Projects
Name
Ubiquitin system and balancing reactive oxygen species levels in multiple myeloma and development of drug resistance
University
Czech Republic (IFMSA CZ) - University of Ostrava, Ostrava
Domain
Hematology
Departement
Department of Hematooncology, University Hospital Ostrava
Head
Prof.Roman Hajek, MD, PhD
Tutor
Prof.Roman Hajek, MD, PhD; Dr.Michal Simicek
Languages
Czech, Slovak, Serbo-Croatian
Duration
4 weeks
Availability
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
No No No No No No Yes No No No No No
Type of Research Project
- Basic science
What is the background of the project?
A rapid increase in reactive oxygen species (ROS) level is one of the main mechanisms of action of the proteasomal inhibitors (PIs) and immunomodulators (IMIDs) that are currently used as a primary regiment in the treatment of monoclonal gammopathies (MGs) and other hematological malignancies. Both types of drugs are targeting components of the ubiquitin-proteasome system (UPS). Thus, mutations and dysregulation of the genes encoding the USP components will likely interfere with the action of PIs and IMIDs. Pacticularly affecting ROS metabolism can have profound concequences and lead to drug resistance or increase sensitivity. How ubiquitination machinery regulates ROS metabolism is highly unknown. Combining next generation sequencing data and expression analysis from patiens with MGs provides interesting hits for further mechanistic investigations that can uncover novel drug targets and/or predictive biomarkers.
What is the aim of the project?
The main aim of the project is investigation of the role of KEAP1 ubiquitin ligase and additional ubiquitin system regulators (esp. deubuquitinases) in the development of PIs and IMIDs resistance in MGs.
What techniques and methods are used?
Cell culture, CRISPR-Cas9 technology, recombinant DNA cloning, western blotting, spectral analysis, flow cytometry, purification of recombinant proteins. Multiple myeloma and acute myeloid leukemia stable cell llines will be genetically modified in order to express specific mutant genes and/or shRNA. Vairous functional assays will be conducted (proliferation, viability, migration, ROS production, drug resistance) to evaluate role of specific gene and mutations on phenotype.
What is the role of the student?
- The student will observe the practical experiments but will be highly involved in the analysis of the results
- If the project includes “lab work”
- the student will take active part in the practical aspect of the project
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Maintaining stable cell lines cultures and performing basic genetic manipulation using electroporation of plasmid DNA and/or siRNA. Protein extraction from mammalian cells and specific protein detection using western blot. Generation of recombinant DNA using polymerase chaing recation based techniques. Sit-directed mutagenesis. Performing and analysis of flow cytometry experiments. Analysis of reactive oxygen species levels in cultured human cell lines using fluorescent dyes and spectral analysis. Fluorescent microscopy with fixed and live cells.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
Yes, before performing any experimental work students will be introduced into the basic of the technique used and overall background of the project will be presented to the student.
What is expected from the student at the end of the research exchange? What will be the general outcome of the student?
- The student will prepare a scientific report
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Basic knowledge of cell and molecular biology. Previous practical experience from molecular biology laboratory. Subjects passed: cell and molecular biology Previous experience with wet lab work required
Are there any legal limitations in the student’s involvement
Yes
Official permission to perform internship at the Faculty Hospital Ostrava.
Hours
6
Type of students accepted
This project accepts:
- Medical students
- Graduated students (less than 6 months)
- Students in biomedical fields
Articles
- Kumar; S.; Rajkumar; V.; Kyle; R.; van Duin; M.; Sonneveld; P.; Mateos; M.; Gay; F. and Anderson; K. (2019). Multiple myeloma. [online] Nature. Available at: https://www.nature.com/articles/nrdp201746 [Accessed 15 Jan. 2019].