Title: Characterization and application of biological Nano particles, Exosomes.

Aim of the study: Exosomes are extracellular nanovesicles, involved in the intercellular transfer of genetic information. In this project, we plan to characterize  and check Bruton's tyrosine kinase (Btk) expression level in B cell derived exosomes. Additionally, we aim to compare two Exosomes harvesting procedure: spin filtration and ultracentrifugation.

Background:Exosomes are nanovesicles (40-100 nm in diameter), involved in intercellular transfer of proteins and genetic information. Exosomes also play a vital role in both normal and pathological processes.

X-linked agammaglobulinaemia (XLA) is a human X-linked genetic disorder. Patients those who are suffering from this disease has a mutation in Btk gene. Btk protein is involved in B cell development and maturation through a signaling effect on the B cell receptor. XLA results in impaired B cell maturation and diminishes B cell mediated immune response. XLA patients are unable to produce mature B cells and exhibit reduced level of Immunoglobulin in serum. The only treatment of this disease is to administration of exogenous antibodies to reduce the severity and number of infections.

In this project, we are interested to check the level of Btk in the exosomes excreted from normal human B cells (Namalwa cell). Furthermore, we indented to transfer Btk containing exosomes from Namalwa cell to XLA B cells to study whether external administration of Btk improves B cell maturation process in XLA.

Impact: The prevalence of X-linked agammaglobulinaemia in male newborns is about 1 in 100,000. Currently, The only treatment of this disease is to administration of exogenous antibodies to fight infection.  Since XLA patients cannot produce mature B cells due to the dysfunctional Btk protein, transfer of canonical Btk through exosomes collected from the cultured normal human B cells can provide alternative therapeutic prospect for XLA.

Method:

We plan to isolate and purify exosomes from Namalwa cells. In this isolation process we plan to employ two different methods; spin filtration and Ultracentrifugation. We plan to confirm the presence of exosomes isolated by both methods using western blot where we will use 1 ° antibody against alix and CD9 as well as 2 ° antibody. We are also interested in the presence of Btk in exosomes that can be easily addressed by Western blot using anti-Btk antibody. To separate exosomes, we will do liquid chromatography separation technique. Then we will analyze the isolated and purified exosomes with electron microscope and NTA (Nanoparticle Tracking Analysis).

Start date: 1 March 2013

Estimated duration: 5 months

Payment: Unpaid


 

Subjects: Biology Pharmacy
Topic: Thesis: Molecular cell biology and gene therapy (Exosomes)
Contact: Mr. Mohammad Khan, Mohammad Abu Quoreshi
Reply to: abu.quoreshi01.khan @ student.sh.se
Institution: Södertörns Högskola / Karolinska Institute
Location: 14152 Huddinge, Stockholm, Sweden. , Röntgenvägen 1 lgh 1,901th