3rd Post 2011!! Photos! April 1, 2011

In this post I will present photos that have been taken during my BioMind’s experience.

Previously I showed photos of me working on the laboratory assembling an electrophoresis. I also showed a photo of the laboratory staff I work with. I have learned so much from every one of them! There is also a photo of the laboratory model species, the sea cucumber Holothuria glaberrima and a photo of where they can be found, La Perla.

Biominds has helped me establish my professional goals by increasing my interest for science and making me see how much is known and how much more is yet to be discovered. It has inspired me to be part of those discoveries. It has also helped me see how important and productive is to work with a team and be organized. Biominds has definitely helped find my truth as what my future goals are. I wish to continue my graduate studies in Medicine and concentrate more on clinical research and volunteer work.

March Post 2011! March 1, 2011

The process of my project has been going slow.  We are having difficulties with the PCR amplification of the rdh gene EST and are in the process of modifying concentrations, temperatures and quantities to obtain the best results possible.  This is a very important step of the experiment because with this results we send to sequence our product to find out how many base pairs does the EST have, how far away we are from having the complete gene, and if it has homology with similar genes of different species. Relating my present achievements with my semester goals, I’m half way.  We have learnt the importance of this gene during development processes and have read many research papers to obtain important information related to the rdh gene.  We have also performed the bioinformatics related to the rdh gene.  Involving the experiments we have extracted RNA and made the reaction to convert it to cDNA.  In the present we are working on the amplification of the cDNA to see if the primers are working.  The obstacles I have had to deal with are the problems we have encounter with our gene and the time management.

Abstract PRISM! February 3, 2011

Characterization and the temporal and spatial pattern of the rdh gene during the intestinal regeneration process of Holothuria glaberrima

University of Puerto Rico, Río Piedras Campus, Faculty of Natural Sciences

Gabriela Rivera-Cardona, Melanie M. Torres-González, José E. García-Arrarás

Regenerative medicine is interested in the characterization of genes involved in regenerative processes, and their temporal and spatial pattern of expression. Our model system, used to characterize regeneration-associated genes is the sea cucumber, Holothuria glaberrima. This organism is an echinoderm, the group of animals within the Deuterostomata with the best capacity to regenerate organs after their loss. Previous experiments using microarrays of H. glaberrima, showed an EST with homology to rdh, which encodes a protein called retinol dehydrogenase, which appeared to be over expressed during regeneration. This protein catalyzes a step of the reaction of retinol to retinoic acid. Retinoic acid is a signaling molecule involved in important developmental processes. This enzyme has been related to proper gut development and differentiation during development. We studied the expression and the spatial distribution of retinol dehydrogenase during intestinal regeneration of the Holothuria glaberrima. The work presented aims to validate the expression of rdh using RT-PCR and characterize its sequence with RACE (Rapid Amplification of cDNA Ends). This was approached by cloning the gene into a vector where it will be expressed. The EST of rdh was analyzed using bioinformatics by (1) comparisons with sequences in the databases and (2) determining if the expected open reading frame was complete. The sequence provides important information on the possible homologies and regeneration functions of rdh. These results provide strong validation for the results of the microarray and serve to further characterize rdh as an important enzyme involved in regenerative processes. This work was partially sponsored by BioMINDS and NIH (1SC1GM084770-01).

November Post!!! Happy Thanksgiving!!!! November 22, 2010

During this semester I started a completely new project with Gabriela Rivera, a fellow Biomind’s student and friend. Gabriela and I have a common interest in regenerative medicine. Regenerative Medicine is the process of creating living, functional tissues to repair or replace tissue or organ function lost due to damage, or congenital defects. This field holds the promise of regenerating damaged tissues and organs in the body by stimulating previously irreparable organs to heal themselves. Regenerative medicine also empowers scientists to grow tissues and organs in the laboratory and safely implant them when the body cannot heal itself. Importantly, regenerative medicine has the potential to solve the problem of the shortage of organs available for donation compared to the number of patients that require life-saving organ transplantation, as well as solve the problem of organ transplant rejection, since the organ’s cells will match that of the patient.

Regenerative medicine is increasingly interested in the characterization of genes involved in regenerative processes, and their temporal and spatial pattern of expression. Our model system, used to characterize regeneration-associated genes is the sea cucumber, Holothuria glaberrima. This organism is an echinoderm, the group of animals within the Deuterostomata with the best capacity to regenerate organs after their loss. Previous experiments in our laboratory using microarrays of H. glaberrima, showed an EST with homology to rdh that appeared to be over expressed during regeneration. The rdh gene encodes a protein called retinol dehydrogenase. This protein catalyzes a step of the reaction of retinol to retinoic acid. Retinoic acid (RA), the active form of Vitamin A (retinol), is a signaling molecule involved in important developmental processes, such as regulation of synthesis of essential proteins for growth and development. The reaction for the conversion of retinol to retinoic acid is a two step reaction. The first reaction involves the conversion of retinol to retinal by the enzyme retinol dehydrogenase (rdh). This enzyme has been related to proper gut development and differentiation during early stages of development. Gabriela and I will study the expression and the spatial distribution of retinol dehydrogenase during intestinal regeneration of the sea cucumber, Holothuria glaberrima.

In this semester, we have concentrated on researching background information of the retinol dehydrogenase gene and the processes it is involved in. We have also use PCR (polymerase chain reaction) for the gene amplification and we are in the process of validating to gene to continue our research. As our future work, we have planned to study the localization and pattern of expression of the gene during intestinal regeneration and determine where in the intestine is the gene being expressed. This will give us important guidelines as to how important is this gene for intestinal regeneration and how the over or under expression of this gene could affect proper intestinal regeneration.

October Post!!! October 21, 2010

The first blog I visited was Maritza Pérez blog. She is a Chemical Engineering student form the University of Puerto Rico, Mayagüez Campus. She is researching Supercritical Fluid Processing and Impregnation of Pharmaceutical Drugs in Bio-Polymer Films.

A previous student made the drug Taxol and Taxorete, which are effective anticancer drugs against breast cancer, lung cancer and prostate cancer. Now, Maritza is analyzing the encapsulation of the drugs made. Her encapsulating agent is Compritol 888, which is a white tasteless non-reactive powder that serves as lubricant and pellets. She elaborated a mix of 50/50 weight of compritol and fluoracil and a mix of 50/50 of compritol with acetaminophen. The encapsulation did not work so she used the supercritical fluid CO2 to encapsulate the anticancer drug. Then she worked in solubility tests to find a solvent that did not dissolve the drug or encapsulation and that is compatible with the human body. She also studied a complex multi-component-system composed of: SCF CO2, co-solvent, anticancer drug and encapsulating lipid biopolymer. She found that ethanol was the best solvent.

Maritza’s research is very interesting and dynamic. I like that she is involved in the making of an anti-cancer drug, specifically in the encapsulation. Her research seems very dynamic because she uses different laboratory techniques like: Diffential Scanning Calorimetry, Nuclear Magnetic Resonance and X-ray diffraction which are techniques science student read about on textbooks constantly and have been key to great discoveries.

The secong blog I visited was Roy Suárez’s Blog. His research area is nanotechnology and bio-engineering. He chose this area because it involves helping the society.

He investigates how to create and install a nanometer scale implant in the brain neurons with multiwalled carbon nanotubes or nanowires used as a bypass between neurons to transmit messages over damaged areas produced by strokes. He is focusing on the synthesis of silver nanowires, their function and characterization. The nanotubes are a better alternative to restore the communication between dysfunctional neurons, than implants, because implants cause injuries to cerebral tissue. His group achieved the function of the nanotube but had problems filtering the carbon nanotube and finding the correct phospholipid for the function of nanowires to ensure attachment between nanowires and neurons. He also had problems separating the nanotbes form the precursor solution but the group overcame it using a filter with extremely small holes. His future project is to measure a voltage and current between cooper wires using a nanostructure solution with brain tissue as a bridge between the wires. Roy learned different techniques including the Scanning Electron Microscope.

I like Roy’s investigation because it involves the use of nanotechnology to transmit messages between neurons in areas of damaged brain tissue. This is a great step in helping people with neurological diseases have an opportunity to live a better life. It also helps other research groups have a broader outlook on alternative technology concerning the repair of damaged tissue function.

September 2010 Blog!!! September 23, 2010

Hi!!!

This semester I will have the pleasure of working with a fellow Biominds’s student and friend, Gabriela Rivera, in Dr. Garcia Arraras neurobiology laboratory. The lab uses the sea cucumber Holothuria glaberrima, as a model system to study different aspects of organogenesis and regeneration as well as comparative neuronal anatomy and evolution. Specifically, my research focuses on the cellular molecular aspects of the intestinal regeneration process and the important genes involved. Gabriela and I will start a completely new project which we are very excited about. It involves the characterization and the study of the gene expression of retinol dehydrogenase during different periods of intestinal regeneration of the sea cucumber. Retinol dehydrogenase is an enzyme that catalyzes the first step of the synthesis of retinoic acid from retinol. Retinoic acid is a potent signaling molecule tht plays important roles in multiple and diverse developmental processes, including intestinal differentiation.

My goal for this semester are:

1. Become an expert in the relationship between retinol dehydrogenase and retinoic acid.

2. Extend the gene EST by designing primers and sequencing

3. Start the experiments to see the expression of retinol dehydrogenase during intestinal regeneration

The project of this semester relates to the last semester’s project in that I will be working with a gene supposedly involved in intestinal regeneration but the retinol dehydrogenase gene is completely different from the gene I was working with last semester. I will be using basically the same techniques as before because I will be applying what I have learned. I will use PCR, electrophoresis, creating primers, sequencing, cultivation of bacteria and use software tools like the “NCBI” website, “4 Peaks” chromatograph program and the “Clustal W” alignment program for the analysis of DNA example.

Third Entry!! April 15, 2010

First, I visited Mariela Fuentes’s blog, from the Medical Sciences Campus. She is involved in the study of the relationship between glutathione reductase and malarial drugs resistance. This study has great medical significance because the parasites agents of malaria have developed resistance to most common drugs for malaria treatment and therefore chemotherapeutic drugs. Her laboratory research has a great future outcome because the study of glutathione reductase amplifies the knowledge of malarial drug resistant and could even lead to the development of a new drug to treat malaria.

I also visited Melvin Caraballo’s blog, form the Mayaguez Campus. He is characterizing microbes form soil samples morphologically, physiologically, biochemically and molecularly. I think this is very important because actinomycetes while many are harmless to animals and higher plants, some are pathogenic for humans and animals. He has also learned many techniques, like PCR and SEM microscopy.

Finally, I visited Jose Grajales’s blog, a fellow student from my campus, Río Piedras. In the Poster Day at our campus, I had the opportunity to hear his presentation and visit his poster and he has definitely accomplished a lot in his project. His project is so interesting; it deals with the Slow Channel Congenital Myasthetic Syndrome, which is caused by mutations in the acetylcholine receptor. His laboratory focuses on drugs that can change the kinetics of the protein and he has studied the effects of fluoxetine (Prozac) on the channel. They have hypothesized that fluoxetine does work as an open channel blocker, so it can be considered as a possible treatment for the syndrome.

I really enjoyed reading the blogs because I have a broader idea of the research done in the UPR system. Also I sense the dedication and passion, we as students have for science. During this semester I have broaden my knowledge in different laboratory techniques like PCR and the bioinformatic aspect of my project. I have noticed improvement in my oral presentation and writing skills. Even more I have learnt to work as a team, to set priorities and manage time effectively. My project has been moving slow because we have encountered problems with the clone of the gene I want to study, Epithelial cadherin (Cdh-1). I’m overcoming this barrier by practically redesigning my plan of work and trying different options, like using different primers to amplify the EST by PCR. My goals for this semester and probably the next involving my project s to hopefully to complete its gene sequence and advance its studies to determine its function during regeneration.

Second entry 2010!!! March 4, 2010

A laboratory technique I have learned during my undergraduate research is PCR (Polymerase Chain Reaction). It is a process of DNA amplification (replication). Polymerase is an enzyme responsible for DNA synthesis, while “Chain Reaction” means exponential growth (at the rate of 2ⁿ where n denotes the number of generation) – hence the PCR name. The process is essential for sequencing a unique piece of DNA. The process is as follows:

1. A target sequence is chosen on the DNA. The sequence had to be known, or at least its two termini.

2. Since DNA replication always runs from the 5′ end to the 3′ end, two DNA primers were synthesized for each strand. The primer is small and complementary to the 3′ end of the original DNA about 20 bp long, it acts as an anchorpoint for DNA polymerase and as initiator of the copying process.

3. Modern technique uses the Taq polymerase (from a microbe in hot springs) to add nucleotide to the new strand. It has the advantage of tolerance to heating up to 94^oC – well beyond the 72^oC for the extension phase.

4. All four DNA nucleotide building blocks are added in sufficient quantity.

5. The sample is heated to a temperature of up to 98^oC to separate the complementary strands. This step is called denaturation.

6. Then the sample is cooled. During the cooling stage, the synthetic primers found complementary sites on the separated DNA strands, whereas the two long DNA strands were unable to find each other because they were present in minute concentration. This process is called primer annealing.

7. The polymerase extended the two primers in opposite directions. As a result, two daughter DNA appeared.

8. Initially, the new DNA carries long single-stranded tail. Only at the 3rd cycle of denaturation-annealing-extension do first authentic copy of the molecules appear. The process can make billions of copies by the 30th cycle.

I use the PCR technique to clone or amplify cDNA from our Holothuria glaberrima cDNA library. Also I use the PCR technique combined with other techniques, like gel electrophoresis to determine gene expression during different stages of the sea cucumber’s intestine regeneration.

In a 1-5 scale, I would rate my progress as a 3, because I have reach approximately 50% of my objectives. I’m already familiarized with the gene I’m trying to sequence and by attending weekly lab meetings and observing my fellow lab partners present research papers and their results, I learned the do’s and don’ts of oral presentations. Nevertheless, I’m having difficulties through the sequencing process of the gene. I have tried different procedures and techniques and still no expression. I consulted my mentor and he suggested a new technique, which I will try next week to see what happens. This is the reason why a rated my progress as a 3, because I have not reached the progress necessary to try new lab techniques, like RACE.

2010 First entry!!! February 6, 2010

This semester I will begin a new project involving the sequencing of the cadherin gene expressed in a normal intestine of the Holothuria glaberrima. In the past project I was confronted with some technical problems, so we decided to leave it in hold for a period of time and concentrate on this new project for now.

Since this is the case, my first objective is to get familiarized with the gene and research information about it. The second objective is to learn new techniques in the cellular molecular part of the laboratory like the RACE technique. The third objective is to fortify my oral presentation skills to present my results to an audience. These objectives do not give direct continuity to the previous project but give continuity to my main goal, which is to get hands on with the techniques in the cellular molecular area.

This semester I will strengthen my skills involving the RT-PCR technique. This technique involves other techniques and procedures, such as electrophoresis, cultivation of bacteria, isolation of small plasmid DNA from bacteria (mini-prep), purification of DNA fragments from PCR reaction, digitalizing electrophoresis results and measuring DNA concentration with the “Nano Drop”. I will also continue to use software tools like the “NCBI” website, the “4 Peaks” chromatograph program and the “ClustalW” alignment program for the analysis of DNA samples. Furthermore, I will strengthen my oral presentation skills and discussion of scientific papers.

Third Entry!!! November 15, 2009

Visiting the BioMinds blogs of fellow researchers was a great experience and opportunity to catch up on the topics of interest of research in the University of Puerto Rico’s system. The three bio-blogs I visited were really interesting and cover diverse disciplines in the biology field. Tatiana Rodriguez from UPR Humacao has been collaborating in the expansion of research directed in the area of collection and identification of mite genera in Puerto Rico, since there is not much information available. I like that the long-term goal of the research has a biomedical application involving the contribution to cure people affected by diseases caused by pathogens. In terms of the skills required to work in a research laboratory, I liked that she mentioned patience and perseverance because I think those two factors are key for a competitive researcher. Jean Cruz from UPR Mayaguez is involved in the Metagenomics field, which is a new discipline where genomic analysis is applied to entire communities of microbes, bypassing the need to isolate and culture individual microbial species. Less than 1% of microbial species can be cultured; Metagenomics will give scientists the access to millions of microbes that have not been studied yet. Jean’s project has important applications, including, biomedical, bioenergy and bioremediation. Diana Rosario from UPR Cayey is a biology student and she is working with a Physics professor. I think this is remarkable because the integration of different fields of science gives a broader outlook to the project been studied. Also, this project has a very interesting biomedical application because the goal is to create an implant coating more compatible with human body. I enjoyed reading the bio-blogs and hope to hear more from the advances made on each project.

During my research this semester I have learned a lot. I have learned and improved different techniques and procedures, especially the RT-PCR technique. This technique involves other techniques and procedures, such as electrophoresis, cultivation of bacteria, isolation of small plasmid DNA from bacteria (mini-prep), purification of DNA fragments from PCR reaction, digitalizing electrophoresis results and measuring DNA concentration with the “Nano Drop”. I have also learned to use software tools like the “NCBI” website, the “4 Peaks” chromatograph program and the “ClustalW” alignment program for the analysis of DNA samples.  Also, I’m in the process of learning immunohistochemistry. Also, I have learned to revise literature to broad my knowledge in my area of research and keep up to date. I have also presented a scientific article in the weekly lab meeting and have presented my results to my lab team. Besides, I have learned the importance of responsibility, time management and teamwork, to continue a research.

During the course of my research I have encounter that sometimes the procedures do not work. Seeking ways to rearrange the procedure so it works overcomes this problem. Sometimes consulting with someone who has more experience doing the procedure and asking for suggestions really helps to solve the problem. Sometimes the procedure has to be repeated in a number of ways until it finally works. This causes the procedure to take more time than you initially expected delaying the project. Like I mentioned before doing research takes time and sometimes it is challenging to manage college work and the research. That is why time management, scheduling everything and organization is very important and essential for research.

My goals for next semester are tož continue immunohistochemistry to study the localization of antigens for the alpha subunits of the proteasome during regeneration. I will carry on doing my college work while continuing my preparation for the MCAT to take the test before summer because I wish to join a summer research program at the United States.