Inhalt

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   Abstract
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   Acknowledgment
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   List of abbreviations
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   List of Figures
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   List of Tables
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  1. Introduction
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  1.1. Systemic sclerosis
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   1.1.1. Definition and epidemiology
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   1.1.2. The pathogenesis of systemic sclerosis
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   1.1.3. Skin fibrosis in SSc
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  1.1.4. Players of skin fibrosis in SSc
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   1.1.4.1. Fibroblast and myofibroblasts
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   1.1.4.2. Oxidative stress
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   1.1.4.3. The TGF-β1 signaling and its role in fibrosis
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   1.1.5. Experimental model of systemic sclerosis
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  1.2. Reactive oxygen species
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   1.2.1. Sources of reactive oxygen species generation
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   1.2.2. ROS in cell signaling
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   1.2.3. Antioxidant studies in vitro, vivo and clinical trials
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  1.3. NADPH oxidases (NOXs)
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   1.3.1. The family of NADPH oxidases
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   1.3.4. Tissue distribution and subcellular localization
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  1.4. NADPH oxidase 4 (Nox4)
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   1.4.1. Structure, function and regulation of Nox4
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   1.4.2. Tissue distribution and subcellular localization
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   1.4.3. Physiological and pathological role of Nox4
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   1.4.4. Pathological role of Nox4 in fibrotic disorders
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   1.5. NADPH oxidase inhibition
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   2. Aim of the study
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   3. Materials and Methods
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  3.1. Materials
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   3.1.1. Chemicals
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   3.1.2. Stimulants and pharmacological inhibitors
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  3.1.3. Media and Buffers
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   3.1.3.1. Sera
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   3.1.3.2. Media
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   3.1.3.3. Buffers
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   3.1.4. DNA ladder and protein markers
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   3.1.5. Consumables and kits
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   3.1.6. Primers
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  3.1.7. Antibodies
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   3.1.7.1. Primary antibodies
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   3.1.7.2. Secondary antibodies
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   3.1.8. Instruments and Laboratory equipment
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   3.1.9. Animals
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  3.1.10. Software and computer programmes
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   3.1.10.1. Online Tools and databases
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   3.1.10.2. Software
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   3.1.11. Cells, cell lines and cell culture reagents
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  3.2. Methods
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  3.2.1. Cell biological methods
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   3.2.1.1. Cultivation of cells
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   3.2.1.2. Freezing of cells and storage
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   3.2.1.3. Generation of Nox4-/--deficient murine fibroblasts
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   3.2.1.4. Generation of skin samples and HDFs from patients with SSc
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  3.2.2. Molecular biological methods
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   3.2.2.1. RNA isolation
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   3.2.2.2. cDNA synthesis and reverse transcription
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   3.2.2.3. Polymerase chain reaction (PCR)
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   3.2.2.4. DNA gel electrophoresis
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   3.2.2.5. siRNA transfection and gene knock-down
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  3.2.3. Biochemical methods
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  3.2.3.1. Preparation of cell extract
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   3.2.3.1.1. Preparation of cell lysate using hot lysis method
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   3.2.3.1.2. Preparation of cell lysates using immunoprecipitation buffer (IP).
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   3.2.3.2. Protein determination
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   3.2.3.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
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   3.2.3.4. Western immunoblotting
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  3.2.4. Immunological methods
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   3.2.4.1. ELISA
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  3.2.5. Cytotoxicity assays
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   3.2.5.1. Crystal violet assay
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   3.2.5.2. XTT test
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   3.2.6. Confocal immunofluorescences microscopy
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   3.2.7. Assessment of dermal thickness.
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   3.2.8. Determination of collagen protein content ex vivo.
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   3.2.9. In silico promoter analysis
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   3.2.10. Measurement of NADPH oxidase activity assay
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   3.2.11. Statistical analysis
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  4. Results
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  4.1. Detection of Nox isoforms and adaptor proteins in HDFs
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   4.1.1. Expression of Nox4 isoform and its adaptor proteins in normal HDFs
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   4.1.2. Expression of Nox4 isoform and its adaptor proteins in affected skin and HDFs from SSc patients
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   4.1.3. Expression of other Nox isoforms, cytosolic subunits and adaptor proteins in normal nHDFs
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   4.1.4. Expression of additional Nox isoform and adaptor proteins in HDFs from SSc patients
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   4.2. Subcellular localization of Nox4 in HDFs
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  4.3. Induction of Nox4 expression and NADPH oxidase activity by TGF-β1 in HDFs
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   4.3.1. TGF-β1-upregulates Nox4 expression at mRNA level
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   4.3.2. TGF-β1 upregulates Nox4 expression at protein level
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   4.3.3. TGF-β1 enhances NADPH oxidase activity
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  4.4. Functional characterization of Nox4 in TGF-β1-mediated activation of HDFs
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   4.4.1. Effect of the pharmacological inhibitor DPI on cell viability and metabolic activity in nHDFs
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   4.4.2. Effect of the pharmacological inhibitor VAS2870 on cell viability in nHDFs
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   4.4.3. Pharmacological inhibition of NADPH oxidase by DPI reduces TGF-β1-mediated fibroblast activation
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   4.4.4. VAS2870 does not reduce TGF-β1-mediated fibroblast activation
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   4.4.5. Genetic knockdown of Nox4 neutralizes TGF-β1-mediated fibroblast activation
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  4.5. Regulation of Nox4 expression by TGF-β1 in HDFs
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   4.5.1. In silico promoter analysis of the human Nox4 gene
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   4.5.2. Nox4 expression is depend on canonical smad3 signaling pathway in HDFs
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   4.6. Pharmacological inhibition of NADPH oxidase activity by DPI reduces cutaneous fibrosis in the bleomycin mouse model of scleroderma
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   4.7. Nox4 is an emerging novel target for antifibrotic agents
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  5. Discussion
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   5.1. Nox4 is abundant in SSc HDFs
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   5.2. Nox4 is colocalized with endoplasmic reticulum in HDFs
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   5.3. Nox4 expression is regulated by TGF-β1 in HDFs
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   5.4. Nox4 expression is regulated by the Smad2/3 pathway in HDFs
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   5.5. Nox4 inhibition prevented experimentally induced skin fibrosis in the bleomycin mouse model of scleroderma
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   5.6. α-MSH suppressed TGF-β1-mediated Nox4 gene expression in HDFs
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   6. Conclusion & Future perspectives
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   7. Pharmacological inhibition of Nox4 by GKT137831 reduces TGF-β1-mediated fibroblast activation
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   8. References
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   Schulausbildung
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   1990-1994 The primary school “Omar Makram” (Alexandria, Egypt)
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   1994-1997 The preparatory school “Ibn Khaldoon” (Alexandria, Egypt)
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   1997-2000 The secondary school “Isis” (Alexandria, Egypt)
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   Studium
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   2000-2004 Bachelor of Science in Biology, Faculty of science, Alexandria
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   University, Egypt
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   2005 Visiting the Pre-Master courses at Faculty of science, Alexandria
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   University, Egypt
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   2005-2008 Master of Science in Microbiology, Faculty of science, Alexandria
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   University, Egypt, with the title (Effect of some Local Probiotics on
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   Immunological and Biological Systems of Experimental Animals)
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   2008-2009 Acquisition of the academic degree M.Sc. (Microbiology).
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   Alexandria University, Egypt
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   March 2010 Acquisition of the DAAD full-PhD scholarship (GERLS)
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   March -June 2010 German courses at Goethe-Institute Alexandria (Alexandria, Egypt)
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   October2010-March 2011
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   German courses at Goethe-Institute (Göttingen, Germany)
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   Promotionsstudium
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   Since April2011 Ph.D student, Department of Dermatology (Prof. Dr.med.Markus Böhm),
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   University of Münster, Münster, Germany
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   with the title. “ The Role of NADPH Oxidase in the Pathogenesis of
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   Systemic Sclerosis”.