Translation control is crucial during virus-host interaction, in which the host relies on the translation machinery to mount an antiviral response or induce the inflammation response to reduce virus spread, while the virus aims to take control of this system to thwart the host defense while producing viral progeny. The field of oncolytic virus (OV) therapy relies on replicating, engineered viruses that preferentially infect tumor cells to induce direct oncolysis or promote an antitumor immune response. Despite the importance of translation control in virus-host interaction, not much has been described on the interaction at the translation level between OV and cancer cells. I propose that this knowledge gap could reveal significant improvements in OV efficacy in treating cancer. In my first study, I set out to characterize the translatome of an infection-resistant breast cancer cell line infected by three clinically advanced OVs to identify residual antiviral activity in cancer cells regulated by translation control. I found the inositol phosphatase Inpp5e to be a novel antiviral gene that is translationally induced during infection via a transcript variant shift. Mechanistically, I showed that the majority of Inpp5e transcripts in uninfected cells contain a long 5’ UTR that harbor four translationally inhibitory upstream reading frames (uORF). Yet, OV infection induced the expression of a shorter 5’ UTR with a spliced intron that removes three uORFs, derepressing the translation of Inpp5e mRNA. CRISPR-Cas9 knockout of Inpp5e also enhanced the infectivity of oncolytic HSV1 and VSV. My study suggests the existence of a class of translationally regulated antiviral genes in cancer cells. In my second study, I sought to adapt the translation of transgenes to the unique translation condition imposed by the infecting virus via the incorporation of a viral 5’UTR. I identified HSV1 5’UTRs by locating the transcription start site of most HSV1 genes using RNA-seq data, then determined the 5’UTR of US11 as a potent translation enhancer during HSV1 infection. Incorporation of this 5’UTR into the transgene expression cassette inserted into the HSV1 genome enhanced transgene expression significantly at the translation level. In my third study, I set out to explore the mechanism of miR-223 mediated inflammation inhibition. miR-223 is a protective miRNA in the context of atherogenesis via suppressing inflammatory signaling. Using transcriptome and translatome profiling (RNA-seq and Ribo-seq), I found that the inhibitory effect of miR-223 on inflammation occurs primarily at the translation level. Overall, my work highlights the importance of translation control in OV-cancer cells interaction, as well as in inflammation-related diseases.

Background: MiRNAs act as negative regulators of gene expression through target mRNA degradation or inhibition of its translation. In cancer, several miRNAs are upregulated and play crucial roles in tumorigenesis, making the inhibition of these oncomiRs an interesting therapeutic approach. This can be achieved by directly complementary single-stranded anti-miRNA oligonucleotides (antimiRs). A major bottleneck in antimiR therapy, however, is their efficient delivery. The nanoparticle formation with polyethylenimine (PEI) may be particularly promising, based on the PEI’s ability to electrostatically interact with oligonucleotides. This leads to their protection and supports delivery. In the present study, we explore for the first time PEI for antimiR formulation and delivery. We use the branched low molecular weight PEI F25-LMW for the complexation of different antimiRs, and analyse tumor- and metastasis-inhibitory effects of PEI/antimiR complexes in different tumor models. Results: In prostate carcinoma, transfection of antimiRs against miR-375 and miR-141 leads to tumor cell inhibition in 2D- and 3D-models. More importantly, an in vivo tumor therapy study in prostate carcinoma xenografts reveals anti-tumor effects of the PEI/antimiR complexes. In advanced melanoma and metastasis, we identify by a microRNA screen miR-150 as a particularly relevant oncomiR candidate, and validate this result in vitro and in vivo. Again, the systemic application of PEI/antimiR complexes inhibiting this miRNA, or the previously described antimiR-638, leads to profound tumor growth inhibition. These effects are associated with the upregulation of direct miRNA target genes. In a melanoma metastasis mouse model, anti-metastatic effects of PEI/antimiR treatment are observed as well. Conclusions: We thus describe PEI-based complexes as efficient platform for antimiR therapy, as determined in two different tumor entities using in vivo models of tumor growth or metastasis. Our study also highlights the therapeutic relevance of miR-375, miR-141, miR-150 and miR-638 as target miRNAs for antimiR-mediated inhibition.

The unveiling of small RNA (sRNA)-mediated gene regulatory pathways has profoundly shaped our understanding of the complexity of gene regulation. In eukaryotes, sRNAs have been found to control cellular metabolism, growth and differentiation, to maintain genome integrity, and to combat viruses and mobile genetic elements. To gain insight into the roles of small RNAs in apple and peach, we conducted sRNA-seq, computational analysis and molecular experiments to genome-widely characterize their microRNAs (miRNAs) and trans-acting siRNA (tasiRNA) pathways. We identified totally 75 miRNAs or families, including 23 conserved, 10 less-conserved and 42 apple-specific ones, and 118 miRNA target genes in apple. Two classical trans-acting siRNA (tasiRNA) pathways, miR390-TAS3 and miR828-TAS4, were characterized with similar but unique tasiRNA biogenesis profiles and target specificities. Importantly, miR159, miR828 and miR858 can collectively target up to 81 MYB genes potentially involved in diverse aspects of plant growth and development. In contrast to the location of the miR159 target site in a sequence-divergent region, the target sites of miR828 and miR858 are located in the region encoding the conserved R3 repeat domain of MYB proteins. 10 out of the 19 miR828-targeted MYBs undergo the biogenesis of various phased siRNA (phasiRNA), which potentially regulate diverse genes outside the MYB family. In peach, totally 94 miRNAs or families and 80 target genes were identified. Similar pathways of tasiRNA (miR828-TAS4 and miR390-TAS3) or phasiRNA (miR828-MYB-siRNA) processing were also characterized in peach. Taking advantage of reverse computation and public available deep-sequencing data, we demonstrated that the miRNA-TAS-PPR-siRNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to instigate phasiRNA production from PPR �genes, which are triggered by different 22-nt miRNAs, including miR7122, miR1509, and fve-PPRtri1/2 and through distinct mechanistic strategies, like miRNA direct-targeting or indirect-targeting through TAS-like genes, one-hit or two-hit, or even two layers of tasiRNA-TAS interactions. We found that the MIRNA genes of these miRNA triggers show great identity with the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Combined results from phylogenetic analyses and conservation extent profiling revealed that the super-miR7122 was potentially evolved from another miRNA superfamily (super-miR4376), which probably originated from the miR390. Additionally, the miR482/2118-NB-LRR-siRNA pathway was found to be conserved, but evolved with distinct features, in apple and peach. Taken together, widespread and complex miRNA and tasiRNA regulatory networks have been adapted in apple and peach. They add another crucial layer of regulation on gene activity and stability, and must exert essential functions in all aspects of plant life. / Ph. D.

No description available.

The two studies follow free nucleic acids in urine in search for biomarkers to distinguish urinary bladder cancer patients from controls. Bladder cancer forms 4 % of newly diagnosed oncological diseases in the Czech Republic. Nowadays, there is no accredited non-invasive method for its diagnosis, which is sufficiently accurate. Urine supernatant, which is washing the bladder mucosa and which does not contain cell debris, seems to be an appropriate source of biomarkers for non-invasive diagnosis. miRNAs, as a non-invasive biomarker of urinary bladder cancer, were studied in one of the studies. miRNAs are short noncoding RNA, which block the process of translation. miRNAs occur in all body fluids and are relatively stable. A study with three phases was assessed to find a suitable miRNA marker. 109 individuals were examined in total (36 controls and 73 bladder cancer patients). The analysis of miRNAs was based on RT-PCR (Reverse Transcription Polymerase Chain Reaction). In the first phase, the urine of 59 individuals was analyzed on TaqMan array card with 381 miRNAs. In the second phase, the results of the first phase were confirmed on the same cohort by a single miRNA assay. In the third phase, a new cohort was used (23 controls and 27 bladder cancer patients), analyzed by a single miRNA assay again....

No description available.

No description available.

Aside from few model organisms, little is known about early embryonic development or sex determination in insects, in particular mosquitoes which are major vectors of worldwide disease. The goals of this work were to investigate a mosquito-specific transcription factor and its intronic miRNA cluster and characterize a novel Y chromosome gene in An. stephensi. The aims of these experiments were to expand on the knowledge of genes involved in embryonic development and sex determination with potential application in vector control strategies. In Ae. aegypti a mosquito-specific bZIP1 transcription factor was demonstrated to be conserved among divergent mosquito species. It was maternally and zygotically-expressed and knock-down of bZIP1 mRNA via siRNA microinjection in the embryo resulted in embryonic death. The expression profile of this gene was determined through the use of RT-PCR and qRT-PCR. Additionally, this gene contains a miRNA cluster that is also relatively conserved amongst members of the Culicidae family suggesting its evolutionary importance. The miRNAs are also maternally and zygotically expressed and are the most abundant embryonic miRNAs as determined by small RNA sequencing and Northern analysis. Promoter activity for bZIP1 was characterized and the promoter was used to direct maternal and zygotic transgene expression in An. stephensi. Y chromosome genes were successfully identified in An. stephensi from Illumina sequencing data. This work focused on a gene unique to the Y 1 (GUY1). It was shown that GUY1 was male specific and linked to the Y chromosome. RT-PCR and single embryo analysis suggested that GUY1 was expressed during the maternal to zygotic transition and was only expressed in male embryos. It was shown in multiple transient and transgenic assays that the ectopic expression of GUY1 can influence the sex of subjected individuals and skew sex distribution to a male bias. There is still much to be investigated before a GUY1-based transgenic line can be tested and implemented for use in vector population control. However, the work in this dissertation represents a major step towards novel mosquito control strategies based on the manipulation of Y chromosome genes. / Ph. D.

The hair follicle (HF) is a cyclic biological system that progresses through stages of growth, regression and quiescence, each being characterized by unique patterns of gene activation and silencing. MicroRNAs (miRNAs) are critically important for gene silencing and delineating their role in hair cycle may provide new insights into mechanisms of hair growth control and epithelial tissue remodelling. The aims of this study were: 1) To define changes in the miRNA profiles in skin during hair cycle-associated tissue remodelling; 2) To determine the role of individual miRNAs in regulating gene expression programs that drive HF growth, involution and quiescence; 3) and to explore the role of miRNAs in mediating the effects of BMP signalling in the skin. To address Aims 1 & 2, global miRNA expression profiling in the skin was performed and revealed marked changes in miRNAs expression during distinct stages of the murine hair cycle. Specifically, miR-31 markedly increased during anagen and decreased during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and mid-anagen phases of the hair cycle resulted in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signalling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes. Luciferase reporter assay revealed that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. In addition, miR-214 was identified as a potent inhibitor of the Wnt signalling pathway in the keratinocytes. Mutually exclusive expression patterns of miR-214 and ¿-catenin was observed during HF morphogenesis. MiR-214 decreases the expression of ¿-catenin and other components of Wnt signalling pathways c-myc, cyclin D1, and Pten in the keratinocytes. Luciferase reporter assay proved that ¿-catenin serves as a direct target of miR-214. In addition, miR-214 prevented translocation of ¿-catenin into the nucleus in response to the treatment with an activator of the Wnt signalling pathway lithium chloride, and abrogated the lithium-induced increase of the expression of the Wnt target gene VI Axin2. This suggests that miR-214 may indeed be involved in regulation of skin development and regeneration at least in part, by controlling the expression of ¿-catenin and the activity of the Wnt signalling pathway. To address Aim 3, the role of miRNAs in mediating the effects of the bone morphogenetic protein (BMP) signalling in the skin was explored. MiRNAs were isolated from the primary mouse keratinocytes treated with BMP4 and processed for analysis of global miRNA expression using the microarray approach. Microarray and real-time PCR analysis revealed BMP4-dependent changes in the expression of distinct miRNAs, including miR-21, which expression was strongly decreased in the keratinocytes after BMP4 treatment. In contrast, miR-21 expression was substantially higher in the skin of transgenic mice over-expressing BMP antagonist Noggin. Transfection of the keratinocytes with miR-21 mimic revealed existence of two groups of the BMP target genes, which are differentially regulated by miR-21. Thus, this suggests a novel mechanism controlling the effects of BMP signalling in the keratinocytes. Thus, miRNAs play important roles in regulating gene expression programs in the skin during hair cycle. By targeting a number of growth regulatory molecules, transcription factors and cytoskeletal proteins, miRNAs are involved in establishing an optimal balance of gene expression in the keratinocytes required for the HF and skin homeostasis.

Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.