[RAF Kinase Inhibitor Protein Expression in Diabetes]

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ACKNOWLEDGEMENT

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible.

DECLARATION

I, [type your full first names and surname here], declare that the contents of this dissertation/thesis represent my own unaided work, and that the dissertation/thesis has not previously been submitted for academic examination towards any qualification. Furthermore, it represents my own opinions and not necessarily those of the University.

Signed __________________ Date _________________

ABSTRACT

Raf kinase inhibitor protein (RKIP) is a member of the PhosphatidylEthanolamine-Binding Protein (PEBP) family. It was originally described as an inhibitor of Ras/Raf/MEK/ERK pathway. Dysregulation of RKIP expression has the potential to contribute to pathophysiological processes including Alzheimer's disease and several types of cancer. It has been believed that oxidative stress plays a role in the pathogenesis in a variety of diseases including cancer and diabetes mellitus. Recently, one study found knocking down RKIP gene in immortalized cell line increased the oxidative stress.

This study is aimed to investigate the expression of RKIP in diabetes mellitus. Diabetic Goto-Kakizaki (GK) rats were used as a model for type 2 diabetes and diabetic Streptozotosin (STZ) rats were used as model for type 1 diabetes. All experimental rats were wounded surgically and left to heal for 7 days. Different tissues (liver, kidney, heart and wounded skin) were removed from all experimental rats. Various oxidative damage markers (dihydroethidium, DNPH, lipid peroxidation) and antioxidant enzymes (catalase, SOD, GSH) were used to verify the status of oxidative stress. RKIP expression was determined by western blot and immunofluorescent assay.

The results showed significant increase in the level of oxidative stress (p<0.05) in all tissues derived from both types of diabetic rats compared to non-diabetic controls. RKIP expression was significantly decreased in all tissues derived from diabetic-GK rats (p<0.05) compared to those of non-diabetic control rats. Meanwhile, diabetic-STZ rats showed no change in the expression of RKIP

RKIP expression is clearly decreased in type 2 diabetic model while it has no change in type 1 diabetic model. Oxidative stress appears to have no effect on the expression of RKIP indicating that RKIP in diabetes may not be associated with oxidative stress.

TABLE OF CONTENTS

ACKNOWLEDGEMENT2

DECLARATION3

ABSTRACT4

CHAPTER 1: INTRODUCTION6

Raf Kinase Inhibitor Protein (RKIP)6

RKIP Functions8

RKIP and the MAPK Pathway8

RKIP and G-protein Coupled Receptor Signaling10

RKIP and NF?B Signaling12

RKIP and the Cell Cycle13

RKIP in Cancer14

RKIP in other cellular processes15

Diabetes15

Diabetes mellitus16

Primary (idiopathic) diabetes mellitus17

Type I Diabetes Mellitus17

Type II diabetes18

Secondary Diabetes Mellitus18

Complications of Diabetes Mellitus19

Metabolic Disturbances In Diabetes Mellitus19

Oxidative stress21

Oxidative Stress Chemistry22

Superoxide radical (O2-.)23

Hydrogen peroxide (H2O2)23

Nitric Oxide (NO)23

Hydroxyl radicals (OH.)24

Cellular Antioxidant Defense24

Dietary Antioxidants25

Reduced Glutathione (GSH)25

Antioxidant Enzymes26

Implications of oxidative stress26

Lipid peroxidation26

Oxidative DNA damage27

Oxidative damage to protein28

CHAPTER 2: HYPOTHESIS AND OBJECTIVES29

Hypothesis29

Objectives29

CHAPTER 3: MATERIALS AND METHODS30

Materials30

Chemicals30

Kits and Assay Solutions30

Antibodies30

Consumables30

Instruments31

Methods31

Experimental animals31

Diabetic Streprtozotocin (STZ) rats- type I Diabetes Mellitus32

Diabetic Goto-Kakizaki (GK) rats- type II Diabetes Mellitus32

Skin wound Induction32

Tissue sampling33

Tissue homogenization for western blot33

Preparation of homogenates for other assays:33

Western Blot and Immunoblot Analysis34

Microscopic sections preparation35

Cryosectioning of the tissues35

Oxidative fluorescent microtopography35

Determination of lipid peroxidation35

Determination of protein carbonylation36

Immunofluorescenthistochemistry36

Total RNA preparation37

DNase digestion37

Reverse Transcription38

Quantitative real- time ...