@phdthesis{Blahetek2024,
  author    = {Blahetek, Gina},
  title     = {The role of alternative intronic polyadenylation on microRNA biogenesis in melanoma},
  doi       = {10.25972/OPUS-25474},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254743},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {mRNA is co- or post-transcriptionally processed from a precursor mRNA to a mature mRNA. In addition to 5'capping and splicing, these modifications also include polyadenylation, the addition of a polyA tail to the 3'end of the mRNA. In recent years, alternative polyadenylation in particular has increasingly been taken into account as a mechanism for regulating gene expression. It is assumed that approximately 70-75 \% of human protein coding genes contain alternative polyadenylation signals, which are often located within intronic sequences of protein-coding genes. The use of such polyadenylation signals leads to shortened mRNA transcripts and thus to the generation of C-terminal shortened protein isoforms. Interestingly, the majority of microRNAs, small non-coding RNAs that play an essential role in post-transcriptional gene regulation, are also encoded in intronic sequences of protein-coding genes and are co-transcriptionally expressed with their host genes. The biogenesis of microRNA has been well studied and is well known, but mechanisms that may influence the expression regulation of mature microRNAs are just poorly understood. In the presented work, I aimed to investigate the influence of alternative intronic polyadenylation on the biogenesis of microRNAs. The human ion channel TRPM1 could already be associated with melanoma pathogenesis and truncated isoforms of this protein have already been described in literature. In addition, TRPM1 harbors a microRNA, miR211, in its sixth intron, which is assumed to act as a tumor suppressor. Since both, TRPM1 and miR211 have already been associated with melanoma pathogenesis, the shift towards truncated transcripts during the development of various cancers is already known and it has been shown that certain microRNAs play a crucial role in the development and progression of melanoma, melanoma cell lines were used as an in vitro model for these investigations.},
  subject      = {Polyadenylierung},
  language  = {en}
}
@phdthesis{Kruber2019,
  author    = {Kruber, Philip},
  title     = {Functional analysis of DROSHA and SIX1 mutations in kidney development and Wilms tumor},
  doi       = {10.25972/OPUS-16141},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161418},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Wilms tumor (WT) is the most common kidney cancer in childhood. It is a genetically heterogeneous tumor and several genetic alterations have been identified in WT patients. Recurrent mutations were found in the homeo-domain of SIX1 and SIX2 in high proliferative tumors (18.1\% of the blastemal-type tumors) as well as in the microprocessor genes DROSHA and DGCR8 (18.2\% of the blastemal-type tumors), indicating a critical role of the SIX-SALL pathway and aberrant miRNA processing in WT formation. Underlined by the fact that a significant overlap between mutations in DROSHA and SIX1 was found, indicating a synergistic effect. To characterize the in vivo role of DROSHA and SIX mutations during kidney development and their oncogenic potential, I analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA or SIX1 carrying a tumor-specific E1147K or Q177R mutation, respectively. The DROSHA mutation E1147K was predicted to act in a dominant negative manner. Six2-cre mediated deletion of Drosha in nephron progenitors led to a lethal phenotype with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-creERT2 resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth-retarded mice died within the first two months. This strong phenotype was consistent with the predicted dominant-negative effect of DROSHA-E1147K. Analysis of the SIX1-Q177R mutation suggested that the mutation leads to a shift in DNA binding specificity instead of a complete loss of DNA binding. This may end up in subtle changes of the gene regulatory capacity of SIX1. Six2-cre mediated activation of SIX1-Q177R lead to a viable phenotype with no alterations or shortened life span. Yet a global activation of SIX1-Q177R mediated by Zp3-cre resulted in bilateral hydronephrosis and juvenile death of the mice. To mimic the synergistic effect of DROSHA and SIX1 mutations, I generated compound mutants in two combinations: A homozygous deletion of Drosha combined with an activation of SIX1-Q177R and a compound mutant with activation of DROSHA-E1147K and SIX1-Q177R. Each mouse model variant displayed new phenotypical alterations. Mice with Six2-cre mediated homozygous deletion of Drosha and activation of SIX1-Q177R were not viable, yet heterozygous deletion of Drosha and activation of SIX1-Q177R led to hydronephrosis, proteinuria and an early death around stage P28. Combined activation of DROSHA-E1147K and SIX1-Q177R under Six2-cre resulted in proteinuria, glomerulosclerosis and lesions inside the kidney. These mice also suffered from juvenile death. Both mouse models could confirm the predicted synergistic effect. While these results underscore the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{Leinders2016,
  author    = {Leinders, Mathias},
  title     = {microRNAs in chronic pain},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144395},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Chronic pain is a common problem in clinical practice, not well understood clinically, and frequently tough to satisfactorily diagnose. Because the pathophysiology is so complex, finding effective treatments for people with chronic pain has been overall less than successful and typically reduced to an unsatisfactory trial-and-error process, all of which translates into a significant burden to society. Knowledge of the mechanisms underlying the development of chronic pain, and moreover why some patients experience pain and others not, may aid in developing specific treatment regimens. Although nerve injuries are major contributors to pain chronification, they cannot explain the entire phenomenon. Considerable research has underscored the importance of the immune system for the development and maintenance of chronic pain, albeit the exact factors regulating inflammatory reactions remain unclear. Understanding the putative molecular and cellular regulator switches of inflammatory reactions will open novel opportunities for immune modulatory analgesics with putatively higher specificity and less adverse effects. It has become clear that small, non- coding RNA molecules known as microRNAs are in fact potent regulators of many thousands of genes and possibly cross-communicate between cellular pathways in multiple systems acting as so-called "master-switches". Aberrant expression of miRNAs is now implicated in numerous disorders, including nerve injuries as well as in inflammatory processes. Moreover, compelling evidence supports the idea that miRNAs also regulate pain, and in analogy to the oncology field aid in the differential diagnosis of disease subtypes. In fact, first reports describing characteristic miRNA expression profiles in blood or cerebrospinal fluid of patients with distinct pain conditions are starting to emerge, however evidence linking specific miRNA expression profiles to specific pain disorders is still insufficient. The present thesis aimed at first, identifying specific miRNA signatures in two distinct chronic pain conditions, namely peripheral neuropathies of different etiologies and fibromyalgia syndrome. Second, it aimed at identifying miRNA profiles to better understand potential factors that differentiate painful from painless neuropathies and third, study the mechanistic role of miRNAs in the pathophysiology of pain, to pave the way for new druggable targets. Three studies were conducted in order to identify miRNA expression signatures that are characteristic for the given chronic pain disorder. The first study measured expression of miR-21, miR-146a and miR-155 in white blood cells, skin and nerve biopsies of patients with peripheral neuropathies. It shows that peripheral neuropathies of different etiologies are associated with increased peripheral miR-21 and miR-146a, but decreased miR-155 expression. More importantly, it was shown that painful neuropathies have increased sural nerve miR-21 and miR-155 expression, but reduced miR-146a and miR-155 expression in distal skin of painful neuropathies. These results point towards the potential use of miRNAs profiles to stratify painful neuropathies. The seconds study extends these findings and first analyzed the role of miR-132-3p in patients and subsequently in an animal model of neuropathic pain. Interestingly, miR-132-3p was upregulated in white blood cells and sural nerve biopsies of patients with painful neuropathies and in animals after spared nerve injury. Pharmacologically modulating the expression of miR-132-3p dose-dependently reversed pain behavior and pain aversion, indicating the pro-nociceptive effect of miR-132-3p in chronic pain. This study thus demonstrates the potential analgesic impact by modulating miRNA expression. Fibromyalgia is associated with chronic widespread pain and, at least in a subgroup, impairment in small nerve fiber morphology and function. Interestingly, the disease probably comprises subgroups with different underlying pathomechanisms. In accordance with this notion, the third study shows that fibromyalgia is associated with both aberrant white blood cell and cutaneous miRNA expression. Being the first of its kind, this study identified miR-let-7d and its downstream target IGF-1R as potential culprit for impaired small nerve fiber homeostasis in a subset of patients with decreased intra-epidermal nerve fiber density. The work presented in this thesis is a substantial contribution towards the goal of better characterizing chronic pain based on miRNA expression signatures and thus pave the way for new druggable targets.},
  subject      = {miRNS},
  language  = {en}
}
@phdthesis{Schwab2022,
  author    = {Schwab, Bernhard},
  title     = {Zirkulierende microRNAs beim komplexen regionalen Schmerzsyndron},
  doi       = {10.25972/OPUS-26695},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-266959},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {CRPS ist eine anhaltende Schmerzerkrankung, die nach Verletzungen auftritt und mit einer variierenden Kombination von Symptomen aus den Bereichen Sensorik, Vasomotorik, Sudomotorik/{\"O}demen und Motorik verbunden ist. Die Physiologie des Krankheitsbildes ist nicht abschließend gekl{\"a}rt, allerdings wird eine komplexe Interaktion mehrerer Pathomechanismen als Ursache angenommen. Deshalb stellt das CRPS sowohl eine diagnostische als auch eine therapeutische Herausforderung dar. miRNAs sind kurze, einstr{\"a}ngige und nicht-codierende RNAs, die durch Inhibierung der Translation und Degradierung von mRNAs an der posttranskriptionellen Regulation der Genexpression beteiligt sind. Sie werden auch von den Zellen durch Exosomen, Mikrovesikel, Lipoproteine und Apoptose freigesetzt, sodass sie in unterschiedlichen K{\"o}rperfl{\"u}ssigkeiten nachgewiesen werden k{\"o}nnen. Bei vielen Erkrankungen kann eine Dysregulation der miRNA-Expression festgestellt werden, weshalb großes Interesse daran besteht, sie als Biomarker oder f{\"u}r therapeutische Ans{\"a}tze nutzbar zu machen. Die miRNAs miR-183, -21, -29b, -144, - 223 wurden bereits im Zusammenhang mit entz{\"u}ndlichen und neuropathischen Prozessen und der Dysruption von Immunobarrieren beschrieben. In dieser Arbeit wurde untersucht, ob bei der Expression dieser miRNAs im Blut von CRPS-Patienten, Patienten mit einem komplikationslosen posttraumatischen Heilungsverlauf und von Kontrollen ohne ein vorangegangenes Trauma Unterschiede bestehen. Die Messungen erfolgten im Plasma, in Leukozyten und Exosomen, um dadurch auch die Regulation in den einzelnen Blutkomponenten vergleichen zu k{\"o}nnen. Tats{\"a}chlich fanden sich unterschiedliche miRNA-Expressionsprofile bei den verschiedenen Biomaterialien. Außerdem konnte bei einzelnen miRNAs ein Einfluss von Alter und Geschlecht auf die Expression nachgewiesen werden. Diese Beeinflussung war dar{\"u}ber hinaus auch abh{\"a}ngig vom untersuchten Biomaterial und vor allem bei den Exosomen besonders ausgepr{\"a}gt. In den Exosomen ergab sich eine signifikante Hochregulation von miR-223-5p bei den FK im Vergleich mit den CRPS-Patienten. Bei der Zusammenfassung der Daten von CRPS und FK fand sich außerdem eine negative Korrelation zwischen der miR-223-5p-Expression und dem CSS, sodass ein erh{\"o}htes Expressionsniveau mit einer milderen Krankheitsausbildung verbunden war. Hinsichtlich der traumanaiven Kontrollen war das Expressionsniveau der CRPS-Patienten hingegen unver{\"a}ndert. Diese Ergebnisse weisen darauf hin, dass beim CRPS im Vergleich mit einem regelrechten Heilungsverlauf eine insuffiziente beziehungsweise ausbleibende posttraumatische Anpassung des miRNA-Spektrums vorliegt. Diese posttraumatische Regulation stellt eventuell eine wichtige Voraussetzung f{\"u}r den Ablauf eines komplikationslosen Heilungsprozesses dar. F{\"u}r miR-223-5p wurde bereits mehrfach eine antiinflammatorische Wirkung durch die Regulation von proinflammatorischen Rezeptoren und die Beeinflussung der Differenzierung von Makrophagen beschrieben. Eine verminderte Expression k{\"o}nnte somit zu einer Disposition f{\"u}r {\"u}berschießende Entz{\"u}ndungsreaktionen f{\"u}hren und dadurch zur Entwicklung von CRPS beitragen. Diese Ergebnisse weisen auf die Beteiligung zirkulierender und vor allem exosomaler miRNAs bei der Pathophysiologie des CRPS hin. Zur weiterf{\"u}hrenden Abkl{\"a}rung der pathophysiologischen Relevanz von miR-223-5p sind jedoch zus{\"a}tzliche Untersuchungen erforderlich. Dabei bleibt es zu pr{\"u}fen, ob eine verminderte miR-223-5p-Expression mit verst{\"a}rkten Entz{\"u}ndungsmarkern und einer verst{\"a}rkten proinflammatorischen Differenzierung von Makrophagen verbunden ist. Eine Abkl{\"a}rung der Herkunft der Exosomen k{\"o}nnte dabei helfen, zwischen einer lokalen und einer systemischen Reaktion zu unterscheiden. Die Beantwortung dieser Fragen k{\"o}nnte zu einem besseren Verst{\"a}ndnis beitragen, warum manche Patienten nach einem Extremit{\"a}tentrauma ein CRPS entwickeln und keinen normalen Heilungsverlauf erfahren.},
  subject      = {miR-223-5p},
  language  = {de}
}