Myotonic Dystrophy Type I (DM1) is a multi-systemic genetic neuromuscular degenerative disease, has a prevalence in most populations of about 1:8000 and is caused by the nuclear retention of pathogenically expanded DMPK mRNA. A previous DM1 RNAi-kinome screen in our lab has identified kinases that reduced both count and area of DMPK mRNA foci in vitro. One such discovered kinase is PACT, which has showed to decrease foci count and area in DM1 fibroblasts by 30-50%. This study explored PACT as well as binding partner DICER involved in cellular RNA processing machinery, to highlight potential therapeutic targets in DM1. DM1 fibroblasts treated with PACT siRNA showed a non-significant trend of upregulation in MBNL1 mRNA and protein expression. PACT knockdown also showed trend of missplicing normalization in SERCA-1, more prominently seen in DM1-2000 human fibroblasts, whereas IR (insulin receptor) splicing remained unaffected. On the other hand, DICER knockdown did not have profound affect on foci integrity as well as MBNL1 RNA and protein xpressions in DM1 fibroblasts. SERCA-1 splicing in DICER siRNA treated samples also remained unchanged. We report here our findings in pursuit of potential therapeutic targets for the treatment of DM1.

The general public is at risk of ionising-radiation exposure. The development of high-throughput methods to triage exposures is warranted. Current biodosimetry techniques are low-throughput and encumbered by time and technical expertise. Although there has been an emergence of gene-profiling tools for the purpose of photon biodosimetry, similar capacities do not exist for alpha-particle radiation. Herein is the first genomic study useful for alpha-particle radiation biodosimetric triage. This work has identified robust alpha-particle induced gene-based biomarkers in isolated, ex-vivo irradiated leukocytes from multiple donors. It was found that alpha-particle and photon radiation elicited similar transcriptional responses, which could potentially be distinguished by aggregate-signature analysis. Although no distinct genes were sole indicators of exposure type, clustering algorithms and principal component analysis were able to demarcate radiation type with some success. By comparing the biological effects elicited by photon and alpha-particle radiation, significant contributions have been made to the field of radiation biodosimetry.

La formation des fruits charnus est un processus de développement impliquant trois stades principaux : (i) la transition fleur/fruit ou nouaison, (ii) la croissance et enfin (iii) la maturation des fruits. Chacune de ces étapes correspond à une transition développementale associée à d’importants changements physiologiques et structurels. Parmi toutes les hormones, l’auxine est connue pour jouer un rôle important dans l‟initiation et la coordination du processus de nouaison et des phases précoces de développement du fruit. La mise en place de la réponse à l‟auxine nécessite l‟intervention de facteurs de transcription appartenant à la famille des ARF (Axin Response Factor) connus pour réguler l‟expression des gènes de réponse précoce à l‟hormone en se liant aux Cis-éléments de type AuxRE (Auxin Response Element) possédant le motif conservé de réponse à l‟auxine. Les ARF sont de ce fait des candidats forts pour faire partie du mécanisme moléculaire par lequel l’auxine intervient dans le processus de nouaison. Le projet de recherche réalisé au cours de la thèse a permis d‟isoler et de caractériser au total 22 gènes Sl-ARF chez la tomate (Solanum lycopersicum), la plante modèle pour l’étude du développement et de la maturation des fruits charnus. Les gènes Sl-ARF montrent des profils d‟expression distincts selon les tissus et organes considérés, suggérant des fonctions spécifiques pour les membres de cette famille multigénique. Il est de plus montré que certains gènes Sl-ARF sont régulés à la fois par l’auxine et par l’éthylène, suggérant qu‟ils participent potentiellement au dialogue entre les voies de signalisation des deux hormones. L’expression transitoire a révélé la capacité des Sl-ARF à agir comme activateur ou répresseur transcriptionnel des gènes de réponse à l’auxine. L‟étude des profils d’expression globale, réalisée par RNA-seq à l‟échelle du génome entier, a révélé pour la première fois l‟existence d‟un niveau important de régulation par épissage alternatif des ARFs pendant la transition fleur-fruit. La localisation nucléaire des protéines Sl-ARF8A / B a été déterminée par fusion avec le gène rapporteur GFP puis expression dans un système "signle cell". L‟étude d’expression a révélé des profils distinctifs entre ARF8A et ARF8B avec une augmentation notable des transcrits Sl-ARF8A suite à la pollinisation des fleurs. Le rôle physiologique du gène Sl-ARF8A a été par la suite abordé par une approche de génétique inverse fournissant un nouvel éclairage sur les événements moléculaires qui sous-tendent la mise à fruit. La surexpression de Sl- ARF8 dans la tomate engendre des phénotypes pléiotropiques touchant la croissance - 4 - végétative (réduction de la taille des plantes, altération du développement racinaire et des tiges latérales) et l‟appareil reproducteur avec la formation de fruits parthénocarpiques (absence de graines). L’analyse histologique a révélé une modification notable du placenta et des ovules chez les lignées de sur-expression de Sl-ARF8 et les études par RNA-Seq ont identifié plus de 2632 gènes différentiellement exprimés chez les surexpresseurs par comparaison avec les lignées non transformées. Au total, l‟étude réalisée au cours de la thèse fournit une description exhaustive de la famille des ARF chez la tomate et une caractérisation fonctionnelle du gène Sl-ARF8 qui souligne son rôle comme figure centrale du mécanisme de contrôle de la nouaison des fruits. / The making of a fleshy fruit is a developmental process involving three main stages known as (i) fruit set, (ii) fruit growth and (ii) fruit ripening each corresponding to a transition step associated with major physiological and structural changes. Among other hormones, auxin is known to play a dynamic role in triggering and coordinating the changes associated with the process of fruit set and early fruit development. Auxin responses are mediated at the transcriptional level by Auxin Response Factors (ARFs) which regulate early auxin-responsive genes by specific binding to TGTCTC Auxin Response Elements (AuxREs). ARFs are therefore good candidates for being among the components of the molecular mechanism by which auxin mediates the fruit set. In the present study, a total of 22 Sl-ARF genes have been isolated and characterized in tomato (Solanum lycopersicum), a model plant for the study of fleshy fruit development and ripening. Expression profiling revealed distinctive patterns for Sl-ARF genes in different tomato tissues. Hormone treatment indicated that Sl-ARFs can be regulated both by auxin and ethylene with Sl-ARF2B, 5 and 9 likely to be involved in the cross-talk between the two hormones. Transient expression using a single cell system uncovered the ability of Sl- ARFs to act either as transcriptional activator or repressor in regulating the expression of auxin-responsive genes. Genome-wide expression profiling performed by deep RNASequencing revealed for the first time the importance of the alternative splicing mode of regulation of ARF genes during tomato fruit set. The physiological significance of two closely related Sl-ARFs, Sl-ARF8A and Sl-ARF8B, was addressed in the present study via a reverse genetics approach providing new insight on the molecular events underlying tomato fruit set. Fusion to GFP reporter gene indicated that both Sl-ARF8A/B proteins are nuclear localized. Expression analysis by RT-qPCR revealed some distinctive features between Sl-ARF8A and Sl-ARF8B with a notable increase in Sl-ARF8A transcript upon flower pollination. Over-expression of Sl-ARF8A/B in tomato resulted in pleiotropic phenotypes, including dwarf plants, altered root and lateral shoot development and parthenocarpic fruits (seedless). Histological analysis revealed altered placenta and ovules development in SlARF8A-OX flowers and RNA-Seq profiling identified over 2632 differentially expressed (DE) genes in SlARF8A-OX flower buds compared to wild type control plants. Considering the dramatic change in gene expression of genes related to auxin, jasmonate and ethylene displayed in SlARF8A-OX lines, these phytohormones are likely to play an active role in coordinating the fruit set process. Altogether, the present - 6 - study provided a comphensive description of the tomato ARF gene family and a functional characterization of Sl-ARF8 defining this ARF member as a central figure of the control mechanism of the fruit set process.

IN ENGLISH LANGUAGE Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmacology and Toxicology Candidate: Mgr. Alžbeta Štefela Supervisor: Prof. PharmDr. Petr Pávek, PhD. Title of the doctoral thesis: Novel bile acid derivatives as promising therapeutic approach Bile acids (BAs) are amphipathic steroidal molecules that are traditionally known to facilitate intestinal digestion and absorption of lipids and fat-soluble substances. On top, the recent findings have revealed that they represent important signaling agents involved in the orchestration of lipid, glucose and energy metabolism and immune response. BAs exhibit these roles by activating intracellular nuclear receptors such as farnesoid X (FXR), pregnane X (PXR) vitamin D receptors. Furthermore, BAs act as endocrine signaling molecules and activate numerous biological cascades via a membrane G-protein-coupled receptor, termed TGR5. Therefore, the extensive modulation of BA scaffold underwent to identify compounds with specific targeting of above-mentioned receptors as a promising therapeutic approach for the treatment of various liver and metabolic disorders including cholestasis, biliary cirrhosis, nonalcoholic steatohepatitis or diabetes. The principal aim of this doctoral thesis was to investigate the structure...

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Transcriptome reconstruction is an important component in the bioinformatical part of transcriptome studies. It is particulary interesting when a reference genome is missing, highly fragmented or incomplete, since in such situations, a simple alignment (or mapping) would not necessarily tell the full story. One species with such a highly fragmented reference genome is the Norway spruce (Picea abies (L.) H. Karst.) -- a conifer, which is very important for Swedish economy. Given its long juvenile phase and irregular cone setting, the demand of cultivated seeds are larger than the supply. This yields a desire to understand the transcriptomal biology behind the cone setting in P. abies. This thesis presents an introduction to this situation, and the biological and bioinformatical background in general, followed by two papers in which this is applied: Paper I introduces a novel de novo transcriptome assembler, with a focus on recovering isoforms, and paper II makes use of this assembler to be able to detect connections between scaffolds in the P. abies genome. Paper I also studies P. abies var acrocona, a mutant with shorter juvenile phase than the wild type, in order to detect how cone setting is initiated.  From differential expression studies of both mRNA and miRNA, a number of genes potentially involved in cone-setting in P. abies were found, and also a set of miRNAs that could be involved in their regulation. / Transkriptomrekonstruktion är en viktig komponent i den bioinformatiska delen av transkriptomstudier. Särskilt intressant är detta när ett referensgenom saknas, är kraftigt fragmenterat eller ofullständigt, ty i dessa situationer skulle inte en vanlig inpassning (eller mappning) kunna berätta allt. En art med ett kraftigt fragmenterat referensgenom är gran (Picea abies (L.) H. Karst.) -- ett barrträd, som är mycket viktigt för svensk ekonomi. På grund av dess långa uppväxtsfas och oregelbundna kottsättning, så är efterfrågan av förädlade fröer större än utbudet. Detta lämnar en önskan att förstå den transkriptomala biologin bakom granens kottsättning. Denna avhandling presenterar en introduktion till denna situation, den generella biologiska och bioinformatiska bakgrunden, följd av två artiklar i vilket detta är tillämpat: Artikel I introducerar en ny de novo transkriptomassembler med fokus på att återskapa isoformer, och artikel II tillämpar denna assembler för att kunna hitta länkar mellan scaffolder (genom-delar som hittills inte kunnat länkas med varandra) i grangenomet. Artikel II studerar även granmutanten acrocona (kottegran), vilken har kortare uppväxtsfas än vildtypen, för att kunna se vad som initierar kottsättning.  Från differentiella expressionsstudier av såväl mRNA som miRNA, hittades ett antal gener potentiellt involverade i granens kottsättning, samt några miRNA som kan vara involverade i dess reglering. / <p>QC 2021-02-12</p>

The "omics" is a concept of biological disciplines that globally characterizes and quantifies biomolecules involved in the key functions of an organism. The "omics" methods are used e.g. in molecular epidemiology, where they help to evaluate potential biomarkers that identify the impact of environmental factors for human health. In this thesis, the "omics" methods were applied in samples collected from newborns born in localities of the Czech Republic mostly differing by pollution levels from industrial sources. The principal aim was to determine whether environmental changes during prenatal development can affect gene expression and its regulation in newborns. The thesis further aimed to evaluate the level of air pollution at the time of biological samples collection. Using the whole genome approach, differentially expressed genes (DEGs) in newborns from districts Karvina and Ceske Budejovice (CB) were identified. In a pilot study of a small group of newborns from districts Most and CB, differentially methylated CpG sites in DNA were assessed. These sites attenuate gene activity and could be responsible for long-term changes at the genetic level. Finally, the aim was to find differentially expressed small non-coding RNA (DE miRNA) in newborns from Most and CB. Samples of umbilical cord blood from...

Sepsis represents a serious medical problem accounting for numerous deaths of critically ill patients in intensive care units (ICUs). An early, sensitive, and specific diagnosis is considered a key element for improving the outcome of sepsis patients. In addition to classical laboratory markers, ICU scoring systems and serum miRNAs are discussed as potential sepsis biomarkers. In the present prospective observational study, the suitability of miRNAs in sepsis diagnosis was tested based on proper validated and normalized data (i.e., absolute quantification by means of Droplet Digital PCR (ddPCR)) in direct comparison to classical sepsis markers and ICU scores within the same patient cohort. Therefore, blood samples of septic intensive care patients (n = 12) taken at day of admission at ICU were compared to non-septic intensive care patients (n = 12) and a healthy control group (n = 12). Our analysis indicates that all tested biomarkers have only a moderate informative power and do not allow an unequivocal differentiation between septic and non-septic ICU patients. In conclusion, there is no standalone laboratory parameter that enables a reliable diagnosis of sepsis. miRNAs are not superior to classical parameters in this respect. It seems recommendable to measure multiple parameters and scores and to interpret them with regard to the clinical presentation.

Cardiovascular disease is a major cause of morbidity and mortality around the globe, which is linked to athero-thrombosis. The risk factors for atherothrombosis, thus cardiovascular disease is impaired anti-thrombotic and antiinflammatory functions of the endothelium. Thrombosis is a hallmark of cardiovascular disease/complications characterised by increased platelet activation and increased secretion of platelet micro-particles that induce angiogenesis. This study determined the role of platelet micro-particles derived microRNA in the regulation of angiogenesis and migration, with a focus on the regulation of thrombospondin-1 release by platelet micro-particles delivered Let- 7a. The role of thrombospondin-1 receptors (integrin beta-1 and integrin associated protein) and downstream caspase-3 activation were explored by Let-7a inhibition prior to PMP treatment. MicroRNA dependent modulation of proangiogenic proteins including monocyte chemoattractant protein-1 and placental growth factor, and recruitment of activating transcription factor-4 protein to their promoter regions were explored. Main findings are: 1. Platelet micro-particles induce angiogenesis, migration, and release of novel cytokine subsets specific to platelet micro-particle’s RNA content. 2. The targeting of thrombospondin-1 mRNA by platelet micro-particles’ transferred Let-7a chiefly modulate the angiogenic effect on endothelial cells. 3. The inhibition of thrombospondin-1 translation enable platelet micro-particles to increase angiogenesis and migration in the presence of functional integrin beta-1 and integrin associated protein, and reduced cleaving of caspase-3. 4. Platelet micro-particle modulate the transcription of monocyte chemoattractant protein-1 and placental growth factor in a Let-7a dependent manner. 5. Let-7a induce angiogenesis ii independent of other platelet micro-particle’s microRNAs. Platelet micro-particle derived Let-7a is a master regulator of endothelial cell function in this model, which presents an opportunity for the development of new biomarkers and therapeutic approaches in the management of cardiovascular disease. Future studies should aim to confirm these findings in-vivo.

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