@phdthesis{Schmidt2022, author = {Schmidt, Jonas Paul Christian}, title = {Nanopore sequencing in laboratory medicine : novel molecular diagnostic prospects for Familial Mediterranean fever and SARS-CoV-2 infections}, doi = {10.26127/BTUOpen-6384}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:co1-opus4-63847}, school = {BTU Cottbus - Senftenberg}, year = {2022}, abstract = {Nanopore sequencing, a third-generation sequencing technique that applies nanometre sized pores to transduce the physical and chemical properties of specific nucleobases into measurable electrical signals, shows attractive advantages over conventional next-generation sequencing techniques. However, primarily due to high sequencing error rates this technique has rarely been used so far in clinical laboratory diagnostics. In this cumulative dissertation Nanopore sequencing was established and validated in clinical diagnostics using the example of the molecular diagnosis of Familial Mediterranean fever (FMF) and SARS coronavirus-2 (SARS-CoV-2) infections. First, a novel data analysis pipeline for accurate single nucleotide polymorphism (SNP) genotyping using Nanopore sequencing data was developed and validated with the corresponding sequencing protocol against conventional Sanger sequencing using 47 samples of patients with clinical suspicion of FMF. This method comparison showed a perfect agreement between both methods rendering current Nanopore sequencing in principle suitable for SNP genotyping in human genetics. The bioinformatic analysis of sequencing data is one of the most challenging parts in Nanopore sequencing experiments and complicates the application in a clinical diagnostic setting. Therefore, six different bioinformatic tools for sequence alignment were evaluated regarding their applicability to Nanopore sequencing data. This evaluation revealed a good suitability of all except one of these tools although differences in quality and performance exist. Since Nanopore sequencing showed a robust performance in SNP genotyping, a SARS-CoV-2 whole genome sequencing (WGS) protocol was established to enable onside viral WGS in a clinical laboratory. This was especially important for viral molecular biological surveillance during the pandemic as shown by analysing viral genetic data over the course of one year. Applying this approach in a clinical research project to investigate host-virus interaction by aggregating for the first time viral genetic data, serological data and clinical data, showed diverse humoral immune responses to SARS-CoV-2, that appear to be influenced by age, obesity and disease severity. Further, even small viral genetic changes may influence the clinical presentation of the associated disease COVID-19. Additionally, a novel reverse transcriptase (RT)- loop mediated isothermal amplification (LAMP) assay for the detection of SARS-CoV-2 was developed and validated for diagnostic use by method comparison with conventional RT-polymerase chain reaction (PCR). In summary, by presenting advancements of sequencing and bioinformatic workflows with the focus on an application in clinical diagnostics, the results of this thesis may pave the way for a broader application of Nanopore sequencing in laboratory medicine in the near future.}, subject = {Nanopore sequencing; Laboratory medicine; Molecular diagnostics; Familial Mediterranean fever; SARS-CoV-2; Nanopore-Sequenzierung; Labormedizin; Molekulare Diagnostik; Famili{\"a}res Mittelmeerfieber; Famili{\"a}res Mittelmeerfieber; Labormedizin; Molekulare Diagnostik; Sequenzierung; SARS-CoV-2; Diagnostics; Molecular medicine}, language = {en} } @phdthesis{Sowa2019, author = {Sowa, Mandy}, title = {Entwicklung und Evaluierung einer Multiparametertechnologie zur serologischen Diagnostik von Autoimmunerkrankungen}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:co1-opus4-51514}, school = {BTU Cottbus - Senftenberg}, year = {2019}, abstract = {Die Zunahme der Komplexit{\"a}t diagnostischer Fragestellungen veranlasst die Entwicklung von innovativen Teststrategien. Multiparameterteste sind eine Antwort auf die sich im Wandel befindende Routinediagnostik von Autoimmunerkrankungen. In der vorliegenden kumulativen Dissertation wurde eine neue multiparametrische Technologie f{\"u}r den Bereich der Autoimmundiagnostik entwickelt und klinisch evaluiert. Diese CytoBead Technologie ist eine Kombination aus Screeningtest, definiert durch die indirekte Immunfluoreszenz als diagnostischen Goldstandard, und einen multiplexen Best{\"a}tigungstest, welcher durch einen Mikropartikel-basierten Immunfluoreszenztest umgesetzt wurde. Zur serologischen Diagnostik von anti-neutrophilen zytoplasmatischen Antik{\"o}rpern (ANCA) assoziierten Vaskulitiden wurde der CytoBead ANCA entwickelt. Dieser Multiparametertest beinhaltet eine Kombination aus indirekter Immunfluoreszenz mit Ethanol fixierten Granulozyten und Mikropartikel-basiertem Immunfluoreszenztest mit Proteinase 3 und Myeloperoxidase. Wesentliche Vorteile gegen{\"u}ber den klassischen Testen sind Zeit- und Kosteneinsparungen, die in der Routinediagnostik unerl{\"a}sslich sind. Unter der Pr{\"a}misse der deutlichen Zeiteinsparung von mehreren Bearbeitungsstunden oder sogar Tagen wurde diese Technologie f{\"u}r verschiedene, krankheitsspezifische Bereiche als Werkzeug der Profildiagnostik entwickelt. Besonders im Bereich der Notfalldiagnostik von Autoimmunerkrankungen z{\"a}hlt jede Stunde, wenn es um das {\"U}berleben der Patienten geht. Die Weiterentwicklung dieser Technologie f{\"u}r die serologische Diagnostik der Rapid Progressiver Glomerulonephritis (RPGN) als Notfalldiagnostik, ist ein wesentlicher Beitrag f{\"u}r eine effiziente in-vitro Diagnostik. Die sich anschließenden klinischen Studien des CytoBead ANCA und CytoBead RPGN wiesen gute bis sehr gute Vergleichbarkeiten zu klassischen Testen auf. Die Studien untermauerten zudem die Wichtigkeit der CytoBead Technologie hinsichtlich Nachhaltigkeit und Patientenmonitoring. Ein wichtiges Hauptaugenmerk der Testentwicklung lag in der Stabilit{\"a}t, Reproduzierbarkeit, Standardisierung und Quantifizierbarkeit von Ergebnissen, die im Rahmen dieser Dissertation evaluiert werden konnten. F{\"u}r eine weitere CytoBead Entwicklung und Evaluierung im Bereich der autoimmunen Lebererkrankungen konnte im Rahmen dieser Dissertation ein wichtiger Grundstein gelegt werden. Es wurde ein Screeningtest auf Basis der indirekten Immunfluoreszenz mit Glykoprotein 2 (Isoform 1,2,3,4) transduzierten HEp-2 Zellen entwickelt und anschließend in einer klinischen Studie evaluiert. Innerhalb dieser Studie konnte eine m{\"o}gliche neue, autoantigene Zielstruktur f{\"u}r Patienten mit Prim{\"a}r Sklerodisierende Cholangitis identifiziert werden. Zusammenfassend stellt sich die Entwicklung der CytoBead Technologie als eine neue, innovative Generation der serologischen Diagnostik von Autoimmunerkrankungen dar, welche zus{\"a}tzlich auf verschiedene Diagnostikprofile angepasst werden kann.}, subject = {CytoBead; Multiparametertechnologie; Autoimmunerkrankungen; Serologische Diagnostik; Autoantik{\"o}rper; CytoBead; Autoimmune diseases; Multiparametric assay; Autoantibodies; Microbeads; Multiparameteranalyse; Autoantik{\"o}rper; Serodiagnostik; Autoaggressionskrankheit}, language = {de} } @phdthesis{Liedtke2023, author = {Liedtke, Victoria}, title = {Lens epithelium-derived growth factor (LEDGF) as an aid in the diagnosis of benign prostatic hyperplasia (BPH) and the molecular principles of LEDGF in human cancer cells}, doi = {10.26127/BTUOpen-6698}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:co1-opus4-66988}, school = {BTU Cottbus - Senftenberg}, year = {2023}, abstract = {Benign prostatic hyperplasia (BPH) is considered an age-related disease of men with unknown etiopathophysiology. Chronic inflammation represents one of the most important pathophysiological mechanisms in this context, and there is also evidence for the involvement of autoimmune responses in an inflammatory environment in the prostate. Conventional therapeutic approaches for BPH are limited; however, immunosuppression by TNF (tumor necrosis factor)-𝛼 blockers seems to reduce symptoms in BPH patients due to the reduction of epithelial hyperplasia and macrophage-triggered inflammation. Therefore, this cumulative dissertation addresses biomarker-based BPH diagnosis by lens epithelium-derived growth factor splice variant 75 kDa (LEDGF/p75). LEDGF/p75 is an autoantigen that is overexpressed in solid tumors and functions as a stress-related transcriptional co-activator. It also plays an important role in cancer and DNA damage repair (DDR). The generation of CRISPR/Cas9-modified HEp-2 LEDGF deficient (KO) and HEp-2 LEDGF/p75 overexpressing (OE) cell lines provided new insights for the diagnosis and therapy of cancer and genetic disorders. Herein, IgG autoantibody reactivity to LEDGF/p75 was detected in patients with prostate cancer (PCa, n=89), bladder cancer (BCa, n=116), benign prostatic hyperplasia (BPH, n=103) and blood donors (BD, n=60) by indirect immunofluorescence assay (IFA) linear immunoassay (LIA) and Chritidia luciliae immunofluorescence test (CLIFT). In addition, proliferation and migration capacity as well as chemosensitivity of genome-edited cells were determined by optimized all-in-one approach, followed by investigation of DDR signaling pathways by Western blot and immunofluorescence. Surprisingly, no increased binding of autoantibodies (autoAbs) to LEDGF/p75 was detected in cancer patients, but autoAb reactivity to LEDGF/p75 O/E cells was unexpectedly increased in approximately 50\% of patients with BPH. At the molecular level, LEDGF-deficient cells exhibit decreased proliferation and migration and increased sensitivity to the chemotherapeutic agent etoposide. A significantly Decreased DNA damage response could also be visualized. New diagnostic capabilities using HEp-2 cells overexpressing LEDGF/p75 or mitochondrial DNA as autoimmune targets could be used to identify BPH patients with autoimmune responses. Furthermore, this Thesis shows that LEDGF is not only involved in the recruitment of DNA damage response proteins, but also affects the proteosomal regulation of DDR signaling molecules.}, subject = {Y-H2AX; LEDGF/p75; CRISPR/Cas9; Prognostic biomarker; DNA damage signaling; Autoimmunity; DNA Reparatur; Darmkrebs; Autoimmunerkrankungen; Autoaggressionskrankheit; Prostatahypertrophie; Autoantigen; DNS-Reparatur}, language = {en} }