MicroRNAs are noncoding RNAs inducing sequence-specific posttranscriptional inhibition of gene expression and represent the major class of small endogenous RNAs in mammalian cells. Over 2,500 of human microRNAs potentially regulating more than 60% of human protein-coding genes have been identified. MicroRNAs participate in the majority of cellular processes, and their expression changes in various diseases, including cancer. Currently, there is no efficient small chemical compound available for the modulation of microRNA pathway activity. At the same time, small chemical compounds represent excellent tools for research of processes involving RNA silencing pathways, for biotechnological applications, and would have a considerable therapeutic potential. The presented work represents a part of a broader project, whose ultimate goal is: (i) to find a set of small molecules allowing for stimulation or inhibition of RNA silencing and (ii) to identify crosstalks between RNA silencing and other cellular pathways. This thesis summarizes results from the first two phases of the project, the development of high-throughput screening assays and the high- throughput screening (HTS) of available libraries of small compounds. To monitor the microRNA pathway activity, we developed and optimized one biochemical...

Abstract Calcific aortic valve disease (CAVD) is the most common valvular heart disease in the Western world. Although it shares mainly the same risk factors as coronary heart disease (CHD), i.e. similar initial events in both diseases but with time, they lead to different clinical outcomes. Thus, when it affects the coronary arteries, the disease leads to an obstructive or rupture-prone plaque whereas in the aortic valve, it causes massive calcification and ossification. This obstructs the blood flow from the left cardiac ventricle, causing myocardial hypertrophy, and if left untreated, heart failure and death. Many of the pathobiological differences between CAVD and CHD remain unknown. Currently, there are no effective lifestyle- or pharmacologic treatments for CAVD and the only therapy is a valve replacement operation. In this thesis, several studies utilizing large-scale methods were undertaken to profile the molecular events leading to CAVD. Surgically removed valves from patients in different stages of the disease were obtained and gene transcripts, microRNA-molecules and several proteins were identified as being differentially expressed. Several of these were investigated further, including two pro-inflammatory CC-type chemokine ligands 3 and 4 (CCL3 and CCL4), microRNA-125b, several granzyme-proteins and heat-shock protein 90. The results of this thesis provide a large dataset of hundreds of molecular changes associated with CAVD. It is proposed that they can be used as a basis for the generation of new hypotheses and assist in the design of experiments to clarify the mechanisms driving CAVD. / Tiivistelmä Aorttaläpän kalkkeutuva ahtauma on länsimaiden yleisin sydänläppäsairaus. Riskitekijät ovat pääosin samat kuin sepelvaltimotaudissa, ja molemmat saavat alkunsa samalla tavalla. Ajan myötä ne kuitenkin johtavat varsin erilaisiin kliinisiin ilmenemismuotoihin: sepelvaltimoihin kasvaa ahtauttavia ja repeytymisherkkiä plakkeja, kun taas aorttaläppään muodostuu runsaasti kalkkia ja luuta. Se haittaa verenvirtausta sydämen vasemmasta kammiosta aorttaan, mikä aiheuttaa sydänlihaksen paksuuntumista. Hoitamattomana tauti johtaa lopulta sydämen vajaatoimintaan ja kuolemaan. Monet syyt eroihin sepelvaltimotaudin ja aorttaläpän ahtauman välillä ovat edelleen tuntemattomia. Tällä hetkellä aorttaläpän ahtaumaan ei ole olemassa tehokasta elintapa- tai lääkehoitoa, ja ainoa hoitomuoto onkin vioittuneen aorttaläpän korvaaminen proteesilla. Tässä väitöskirjatyössä tehtiin useita laaja-alaisia molekyylitason profilointitutkimuksia, joilla selvitettiin aorttaläpän ahtaumaan mahdollisesti johtavia mekanismeja. Aineistona oli leikkauksessa potilailta poistettuja, erilaisissa taudin vaiheissa olevia aorttaläppiä. Niistä kerättiin tietoja kaikkien geenien ilmentymisestä, mikroRNA-molekyyleistä sekä koko proteomitason muutoksista. Useat tunnistetuista molekyyleistä valittiin jatkotutkimuksiin niiden tarkempien ominaisuuksien selvittämiseksi. Näitä olivat tulehdusta välittävät kemokiinit CCL3 ja CCL4, mikroRNA-125b, useat grantsyymiproteiinit sekä lämpöshokkiproteiini 90. Väitöskirjatyön tuloksista voidaan muodostaa ainutlaatuinen aineisto sadoista erilaisista aorttaläpän ahtaumaan johtavista molekyylitason muutoksista. Sitä voidaan hyödyntää uusien tutkimushypoteesien muodostamisessa sekä aorttaläpän ahtauman tarkempien mekanismien selvittämiseen tähtäävien kokeellisten tutkimusten suunnittelussa.

Oncogenic microRNAs (miRNAs) are small RNA molecules that inhibit post-translational regulatory mechanisms at the epigenetic level. miRNAs are often deregulated in malignancies and due to their stability are detectable in non-cellular fractions of peripheral blood. In our laboratory, we have performed several studies that have investigated and utilized miRNAs as biomarkers for various hematological tumors (e.g., chronic lymphocytic leukemia, Hodgkin`s lymphoma) and solid tumors (e.g., breast cancer). The aim of these studies was to find the association of miRNAs with pathophysiological and clinical aspects of each disease. Here, we confirmed the importance of particular miRNA or its complex during disease monitoring. Combining clinical, molecular biological and statistical analyses, we were able to find miRNA sets that fulfilled not only a diagnostic role but also a prognostic role beyond expectations. The main focus of this thesis is on the investigation of microRNAs in the diagnosis of a hematological malignancy - primary cutaneous T-cell lymphoma (CTCL). Tumor specificity of some miRNAs has been demonstrated. Their aberrant expression in tissue samples of CTCL patients obtained from skin biopsies, correctly distinguished malignant disease from control samples of benign skin lesions. Here, we...

miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...

MicroRNA (miRNA) are short non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Many miRNAs are involved in the pathogenesis of cardiovascular diseases, which is associated with altered gene expression. This work compares miRNA gene expression profiles among various biological sources - whole peripheral venous blood (whole PB), plasma and plasma exosomes. For all tested groups combined, the expression levels of miRNA were maximal in whole PB and lowered in plasma and plasma exosomes, and the expression levels of miRNA were higher in plasma than in plasma exosomes, except miR-126-3p, which had a higher level detected in plasma exosomes compared to plasma. This work also compares expression levels of cardiovascular miRNA between women with anamnesis of gestational diabetes mellitus (GDM) and physiological gravidity 3-11 years postpartum in whole PB, plasma and plasma exosomes. In whole PB, 12 of 29 tested miRNAs were up-regulated in women with prior exposure to GDM. MiR-181a-5p was up-regulated in plasma exosomes and miR-499a-5p in plasma in women with prior exposure to GDM. The changes in whole peripheral venous blood seem to reflect the complex systemic response to the changes that occurred during the onset of GDM. Women with aberrant epigenetic profiles may...

Deregulation of gene expression caused by genetic or epigenetic changes plays an important role in pathogenesis of cancer. The thesis is a commented collection of ten publications dealing with the molecular biology of tumours. The author has significantly contributed to all of them. All the articles contained in the thesis are linked to the topic of assessment of molecules involved in gene expression regulation (microRNAs) or DNA alterations that affect gene expression (promoter methylation, presence of a fusion gene). MicroRNAs are short single-stranded RNA molecules involved in posttranscriptional regulation of gene expression by triggering mRNA degradation or inhibiting translation. It is a basic mechanism with an impact on all cellular processes including the pathogenesis of various diseases. MicroRNAs can either act as oncogenes by decreasing the expression of tumour-suppressor genes or as tumour-suppressor genes by decreasing the expression of oncogenes. However, the network of microRNA - RNA interactions is much more complex. Our published results that are part of this thesis are focused on colorectal carcinoma (CRC), prostate cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer and non-small cell lung cancer (NSCLC). In patients with CRC, we demonstrated the prognostic...

Deregulation of gene expression caused by genetic or epigenetic changes plays an important role in pathogenesis of cancer. The thesis is a commented collection of ten publications dealing with the molecular biology of tumours. The author has significantly contributed to all of them. All the articles contained in the thesis are linked to the topic of assessment of molecules involved in gene expression regulation (microRNAs) or DNA alterations that affect gene expression (promoter methylation, presence of a fusion gene). MicroRNAs are short single-stranded RNA molecules involved in posttranscriptional regulation of gene expression by triggering mRNA degradation or inhibiting translation. It is a basic mechanism with an impact on all cellular processes including the pathogenesis of various diseases. MicroRNAs can either act as oncogenes by decreasing the expression of tumour-suppressor genes or as tumour-suppressor genes by decreasing the expression of oncogenes. However, the network of microRNA - RNA interactions is much more complex. Our published results that are part of this thesis are focused on colorectal carcinoma (CRC), prostate cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer and non-small cell lung cancer (NSCLC). In patients with CRC, we demonstrated the prognostic...