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Technical Background
7
Abir Rahman - Technical Background
Presentation
7/15/2020
Introduction
Abir Ashfakur Rahman
Ph.D. in Biomolecular Sciences
Boise State University, Boise, Idaho
Introduction
Current affiliation: Tulane University School of Medicine, Bix Lab
Current Postdoctoral work
Postdoc work
Novel therapeutics for ischemic stroke
Tandem transient middle cerebral and common carotid artery occlusion (tMCAO)
Occlude both the common carotid artery and the middle cerebral artery of mice at the same time, for one hour.
In vivo
Cell lines and procedures
bEnd.3
In vitro
bEnd.3 cells - Mouse Brain Endothelial Cells
- Procedure: 8-hour Oxygen-Glucose Deprivation and 18-24 hour reperfusion
- Readouts: Western blot and Immunocytochemistry
Primary Mouse Neural Stem Cells (Neurospheres)
- Neurogenesis questions:
- Stem cell proliferation
- Neural differentiation
- Neuroblast migration
Primary microglia: Morphological characterization and proteomic profiling.
Neurospheres
Neurospheres
(adhered and cells migrating)
Autophagy dysfunction as a mechanism of Parkinson's Disease
Doctoral work
The role of the VPS35 D620N mutation
Autophagy
Mechanism of cellular homeostasis
- Protein turnover
- Mitochondrial turnover
- Endoplasmic reticulum turnover
- Lipids turnover and more
Autophagy
Source: Novus Biologicals
Measuring Autophagic Flux
Measuring Autophagic Flux
Source: Stress Marq Biosciences and Pieurucci et al. Neuropharmacology (2017)
VPS35 D620N
Originally identified by two independent studies in 2011 as a causal mutation.
VPS35
Williams E.T., Chen X., and Moore D.J., Journal of Parkinson’s Disease (2017).
VPS35 D620N inhibits autophagy
Autophagy defect
We used an overexpression construct, either overexpressing the WT allele, or the D620N mutant allele.
We showed that autophagic flux was reduced in VPS35 D620N mutant cells
Lentivirus mediated gene transduction
Cell line generation
For generating the WT and VPS35 D620N mutant transgenic cells,
- We used Lentivirus to deliver the transgenes
- HEK 293T cells were first transfected with viral component coding DNA, along with our transgenic construct, which also contained a puromycin resistance gene
- The virus containing media was then collected and administered to SH-SY5Y cells
- Cells were selected by puromycin treatment and validated via PCR and sequencing
Source: Sigma-Aldrich
Source: Elegheert et al. Nature Protocols (2018)
CRISPR/Cas9 mediated mutagenesis
- Used Origene kit and Synthego gRNA, to induce a G to A point mutation at base position 1858.
CRISPR/Cas9
The transcriptome is altered in the VPS35 D620N mutant background
ECM receptor interaction pathway
Transcriptomic changes
KEGG pathway analysis revealed potential altered pathways
Akt signaling pathway
Proposed pathway
Hyaluronan Signaling
High molecular weight HA inhibited autophagy
HA and Autophagy
CD 44 and HMMR expression altered in VPS35 D620N
HA receptors
Phosphorylation of AKT
AKT Phosphorylation
Other previous work
Other work
Confocal microscopy/ fluorescent microscopy: Took a course in bioimaging
Worked as core facilities intern
Worked with C. elegans and with D. melanogaster
Team management:
- trained and worked with many undergraduate students
- taught lab sections of undergraduate courses
- trained fellow postdoc and lab technician tissue culture and related in vitro work
Acknowledgements
Acknowledgements
Members of the Bix Lab:
Dr. I. Joachim Biose, Dr. Amruta Narayanappa, Mr. Kyle Esteves and Dr. Greg Bix.
My thesis committee:
Dr. Brad Morrison, Dr. Allan Albig, Dr. Julia Oxford and Dr. Matthew Ferguson
BMOL Program Director: Dr. Denise Wingett and
Program administrator: Beth Gee
Dr. Bill Walthall, Dr. Ananias Escalanate
Dr. Maria Andreina Pacheco Delgado and so many more.