Proper understanding of ovarian follicular and luteal development is essential to improve and optimally control reproductive function in domestic animals and to unravel causes of infertility in animals and humans. MicroRNAs (miRNAs) are key post-transcriptional gene regulators in multiple processes involving tissue growth and differentiation. The studies in this thesis were carried out to identify and characterise expression profiles of miRNAs and their potential roles during ovarian follicular development in the cow. The first study aimed to identify expression profiles of miRNAs during antral follicle growth. Follicles were collected from abattoir ovaries and their status (healthy/atretic) was assessed by measuring steroid levels and aromatase expression. An heterologous microarray approach followed by RT-qPCR validation was used to identify and compare miRNA profiles between large (13–16 mm) healthy and small (4–8 mm) follicles. A unique subset of 7 miRNAs (miR-144, miR-202, miR-210, miR-451, miR-652, miR-873 and miR-876) was identified that were enriched in the Large Healthy follicles relative to small follicles and Large Atretic follicles. Further characterisation revealed that many of these miRNAs were highly expressed in the granulosa compartment of the follicle, predominantly in the mural granulosa cells, indicating their potential involvement in regulating granulosa cell function. Expression patterns of miRNAs in the ovarian tissues were generally reflected in the follicular fluid, thus follicular fluid may be used to monitor follicular health. Finally, tissue-wide screening of miRNAs identified miR-202 as a gonad-specific miRNA in the cow. The aim of the second study was to identify potential roles of the miRNAs enriched in Large Healthy follicles. Using in silico approaches, about 1700 bovine genes were identified as the predicted targets of those miRNAs. Putatively targeted signalling pathways are primarily involved in cell survival, proliferation and differentiation. Further, as expected, top predicted gene targets (SPRED1, ATG7, TGFBR2) showed an expression pattern in follicular tissues that was opposite to the patterns of the targeting miRNAs. However, expression patterns of miR-202 or miR-210 during follicular growth could not be recapitulated in gonadotrophin-stimulated cells in vitro and moreover, over-expression or inhibition of miR-202 in these cells did not result in changes in target mRNA levels. The third study investigated the expression profiles of miRNAs during follicle-luteal transition. Microarray analyses identified 9 miRNAs that were upregulated and 14 miRNAs that were downregulated between Large Healthy follicles and early corpus luteum in the cow. Predicted targets of these miRNAs were associated with signalling pathways involved in cell survival, proliferation and differentiation, indicating that these miRNAs might play significant regulatory roles during ovulation and early luteal development. Further, expression of some of these miRNAs and their putative targets during luteinisation in vivo could be recapitulated in cultured granulosa cells providing a system to study the functional roles of these miRNAs during follicle-luteal development. In summary, this is the first study identifying unique subsets of miRNAs associated with follicular development and the follicle-luteal transition in the cow which may be important for female fertility.

Kaposi’s Sarcoma-associated Herpes Virus (KSHV) is a human herpesvirus associated with cancers. To date, KSHV miRNAs have been mostly identified via analysis of cells that are undergoing latent infection. This work presented here is a novel approach to profile small RNAs from populations of cells undergoing predominantly lytic infection. Using two different next generation sequencing platforms, I cloned and sequenced both pre-microRNAs and derivative microRNAs (miRNAs). This analysis shows that the vast majority of viral and host 5p miRNAs are co-terminal with the 5 prime end of the cloned pre-miRNAs, consistent with both being defined by microprocessor cleavage. I report the complete repertoire (25 total) of 5p and 3p derivative miRNAs from all 12 previously described KSHV pre-miRNAs. Two KSHV pre-miRNAs, pre-miRs-K8 and K12, encode abundant derivative miRNAs from the previously unreported strands of the pre-miRNA. I identify several novel small RNAs of low abundance, including viral microRNA-offset-RNAs (moRNAs), and antisense viral miRNAs (miRNA-AS) that are encoded antisense to previously reported KSHV pre-miRNAs. This work also shows that much of the KSHV genome is transcribed in both the top and bottom strand orientations during lytic replication. Despite the enormous potential to form double-stranded RNA in KSHV-infected cells, I observe no evidence for the existence of abundant viral-derived small interfering RNAs (siRNAs). From the small RNA deep-sequencing, I also detected a low abundant small RNA fragment (23 nt) that maps to a putative hairpin structure (named hairpin K) within the KSHV PAN transcript. I demonstrate that hairpin K is a cis-negative regulatory element in PAN. It is well-appreciated that viruses utilize host effectors for macromolecular synthesis and as regulators of viral gene expression. Viruses can encode their own regulators, but often utilize host-encoded factors to optimize replication. This work shows that Drosha, an endoribonuclease best known for its role in the biogenesis of miRNAs, can also function to directly regulate viral gene expression. Kaposin B (KapB) is a KSHV-encoded protein associated with cytokine production and cytotoxicity. I demonstrate that in addition to previously known transcriptional mechanisms, differences in Drosha levels contribute to low levels of KapB expression in latency and robust increases in expression during lytic replication. Thus, KSHV modulates Drosha activity differentially depending on the mode of replication. This regulation is dependent on Drosha-mediated cleavage, and KapB transcripts lacking the Drosha cleavage sites express higher levels of KapB resulting in increased cell death. This work increases the known functions of Drosha and implies that tying viral gene expression to Drosha activity is advantageous for viruses. / text

Apolipoprotein B (apoB) is a key structural and functional protein of lipoproteins and is synthesized constitutively in the liver. This study investigated the role of microRNAs (miRNAs) in translational control of apolipoprotein B (apoB) mRNA and protein synthesis. Using bioinformatic analysis, I identified two specific miRNAs namely, miR-544 and miR-1202 with potential to interact with 3’ and 5’ UTR of apoB, respectively. Using HepG2 cells as the model system, the effects of transfection of exogenous miRNAs and inhibition of endogenous miRNAs were assessed on the expression of apoB mRNA and protein synthesis, as well as apoB mRNA traffic into cytoplasmic P-bodies. miR-544 induced a significant reduction in apoB mRNA expression and protein synthesis while increasing the co-localization of apoB mRNA into P-bodies. In contrast, transfection of miR-1202 increased apoB mRNA expression and protein synthesis. In summary, these data demonstrate that specific miRNAs are involved in translational control of apoB mRNA.

Leveraging miRNA-Seq data and the 1000 Genomes imputed genotypes, we identified rs174561-C as a strong miRQTL for circulating miRNA-1908-5p (P=4.8x10-31) which has an inverse relationship with circulating LDL-C, fasting glucose and A1c. Here I investigated the molecular mechanism(s) linking miR1908-5p to cholesterol metabolism. First, by overexpression experiments in HuH-7 cells demonstrate that the presence of the C allele, associated with lower LDL-C levels, significantly increases miR-1908-5p by 2.15-fold relative to the T allele. Further experiments revealed that 72-hour cholesterol depletion increases miR-1908-5p expression (2.11-fold) whereas cholesterol loading decreases miR-1908-5p expression (0.69-fold). Differential miR-1908-5p expression was then used to profile genes involved in lipoprotein signaling and cholesterol metabolism using a PCR array to identify LDLR as a gene of interest. Although total RNA and protein expression of LDLR was unchanged in response to differential miR-1908-5p expression, the ratio of the mature form to the cleaved form of LDLR decreased following miR-1908-5p inhibition (0.85-fold) and conversely, increased with mimic treatment (1.63-fold). Cleavage of the mature LDLR is known to reduce cell surface affinity for LDL. These findings uncover a potential mechanism linking miR-1908-5p to lower LDL-cholesterol levels through reduced LDLR cleavage.

Stress signaling pathway regulates proteins which are critical for reprogramming of metabolic synthesis and gene expression to achieve homeostasis and cellular stability under stress conditions. The understanding of stress signaling mechanism and response will increase the ability to improve plant‘s oralgal resistance to stress. The theoretical part summarizes the current knowledge of biosynthetic pathway and miRNA roles and presents selected model organisms (Chlamydomonas, Arabidopsis). As well, it introduces abiotic stress and seeks links between abiotic factors, secondary metabolites and miRNAs. The experimental part deals with selected abiotic factors [lycorine, UV-C radiation, ZnSe quantum dots (QDs), CO2] and their influence on Chlamydomonas reinhardtii and Arabidopsis thaliana. It has been shown that 100 and 250µM ZnSe QDs in the form of foliar feeding caused oxidative stress in the leaves without morphological changes in Arabidopsis thaliana. Additionaly, 250µM concentration inhibited the viability of Agrobacterium tumefaciens, a Gram-negative bacterium infecting plants, by 60 %. Furthermore, the regulatory biochemical, molecular and post-transcriptional pathways of Chlamydomonas reinhardtii response to UV-C radiation and lycorine were identified. Further, three specific miRNAs (Cre06.g281600, Cre06.g30900, Cre16.g662600) have been studied in connection with carbon concentrating mechanism (CCM) in Chlamydomonas reinhardtii. Expression of mentioned miRNAs had a positive correlation with target mRNA and revealed the potential regulatory role of miRNAs during CCM adaprion and possibility to increase the efficiency of photosynthesis. Despite, the role of epigenetics including miRNA interference is still unclear in the unicellular algae.

This work deals with the study of the possibility of creating an electrochemical biosensor for the detection of specific nucleic acid sequences. The theoretical part describes various types of RNA, with a special focus on miRNAs and possibilities of its detection. It also describes various electrochemical detection approaches, both nucleic acids, and more specifically miRNAs. miRNA is a potential biomarker that can be used for early and non-invasive diagnosis of cancer. The experimental part is aimed at designing, optimizing and preparing a sensitive biosensor for the detection of miRNA-21. A biosensor was prepared to detect the lowest concentration of miRNA-21 in the sample. A linear concentration range for the calibration curve from 1 nM to 1 µM concentration was measured. We measured LOD of 1 fM and by calculation from the regression equation to 3.2 zM and LOQ was 10.8 zM. Subsequently, samples were measured in artificial urine to verify the functioning of the sensor in real conditions. The results showed a minimal effect of the matrix on the determination of the target miRNA.

Reticulitermes flavipes is one of the most common termite species in the world, and has been an intriguing research model due to its ecological and biological and economic significance. The fundamental biological question addressed by this study is to elucidate the role of miRNAs in termite development and how miRNA can influence labor division. miRNAs are short non-coding RNAs that have an important role in gene regulation at post-transcriptional level, and can potentially be involved in the regulation of caste polyphenism. Using a computational approach, I identified 167 conserved and 33 novel miRNAs in the dataset. miR-iab-4 and 19 other miRNAs showed highly differential expression between worker and soldier, and their possible roles in termite biology are discussed. To reliably quantify miRNA expression in experiments, I tested the stability of 10 miRNAs as reference gene using quantitative real-time PCR. miR-8_3, bantam and miR-276a-3p are the most stable miRNAs in different castes, pre-soldier formation, and different tissues, respectively. Lastly, the predicted miRNA expression is verified by the qRT-PCR for 8 miRNAs. Overall, this study shows that miRNA plays a role in mediating the work-soldier transition in R. flavipes.

<p>miRNAs therapeutics have emerged as a potential cancer therapeutics due to their unique ability to target multiple genes, allowing a single miRNA to act as a multi-drug cocktail. However, toxicity associated with current delivery vehicles as well as the sensitivity of the miRNA to serum nucleases are critical hurdles that stand against their clinical utility. Ligand-targeted delivery approaches such as small molecules, aptamers, antibodies or glycoconjugates provide a promising strategy to achieve specific delivery of the therapeutic cargo to the intended targeted cells without any toxicity. We hypothesized that combining both ligand targeted approach along with modifying the miRNA would provide a perfect delivery system which does not only achieve specific delivery but also reduce the therapeutic dose. The data presented in this dissertation shows (i) that miR-34a delivered using a combination of targeting ligand (DUPA) and an endosomal escape agent (nigericin) enables selective delivery of miR-34a to prostate cancer cells, (ii) that the use of full chemical modification approach enhances miR-34a stability and activity both in vitro and in vivo. Overall, these results provide an advancement in the miRNA delivery field and will help the development of miRNA based therapeutics against cancer.</p>

No / MicroRNAs are a relatively new class of small, noncoding RNA species that represent a cornerstone of cell biology, with diverse roles ranging from embryonic development to aging. miRNAs function to regulate posttranscriptional gene expression, are critical to the normal function of cells, and as such are frequently dysregulated during disease processes. In this chapter, we discuss the biogenesis and mechanism of action of miRNA and their role in cancer initiation, promotion, and progression. In addition, we discuss the most recently identified dual roles of miRNA in epigenetic gene regulation; how they are both regulators and regulated. Finally, we discuss the emerging roles of miRNA as epigenetic anti-cancer therapeutics, the current research examining inhibition of oncogenic miRNAs, and studies now establishing the potential of replacing lost, tumor-suppressive miRNA.

Die Inzidenz chronischer Lebererkrankungen und des hepatozellulären Karzinoms (HCC) mit nutritiv toxischer Genese ist in den vergangenen Jahren in den Industrienationen stetig angestiegen. Daher wird bereits seit längerem über eine Stadien-basierte Therapie bis hin zur Lebertransplantation intensiv diskutiert. In diesem Kontext zeigt die Stadien-spezifische Analyse der MikroRNA-(miRNA)-Profile ein großes Potenzial. In der vorliegenden Studie wurden erstmals die miRNA-Profile in Gewebeproben von Patienten mit einem hepatozellulärem Karzinom (HCC) und Leberzirrhose analysiert. Von 189 lebertransplantierten Patienten zeigten 93 eine nutritiv toxische Genese. Die Aufteilung der Patienten in zwei Studiengruppen erfolgte nach histopathologischer Beurteilung. So konnte bei 59 Patienten eine äthyltoxische Leberzirrhose ohne HCC (Gruppe A) und bei 34 Patienten ein HCC in äthyltoxischer Leberzirrhose (Gruppe B) nachgewiesen werden. In Gruppe B erfolgte die Probenentnahme aus dem HCC-Tumor („T“) und dem umgebenden Leberzirrhosegewebe („sT“). Zur Extraktion der Gesamt-RNA aus den Gewebeproben von 30 Patienten mit nutritiv toxischem Leberschaden, wurde Trizol® genutzt (16 Gruppe A „Ex“, 14 Gruppe B „sT“, 7 Gruppe B „T“). Die miRNA-Analyse dieser Gewebeproben wurde mittels miRCURY LNA Array (6. und 7. Generation) und Agilent G2565BA Microarray Scanner durchgeführt. Die Untersuchung der laboranalytischen Parameter und des präoperativen Lab-MELD ergab keinen statistisch signifikanten Unterschied zwischen beiden Untersuchungsgruppen. Die Patienten der Gruppe A war signifikant jünger als die Patienten der Gruppe B. 40 miRNA zeigten einen signifikanten Unterschied zwischen T- und sT-Gewebe, 29 miRNA zwischen T- und Ex-Gewebe und 56 miRNA zwischen Ex- und sT-Gewebe. Unter Berücksichtigung der bekannten Zielproteine der identifizierten miRNA wird das diagnostische Potenzial der miRNA-Profiländerungen bei der nutritiv toxischen Genese der Leberzirrhose mit und ohne HCC diskutiert.