>Magister Scientiae - MSc / Prostate Cancer is the leading cause of cancer-related death in males in the Western world. It is a common biological disease originating from the reproductive system of the male namely, the prostate gland, usually in older patients (over the age of 50) and with a family history of this disease. The disease shows clinical aggressiveness due to genetic alterations of gene expression in prostate epithelial cells. Prostate cancer is currently diagnosed by biopsy and prostate cancer screening via the Prostate-Specific Antigen (PSA) blood test. Early detection is critical and although PSA was discovered to aid in the diagnoses of this cancer at its early stages, it has a disadvantage due to its low specificity thus causing unnecessary biopsies of healthy individuals and overtreatment of patients. Although various studies and efforts have been made to identify the ideal biomarker for prostate cancer and many even being applied to clinical use, it is still challenging and has not replaced the best-known biomarker PSA. PSA test has minimal invasive characteristics, at relatively low cost together with high sensitivity but low specificity. Biomarker discovery is a challenging process and a good biomarker has to be sensitive, specific and its test highly standardized and reproducible as well as identify risk for or diagnose a disease, assess disease severity or progression, predict prognosis or guide treatment. Computational biology plays a significant role in the discovery of new biomarkers, the analyses of disease states and the validation of potential biomarkers. Bioinformatic approaches are effective for the detection of potential micro ribonucleic acid (miRNA) in cancer. Altered miRNA expression may serve as a biomarker for cancer diagnosis and treatment. Small non-protein coding RNA, miRNA are small regulatory RNA molecules that modulate the expression of their target genes. miRNAs influence numerous cancer-relevant processes such as proliferation, cell cycle control, apoptosis, differentiation, migration and metabolism. Discovery and existence of extracellular miRNAs that circulate in the blood of cancer patients has raised the possibility that miRNAs may serve as novel diagnostic markers. Since a single miRNA is said to be able to target several mRNAs, aberrant miRNA expression is capable of disrupting the expression of several mRNAs and proteins. Biomarker discovery for prostate cancer of mRNA and miRNA expression are strongly needed to enable more accurate detection of prostate cancer, improve prediction of tumour aggressiveness and facilitate diagnosis. The aim of this project was to focus on functional analyses of genes and their protein products regulated by previously identified miRNA in prostate cancer using bioinformatics as a tool. Most proteins function in collaboration with other proteins and therefore this study further aims to identify these protein-protein interactions and the biological relevance of these interactions as it relates to Prostate cancer. Various computational databases were used such as STRING, DAVID and GeneHub-GEPIS for functional analyses of these miRNA regulated genes. The main focus was on the 21 genes regulated by several miRNAs identified in a previous study. Results from this study identified six genes; ERP44, GP1BA, IFNG, SEPT2, TNFRSF13C and TNFSF4, as possible diagnostic biomarkers for prostate cancer. These results are promising, since the targeted biomarkers would be easily detectable in bodily fluids with the Gene Ontology (GO) analysis of these gene products showing enrichment for cell surface expression. The six genes identified in silico were associated to transcription factors (TFs) to confirm regulatory control of these TFs in cancer promoting processes and more specifically prostate cancer. The CREB, E2F, Nkx3-1 and p53 TFs were discovered to be linked to the genes IFNG, GP1BA, SEPT2 and TNFRSF13C respectively. The expression of these TFs show strong association with cancer and cancer related pathways specifically prostate cancer and thus demonstrates that these genes can be assessed as possible biomarkers for prostate cancer. The prognostic and predictive values of the candidate genes were evaluated to assess their relationship to prognosis of this disease by means of several in silico prognostic databases. The results revealed expression differences for the majority of the candidate genes were not significantly sufficient to be distinguished as strong prognostic biomarkers in several prostate cancer populations. Although one marker, GP1BA was supported as having prognostic value for prostate cancer based on it's statistical pvalue in one of the prostate cancer patient datasets used. Another candidate gene SEPT2 showed promise as it has some prognostic value in the early stages of the disease. Although the results yielded, based on the in silico analysis, were not the discovery of an ideal diagnostic marker based on the set criteria in this study, further analysis using a molecular approach qRT-PCR can be considered for a detailed followup study on selected candidate genes to evaluate their roles in disease initiation and progression of prostate cancer using cell lines as well as patient samples. / CSIR

MicroRNAs (miRNAs) have now been identified in a vast array of organisms and a great deal of research has been carried out to elucidate the role they play. The dysregulation of miRNA expression has been implicated in a number of disease states and their importance has been highlighted by the beginning of their utilisation as therapeutics. The focus of this study was to identify the role played by miRNAs encoded by the Marek’s disease vaccine viruses, Marek’s disease virus serotype 2 (MDV-2) and Herpesvirus of turkeys (HVT). In order to better understand the functions of these miRNAs we wanted to identify their targets within the host cell. Using a combination of bioinformatic and biochemical approaches we were able to build up a library of potential targets. Three viral miRNA targets; AKT3, RAP1A and DEK, were further validated using dual-luciferase assays to highlight the exact site of miRNA targeting, and western blots to demonstrate an effect of miRNA targeting on protein abundance. An attempt at using label-free proteomics to observe the viral miRNA mediated changes in the host proteome is also described, however this proved to be unsuccessful. Additionally the function of one particular MDV-2 miRNA, mdv2-miR-M21, was explored in more detail, describing its role as a potential ortholog of the host miRNA; gga-miR-29b. By using the observation that the viral miRNA contained an identical 'seed' region to the host miRNA, we were able to use the data collected from existing studies on miR-29b to search for targets of mdv2-miR-M21. We demonstrated that mdv2-miR-M21 targeted DNMT3B, crucial for epigenetic modification of the genome. The final part of this study aimed to understand the wider context the viral miRNAs played in the viral biology and protective ability of the vaccine viruses. The miRNAs were deleted from the viruses, and then the miRNA-deletion viruses were used to vaccinate birds before challenge with the oncogenic Marek's disease virus serotype 1 (MDV-1), survival rates to the 'wild-type' MDV-2 and HVT vaccine viruses were then compared.

Invasive lobular carcinoma (ILC) is the second most common form of breast cancer. ILC presents at later stages with many challenges, therefore improved diagnostic and therapeutic targets are needed. A microRNA (miRNA) genome analysis identified miR-23c and miR-23b-3p as possible regulators of ILC invasion due to their significantly increased expression in invasive compared to minimally invasive ILC cell lines. By decreasing the levels of miR-23c and miR-23b-3p using hairpin inhibitors, the invasive MDA-MB-330 cell line had significantly reduced invasion, while overexpressing these miRNAs using mimics in the minimally invasive MDA-MB-134VI cell line increased invasion. During the course of this study, it became apparent that limited tools exist for studying invasive ILC. Therefore, two more invasive ILC cell line models were created by isolating and expanding MDA-MB-134VI cells that had invaded through Matrigel® coated invasion chambers. This thesis has thus created new models of invasive ILC as well as identified miR-23c and miR-23b-3p as regulators of MDA-MB-330 and MDA-MB-134VI cell line invasion.

Background: MicroRNAs (miRNAs) are small non-coding single-stranded RNAs involved in the posttranscriptional inhibition of gene expression and thereby regulating all cellular functions. Their dysregulation contributes to the pathophysiology of many diseases, including rheumatic diseases. MiRNAs can also be found extracellularly in body fluids and represent promising diagnostic and prognostic biomarkers. Our study aimed to investigate miRNAs as biomarkers of stage and activity and predictors of therapeutic response of two most common inflammatory rheumatic diseases: spondyloarthritis (SpA) and rheumatic arthritis (RA). Results: We found several circulating miRNAs differentially expressed in SpA patients reflecting the severity of axial involvement and/or disease activity. The decrease in circulating miR-145 in plasma of patients with ankylosing spondylitis 3 months of anti-TNF therapy predicted a good therapeutic response and low disease activity after a year of therapy. Circulating and intracellular expression of miR-125b in peripheral blood mononuclear cells (PBMC) was lower in treatment-naïve patients with early RA than in healthy controls. Baseline expression of miR-125 in PBMC predicted a (non)adequate therapeutic response. We also found the increased expression of miR-451 in PBMC in...

Several disease processes taking place in the cells are characterized by an increase of the concentration of nucleic acids, in particular micro RNAs (miRNAs). A detection system that could selectively detect the increased presence of the miRNAs directly in the living cells in real time with nanoresolution is therefore highly desired. Fluorescent nanodiamond particles are considered promising candidates thanks to their biocompatibility, small size, allowing them to penetrate the cell membrane, and stable fluorescent defects in the crystal lattice, namely nitrogen-vacancy (NV) centres. The NV centres are the most studied colour centres of nanodiamonds due to their unique room-temperature optical properties, allowing for highly sensitive detection of changes in the magnetic field (magnetic noise) with quantum sensing techniques. For instance, the length of the T1 relaxation time NV electronic spin is greatly influenced by the presence of paramagnetic species, which causes a shortening of the T1 relaxation time depending on the proximity to the NV centres. However, for selective quantum sensing with nanodiamonds, the use of molecular transducers is necessary to bind targeted molecules with high specificity and allow their detection via the change of the NV spin properties. In this work,...

Low-cost, highly-sensitivity, and minimally invasive tests for the detection and monitoring of life-threatening cancers can reduce the worldwide disease burden. The disease diagnosis community is constantly working to improve the detection capabilities of the deadly cancers (e.g.: pancreatic and lung) at their early stages. Still there were many cancers cannot be detected at their early stages due to lack of early diagnosis techniques. One of the reason being, many cancers that occur in the body release minute amounts of biomarker molecules during the initial stages (e.g.: DNA, RNA, miRNA and antigens) in the body fluids such as blood and serum. Since the traditional bio-sensing techniques have reached their maximum capacity in terms of critical performance parameters (sensitivity, detection time, reproducibility and limit of detection) there is an urgent need for innovative approaches that can fill this gap. To address this unmet need, here we report on developing a novel bio-sensing technique for detecting and quantifying biomolecules from the patients’ plasma/serum samples at point-of-care settings. Here we have investigated the novel interactions between biomolecules and externally applied fields to effectively manipulate and specifically concentrate them at a certain detection spots near electrodes on the detection device. Then the near-field interactions between the fluorophores and the free electrons on metal surfaces were successfully integrated with the externally applied low frequency (<10MHz) electric field, to achieve maximum florescence enhancement, that produces the detection limit of target-biomolecules in the rage of femto molars (fM). Moreover, the externally applied electric potential produces dielectrophoretic and thermophoretic force on the biomolecules, together with these forces we were able to separate the fluorophore-labelled rare target-biomolecules from the others in a sample. The novel integrated technique is tested and proved to be superior to the current gold standards (qRT-PCR and ELISA) for target-biomolecules detection in critical performance parameters. Finally the technique was used to analyze healthy and pancreatic cancer patients’ samples and further it has been proved that we can differentiate the healthy individuals and cancer patients. In addition, this technique is being applied to the other diseases such as obesity, opioid addiction and other types of cancers.

Regulation and function of protein-coding genes are increasingly well-understood, but no comparable evidence exists for non-coding RNA (ncRNA) genes, which appear to be more numerous than protein-coding genes. We developed a novel machine-learning model to distinguish promoters of long ncRNA (lncRNA) genes from those of protein-coding genes. This represents the first attempt to make this distinction based on properties of the associated gene promoters. From our analyses, several transcription factors (TFs), which are known to be regulated by lncRNAs, also emerged as potential global regulators of lncRNAs, suggesting that lncRNAs and TFs may participate in bidirectional feedback regulatory network. Our results also raise the possibility that, due to the historical dependence on protein-coding gene in defining the chromatin states of active promoters, an adjustment of these chromatin signature profiles to incorporate lncRNAs is warranted in the future. Secondly, we developed a novel method to infer functions for lncRNA and microRNA (miRNA) transcripts based on their transcriptional regulatory networks in 119 tissues and 177 primary cells of human. This method for the first time combines information of cell/tissueVspecific expression of a transcript and the TFs and transcription coVfactors (TcoFs) that control activation of that transcript. Transcripts were annotated using statistically enriched GO terms, pathways and diseases across cells/tissues and associated knowledgebase (FARNA) is developed. FARNA, having the most comprehensive function annotation of considered ncRNAs across the widest spectrum of cells/tissues, has a potential to contribute to our understanding of ncRNA roles and their regulatory mechanisms in human. Thirdly, we developed a novel machine-learning model to identify LD motif (a protein interaction motif) of paxillin, a ncRNA target that is involved in cell motility and cancer metastasis. Our recognition model identified new proteins not previously known to harbor LD motifs and we experimentally confirmed some of our predicted motifs. This novel discovery will expand our knowledge of cancer metastasis and will facilitate therapeutic targeting linking specific ncRNAs via paxillin proteins to diseases. Finally, through bioinformatics approaches, we identified lncRNAs as markers that distinguish classical from alternative activation of macrophage. This result may have good use in the diagnosis of infectious diseases.

MicroRNAs are small non-coding RNAs that regulate gene expression through RNA interference. These small RNAs enact gene silencing by forming a RNA-inducing silencing complex together with the effector protein Argonaute. The function of the Argonautes in the social amoeba Dictyostelium discoideum is not yet fully understood. In this study, we look closer at Argonaute B by investigating if it is possible to extract the protein from the cells by the addition of a polypeptide protein tag called 3xFlag. At the same time, we also look into if Argonaute B is important for cell growth. Sequences of the 3xFlag tag with or without the Argonaute B gene (agnB) attached had previously been cloned into a vector and transformed into Dictyostelium discoideum cell. The 3xFlag::agnB sequence was confirmed in wild type and agnB knock-out strains through polymerase chain reaction. We then verified the expression of the fusion protein in the cells by western blot. The cell growth was measured by how the number of cells changed over time. The experiment suggested that Argonaute B is important for growth. Our result show that the construct 3xFlag::agnB sequenced had correctly been transformed into the strains and is highly expressed under tested conditions. We could also see that Argonaute B is an important factor in cell growth.

MicroRNAs regulate pathways contributing to oncogenesis, and thus the mechanisms causing dysregulation of microRNA expression in cancer are of significant interest. Mature mir-29b levels are decreased in malignant cells, and this alteration promotes the malignant phenotype, including apoptosis resistance. However, the mechanism responsible for mir-29b suppression is unknown. Here, we examined mir-29 expression from chromosome 7q32 using cholangiocarcinoma cells as a model for mir-29b downregulation. Using 5′ rapid amplification of cDNA ends, the transcriptional start site was identified for this microRNA locus. Computational analysis revealed the presence of two putative E-box (Myc-binding) sites, a Gli-binding site, and four NF-κB-binding sites in the region flanking the transcriptional start site. Promoter activity in cholangiocarcinoma cells was repressed by transfection with c-Myc, consistent with reports in other cell types. Treatment with the hedgehog inhibitor cyclopamine, which blocks smoothened signaling, increased the activity of the promoter and expression of mature mir-29b. Mutagenesis analysis and gel shift data are consistent with a direct binding of Gli to the mir-29 promoter. Finally, activation of NF-κB signaling, via ligation of Toll-like receptors, also repressed mir-29b expression and promoter function. Of note, activation of hedgehog, Toll-like receptor, and c-Myc signaling protected cholangiocytes from TRAIL-induced apoptosis. Thus, in addition to c-Myc, mir-29 expression can be suppressed by hedgehog signaling and inflammatory pathways, both commonly activated in the genesis of human malignancies.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21020号 / 医博第4366号 / 新制||医||1028(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 椛島 健治, 教授 三森 経世, 教授 小柳 義夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM