Deciphering the Mechanism of Immune Dysfunction in Vici Syndrome

Inhaltsverzeichnis (Table of Contents)

• Chapter 1 - INTRODUCTION.
  • 1.1) Vici Syndrome
  • 1.2) EPG5
  • 1.3) Autophagy and endocytosis
  • 1.4) Pathogenesis of Vici Syndrome
  • 1.5) Toll-like protein receptors (TLRs)
• Chapter 2 - AIM OF THE WORK..
• Chapter 3 - MATERIALS AND METHODS..
  • 3.1) Materials
    • 3.1.1) Patients clinical data
    • 3.1.2) Cell Cultures
  • 3.2) Methods
    • 3.2.1) Isolation of Peripheral Blood Mononuclear Cells (PBMCs)
    • 3.2.2) Generation of a lymphoblastoid cell lines
    • 3.2.3) PBMCs stimulation with TLR2-9 agonists
    • 3.2.4) Fibroblast stimulation with CpG or IL-1ẞ and NF-kBp65 translocation
    • 3.2.5) CpG-FITC internalization in lymphoblastoid cell lines (LCLs)
    • 3.2.6) Immunofluorescence
    • 3.2.7) Transient gene silencing by siRNA
    • 3.2.8) Transient gene transfection by lipofection
    • 3.2.9) Transient gene transfection by electroporation
    • 3.2.10) Extraction of genomic DNA
    • 3.2.11) Extraction and quantification of the RNA
    • 3.2.12) Reverse transcription and amplification (RT-PCR)
    • 3.2.13) Electrophoresis, extraction from the agarose gel and sequencing of PCR products
    • 3.2.14) Amplification and quantification by real-time PCR
    • 3.2.15) Protein extraction and quantification
    • 3.2.16) Electrophoresis of proteins by SDS-polyacrylamide gel
    • 33.2.17) Western blotting
    • 3.2.18) Plasmid constructs and transformation of bacterial cells
    • 3.2.19) Preparation of plasmid DNA
    • 3.2.20) DQ-BSA internalization
• Chapter 4-RESULTS.
  • 4.1) Molecular genetic analysis
  • 4.2) Analysis of the effects of mutations on the transcribed EPG5
  • 4.3) Analysis of the effects of the mutations on protein EPG5
  • 4.4) Stimulation of PBMC in vitro with TLR2-9 agonists
  • 4.5) Detection of NF-kB p65
  • 4.6) Analysis of intracellular transport of DQ-BSA
  • 4.7) Analysis of intracellular transport of CpG (EAA1, LAMP1, LAMP2)
  • 4.8) Internalization of CpG-FITC in the patient's and control fibroblasts
  • 4.9) Transient silencing of EPG5 and internalization of CpG-FITC
  • 4.10) Cellular localization of EPG5 in HEK293T cell lines
• Chapter 5-DISCUSSION.

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This Ph.D. thesis aims to investigate the mechanisms of immune dysfunction in Vici syndrome, a rare genetic disorder characterized by severe immune deficiency, developmental delay, and neurological abnormalities. The study focuses on the role of the EPG5 gene, which encodes a protein involved in autophagy and endocytosis, in the pathogenesis of the disease.

• The role of EPG5 in immune function and its association with Vici syndrome.
• The impact of EPG5 mutations on autophagy and endocytosis pathways.
• The effects of EPG5 dysfunction on TLR signaling and immune responses.
• The potential therapeutic implications of understanding the molecular mechanisms of Vici syndrome.

Zusammenfassung der Kapitel (Chapter Summaries)

• Chapter 1: Introduction provides an overview of Vici syndrome, including its clinical presentation, genetic basis, and pathogenesis. This chapter also delves into the roles of EPG5, autophagy, endocytosis, and TLRs in the context of the disease.
• Chapter 2: Aim of the Work outlines the specific objectives and research questions addressed in the study.
• Chapter 3: Materials and Methods details the experimental design, including the patient cohort, cell lines, and techniques employed to investigate EPG5 function and its role in immune dysfunction.
• Chapter 4: Results presents the findings of the study, covering molecular genetic analysis, the impact of EPG5 mutations on protein function, and the effects of EPG5 dysfunction on immune responses.

Schlüsselwörter (Keywords)

Vici syndrome, EPG5, autophagy, endocytosis, TLR signaling, immune dysfunction, genetic disorder, rare diseases, molecular mechanisms, therapeutic implications.