Charles University, Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Daniel Diviš Supervisors: Prof. Dr. Florence Apparailly, Directrice de Recherche Prof. PharmDr. Petr Pávek, Ph.D. (formal tutor) Title of diploma thesis: The role of miR-150 in the physiopathology of oligoarticular juvenile idiopathic arthritis Juvenile idiopathic arthritis (JIA) is the most common chronic rheumatoid disease affecting children, and its pathological mechanisms are still poorly understood. Innate and adaptive immunity including myeloid cells play a major role in these processes. Epigenetic deregulations along with non-coding microRNAs have been reported in many inflammatory diseases. Moreover, preliminary results obtained by the research group of Prof. Florence Apparailly showed accumulation of intermediate monocytes along with the high expression of miR-150 in the synovial fluid of children affected by oligoarticular JIA. Based on these findings a hypothesis has been postulated suggesting that miR-150 could have a role in the pathogenesis of this disease and in the regulation of monocyte differentiation and function. To study the impact of miR-150 on monocytes from the peripheral blood of healthy donors, transfection experiments were performed to neutralize miR-150. The...

Toxoplasma gondii is an obligate intracellular parasite, that is able to infect any nucleated cell. An important global pathogen, T. gondii can cycle between primary and secondary hosts, thus enabling widespread penetrance. Within its intracellular niche – a membrane-bound parasitophorous vacuole – T. gondii is nevertheless able to subvert a variety of host cell processes to allow its continued survival and replication. This includes modulation of host signalling processes as well as the scavenging of nutrient macromolecules. In recent years, microRNAs have emerged as important regulators of cellular processes including inflammation, tumorigenesis and metabolism, as well as development. It has become increasingly clear that this species of non-coding RNA is of great importance in ‘fine tuning’ many cellular responses. I hypothesise in this work that host cell miRNAs may be yet another means by which T. gondii manipulates its host upon infection. Using high-throughput-sequencing, I examine host cell transcriptional responses to infection both at the mRNA and microRNA level, using two strains of T. gondii at a variety of Multiplicities of Infection over a time course of 43 hours. Through these transcriptional analyses I identify a number of dysregulated pathways common in tumorigenesis, and contemplate the hypothesis that T. gondii may be behaving as an ‘intracellular tumour’, subverting host cell metabolic processes to mimic a long-known feature of cancer metabolism – that of aerobic glycolysis (the Warburg effect) – in order to satisfy its own energetic and metabolic needs.

Introduction Chikungunya virus (CHIKV) is an alphavirus in the family Togaviridae. Since 2005 the virus has caused a major epidemic of disease in humans, ranging from Central Africa, South-East Asia, Caribbean and more recently the Americas. The virus is spread by mosquitoes, most notably Aedes aegypti and Ae. albopictus. CHIKV causes an acute disease in humans, which is characterised by a rapid onset of high fever, rash, myalgia and arthralgia. The symptoms typically resolve within a week. Remarkably, up to a third of patients who recover from acute chikungunya develop chronic arthritis/arthralgia, which may last for months or years and has a large negative impact on the quality of life. The mechanism by which this occurs is not yet fully understood. CHIKV can infect human monocytes, and macrophages positive for CHIKV antigen have been observed in joint tissue from patients recovered from acute CHIKV infection but with chronic arthritis. Furthermore, it has been demonstrated that macrophages can be infected with CHIKV in vitro by a mechanism involving apoptotic debris from CHIKV-infected cells. Hypothesis and aims Infection of monocytes and macrophages with CHIKV contributes to clinical disease and virus persistence in vivo. The aim of this project was to investigate the mechanism by which alphaviruses infect macrophages in vitro, and to generate a CHIKV which is unable to replicate in monocytes and macrophages in vitro, and to study its pathogenicity in vivo. Materials and methods HeLa cells were infected with Semliki Forest virus (SFV), an alphavirus closely related to CHIKV, or SFV replicon particles (SFV VRP). Following cell death, whole cell supernatant or clarified cell supernatant from SFV- and SFV VRP-infected cells was passaged onto human monocyte-derived macrophages (MDMs). These cells were observed microscopically for expression of the fluorescent marker encoded by the SFV. Virus and VRP-infected apoptotic debris were inspected for the presence of alphavirus replication complexes by electron microscopy. Subsequently, a recognition element (RE) for a haematopoietic-specific miRNA (miR-142-3P) was incorporated into the genome of SFV (proof-of-concept) and CHIKV to investigate if blocking virus replication in cells of the mononuclear phagocyte system altered virus kinetics in vitro. The replication of the modified viruses was investigated in macrophage/monocyte cell lines Thp-1 and IC-21, and in HEK 293 cells modified to express miR-142-3P under the control of an inducible tetracycline promoter. Modified viruses were tested in animal models of disease (mouse for SFV and non-human primate for CHIKV) to investigate the pathogenicity of these viruses in vivo. Results The presence of apoptotic debris from SFV-infected cells was required to infect MDMs with SFV. The presence or absence of infectious virus particles in the apoptotic debris did not affect the infection rate. Intact alphavirus replication complexes were found within the apoptotic debris. MiR-142-3P RE was successfully incorporated into the genome of both SFV and CHIKV. RE-virus replication in all cells expressing miR-142-3P was reduced by 90-99% when compared to control viruses. RE-virus replication was not affected in cells which did not express miR- 142-3P. In interferon-α/β receptor knockout mice, RE-SFV generated viraemia comparable to the control virus, but could not infect efficiently the population of macrophages resident in the marginal zone of the spleen. RE-CHIKV was found to be genetically stable in vitro following multiple passages on BHK-21 cells in the absence of a selective pressure from miR-142-3P. RE-CHIKV was inoculated into two cynomolgus macaques. The data from this experiment are not yet available. Conclusion SFV was shown to infect MDM via apoptotic debris containing intact alphavirus replication complexes, which were the most likely infectious agent. SFV and CHIKV unable to replicate in haematopoietic cells in vitro were successfully engineered. The pathogenicity of modified SFV and CHIKV was investigated in vivo.

In recent years, LIM domains-containing protein 1 (LIMD1) has been identified as a critical component in microRNA (miRNA)-induced silencing complex (miRISC) to regulate miRNA-mediated gene silencing. Human Argonaute (AGO) 2 with its family members (AGO1-4) are critical for the biogenesis of miRNA and thus miRNA-mediated gene silencing. In this study, we have investigated the direct interaction interfaces between LIMD1 and AGO2. A distinct interface within LIMD1, amino acid (a.a) 140-166, is identified to be responsible for the binding to AGO2 and other members of AGO family. Furthermore, the Linker-2 (L2) domain within AGO2 is identified to be responsible for LIMD1 binding and its dependency on the phosphorylation at serine 387 (S387) residue within the L2 domain of AGO2. The phospho-mimic mutant (S387E) enhances the binding of AGO2 to LIMD1, whereas the phospho-deficient mutant (S387A) attenuates AGO2-LIMD1 interaction. In addition, the association of LIMD1 with other AGOs is also dependent on the phosphorylation at the equivalent conserved serine residue within the L2 domain on other AGOs. In addition to the above aspects, LIMD1 is a tumour suppressor gene frequently down-regulated in more than 75% human lung tumours. Because of their loss of expressions or functions, it is of the inherent difficulty in targeting tumour suppressor genes to treat cancers. In this study, the concept of synthetic lethality was used to identify possible protein kinases, the ablation of which are synthetically lethal to LIMD1 negative cancer cell lines. As a result, drugs that target these kinases may represent novel targeted therapies for LIMD1 negative lung tumours. ACVR2B and STK39 are validated to be synthetically lethal with LIMD1 loss. Additionally, the complete loss of LIMD1 expression causes a dramatic increase of STK39 expression due to miRNA-mediated gene silencing pathway. The inverse relationship between LIMD1 and STK39 may represent a conserved and fundamental signalling response and may be a predictive marker for STK39-targeted therapy.

Malignant mesothelioma (MM) is a locally invasive and highly aggressive cancer arising on the mesothelial surface of organ cavities (mainly pleural) as a direct result of asbestos exposure. The latency period of MM is long (20-50yrs) after initial asbestos exposure, and the prognostic outcomes are dismal with median life expectancy of 6-12 months post-diagnosis. There are no useful biomarkers for early MM diagnosis, no successful therapeutic interventions. These vast voids of knowledge led to our hypotheses that secreted vesicles, termed exosomes, play an important role in MM development and tumorigenic properties. Exosomes are nano-sized particles secreted from all cell types and carry biologically active cargo in the form of proteins, RNA, and lipids that can potently act as intercellular messengers in both healthy settings and disease states. We are the first to have conducted studies implicating the roles of exosomes in MM pathogenesis. Firstly, we analyzed the proteomic signature of exosomes from asbestos exposure models, in vitro and in vivo. Our in vitro data demonstrated that asbestos exposed lung epithelial cells and macrophages secrete exosomes with differentially abundant proteins compared to non-exposed controls and some of these proteins are relevant to asbestos exposure toxicology and MM development. Additionally, the exosomes from asbestos exposed cells significantly modulated the gene expression of target mesothelial cells in a way that reflected epithelial to mesenchymal transition and other tumorigenic properties. The in vivo mouse studies illustrated that mouse serum exosomes house differentially abundant proteins after asbestos exposure and this is measurable at an organism wide scale. Secondly, we assayed the miRNA composition of MM tumor exosomes compared to healthy mesothelial cell exosomes and found signature differences in miRNA abundances, particularly that MM tumor cells had significantly higher amounts of tumor suppressor miRNA, particularly miR-16-5p, in their exosomes. This led to the hypothesis that MM tumor cells preferentially secrete tumor suppressor miRNAs via exosomes to rid themselves of the anti-tumor effects. We employed exosomes secretion inhibitors and exosome force-feeding to demonstrate that MM cells do in fact secrete miR-16-5p (along with other tumor suppressor miRNAs) through exosomes and that this property can be targeted as a potentially novel therapeutic advance. Furthermore, we identified a mechanism of miR-16-5p loading into exosomes by the RNA binding protein HuR, and this mechanism is interestingly regulated by miR-16-5p itself in a negative feedback loop. Our studies thus far provide intriguing evidence on the role of exosomes in asbestos exposure and MM biology. We demonstrated the potential for exosomes as protein biomarkers in asbestos exposure and conduits of tumorigenic information to mesothelial cells. In addition, we incriminate exosomes as vehicles of tumor suppressor removal from MM tumor cells and we can target this as a potential n MM therapy.

In 2017, a novel miRNA was found at the MLTU-region of adenoviral genome, termed as MLP-TSS-sRNA. This current study started with performing a series of mutations in the MLP-TSS-sRNA in order to investigate how the MLP-TSS-sRNA as a single stranded small RNA was protected from rapid RNA degradation in transfected cells (in vivo). Since the hairpin structure of this small RNA was considered to be the reason to its high stability, the deletions of nucleotides were occurred inside the complementary region and the loop of the hairpin structure. Three variants of MLP-TSS-sRNAs were therefore transfected into the A549-lung epithelial cancer cell line and measured during times series studies. The results showed that the wild type form of this small RNA has the highest stability. Subsequently, a panel of different synthetic single-stranded RNAs, in which the MLP-TSS-sRNA sequence was modified to target different genes of interest, was used to compare its suppressive efficiency to the more traditional double stranded small interfering RNA “siRNA” or miRNA mimics. To this, the MLP-TSS-sRNA sequence was modified in such a way that it targeted the Dicer mRNA, thus termed as 3s-dicer-miRNA. Successful suppression of the Dicer mRNA as a consequence of using this modified 3s-dicer-miRNA sequence could emphasize that, theoretically, any possible mRNA of interest could be targeted. To express this miRNA inside a host cell, its sequence was incorporated in a CMV-driven plasmid vector system, upstream of the gene encoding for the HDV-ribozyme, which showed to be functional in vitro, but not in vivo. On the other hand, the vector system showed a clear tendency of being functional even in vivo, once it was put into the test by co-transfecting it with a Dicer plasmid inside 293-cells.

Transposable elements (TEs) are acknowledged sources of genetic change within organisms. The effects of transposition can range from the disruption or creation of a single gene to large-scale genome rearrangements. Transposition events can result in beneficial mutations which allow an organism to adapt to a new environment. In the last three years, several studies have reported that some miRNAs, small RNAs involved in post-transcriptional gene regulation, have evolved from TEs. miRNAs play an important role in the stress responses of many organisms. Interestingly, TEs are derepressed under the same stress conditions that miRNAs are known to ameliorate. The observation that miRNAs are known to evolve from TEs and that TEs are derepressed under stress conditions lead me to question whether TEs play a role in environmental adaptation through the creation of small RNA networks. To test this idea, Chlamydomonas reinhardtii cultures were grown under low carbon, nitrate enriched conditions and the small RNA pool was analyzed. I found that these conditions do stimulate the expression of novel small RNAs and that some of these RNAs and their targets are derived from transposition events.

Les exosomes sont des vésicules d’origine endosomale sécrétées par les cellules dans leur environnement après fusion à la membrane plasmique des endosomes multivésiculés. Les exosomes représentent un nouveau mode de communication entre les cellules en permettant un transfert direct de protéines, de lipides et d’ARN. L’objectif de ma thèse était d’étudier le rôle des exosomes dans la communication entre les neurones. Précédemment, le laboratoire a montré que les neurones sécrètent des exosomes de manière régulée par l’activité synaptique. Nous avons observé que les exosomes neuronaux ne sont endocytés que par les neurones. Après avoir montré qu’ils ne contiennent que des ARN courts, nous avons réalisé un séquençage complet de leurs microARN et observé que ces microARN étaient sélectivement exportés dans les exosomes. Nos observations suggèrent que les microARN contenus dans les exosomes peuvent modifier la physiologie des neurones receveurs. Nos résultats renforcent l’hypothèse du rôle des exosomes dans la communication entre les neurones via le transfert de microARN. / Exosomes are vesicles of endocytic origin released by cells into their environment following fusion of multivesicular endosomes with the plasma membrane. Exosomes represent a novel mechanism of cell communication allowing direct transfer of proteins, lipids and RNA. The goal of my PhD thesis was to study that exosomes represent a novel way of interneuronal communication. Our team has previously reported that neurons release exosomes in a way tightly regulated by synaptic activity. We observed that exosomes released by neurons are only endocytosed by neurons. We found that exosomes contain only small RNA and did a deep sequencing of all their microRNA. MicroRNA are selectively exported into exosomes. It seems that exosomal microRNA can modify the physiology of receiving neurons. Our results strengthen the hypothesis of the role of exosomes in the interneuronal communication by the way of microARN transfert.

Les laminopathies regroupent des pathologies liées aux lamines. La Progeria est due à une mutation du gène LMNA entrainant la synthèse d’une protéine anormale : la progérine. Elle s’accumule dans le noyau et entraîne des dommages cellulaires aboutissant à une sénescence prématurée, à l’origine d’un vieillissement prématuré et accéléré des patients dont le décès survient vers l’âge de 14 ans. Les microARNs (miRs) sont des petits ARNs non-codants régulant l’expression des gènes. Dans le projet principal de ma Thèse, nous avons identifié par un miRNome en RT-qPCR, 14 miRs différentiellement exprimés dans les fibroblastes HGPS. Certains d’entre eux appartenant à la région 14q32.2-14q32-3, sont surexprimés à cause de modifications chromatiniennes. Nous avons ensuite étudié l’impact de la surexpression des miR-376a-3p et miR-376b-3p sur la régulation de l’autophagie. L’inhibition de ces miRNAs entraine une augmentation du niveau d’autophagie, associée à une diminution de la progérine. Une 2ème étude miRNome réalisée en NGS, a permis d’identifier d’autres miRNAs potentiellement impliqués dans la Progeria. Dans un second projet, nous avons analysé l’expression des lamines de type A dans des cellules tumorales métastatiques issues d’épanchements pleuraux de patients atteints d’adénocarcinome bronchique. Nous avons démontré que la diminution d’expression de la lamine A chez un groupe de patient est corrélée à un mauvais pronostic. Cette diminution pourrait être due à miR-9 qui cible directement l’ARNm de la prélamine A. Ces travaux de Thèse illustrent le rôle fondamental des lamines et suggère une place importante des miRNAs dans la physiopathologie de ces 2 types de laminopathies / Laminopathies are diseases linked to lamins. Progeria (HGPS) is a genetic disease caused by a mutation in LMNA gene leading to an abnormal protein called progerin. It accumulates in nucleus and causes cell damages leading to a premature senescence. Patient die around 14 years old. miRNAs are small non coding RNA regulating gene expression. In my main project, I identified with a miRNome approach by RT-qPCR, 14 differentially expressed miRNAs in dermal HGPS fibroblasts. We demonstrated that the overexpression of the miRNAs that belong to the 14q32 region was caused by chromatin modulation. Next, we studied the role of miR-376-3p and miR-376b-3p on autophagy and demonstrated that the inhibition of their overexpression increases autophagy and decreases progerin. A second miRNome by NGS identified other miRNAs potentially linked to HGPS pathophysiology. In my second project, I studied lamins expression in metastatic cells from pleural effusion of lung adenocarcinoma patients. We showed that the decreased expression of lamin A in a group of patients was correlated with poor prognosis, which could be linked to miR-9 expression. This thesis illustrates the fundamental role of lamins and suggest the role of miRNAs in the pathophysiology of this to types of laminopathies.

Head and neck cancer is the 6th most common malignancy worldwide, with tumours of the tongue being one of the most prevalent sites. Despite advances in surgery and radiotherapy, the five-year survival has not changed during the last decades and remains at approximately 50%. Identification of novel biomarkers for more personalized treatment is important for increasing survival in these patients. One of the most commonly used methods in the search for new biomarkers is microarray analysis. A substantial limitation with this technique is the requirement for fresh frozen samples from which high quality RNA can be extracted. This becomes particularly problematic when attempting to discover differences associated with individual sub-types or rare cancers. Recent developments, including the DASL microarray platform, have provided the possibility of analysing RNA of poorer quality from formalin fixed paraffin embedded (FFPE) samples. FFPE is the standard way of preserving tissue from patients and millions of samples are stored around the world. In this thesis we have evaluated the use of FFPE samples and global gene expression profiling for increasing basic knowledge in a subgroup of oral cancer patients with tumours of the tongue. As confirmation of microarray results using qPCR is of outmost importance for conclusive data evaluation, we first aimed at finding a housekeeping gene stably expressed across malignant and non-malignant FFPE oral tissue. TUBA6, which belongs to the tubulin family was detected as being the most stable out of eight possible genes and was thus used for qPCR normalization throughout the following studies. We have performed three separate microarray experiments. Initially only a focused DASL array covering 502 cancer related genes was available and we used it to analyze a smaller cohort of patients and controls (n=36). A similar cohort (n=29) was also analyzed for expression of 836 micoRNAs. In 2009 a whole genome DASL array was launched, covering over 20,000 genes, and all tongue tumour samples available between 1997 and 2010 (n=87) were analysed using this array. Similar to other research groups we observed very high replicate reproducibility using both DASL arrays. When using the microRNA array and the whole genome DASL array an effect of sample quality on the detected expression level of individual genes was noticed. While the expression of some genes severely decreased with a decrease in sample quality others were not changed. This will impair normalization, leading to a residual non-biological variation within the data. Based on our findings we have presented some recommendations for minimizing the effect of sample quality and maximizing the level of biologically relevant information obtained from these experiments, e.g. ensuring that samples in groups to be compared are of the same quality range. For the microRNA data we also introduced an additional normalization step to the standard normalizations. We could show that lists of differentially expressed genes generated when taking these precautions were enriched for genes involved in cancer related processes and contained for tongue carcinoma previously identified changes. A number of differentially expressed genes, novel for tongue carcinoma, were also confirmed in high quality fresh frozen samples, including BCL2A1 (apoptosis), CXCL10 (immune response), SLC2A6 (energy transport) and miR-424 (angiogenesis). In conclusion microarrays can be used to analyze FFPE samples but should be performed with care. Standard normalization methods will not remove the variation introduced by samples being of different quality, leading to spurious results. Taking a few precautions, however, led to the identification of differentially expressed genes relevant in tumour development and maintenance. The recommendations we make can facilitate design of future studies using FFPE samples. The genes we identified as being differentially expressed in tumour tissue now need to be further evaluated for their potential as biomarkers in tongue carcinoma.