Workshops:

Fish DNA Barcoding, Protein Profiling Workshop:

The purpose of this laboratory is to learn the tecniques that could be a solution to the mislabeling of sea food. The mislabeling of seafood is a problem and it may expose consumers to serious health risks. As an example, some of these organisms can contain neurotoxins, which are neurologically destructive. Because only one third of the marine species have been identified, it is very essential to discover new ones. Therefore, it is important to identify species by their physical characteristics, but it is also more accurate to use the DNA Barcoding. The main steps of DNA Barcoding are to isolate, purify, amplify, and sequence DNA. Isolation and purification are part of DNA extraction. In this first step we added resuspension, lysis, neutralization, matrix, and wash. The use of Lysis is to loosen the cells and therefore make it easier to extract the DNA. After the extraction of DNA from fish tissue, we added different buffers in order to release the DNA from the cells. The DNA was then bound to the matrix suspension in a spin column. The impurities were washed away. This DNA was then used for PCR amplification. Next, we made copies of the COI target sequence and the PCR product was placed in the gel electrophoresis. The last step was sequencing. Here the PCR products were sent to a sequencing facility to be purified and sequenced. The results are still pending. These studies are very important because proteins are in charge of a large range of functions in the body. Proteomics is the study of the importance and diversity of proteins. It also studies proteins function, protein-protein interactions, cellular locations, expression levels, and posttranslational modifications of all proteins. During our workshop proteins were extracted and separated by polyacrylamide gel electrophoresis. We also talked about Nemerteans, and Dr. Jon. Norenburg explained how these worms help in regeneration. The study of regeneration is very important, because many humans suffer from injuries in the nervous system.

Microscopy and Photomicrography:

During Dr. Robert Ross’s workshop, we worked with different microscopies and micro techniques. Some examples of the microscopies are Bright-field, Phase Contrast, and Dark-Field. Dr. Ross explained the function and use of Scanning Electron Microscopy and the Transmission Electron Microscopy. He also explained micro-techniques such as whole mount, cross section,longitudinal-section, and squash. As part of this workshop, we used the Dissecting Scope, Phase Contrast Microscope and the Bright Field Microscope. Dr. Ross taught us a little about the history of the different microscopes. Later, each one of us instructed our partner in the usage of a particular microscope. In my case, I learned how to use the Phase Contrast Microscope and then I taught Felix how to use it. By using the Phase Contrast, you can discover structures that are not visible with a Bright-field microscope. Fernando taught me how to use the Dissecting Scope. I looked for the fern Polypodium, and then I used a blade to cut a small piece of the spores and a piece of the entire leaf with all the sporangia in order to observe it under the microscope. Next, I compared my pictures with Internet images. As part of the results, I did not find in Internet a Phase Contrast image of Polypodium. Instead, I looked for an analogous picture with a similar magnification. The usage of microscopes is very important, because with it we can observe and investigate many things such as microorganisms and parts of cells. Microscopes also help in the

discovery of diseases and in how they can be treated, because diseases are too small to be seen by the human eye. Microscopes provide us the ability to see at a cellular and a molecular level.

Nanotechnology and Electron Microscopy:

The RISE students took a workshop about Nanotechnology. As Dr. Wilfredo Otaño explained nano means dwarf, and the importance of using nanotechnology is that you can use or see things at a very small scale. Nanotechnology helps in the manipulation of the physical and technological characteristics of matter in an atomic, molecular, and super molecular scale. The objects need to have a scale of 100 nm or less. During the process, the first step was to prepare a solution of Polyethylene Oxide (PEO). This was left for 24 hours in magnetic stirring. Then we took the solution and used the Electro spinning to create the fibers. Electro spinning is an efficient technique that is used to make fibers. Therefore, this process helps in the formation of fibers at a nanometric scale. An electrical field is utilized to make a charged jet of polymer solution. As this jet travels in air, the solvent evaporates leaving behind fiber that can be electrically deviated or collected on a metal screen. Then in the Scanning Electron Microscope (SEM) the morphology and structure of the fibers are observed. The fibers are taken to the Sputtering machine which uses ions to attract Palladium. Palladium covers the fibers and this is taken to an oven in which the fibers disintegrate and the remaining material is a Palladium shell. The purpose of the lab techniques is to create Palladium shells. This is then taken to the SEM. Some advantages of nanotechnology are a major superficial area, domain of superficial forces, quantum confinement effects, and biomedical applications. In addition, when something is divided into little pieces it will increase in its surface area ratio to its volume. The larger something is the lower its surface area ratio to its volume is. The cohesive forces between the molecules inside a liquid are shared with all the near atoms. The superficial molecules do not have atoms at the top of them and that is why they present stronger attractive forces with the molecules that are by the side. Future uses of nanotechnology are to create better prosthesis that does not break. For other future studies, these shells serve as hydrogen sensors and can be used to detect gas leaks. These fibers can function as drug delivery systems because they stick to cancerous tumors. Since they are hydrophobic like the cell membrane, they enter the cell with the drug and reduce the tumors. Nanoparticles can be programmed to penetrate tumors. Nanoparticles are also used in technology to make it more effective by increasing sensitivity as well as the response time. This creates faster processing products.

Water Sampling and Testing:

Dr. Arce explained to the RISE students his work in the research laboratory. The purpose of his investigation is to find out how the quality of water has changed throughout time as a cause of variations in the soil composition. These changes in soil composition are related to alterations in soil territory, the management of hydraulic systems and precipitation patterns. In addition, Jenifer Paredes Lopez helps Dr. Arce in his investigation. After Dr. Arce’s presentation, she spoke about different fecal indicators of pathogens in water. As a result of the pollution of water, there are 3.4 million deaths annually, due to Salmonella. Salmonella is a pathogen that can also be found in these same samples because it uses the same environmental conditions as E. coli. The objective of this

practical part of the workshop is to find out if water is contaminated by animal feces. In this case, E. coli is used as a fecal indicator and it identifies coliphages. A coliphage is a type of bacteriophage that infects E. coli. So if E. coli is infected that means that there are coliphages in that sample. E. coli is important because it can be helpful or harmful to the human health. If there are high concentrations of E. coli in our intestines it is harmful but if there are normal concentrations then it can be helpful by helping in the digestion and absorption of foods and nutrients. Drinking contaminated water can lead to many symptoms, one being diarrhea which is the second leading cause of death among children around the world. Deaths caused by pathogens can be prevented by taking samples and analyzing them. Other types of water sampling use thermo tolerant coliforms which are poor indicators. On the other hand, there are different methods used to identify coliphages in water. One is the SODIS Method. Water can be disinfected by using the rays of the sun and in this way it is made drinkable. The other one is the Phages Method. This one is fast, cost effective, simple and uses bacteriophages which have a long life span. As part of the Phages Method, we mixed 2 mL of the bacteria E. coli, 100 mL of the sample of water and 100 mL of the food for the bacteria. Later, the mixture was placed in eight petri dishes. The plates were incubated at 36 ºC for 24 hours. The next day there were no positive results, because no coliphage was found.

Experiments with Membranes, Osmosis& Selective Transport :

During this workshop we mimicked cell membrane bilayers by using droplet interface bilayers (DIBs). DIBs are formed when two aqueous solutions of lipids are in oil. This happens because there are vesicles inside the droplet, which have lipids that can form the monolayer of the membrane. The purpose of this workshop was to make contact between two droplets and to form a lipid bilayer. Depending on the substances inside the droplets, osmosis or transport of substances occurred. Osmosis is the diffusion of free water across a selectively permeable membrane into a region of higher solute concentration. This is done in the direction that tends to equalize the solute concentrations on the two sides. On the other side, active transport uses energy to move substances against a concentration gradient or across a partially permeable membrane.Big molecules and charged ions cannot simply cross the plasma membrane, while some small molecules and no charged ions can cross easily. When the membrane allows some things to cross and keep others out, it is called semi-permeability. Molecules that freely cross cell membranes do so through the process of simple diffusion. In the procedure we put the chip at the top of the microscope and then filled the reservoir with oil or hexadecane. Subsequently, we inserted the different solutions in the oil. We made five rows because in the first row droplets from vesicle A and B mixed before time. Two rows had vesicles B (VB) and two others had vesicles A (VA). After a certain time, we used a tip to make contact with the two different type of vesicles. This made a lipid by layer with the two droplets. By osmosis, we proved that the bubbles with a higher solute VB became bigger than the ones with less solute or salt VA. As part of the results, water from VA passed to VB. During the time lapse of 20 minutes, many droplets got united and images were collected. Then, we created a row of dyed vesicles. The dye had a color and it was also fluorescent. As part of the results, when this was illuminated we saw dye concentrations in the vesicles that were without dye. This showed that the dye crossed the membrane. This method can be used for future studies by creating

new medications that can pass easier through the cell membranes and therefore provide more rapid delivery and possible cure for diseases.

Protein Isolation and PAGE Electrophoresis:

The purpose of this workshop is to isolate plasminogen activator (TPA). The TPA is taken from the cell culture broth. This broth has different proteins and other substances, but we are looking for plasminogen activator. In our process, we used magnetic nanoparticles. In this less expensive process the particles can be reusable and the proteins are easier to separate. At first, the laboratory procedure was to equilibrate the nanoparticles that have affinity for the plasminogen activator. Then, we placed the broth in the micro tube, which has plasminogen activator and also the buffer used to equilibrate the nanoparticles. The rocker was used to attach the nanoparticles and the plasminogen activator. Later, we used again the magnet to attach the nanoparticles with TPA and we threw out all the other substances. We washed the mixture to keep out all the other substances that were not needed. The more we repeated the process of washing, the purer the result of the protein. In addition, we also made three elutions to change the pH to a lower state and cause the nanoparticles to release the TPA. On the second day, we prepared the gel and used zymography. Zymography helps to differentiate proteins using enzymatic activity. We added casein and plasminogen to the gel. If there is presence of TPA in our samples, TPA is converted into plasmin and then plasmin brakes casein in the gel. The gel was dyed blue. As a consequence, if there is no blue area there is no protein. There were five lanes. The first two contained load, spent, and proteins. In the last three sample of the elutions there were no proteins. A possible reason is that the elution buffer was not functioning correctly. Furthermore, the human cells secrete this protein and this activates other proteins in our body. TPA converts plasminogen into plasmin and plasmin breaks clots that are formed in the circulatory system. As part of the implications of the workshop, new medications can be made using TPA. This medications help in the prevention of blood clots and strokes.