Anxiety is characterized by cognitive, somatic, emotional, and behavioral components, which combine to evoke fear, apprehension, or worry. Neural circuitry involving the amygdala and hippocampus is thought to underlie anxiety. Pharmacological agents to treat anxiety disorders include Benzodiazepines and Non- Benzodiazepines drugs. The first group of drugs is extremely addictive and the later requires long periods of time to function. The latest pharmacological trend to treat anxiety disorders is the use of neurologically active peptides. For anxiety, Neuropeptide-Y has risen as a potential target. NPY is a 36 amino acid peptide neurotransmitter found in the brain and Autonomic Nervous System (ANS), has been associated with the regulation of energy balance, memory and learning, and epilepsy. Using C. elegans as a model organism we hypothesize that NPY will activate a signal pathway which ultimately leads to a loss of anxiety. The main goal of the project is to test the use of NPY as a future pharmacologically available anxiolytic drug. We have deduced a signal transduction pathway which includes Phospholipase C activation and ultimately CREB-mediated gene expression. We have concluded that E.coli strain OP50 successfully grew on Nematode Growth Agar (NGA). C.elegans successfully grew with the OP50 strain in NGA. In addition, Solid-Liquid Nematode growth promoted C.elegans growth and the multiplication of fungal contaminants. A solution of 10% Sodium Hypochlorite did not remove the fungal contaminants. After the usage of 1/1000 Antifungal, large liquid cultures were maintained and whole organismal proteins were isolated and quantified via Bradford Assay. These samples were later run on an SDS PAGE for quality analysis.

Final post after two years of participation within the Amgen Foundation BIOMINDS Program, first cycle. First off, I would like to thank everyone not only @ AMGEN but also everyone @ UPRM Dr. Buxeda, Deyka Lopez, and Lizzy Muniz. It has been a long two years but very fruitful and enjoyable. 

Last month we had our annual Poster Presentation Day. All in all, it was a very nice event, we got to me all the other members, observe their work and create close relationships. Seminars offered presented new outlooks on aspects and were interesting. In addition to the activity we were each assigned three posters which we evaluated and observed. I was assigned F-7 Juliani Rivera Calo, H-1 Diego Hernandez, and K-1 A. Lopez Cruz.

Mr. Lopez offered a very interesting poster on the applications of organic chemistry to nanotechnology. This poster I found particular, since I am a biologist and this was the only poster which I had to evaluate and not only was it organic chem, but also with nanotech. Although not my area, I found it so nice to find applications for organic chemistry. His results were very positive and he explained very well, so he knew his material very well. 

Later I evaluated, Mr. Hernandez’s poster in molecular microbiology. Gave me a flashback of days @ the Parguera with the bioluminescent bay, because his project was amplifying the LuxH 16S region from entire bacterial libraries. He had done a great deal of work and had already finished 50% of all his samples with primer design and all. It was a project with alot of biotech potential, it would provide another source of fluorescent marking with new parameters and uses. 

Finally, I evaluated Ms. Juliani Rivera, a project in general microbiology. She was on the search for new species and quantifyable data on extremophiles. In this case, salinity, from southern mangroves. She tested to quantify the bacteria a different distances from the shore and at different depths. She had very conclusive results on both aspects and now has begun to work on the identificacion of these. 

All in all, it was a great day and alot was learned.

This semester as my final semester within the AMGEN BIOMINDS program I will be concluding my participation in my work with Dr. Ricardo Chiesa. This semester we will have three other students working with us, two freshmen from the MBRS-RISE program and another student from the BIOL 4990 intro to research course.

At the end of the last semester, we froze the C. elegans in -80 with 30% glycerol. Our first goal was to “revive” these nematodes and make sure that are functional. We have already began this processes, we defrosted, centrifuged, and resuspended. Then we transfered to plates previously seeded with OP50. These have been observed and all seems to be working fine. 

Our next goal was to identify strains lacking possible target genes. I was able to do this last week and the strains have been identified and purchased, arriving sometime this week. Our goal for the semester is to grow these nematodes in larger quantities and isolate their proteins for western blot analysis of the possible effects of NPY in the CNS. These results will be validated comparing between the wild strain and the KO’s since they lack proteins we believe are needed for the pathway. 

This semester is different from the last because, we now know how to culture the nematodes and this facilitates the protein extraction processes. We will use new techniques such as protein isolation, quantification and western blotting for identification.

Well….this is it, we are now 2/3 of the way there!

Last semester was the first time we worked on the project, which was also new for the Chiesa lab. Our final results last semester concluded that we needed Fungicides in Nematode Growth Media, liquid and solid to properly replicate them and create a stable line. This semester we grew them in Amphotericin B 1/1000 with respect to the liquid volume. We observed no adverse effect of the fungicide on the nematodes and there were no traces of live fungi. Some traces of fungi which came when transferring, showed dead fibers and no progression. Once we established a stable line, they replicated like crazy! Leaving about 10 on friday, gave 1000 on monday! This was incredible. We continued to subculture these to reach the goal number of approximately 8000, 4000 for liquid culture containing our active neuropeptide, Neuropeptide-Y and 4000 for controlled liquid growth. We administered NPY to the experimental group at a final concentration of 1 uM and left it for 24 hours. We then collected the worms from the liquid media and isolated whole-organism proteins by sonication protein collection. These are now stored in -80 for future western blotting and protein characterization to see if NPY can activate anxiolytic molecular pathways in the model organism. We have also identified other proteins of interest which may be involved in the propagation of these pathways through our lab meetings and presenting papers. For next semester, we would like to western blot for NPYR2, CREB and ERK proteins to see if NPY can induce expression of this proposed pathway. We would also like to prove that any results are directly from NPY by adding naloxone, flumazenil, and CTAP to the media since they are pharmacological activators or blockers of the NPY receptor. 

Although I did not go to ABRCMS 2008 through Biominds, I did go through the MBRS-RISE program and must say it was an enjoyable experience. I presented my research in electrophysiology from Harvard Medical School in the Biophysics area and attended a number of incredible seminars. There were also a great number of exhibitor sessions where we were able to obtain information from schools.

As for my interviews and everything. I interviewed for the MD/PhD program at New Jersey Medical School on Oct. 17 and they told me they would respond in 4-6 weeks, so that means soon. I have an interview for the MD program at University of Puerto Rico Medical Sciences Campus on the 15th of December. And my next US interview is for the MD/PhD MSTP Program at Tufts School of Medicine in Boston. I am dying to know what will be of my future….and where I’ll be in a few months….so nervous….lol.

25/10/2008

Progress Report: 2/5 with respect to proposed goals in August.

Note: Due to a personal and professional offense previously posted this post has been edited 9/12/2008

One major goal that I did accomplish was not in the lab but actually cleaning up our model alot. My project has mostly been geared by myself and my model for Neuropeptide-Y function was way to broad including many Kinase Families such as ERK, CamKII and PKA. Yet I found an awesome article where they were analyzing pathways in a very similar receptor and they used a number of techniques to reduce these Kinases to only one family, ERK. This really helps me, because since I am characterizing the receptor by intracellular signaling, I now have more detailed target proteins to Western Blot for.

As for the wet lab work. I have made plates for both bacterial and nematode growth, but I ordered both a while ago and still haven’t received them. In addition, last semester we concluded that we needed antifungals to reduce contaminations.

can’t believe I forgot the 25th of every month rule! With sooo many tests in a row, anyone can go crazy. Late, but sure…

The main focus of this post is a 0-5 rating on project progress. 

Our progress has been like a 2 or 3. Although we haven’t gotten much done in the lab mostly because I sent my requisition to Ms. Muniz and haven’t gotten a response yet, we did get some background done. We had developed our own theory on how the NPY receptor works in Anxiety models yet, it was too broad of a model using PKC, CamKII and ERK as possible modifiers. Based on a new paper published this month (using a receptor similar) we found that it may be just ERK. That really helped our model along. 

This month we found out that 3 more students will be joining the lab next semester so that is good, you can never have enough helping hands!

Well…. as they say all good things must come to an end. But wow is it being of a year! Starting the year off with a second publication. This time it was based off of my direct lab work. I feel so proud of this paper. I added it to the available links. 

Classes are coming along great. I am taking Anatomy and Physiology I + Lab, Zoology + Lab, Clinical Psychology, Occidental Cultures III, and History of Puerto Rico. I continue work as a tutor/mentor for the Biology students at the Center for Student Aid. And of course I am still working with Dr. Chiesa on our project on molecular mechanisms of Neuropeptide Y in anxiety. This year we have a fellow student, Maralisi, working with us through the AMP program. 

In addition, this past saturday the RISE program gave awards to the best AMGEN posters and the Best Summer Research posters. I won second place in the AMGEN posters, which entailed a $30 prize. yay. And for the summer research I won First Place in the Biology Second or More Experienced Students. That prize included an all expenses paid trip to the ABRCMS meeting being held in Orlando, Fl this year! So exited.

     During the 2008 Summer, I had the opportunity to participate in the Harvard Medical School SHURP Program. I worked in the Boston Children’s Hospital in Dr. David Clapham’s electrophysiology lab. I worked characterizing the non-selective cation Transient Receptor Channel 7. I learned alot about electrophysiology, including Calcium Imaging and Whole Cell Patch clamp. Although these techniques require instrumentation that is every expensive which means I cannot do this in PR, I did learned a lot about organization and work ethic that I will be able to apply not just in my Biominds Lab but also in any lab in life. 

    For my second semester in the Biominds program I wish to accomplish a number of things. Last semester I worked on the culturing of the model organism for my studies, C. elegans. For this semester, I have a fellow undergraduate student working with me, and she has prior experience working with nematodes which I do not have since most of my work has been molecular and biochemical. After culturing these, I wish to extract and quantify the whole cell proteins of the nematode. Once I use the bradford assay to measure the concentrations, I need to identify the target proteins through Western Blotting proteins from our proposed anxiety model. 

     My work for this semester is a direct continuation from last semester work because, I had never worked with C. elegans before and last semester I became familiarize with this organism. And I learned that I am not very good at culturing this organism and this is the reason Dr. Chiesa has contracted this new student which has worked with nematodes before, so I can learn from her. This semester we will culture the worms as I did last semester and isolate thier proteins for immunoassaying. 

     This semester I hope to fully develop techniques related to the culturing since I have already isolated proteins and western blotted before. Through this program I have created a close relationship with my mentor who was my professor for two of my upper level concentration classes and I hope to continue to build this relationship and become a greater person through this last year at the UPR-Cayey and the Biominds program.

He aprendido mucho a travez de esta investigacion. Paciencia es una virtud!!! lol! Nematodo no es un modelo tan facil para utilizar. En los libros todo se ve tan lindo pero en la vida real no es asi. Eso es una leccion muy importante. Aprendi sobre el ciclo de vida y requisitos para mantener este organismo. Aprendi tambien como aislar proteinas del C. elegans, aunque lo habia hecho antes con otros modelos, es muy diferente en nematodo. He superado mis dificultades en varias reuniones con mi mentor, Dr. Chiesa. Hemos leido varios articulos sobre nuestra investigacion y diferentes protocolos para llegar a nuestra meta. En este semestre hemos llegado a nuestros short term goals que eran creecer el organismo y aislar sus proteinas. Para el semestre que viene, queremos identificar estas proteinas asociadas a la ansiedad por medio del Western Blot. Ha sido todo una experiencia.