Cell-free production and purification of mitochondrial carriers

Scientific Background

Mitochondria are double-membrane organelles and the powerhouse of the cell. The respiratory chain in the inner mitochondrial membrane (IMM) generates a proton gradient and its dissipation is coupled to the production of adenosine triphosphate (ATP) to supply energy for cellular processes. Mitochondrial uncoupling is the dissipation of the proton gradient without ATP production. Uncoupling protein 1 (UCP1) is a mitochondrial carrier that mediates non-shivering thermogenesis in brown fat by mitochondrial uncoupling. It is activated by free fatty acids and inhibited by purine nucleotides. To directly study the function and mechanism of mitochondrial carriers, we employ biomimetic systems with recombinant UCP1 reconstituted into lipid bilayer membranes. This approach requires correctly folded protein with highest purity to conclude observations with the function of UCP1.

Here, we attempt to establish a novel protocol, which employs the cell-free production of UCP1 The project will include protein engineering, up-to-date purification procedures and reconstitution of UCP1 into liposomes.

Tasks of the student

We seek for a highly motivated bachelor or master student who is interested in learning cell-free production and purification of mitochondrial membrane proteins. The candidate will learn how to prepare and conduct the cell-free production of UCP1 and its purification by metal-ion chromatography and size-exclusion chromatography. The candidate will explore different approaches and optimization procedures to maximize protein yield and purity. The student will also learn to perform transport assays to test the correct function of the cell-free produced UCP1. Finally, the candidate will apply the protocol to produce further mitochondrial carriers, such as the ADP/ATP carrier and the aspartate/glutamate carrier.

Application: Please send your application, including a motivation letter, your CV and a transcript of records to Juergen.Kreiter@vetmeduni.ac.at until 31.12.2024. If you receive no answer within 2-3 days, please send your application to Elena.Pohl@vetmeduni.ac.at.

Location: Institute of Physiology and Biophysics, Department of Biomedical Sciences, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna.

Duration: Minimum of 9 months, start as soon as possible.

Project description

Cell life is organized on a strictly controlled Ca²⁺ homeostasis in which the mitochondrial Ca²⁺ handling is essential. Disturbance of mitochondrial Ca²⁺ levels affects cytosolic Ca²⁺ buffering, and Ca²⁺ signalling, which crucially regulate mitochondrial and cellular metabolism and cell death decisions.

The risk of lung and metabolic diseases has increased significantly, as evidenced by the last pandemic and the rising number of obesity cases. In this project, we aim to elucidate the metabolic role of Ca2+ homeostasis in lung health and the effects of nutritional conditions on metabolic stress and adaptation from the perspective of mitochondrial functions.

In the first part of the project, we will investigate the effect of mitochondria Ca²⁺ handling in Drosophila trachea, which has become an excellent model system for pulmonary disease research, specifically for COPD and SARS-CoV-2. The master student will focus on the newly identified mitochondrial Ca²⁺ transporter TMBIM5 and generate Drosophila conditional trachea-specific TMBIM5 knockdown fly lines to investigate cellular viability, mitochondrial functions, metabolism and immunity. Experimental approaches include molecular and cell biology, biochemistry, and microscopy.

In the second part of the project, the role of mitochondria in response to metabolic stress will be studied. We will use replicated populations of Drosophila that are adapted to different diet (protein-rich and carbohydrate-rich) and investigate the mitochondrial functions, and life history trait of individuals adapted to different diet.

This master project will shed light on the role of mitochondria in metabolism in response to diseases and diet.  

Candidates

We are looking for a highly motivated and talented Master student with solid knowledge in cell or molecular biology and good interpersonal skills. 

Offer

The Master's project is highly interdisciplinary and, led by a collaborative team, offers the opportunity to combine different areas of expertise and explore the intersection of Drosophila and mitochondrial biology in a cutting-edge research topic, with the chance to contribute to a major scientific publication.

We offer a scholarship of around 450 €.

Application

Please send your application including CV to Neda Barghi and Karin Nowikovsky (barghi.neda@gmail.com, karin.nowikovsky@vetmeduni.ac.at)

We are looking for Master and Bachelor students.

Job description

Diploma Thesis to assign.

More information here

We have a thesis to assign.

Deutsch

Englisch

The Mitochondria Biology Group offers a Master Thesis position.

Mitochondria perform essential functions for cellular metabolism and play a pivotal role in most areas of medicine.

Candidates holding a bachelor degree in Life Science are encouraged to apply. Starting date as soon as possible.

Please send your application including CV, motivation letter and contact detail of a referee to Karin.Nowikovsky@vetmeduni.ac.at.

PDF with full advertisement

Students who are interested in completing a thesis or internship are always welcome to apply. The topics come from two areas:

Membranbiophysics Head: Univ.-Prof. Dr. Elena Pohl

Environmental Health Head: Dr. Alois Schmalwieser

Interested students are kindly invited to send an email or write to the head of the respective unit. Applications should consist of a letter of application, a CV and a letter of motivation.