to restore the function of the LDLR in the receptor-deficient cell line CHO-ldlA7. RESULTS: The introduced vector p3.7LDL produced functionally active LDL receptors in the receptor-deficient cell line CHO-ldlA7 during the 32-week period of observation as determined by the internalisation assay with the labelled LDL particles. CONCLUSION: Bovine papillomavirus type-1 (BPV-1)-derived vectors could be suitable for gene therapy due to their episomal maintenance at intermediate to high copy number and stable, high-level expression of the gene products. The constructed BPV-1 based vector p3.7LDL produced functionally active LDL receptors in the LDLR-deficient cell line CHO-ldlA7 during the 32-week period of observation. In vivo experiments should reveal, whether 1–5% transfection efficiency obtained in the current work is sufficient to bring about detectable and clinically significant lowering of the amount of circulating LDL cholesterol particles

Maanitsus : Petserimaa eestlastele kimmäse sõpru poolt kirotet

http://tartu.ester.ee/record=b1770657~S1*es

Analysis of the use of electricity in a private house

Bakalaureusetöö Tehnika ja tehnoloogia õppekavalTänapäeval on väga aktuaalseks teemaks energiasäästlik ja roheline ühiskond. Bakalaureuse töö käsitleb eramaja elektrienergia tarbimist ning selle analüüsi. Antud majapidamise aastane elektritarbimine on autori hinnangul liiga suur. Töö eesmärk on arendada meetodeid, millega tarbija teadlikkust enda elektritarbimise suhtes tõsta. Andmete kogumiseks kasutati Eesti Energia andmebaasi ning neid töödeldi Microsoft Exceli tabelarvutus programmis. Töö tulemusena leiti suurema elektritarbimisega perioodid ja oletatavad tarbimistippude tekitajad. Lisaks pakuti välja alternatiivenergia allikas päikesepaneelide näol, millesse investeering võiks tasuda ära vähem kui kümne aastaga. Elektritarbimise analüüsi tulemuste kasutamiseks oleks vajalik välja töötada mobiilirakendus, kus kuvatakse tarbija enda sätestatud tingimustel elektrikasutuse jooksva keskmise andmeid iga tunni kohta. Rakendus kuvab märguandeid, kui kodune elektritarbimine on ületanud viimasel tunnil eelnevalt määratud piiri. Rakenduse abil saab tarbija teadlikumalt jälgida ning juhtida igapäevast elektrikasutust ning seeläbi tarbida säästlikumalt.Nowadays, an energy-saving and green society is a very topical issue. This thesis concentrates on the use of electrical energy in a private house and also the analysys of it. According to the author, the annual electricity consumption of this household is too high. The aim of this thesis is to develop new methods to raise the consumers awareness of their own electricity consumption. The data was collected from Eesti Energia´s database and was processed in a Microsoft Excel spreadsheet program. As a result of the work, periods with higher electricity consumption and supposed consumers of consumption peaks were found. In addition, an alternative source of energy in the form of solar panels was proposed, in which the investment could expectedly pay off in less than 10 years. In order to use the results of the electricity consumption analysis, it would be necessary to develop a mobile application that shows the current moving average hourly data of electricity usage per hour under the conditions set by the consumer. The application displays alerts when home electricity consumption has exceeded the predefined limit in the last hour. With the application, the consumer can better monitor and control the daily use of electricity and thereby consume more economically

HPV-18 genoomi jagunemine ja säilumine rakkudes

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Pärast papilloomiviiruste (HPV) ja emakakaelavähi vahelise seose avastamist 1980ndate aastate lõpust on HPV alane uurimustöö olnud suunatud nende viiruste poolt indutseeritavate vähkkasvajate tekke molekulaarsete mehhanismide väljaselgitamisele. Preventatiivsete vaktsiinide väljatöötamise järgselt on võimalik HPV nakkust vältida. Samas on suur hulk inimesi, kes praeguseks on HPV-ga juba nakatunud ning neil on reaalne risk saada vähkkasvaja oma hilisematel eluetappidel. Kahjuks pole HPV levikut suudetud siiani efektiivselt veel piirata. Seetõttu on jätkuvalt aktuaalne viirusvastaste ravimite väljatöötamine, mis võimaldaks viirusnakkust efektiivselt elimineerida. Sealjuures on põhirõhk leida ühendeid, mis suudaksid peatada viiruse DNA replikatsiooni või takistada viiruse genoomi säilumist jagunevates rakkudes. Käesolevas doktoritöös anname ülevaate meie tööst - erinevate HPV subtüüpide võimest replitseeruda inimese osteosarkoomi rakuliinis U2OS, kirjeldame HPV-18 transkriptsioonikaarti ning näitame, et HPV replikatsioon ning transkriptsioon U2OS rakkudes on otseses vastavuses varasema informatsiooniga HPV genoomi replikatsiooni ning transkriptsiooni kohta. See annab alust järelduseks, et U2OS rakuliin on relevantne süsteem HPV genoomi funktsionaalseteks uuringuteks. Minu doktoritöö viimases osas esitame tulemused HPV-18 DNA stabiilse säilumise uurimisest, kus me kirjeldame HPV-18 DNA säilumiseks vajalike viiruslike elemente ning valideerime neid HPV-18 genoomi kontekstis. Kokkuvõtvalt, meie töö tulemusena kirjeldame me rakuliini, mille abil on võimalik luua süsteem viirusvastaste replikatsiooni ja HPV-18 DNA säilumist inhibeerivate ühendite skriinimiseks.After the discovery of the link between papillomaviruses (HPV) and cervical cancer in the 1980’s, papillomavirus research has been directed towards the studies of the molecular mechanisms of induction of cervical cancer by the papillomaviruses. The preventive vaccine against HPV makes it is possible to avoid HPV infection, however a large number of people are still infected and HPV infection has not been effectively controlled yet. Thereby it is still important to continue to pursue HPV antivirals that could either block the viral DNA replication or stable maintenance of the viral genome in infected cells. In this thesis we describe immortalized human osteosarcoma cell line U2OS that is capable of supporting replication a number of different HPV subtypes. We describe further the transcriptional map of HPV-18 in U2OS cells and verify the relevancy of this system as the information obtained from these studies correlates with the previous findings of HPV-18 in primary keratinocytes. In the last part of this thesis we go in depth of the stable maintenance and segregation of HPV-18 DNA as we describe the cis-elements crucial for this function. We further validate this information in the context of the U2OS cells. In conclusion we describe a cell line suitable for developing screening system for antivirals against HPV and we go in depth of describing crucial elements of HPV-18 DNA segregation

Differential phosphorylation determines the repressor and activator potencies of GLI1 proteins and their efficiency in modulating the HPV life cycle

The Sonic Hedgehog (Shh) signalling pathway plays multiple roles during embryonic development and under pathological conditions. Although the core components of the Shh pathway are conserved, the regulation of signal transduction varies significantly among species and cell types. Protein kinases Ulk3 and Pka are involved in the Shh pathway as modulators of the activities of Gli transcription factors, which are the nuclear mediators of the signal. Here, we investigate the regulation and activities of two GLI1 isoforms, full-length GLI1 (GLI1FL) and GLI1ΔN. The latter protein lacks the first 128 amino acids including the conserved phosphorylation cluster and the binding motif for SUFU, the key regulator of GLI activity. Both GLI1 isoforms are co-expressed in all human cell lines analysed and possess similar DNA binding activity. ULK3 potentiates the transcriptional activity of both GLI1 proteins, whereas PKA inhibits the activity of GLI1ΔN, but not GLI1FL. In addition to its well-established role as a transcriptional activator, GLI1FL acts as a repressor by inhibiting transcription from the early promoters of human papillomavirus type 18 (HPV18). Additionally, compared to GLI1ΔN, GLI1FL is a more potent suppressor of replication of several HPV types. Altogether, our data show that the N-terminal part of GLI1FL is crucial for the realization of its full potential as a transcriptional regulator