The role of SSAO in different cells/tissues

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Table of contents

I. Introduction
1. Amine oxidases
1.1. Amine oxidases
1.1.1. FAD-containing amine oxidases
1.1.2. TPQ-containing amine oxidases DAO Lysyl oxidase
1.2. The semicarbazide-sensitive amine oxidase
1.2.1. Gene and protein structure of SSAO
1.2.1.1. Cloning of the gene
1.2.1.2. Protein domains and active sites
1.2.1.3. Soluble forms of SSAO/VAP-1
1.2.2. TPQ and enzymatic reaction mechanisms
1.3. Enzymatic reaction of SSAO
1.3.1. Substrates (MA, BZM)
1.3.2. Products of SSAO reaction
Aldehydes
Hydrogen peroxide
Ammonia
1.3.3. Inhibitors
1.4. Protein expression of SSAO in different tissues
1.5. The role of SSAO in different cells/tissues
1.5.1. Adipocytes (glucose uptake and cell differentiation)
1.5.2. Vascular smooth muscle cells
1.5.3. Endothelial cells of high endothelial venules
1.5.4. Chondrocytes
1.6. Expression and activity of SSAO in pathological conditions
Diabetes
Atherosclerosis
Other diseases associated with SSAO
2. Cartilage
2.1. Physiology of cartilage
2.2. Articular cartilage function
2.3. Structure of the hyaline cartilage
2.3.1. Chondrocytes
2.3.2. Extracellular matrix (ECM) components
2.3.2.1. Collagens
2.3.2.2. Proteoglycans
2.4. Cartilage formation
2.4.1. Long bone development
2.4.2. Markers of chondrocyte differentiation
Condensation
Proliferation
Maturation and hypertrophy
Apoptosis
2.4.3. Transcriptional factors of chondrogenesis
2.4.3.1. Sox9
2.4.3.2. Runx2
2.5. Diseases of joints and cartilage degradation
2.5.1. Osteoarthritis (OA)
2.5.3. Animal models of cartilage diseases
3. Vascular Part
3.1. General structure and function of blood vessels
3.1.1. Vein
3.1.2. Artery
3.1.3. Capillaries
3.2. Structure of an aorta
3.2.1. Intima
3.2.1.1. EC
3.2.2. Media
3.2.2.1. Vascular smooth muscle cells
3.2.2.2. vSMC differentiation and phenotype changing
3.2.2.3. ECM
3.2.3. Adventitia
3.3. Remodeling of the arterial wall
3.4. Diseases of the vascular system
3.4.1. Atherosclerosis – Inflammatory disease of arterial blood vessels
3.4.1.1. Endothelial cell dysfunction
3.4.1.2. Cell types involved in atherosclerosis
3.4.1.3. Atherosclerotic plaque characterization
3.4.1.4. Animal models of atherosclerosis – ApoE-/- mice
II. Working hypothesis and objectives
III. Materials and methods
1. Models
1.1. Human cartilage
1.2. Animals
1.2.1. Normal rats
1.2.2. Models of cartilage degradation: MIA
1.2.3. WT and knock out SSAO-/- mice
1.2.4. Model of atherosclerosis: ApoE-/- SSAO-/ -double knock out mice
1.3. Cells: models of hypertrophic differentiation
1.3.1. Primary cell culture of rat chondrocytes and hypertrophic differentiation protocol
1.3.2. Progressive passage of rat chondrocytes
1.3.3. ATDC5
2. Common methods
2.1. Histology and Immunohistochemistry
2.2. Western blot
2.3. RNA isolation
2.4. RT- and real time qPCR
3. Specific methods
3.1. SSAO activity
3.1.1. Homogenates preparation for SSAO activity measurement
3.1.2. SSAO activity measurement
3.2. Measurement of 2-Deoxy-D-glucose uptake
3.3. MTT
3.4. Atherosclerotic lesion quantification in aorta
3.5. Atherosclerotic plaque quantification in the aortic sinus
3.6. Mesurement of cytokines
3.7. Mice genotyping
3.8. Experiments performed in collaboration
3.9. Statistical analysis
IV. Cartilage and chondrocyte differentiation
1. Hypothesis
2. Objectives and strategies
3. Results
3.1. The presence of SSAO in rat cartilage
3.1.1. SSAO expression in healthy cartilage of Wistar rats.
3.1.2. SSAO is present in the rat growth plate
3.2. The role of SSAO in cartilage
3.2.1. Model of chondrocyte terminal differentiation
3.2.1.1. ATDC5
3.2.1.2. Primary rat chondrocytes culture
3.2.2. Expression and enzyme activity of SSAO
3.2.2.1. SSAO in ATDC5
3.2.2.2. SSAO expression and activity during primary chondrocyte differentiation.
3.2.3. Effect of SSAO inhibition on terminal chondrocyte differentiation
3.2.4. The role of SSAO in terminal chondrocyte differentiation
3.3. The presence of SSAO in human OA cartilage
3.3.1. SSAO in human more and less diseased cartilage
3.3.2. Expression and activity of SSAO in human more and less diseased cartilage
3.4. Presence of SSAO in cartilage of the MIA rat model of cartilage degradation
4. Discussion
5. Conclusions
6. Perspectives
V. Vascular and atherosclerosis studies
1. Hypothesis
2. Objectives and Strategies
3. Results
3.1. Role of SSAO in plaque development: Invalidation of SSAO in ApoE-/- mice
3.1.1. Lipid profile in ApoE-/- and ApoE-/-SSAO-/- mice
3.1.2. Kinetics of disease development
3.1.3. Phenotype of atherosclerosis plaques and media
3.1.3.1. α-actin expression in media of aortic sinus
3.1.3.2. Lymphocyte infiltration in media of aortic sinus
3.1.3.3. Monocyte/macrophage presence in media of aortic sinus
3.1.3.4. Collagen content in the media and the plaque of the aortic sinus
3.1.4. Exploration of pro- and anti-inflammatory profiles – in spleen – in aorta
3.1.5. Exploration of the VSMC phenotype
3.1.6. Role of SSAO in cellular trafficking in ApoE-/-SSAO-/- mice vs ApoE-/- mice – in mice under HFD
4. Discussion
5. Conclusion
6. Perspectives
VI. General discussion
VII. References