Friday, August 10, 2012

Week 12-Conclusion to my Summer Blogging!

This week was supposed to be the last official week of summer research (as per the UH academic calendar); however, I am so pleased with my experience in Dr. McConnell's lab that we both agreed to continue my position in his lab throughout the fall and spring semesters (at reduced hours and reduced days, of course). This is important to me because I want to apply and continue what I have learned during this internship to my own independent research project (and perhaps honors thesis) in the future. I feel as if the skills and technique I have picked up during this experience are extremely invaluable to any lab setting I may encounter, whether it be a course-required lab or a lab used in the institutional setting. One thing I really enjoyed learning was the actual practice of asceptic technique in a laminar flow hood, something that my supplemental studies indicated was extremely important in the pharmacy setting. My graduate student mentor will still be working towards her Ph.D. and the rest of the research team will still be continuing their experiments by the time the fall semester kicks up, so I will be stopping by to help them with the same things I have learned this summer.

For the next few work-days, I will be helping one of the members of the research team with running cAMP and PKA (with PKI) assays. This will be used to determine cAMP (cyclic AMP) levels in our tissues of interest.

My graduate student mentor has also been showing me her technique for calcium imaging to measure the intercellular calcium release in our cells of interest.

The lab has been quite hectic because we are within weeks of revising and submitting over six papers for publication. Despite this though, it is the unity that comes about in times like this that never ceases to make me proud that I am a part of such a hard-working lab.

Thank you to my Tier One Scholarship, the University of Houston, and Dr. McConnell for this amazing opportunity!

Our Lab's Updated Roster

Friday, July 27, 2012

Week 10 & 11-The Research Continues

This week has been the busiest by far for me. Since I'll be out of town next week, I have ensured that all of my duties have been performed in such a way to last an extra week. Western blots, cardiomyocyte isolations, buffer preparation, lipid lesion analysis, and surgeries on our specimen are still running rampant in the lab.

After watching my DNA Gel Electroporesis video, Dr. McConnell, my PI, suggested that I create a library of lab videos to show incoming students as well as students who are undergoing their rotations within the department the proper technique for basic lab functions.

Storing Western Blot Membranes-Lauren Tolat from Lauren Tolat on Vimeo.

How to: Standardize and Use the pH Meter - Lauren Tolat from Lauren Tolat on Vimeo.

The first video demonstrates the technique (and some tips) involved in storing Western Blot membranes. A common way to store the membranes is by transferring them to a polyethylene bag and keeping them saturated in TBST. The final product is stored in the refrigerator (2 to 8 degrees Celsius).

The second video is a technique used in general lab settings-standardizing and using the pH meter. When preparing a high volume of buffer solutions that need specific pH and temperature adjustments, the pH meter is a lifesaver. However, many lab students are unaware of the lengthy procedure involved in standardizing and cautiously using the meter for their own measurements.

Overall, I am beyond ecstatic about ALL of the opportunities that I have been able to experience during my undergraduate research and I feel incredibly blessed to be here at the University of Houston.

Friday, July 20, 2012

Week 9-DNA Gel Electrophoresis

This week has been super busy! Among cardiomyocyte isolations, western blots, protein assays, operations on our live specimen, and a potluck party thrown in honor of my graduate student mentor's successful research proposal, I had the opportunity to perform my very own DNA Gel Electrophoresis. Electrophoresis is a method used in this lab for genotyping among our six different experimental groups. Specifically, it is used to determine whether our gene of interest is available in a particular sample after PCR has been performed.  It involves four steps: 1) preparing the gel; 2) loading the gel; 3) running the gel; and, 4) visualizing. With the lovely help of my multi-talented research team, I created a video of me performing these steps.

An Assortment of Food-Potluck Party

The Team-Potluck Party

The Team-Potluck Party

Cori and Fan

Western Blots

An Alternative to Milk in Western Blotting-BSA

Lauren's Homemade Stripping Buffer :)

Casket used to Mold Gel

Running the Gel

Protein Electrophoresis-Loading Samples

Checking the Current

Light Cabinet-Visualizing 
Light Cabinet-Visualizing

Software used in Electrophoresis

Visualizing the Bands

Choosing a Filter to View the Results of Electrophoresis

Transferring Membranes to Polyethylene Bags

Transferring Membranes 
Storing Membranes

Preparing Hypotonic Buffer for Cell Culture

Friday, July 13, 2012

Week 8-Solutions, Buffers, and the Joy of Scientific Replication

Congratulations are in order for Ms. Qiying Fan, the hard-working and dedicated Ph.D. graduate student, who successfully completed her graduate research proposal this week. The entire research team is so proud of her and we couldn't have asked for a more positive response from the committee.

Congrats to Fan!
The entire research team and our PI have been pretty busy-writing grants and conducting/planning experiments. 
Tony Ohonba-Pharmacy Student 
My Graduate Student Mentor and I
Andrea has been planning experiments and the next steps for her project which is likely to span months. During the previous weeks, she had been refining her skills at inducing myocardial infarctions on her live specimen (mice). Now, with her technique finalized, she is officially ready to begin the actual experimental part of the project. She will be injecting certain stem cells into her specimen to test whether these cells improve cardiac function after the infarction. Her overreaching goal is to figure out if these stems cells aid in the recovery of localized damaged tissue in the heart.

Abeer has been continuing her experiments involving cardiomyocyte isolations. Once she obtains the cardiomyocytes (the cells that comprise cardiac muscle), she performs measurements to figure out the calcium ratio present using a specialized miscroscope called the Ionoptix. The calcium ratio will determine the degree of contractility in the cardiomyocytes.

Fan, while still recovering from a stressful week, has been continuing the development of Western blots to detect different treatments of Bapta-AM, a calcium inhibitor (used because calcium is involved in the specific pathway she is trying to identify). She has been treating VSMC (vascular smooth muscle cells) to see how specific growth factors affect VSMC intracellular signaling. Overall, this will further her understanding of how calcium, and how much with her different experimental groups, is involved in the pathway.

I had to opportunity to make my own Western Blot Stripping Buffer using substrates from the lab. A stripping buffer is used in Western blots before the blocking stage to remove primary and secondary antibodies from the PVDF membrane and allow chemiluminescent Western blots to be reprobed. This saves time and is more efficient than continuously performing gel electrophoresis and duplicate immunoblot assays. In other words, it allows the experimenter to reuse a PVDF blot to detect a different target with a different primary antibody.

Effect of Stripping Buffer on Membranes
Mild Stripping Buffer-Protocol

Glycine-15 g

SDS-1 g

Tween 20-10 mL

Adjusting the pH to an Acidic Level

Adjusting pH

The Finished Product

Aluminum Foil used to Protect Against Light Sensitivity 
Commercial Stripping Buffer

So, instead of spending $134 to buy a commercial buffer, why not recreate it on your own? :)

Other activities this week included preparing transfer buffer, running buffer, developing Western blots, transferring tissue to histology cassettes, and autoclaving. 

Friday, July 6, 2012

Week 7-The Research Proposal

I hope everyone had a wonderful Independence Day! 

This week has been action-packed, tension-filled, and stressful! My graduate student mentor, Qiying Fan, is ambitiously preparing for her research proposal and defense that she will soon present to a committee. This is extremely important because it partially defines her Ph.D. candidacy and she must prepare for a variety of questions to be thrown at her--including those from specialized experts hailing from Houston's medical district. Everyone has been trying to help out with her experiments as much as possible in order to allow her adequate preparation time. For practice, she will be presenting her proposal and defense to me. Her presentation includes a lot of information about the initiation and procession of atherosclerosis and the necessity to continue developing the relationship between a particular protein and the onset of atherosclerosis. It also includes a lot of information about the angiotensin II signaling pathway and A Kinase Anchoring proteins. Dr. McConnell told me to critique her analysis and her explanations about her presentation so that she will be better prepared when the real time comes!

One thing that Fan's research presentation showed me was the importance of having a strong connection between the background and the preliminary data. The background indicates the significance of the problem to be examined in the research project while the preliminary data targets  a specific aspect of the problem. In this specific situation, Fan needed to make a stronger connection between her notes about atherosclerosis and the protein that she is investigating in the signal transduction pathway.

Additionally, two graduate students from TSU have been touring the facility lately. Dr. McConnell said that they are prospective lab members and are in the process of learning specific lab protocols/techniques. I was asked to introduce these students to what I've learned over the course of my stay here in the lab, including: autoclaving, filtration, and Western blots. How exciting is that? Just a few weeks ago I was in the process of being trained, and now, I get the opportunity to instruct. It's such a rewarding feeling.

This week I participated in developing Western blots, transferring heart tissue into histology cassettes, and preparing buffers for cardiomyocyte isolation. One buffer I had a fun time making was the Fura 2-AM loading buffer (cardiomyocyte perfusion buffer). It was difficult making this buffer because the substrates do not fully dissolve in the solution unless the pH is adjusted. The contents make the solution basic so hydrochloric acid needs to be added in order to reach proper homogeneity. Fura 2-AM is an intracellular calcium indicator that is ratiometric and UV light excitable. At low concentrations of the indicator, accurate measurements of the intracellular Ca2+ concentration are able to be measured.  
Cardiomyocyte Buffer-Preparation Sheet

Cardiomyocyte Buffer-Preparation

Sonal Performing Cardiomyocyte Isolation



Cori and I
Solution Making

Adjusting the pH

Cori :)

The Finished and Filtered Buffer!

Fan Preparing Her Research Proposal

Fan-Practicing Her Presentation

Friday, June 29, 2012

Week 6-Seeking the Bigger Picture & Meeting the Team

After hearing about a variety of research projects that are underway and have been completed on this campus, I am now even more excited about continuing my ongoing project on heart disease. With the legitimate concerns about pandemics of AIDS and behavioral disorders, it's easy to forget that cardiovascular disease kills more people each year worldwide than any other disease. We've grown so accustomed to hearing about heart disease that we even consider it a natural cause of death and tend to disregard a comprehensive solution to an obvious growing issue. I can even attest to the effects of heart disease, as it has personally plagued loved ones and family friends. Some of the most recent well-publicized deaths are even linked to such: Whitney Houston suffered from atherosclerosis, a disease of the arteries as a result of fatty materials such as cholesterol. The best part about joining this lab and its ongoing project is the fact that I feel like I am tackling two developments at the same time--discovering the role of a particular protein in heart disease and finding a solution to modulate this protein and, perhaps, a solution to heart disease in general through pharmacodynamics. I am ecstatic that this isn't just a project that stops at the discovery of a particular thing--it seeks to improve the human condition through application.

Even more exciting is the fact that one of my research team members, Ashley, is on the brink of submitting a huge paper that includes data/data analysis dating back (whew..a mouthful) to 2010! A lot of what she is including in her paper predates the contributions I have made since joining the lab; however, the implications of a successful publication are enormous for the rest of the lab work to follow.

Speaking of my research team, allow me to introduce you to some of them! They have been a part of Dr. McConnell's lab for as long as he has been a PI, and I can assure you they are a TREMENDOUS help (and motivation) to my research experience:

Name: Cori Wijaya
Education: B.S. in Biochemistry-University of Houston
What sparked her interest in researchShe started doing research work during one summer in her undergraduate senior year. She found that doing hands-on work in the lab helped her with her studying: what she read in her textbooks actually started to make sense! As she graduated and started working in the lab, she found it exciting to do experiments that helped to elucidate the questions and hypothesis of the research work.
The best part about her experience in Dr. McConnell's lab: Getting to know people who share her knowledge and skills
Advice she gave me about continuing to pursue research: Keep a curious and open mind. Research is a long process with so many little things to take care of-but be glad that you are a part of it. Spend a long enough time in one lab to learn something, but also give yourself a chance to try a different lab and see how other labs work differently.

Name: Abeer Rababa'h
EducationMaster degree in Clinical Pharmacology/ Jordan University of Science and Technology
What sparked her interest in researchShe has an interest in correlating and interpreting data into biological facts that help downstream in achieving new clinical aspects.
The best part about her experience in Dr. McConnell's labResearch with Dr. McConnell gives her the opportunity to interpret many findings in a logical way, find explanations for every single piece of data, convert bored data into exciting findings, look for something from a different point of view in a comfortable friendly research environment.
Advice she gave me about continuing to pursue research: My advice is to get as much experience as you can from the lab. Every small contribution may participate in a big achievement in your career goal. Challenge yourself and be patient. Being hard working is a requirement for the research road.

Name: Andrea Diaz Diaz
EducationB.S. in Biology and Biochemistry-University of Houston
What sparked her interest in research: She took some classes in college with a wonderful teacher that motivated her to do research.
The best part about her experience in Dr. McConnell's lab: She gets to do surgeries (on mice, that is!)
Advice she gave me about continuing to pursue research: It is a very interesting career where you can learn many things about any topic you are interested in!

X-Ray Film Processor

Developing the Film-Western Blot

Labeling the Film

Discovery Workshop-Using EndNote for your References

Program: EndNote

Heart Tissue Stored in Ethanol

Collected Heart Tissue

Moving Tissue to Histology Cassettes

Moving Tissue to Histology Cassettes