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Dissertation
Bovine leukemia virus antisense transcription is essential for viral replication and oncogenesis
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Year: 2023

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Bovine Leukemia Virus (BLV) is a retrovirus that naturally infects the B lymphocytes of different species of the Bovidae family. The virus integrates into the host genome as a provirus, resulting in a lifelong infection. BLV induces a fatal disease called Enzootic Bovine Leukosis (EBL). Shortly after infection, viral sense transcription (from the 5’LTR to the 3’LTR) is strongly repressed by epigenetic mechanisms. However, the provirus still constitutively transcribes non-immunogenic microRNAs and RNAs via antisense transcription (from the 3’LTR to the 5’LTR). The biological function of antisense transcription is currently unknown. In this context, the objective of this thesis is to evaluate the relationship between antisense transcription, viral persistence, and oncogenesis. A reverse genetics approach was implemented, exploiting a mechanistic difference during the initiation of sense and antisense transcription. A mutation of only two base pairs in the provirus (A1) impaired antisense transcription in cell culture and sheep. RNA sequencing, confocal microscopy, and immunochemistry data revealed that expression of the HMCN-1 gene encoding the Hemicentin-1 protein was upregulated in B lymphocytes infected by the provirus carrying the A1 mutation. RNA interference showed that HMCN-1 was involved in cell adherence, migration, and death. In contrast to the wild-type, the A1 provirus did not induce leukemia/lymphoma. In conclusion, this thesis highlights the role of antisense transcription in viral persistence and oncogenesis induced by BLV. These observations may be helpful to understand better the pathogenesis of the Human T-cell Leukemia Virus type 1 (HTLV-1).

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Dissertation
Berlin as a new global start-up hub

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Dissertation
Bovine leukaemia virus antisense transcription is required for viral replication
Authors: --- --- --- --- --- et al.
Year: 2019 Publisher: Liège Université de Liège (ULiège)

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The bovine leukaemia virus (BLV) is a deltaretrovirus naturally infecting B-cells in cattle, which can lead in 5% of cases to an aggressive condition called enzootic bovine leukosis. The recent discovery of antisense transcripts, continuously expressed in both asymptomatic and leukaemic cells, opens new perspectives for understanding viral replication or pathogenesis. The objective of this work is to evaluate the importance of BLV antisense transcription in viral replication. Results show that a two-nucleotides mutation of the SP1 binding site drastically inhibits the transcriptional activity of the minimal 3’LTR promoter. In the context of a full-length provirus, this mutation induces a significant decrease of antisense transcripts in cell culture, and in a drastic reduction of proviral loads in infected animals. Antisense transcription thus plays a crucial role in viral replication.


Dissertation
Bovine leukemia virus antisense transcription regulates viral and host genome expression
Authors: --- --- --- --- --- et al.
Year: 2021 Publisher: Liège Université de Liège (ULiège)

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Bovine leukemia virus (BLV) is a deltaretrovirus that naturally infects B-cells of cattle, leading to a widespread lymphoproliferative disease termed enzootic bovine leukosis. In infected individuals, the virus is able to escape from the host immune response by viral silencing while maintaining its replicative and oncogenic potential. The recent discovery of BLV-associated antisense transcripts continuously expressed in both non-malignant and leukemic cells have raised the opportunity to unravel key mechanisms behind viral replication and pathogenesis. The objective of this work is to evaluate the importance of BLV antisense transcription in viral expression and replication, as well as its influence on host genome expression. Data show that mutation of two Sp-1 binding sites inhibits the antisense transcriptional activity of the 3' long terminal repeat (LTR) minimal antisense promoter in vitro. The mutation carried out in the U5 region on a full-length LTR also induces a reduction of antisense transcription, but consequently increases sense transcriptional activity. In the context of a mutated full-length provirus, viral sense and antisense expression are dysregulated in infected animals while proviral loads decrease drastically. Furthermore, the expression of host genes, especially TPPP and HMCN1, is influenced by altered antisense transcriptional activity. Antisense transcription is therefore able to control viral replication by mediating sense transcription and interferes with host genome expression.


Dissertation
Bovine Leukemia virus antisense transcription is required for efficient replication in vivo
Authors: --- --- --- --- --- et al.
Year: 2022 Publisher: Liège Université de Liège (ULiège)

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The Bovine leukemia virus (BLV) is an oncogenic deltaretrovirus that induces enzootic bovine leukosis. Most of the infected bovines are asymptomatic, but after a long latency period, approximately 5% of them end up developing a leukemia/lymphoma. How the virus promotes the evolution of the disease is still not fully understood. Nevertheless, recently discovered antisense RNAs (AS1-S/L and AS2) are likely to have an important role in viral propagation and malignancy. The aim of work is to assess the involvement of antisense transcription in viral infectivity, replication and B cell homeostasis. Based on a reverse genetics approach, results show that a two base pair mutation in the promoter sequence affects negatively viral antisense transcripts levels in vitro, and that viral replication, in contrast to infectivity, requires expression of antisense RNAs in vivo.


Dissertation
Ontwikkeling van een FPGA gebaseerd acquisitiesysteem voor aandrijflijnfouten in automotive testing

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Het doel van deze thesis is het ontwikkelen van een hardwareoplossing die nauwkeurige en betrouwbare registratie van encoderdata mogelijk maakt en deze efficiënt via EtherCAT verstuurt. Dit is een veelgebruikt industrieel netwerkprotocol dat bekend staat om zijn goede prestaties in realtime communicatie. Deze encoders, geïntegreerd in een transmissie, worden gebruikt om de transmissiefout te meten. Deze metingen zijn cruciaal voor de validatie van simulatiemodellen die KU Leuven in samenwerking met Toyota Motor Europe ontwikkelt. Het ontwikkelingsproces omvatte het opstellen van de vereisten, ontwerpen van de hardware en PCB, prototyping, testing en de ontwikkeling van een FPGA implementatie. Hierbij werden geavanceerde ontwerptools, simulatiesoftware en meetapparatuur ingezet om de nauwkeurigheid en betrouwbaarheid van het systeem te waarborgen. Deze nieuwe ontwikkeling vervangt de huidige, minder effectieve oplossing, waarbij verschillende encoderkaarten rechtstreeks met de EtherCAT-bus verbonden zijn. Aangezien deze kaarten afzonderlijke systemen zijn en hun klokken niet perfect synchroon lopen, levert de huidige oplossing niet de gewenste resolutie. De nieuwe hardwareoplossing lost dit probleem op door alle encoders op één systeem te integreren en simultaan met dezelfde systeemklok te verwerken. Deze parallelle dataverwerking maakt een FPGA een ideale verwerkingsunit. Hoewel de oplossing veelbelovend is, zijn er enkele problemen opgetreden bij de integratie van het EtherCAT IP van Beckhoff, waardoor de communicatie nog niet volledig functioneert. Er is contact opgenomen met de support van Beckhoff en de EtherCAT Technology Group om dit probleem op te lossen. De rest van het systeem werkt zoals vereist. Deze oplossing is ontwikkeld als basis en test voor de ontwikkeling van andere EtherCAT-slaves. Het doel is een herbruikbare basis te creëren die kan worden toegepast in combinatie met klantspecifieke I/O-oplossingen. Hierdoor ontstaat een flexibel platform dat kan dienen als oplossing voor vele andere toepassingen.

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