General Objective To Investigate The Bioenergetic Profile Of Fibroblasts In Selected Energy Deficiency Type Of IEM Patients. Specific Objective(s) 1. To Identify The Gene Of Mutation In Energy Deficiency Type Of IEM Patients. 2. To Evaluate The Bioenergetics Profile Using Real Time Analysis Assays. 3. To Determine The Mitochondrial Morphology. 4. To Determine The Dynamic Regulation Of Mitochondrial Morphology. Study Design This Observational, Lab-based Study That Meet Strict Inclusion Criteria. Sample Prep: Cryopreserved Patient And Control Fibroblasts Are Cultured And Expanded For Analysis. Mutation Analysis: DNA Is Extracted, Sequenced, And Analyzed For Relevant Mutations. Bioenergetic Profiling: Mitochondrial And Glycolytic Functions Are Measured And Evaluated. Parameters Include Oxygen Consumption And ATP Production. Morphology Assessment: Mitochondrial Structure Is Visualized With Immunofluorescence Microscopy. Dynamics & Protein Assessment: Fusion And Fission Regulatory Proteins (MFN1, MFN2, DRP1, OPA1) Are Measured By Western Blot And Correlated With Functional Findings.
Mitochondrial Oxidative Phosphorylation (OXPHOS) Is Vital For ATP Generation, And Its Disruption Is A Hallmark Of Mitochondrial Diseases That Affect Highly Energy-dependent Tissues. Patient-derived Skin Fibroblasts Provide A Convenient Model To Study Mitochondrial Dysfunction, Yet Little Was Known About How OXPHOS-defective Fibroblasts Adapt Under Metabolic Stress. This Project Aimed To Develop And Validate An Optimized Culture System To Assess Oxidative Capacity In Fibroblasts With OXPHOS Defects. By Optimizing Metabolic Conditions And Performing Quantitative Bioenergetic Assays, The Study Established A Reliable Framework To Differentiate Normal And OXPHOS-impaired Mitochondrial Function At The Cellular Level. Objectives: The General Objective Was To Investigate The Mitochondrial Oxidative Capacity Of Cultured Skin Fibroblasts With OXPHOS Defects. Specifically, The Study: Established Optimum Conditions To Stimulate Oxidative Phosphorylation By Varying Medium Composition And Oxygen/glucose Availability. Compared Growth And Proliferation Between Normal And OXPHOS-defective Fibroblasts Under OXPHOS-dependent Culture. Characterized Oxidative Capacity Through Intracellular ATP And Lactate-to-pyruvate Measurements. Methodological Approach: Primary Fibroblasts From Healthy Controls And Patients With Confirmed OXPHOS Defects Were Cultured Under Defined Metabolic Conditions. Sequential Optimization Determined Suitable Media And Environmental Parameters That Maximized Mitochondrial Respiration. Cell Proliferation Was Assessed Via Cell Counting, MTT, And EdU Assays. Intracellular ATP Was Quantified Using Luminescence-based Methods, While Lactate And Pyruvate Concentrations Were Determined Enzymatically. Lactate-to-pyruvate Ratios And ATP Levels Were Used As Indices Of Mitochondrial Efficiency. Results And Significance: The Project Successfully Optimized OXPHOS-dependent Conditions And Demonstrated That OXPHOS-defective Fibroblasts Exhibited Slower Proliferation, Reduced ATP Production, And Elevated Lactate-to-pyruvate Ratios Compared To Normal Controls. These Findings Confirmed A Metabolic Shift Toward Glycolysis, Consistent With Mitochondrial Impairment. The Optimized System Provided A Reproducible In Vitro Model For Evaluating Mitochondrial Energy Defects Using Patient-derived Fibroblasts. It Offers A Practical Platform For Future Diagnostic, Therapeutic, And Mechanistic Studies In Mitochondrial Disease Research.