SIMI 2019

Ali Mohd
Under Dr. Kiela and Dr. Da Paz
Gastroenterology/Nutrition
Lab’s Main Focus
Dendritic cells (DC) in the immune
system are a key factor for tolerance in
the intestines as there are millions of
healthy commensal bacteria that won’t
cause disease.

However, in an autoimmune disease like

IBD, the immune system is unable to
differentiate between healthy bacteria
and pathogens.
Dab2
Disabled homolog 2 (Dab2) is a
adapter protein that regulates cell
growth and inflammatory
response in DCs.
My lab focuses on this protein to
see how much of a role Dab2
plays during IBD.
Initial Questions
What will I be doing?
Will I contribute to something real?
How will this program help me
grow?
What have I been doing?

Protein Western
Assays Blot
Cell Culture

DC2.4
Passage 5
1:10
7/1/19
10X objective zoom

Immortalized
mouse DCs
What did I contribute?
The work I did was a preparatory procedure
for a follow-up experiment.

In February, the lab published a paper

describing the effects of downregulation of
Dab2 in dendritic cells.
Lab Project: Intro

Dab2 is a protein that regulates signalling pathways

between cells, including ones that mediate cell
growth and inflammatory response.

The lab analyzed the expression of Dab2 in

dendritic cells during bacterial exposure(LPS).
Intro Cont.

LPS was added to BMDCs at different times to

examine Dab2 expression over time of exposure.
They quantified Dab2 using Western blot.

They also looked at Dab2 mRNA presence over time

of exposure using RT-PCR.
Western Blot Results

RT-PCR
Intro Cont.
It was concluded that exposure to bacterial
stimuli decreases Dab2 protein and Dab2 mRNA
expression in DCs, leading to more severe
inflammatory responses.
Lab Project: Content
Now we have to ask: What is the molecular
mechanism causing Dab2 to drop in DCs?

A cell line called DC2.4 was tested to see if they

shared the property of down regulating Dab2
under bacterial stimuli.
Why DC2.4?
- Cheaper than
BMDCs
- Easy to reproduce
- Immortalized cells
- There is no
sacrificing of mice in
producing samples
DC2.4 Passage 5 1:10 7/1/19 10X objective
Content Cont.
Cells were cultured and plated in 12-well plates;
6 groups of three were made.
LPS was added at either 24, 16, 6, 3, or 2 hours
with one set left untreated.
Content Cont.
Cells were then lysed for either protein detection
or RNA analysis.

Protein- Western Blot

RNA- RT-PCR
Protein Quantification

r2= 0.999
y= 0.000556x + 0.128
Western Blot Results

BMDCs
DC2.4
 Real Time PCR Results (RT-PCR)

BMDCs
DC2.4
DC2.4

BMDCs
Conclusion
Based on the results of this experiment, this line
of cells is not viable for experimentation since
they don’t show a direct correlation between
bacterial exposure and Dab2 expression.
Unfortunately, this means that mice will still be
needed for future experiments.
What I’ve Learned
I have learned many different techniques used in
the lab and have been able to participate in a lot
of them. I completed this experiment relatively
independently and may have made mistakes
resulting in unwanted results, but I have been
given what isn’t taught in school.
How has this program helped me?
This program has introduced me to an
environment that I would never have seen during
high school. I’ve learned more in 7 weeks than I
have in a semester in school. I am also grateful
for being part of such a welcoming lab.
Additional Questions
How can I get more experience in the lab during
the school year?

Who else can help me decide on a career to

pursue?

How else can this program help me?

How will I answer these questions?
I’ll stay in contact with my lab and my mentor
and continue to learn outside of school about my
interests.
I’ll also apply for this program next year to
continue my experience in research.
What are some questions
you have?