The aim of this thesis (Apoptosis Regulation via the Mitochondrial Pathway: Membrane Response upon Apoptotic Stimuli) was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems.The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described….
Contents
I Apoptosis Regulation via the mitochondrial pathway: Introduction
a) Mitochondria
Structure and internal organization
Apoptosis regulation via the mitochondrial pathway
The use of isolated mitochondria in apoptosis studies
b) Bcl2 proteins regulation
The Bcl2 family: function, location, structure
Interplay at the mitochondrion
The use of Bcl protein in disease treatment
c) Aims and strategy
II BH4, TAT-BH4 and Bax-α1 peptide production
a) Solid Phase Peptide Synthesis (SPPS)
Chemistry
Cleavage
b) Purification (RP-HPLC) and characterization (MALDI-TOF)
High Performance Liquid Chromatography (HPLC)
Matrix Assisted Laser Desorption/Ionisation mass spectroscopy coupled with Time-Of-Flight detector (MALDI-TOF)
c) Trifluoroacetic acid (TFA) contamination
Risk for experiments
Detection and removal
d) Results & Comments
III Interaction with membranes: methodology and applications
a) From the peptide side33
Circular dichroism spectroscopy (CD)
Attenuated total reflection spectroscopy (ATR)
b) From the lipid side
Phospholipid bilayer polymorphism
Nuclear magnetic resonance (NMR)
Investigation of the headgroup region: P NMR and N NMR
Investigation of the lipid hydrophobic core: 2H NMR
Differential scanning calorimetry (DSC)
c) Results & comments
IV Tracking lipid interactions in living mitochondria
a) Isolation of mitochondria- “J’ai la patate”
b) P NMR ex vivo experiments – Set up
– Application: mitochondrial membrane response upon calcium stress….
Author: Sani, Marc-Antoine
Source: Umea University
Download URL 2: Visit Now