Rakshya Pokharel

Investigating the role of a different protein in neurodegenerative disease models. Design and execute experiments with various assay techniques and research development including immunostaining, immunoblotting, protein quantification and detection assays. Aim is to target different protein pathway activation in disease cell model and diseased brain tissue, and determine the role of different compounds/molecules with various selectivity profiles onto pathological cascade in vitro by the use of iPSC-derived cells with familial Alzheimer Disease mutations obtained commercially. Treated different cell lines with the different compounds to test for the protein activity and compare to the wild type control groups.

Cryoprotection of mouse brain, flash freeze and cryosectioning of fixed mouse brain using a microtome. Followed by tissue processing, and IHC for detection under light and confocal microscopy for research to understand the distribution and localization of various neuronal cells, biomarkers and overall parts of the brain in both healthy and diseased mice.

Key Accomplishments:

• Screened various compounds with a vehicle control and a positive control involved in level of expression of a kinase and its involvement in tauopathy and compare to the non-disease tissue, assess correlation to pathological amyloid, tau neuroinflammation.
• Testing different antibodies to test the level of different proteins in different fAD mutation cell lines.
• Testing different antibodies of a protein and its isoform to test levels in different fAD mutation cell lines.
• Stored conditioned media from each treatment to screen for amyloid beta levels via ELISA assay, kinase activation measured by alphaLISA, immunoblot and IHC. And examine the roles of stressors to amplifying its activities in disease conditions in vitro.
• Three compounds were tested in different dosages to test the appropriate dose to repeat studies with stressors on.
• Using the cell culture system to harbor increased levels and/or activity to examine the inhibitor compounds on the activity level and assess the levels of its phosphorylated isoforms.
• Evaluate the kinase in human disease brain tissue and assay the mRNA level and related family members and protein in the cortex of human brains from those with AD, AD in DS, FTD-tau patients and their non-demented controls. The preliminary results give evidence that relative to non-demented controls the level of this kinase products increased in AD males and in AD-DS both males and females. Standard western blot was carried out to evaluate the target proteins.

Produce research and clinical grade components such as NTPs. Performing wet chemistry reactions such as synthesis, phosphorylation, quenching, purifications, precipitation and the finalization, with quality control throughout each processes by utilizing pilot grade systems of purification, QA, analytics and GDP. Prior to setting up any reactions or processes, performing equipment specifications. Maintaining good lab practices such as: cleaning, operating, maintaining the equipment in reliable working order, recording and documenting, and following laboratory safety guidelines and rules.

Key Accomplishments:

• Preparing for production by reviewing production schedule; studying and clarifying specifications; calculating requirements; assembling materials and supplies.
• Providing written and verbal updates for supervisors or other department managers.
• Ensuring timely completion and compliance of all other relevant company training requirements.
• Writing and executing batch records, authoring and reviewing Standard Operating Procedures, and maintaining quality documents associated with manufacturing.
• Participating in the maintenance and overall cleanliness of the workspace.
• Keeping equipment operating by following operating instructions and troubleshooting breakdowns.
• Interfacing with manufacturing support groups to align on production requirements.
• Use of LS/MS and HPLC system for chromatographic analysis purposes.

• Inability to work in the lab due to COVID-19 (2020/2021). Unfortunately, the start date was postponed until further notice.

Performed various microbiology techniques such as cloning, DNA/RNA extraction, Polymerase Chain Reaction (PCR), colony & RT-PCR, and qPCR. Mentoring and training students with research projects and biological protocols. Design and execute experiments with various protein detection assay. Target host-seeking and appetite suppression in mosquitos that transmit human pathogens to control mosquito-borne disease. Investigate Helper Genes impact on potency of effector molecules that can suppress viral pathogens. Study various small peptides to identify greatest binding strength with neural receptors to reduce mosquitos’ impulse to host seek for bloodmeal. Prepare regulatory documentation and presentations. Oversee bio-chemical inventories to support numerous research projects.

Key Accomplishments:

• Cloned genes involved in RNAi expression vectors with E. coli, C. Elegans, and Pseudomonas species to knock down, suppress, or neutralize viral expression.
• Engineered transgenic bacteria for supporting infectious disease control research - activated the G protein-coupled neuropeptide Y (NPY)- like receptors (NPYLR7) which suppress desire of mosquitos to feed in several species.
• Collected, analyzed, and documented experimental data with clustering and random sampling to contribute to organization’s data pool.
• Reared in-house mosquitoes to screen bacterial constructs.
• Contributed to development of patent currently under review.

• Took part in microalgal project - executed algal optical density measurements to log growth curve with Spectrophotometer. Earned full time position after three months.
• Developed strong relationships with external partners, facilitating effective collaboration on multi-institutional projects.
• Assisted in the experimental process, contributing in securing funding.