Investigating the innate immune response towards necrotic cell death
Belgium (BeMSA) - KU Leuven, Leuven
Department of Microbiology, Immunology and Transplantation, Rega Institute
Prof. Pedro Marques
Prof. Pedro Marques
4 weeks
Cities/Months Jan Feb Mar Apr May Jun Jul Augt Sep Oct Nov Dec
No No No No No No No Yes No No No No
Type of Research Project
- Basic science
What is the background of the project?
Necrotic cell death is a powerful inflammatory stimulus. It promotes the release of a myriad of damage-associated molecular patterns (DAMP) and proinflammatory mediators. Soon after release, molecules such as deoxyribonucleic acid (DNA), histones, actin, chemokines, and many others may be deposited in the tissue, initiating a massive inflammatory response that often harms the tissue further. There is a broad interest in understanding how such an inflammatory process is initiated and sustained, and above all, to find means to inhibit the collateral tissue damage. Our lab investigates the molecular cues that drive inflammation. Here, we use biochemistry and imaging approaches to study how inflammatory mediators are released and modified, both in vitro and in vivo disease models. Moreover, we aim to understand how inflammation drives tissue regeneration by stimulating the removal of necrotic debris from tissues.
What is the aim of the project?
The aim of this project is to determine how necrotic cell debris such as DNA and histones are cleared from sites of tissue injury.
What techniques and methods are used?
In this study, the student will be working with in vitro biochemical assays for cytokines and chemokines. An example of one of the biochemical assays is Enzyme-Linked Immuno Sorbent Assay (ELISA). This technique detects and quantifies substances, which in this case are the relevant cytokines and chemokines involved in the inflammation process. The main mechanism of this technique is the highly sensitive antibody-antigen interaction in which the antigen (possibly present in the sample) is attached on a solid base. The antibodies are added to the array in order to bind the antigen. The signal intensity is in direct relation with the amount of antibody-antigen interaction. In vitro DNA binding assays are assessed by means of fluorescence and microscopy. The assays make use of the integration of fluorophores into the binding process which emit fluorescence at a certain nanometer (nm). This will be visualized by means of fluorescence microscopy. The student, who should be in the possession of a Federation for Laboratory Animal Science Associations (FELASA) B certificate, is expected to provoke inflammation in in vivo models. The experimental laboratory animals used for this study are mice in which the liver and skin form the targets for the inflammation. The liver and skin tissues injured by inflammation will be collected on which immunostaining will be applied. Immunostaining, a technique which also includes the use of antibodies, will be applied on tissue sections and the cells of the collected liver and skin tissues. After immunostaining, the detection will happen by means of in vivo (intravital) and super-resolution microscopy (SRM). In vivo microscopy involves tracking of the inflammation process in live animals by which individual cells are detectable. SRM is a unique detection method since it makes use of a physical barrier which restricts the optical resolution up to 250 nm. This technique allows visualization on subcellular level.
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
- The tasks will be done under supervision
What are the tasks expected to be accomplished by the student?
Active participation in laboratory meetings and discussions. Active participation in experiments happening in the laboratory, such as experiments performed by senior students of the lab for their own projects. Students will receive training and will have to practice some of the laboratory’s standard techniques, such as cell culture, immunostainings, and microscopy. Planning and execution of the student’s own experiments after discussion with the supervisor. Scientific data analysis and interpretation of the results of the student’s experiments using FIJI (Fiji Is Just ImageJ) also known as ImageJ which is an image processing program.
Will there be any theoretical teaching provided (preliminary readings, lectures, courses, seminars etc)
The student will be exposed to explanations and discussions from the lab members. The student will also be encouraged to pursue the relevant knowledge for his study in the scientific literature and protocols There will be no formal teaching.
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 presentation
What skills are required of the student? Is there any special knowledge or a certain level of studies needed?
Subjects passed: Immunology, Cell Biology, Practical laboratory courses. Motivation, scientific curiosity, scientific method, university-level knowledge of immunology and cell biology, basic laboratory skills (pipetting, weighing, preparation of solutions, calculations).
Are there any legal limitations in the student’s involvement
In case the student lacks a Federation of European Laboratory Animal Science Associations B (Felasa B) certificate to work with laboratory animals, the laboratory work will be restricted to in vitro experiments.
Type of students accepted
This project accepts:
- Medical students
- Graduated students (less than 6 months)
- Pre-Medical students from the American-British system
- Students in biomedical fields
- Hepatic DNA deposition drives drug-induced liver injury and inflammation in mice. Marques PE; Oliveira AG; Pereira RV; David BA; Gomides LF; Saraiva AM; Pires DA; Novaes JT; Patricio DO; Cisalpino D; Menezes-Garcia Z; Leevy WM; Chapman SE; Mahecha G; Marques RE; Guabiraba R; Martins VP; Souza DG; Mansur DS; Teixeira MM; Leite MF; Menezes GB. Hepatology. 2015 Jan;61(1):348-60. doi: 10.1002/hep.27216. Epub 2014 Jul 31.
- Chemokines and mitochondrial products activate neutrophils to amplify organ injury during mouse acute liver failure. Marques PE; Amaral SS; Pires DA; Nogueira LL; Soriani FM; Lima BH; Lopes GA; Russo RC; Avila TV; Melgaço JG; Oliveira AG; Pinto MA; Lima CX; De Paula AM; Cara DC; Leite MF; Teixeira MM; Menezes GB. Hepatology. 2012 Nov;56(5):1971-82. doi: 10.1002/hep.25801. Epub 2012 Aug 21.