Being a CBST intern this summer has been such an amazing, unforgettable experience. During the first week, the other interns and I participated in lab training and bonded as a group by completing some fun activities together. Then, I began my research with my primary mentor, Dr. Sebastian Wachsmann-Hogiu. I have learned so much from working with Sebastian; he is an extraordinary mentor. Throughout the summer, he was always happy to help me if I had any questions, and he inspired me to persevere and continue my research despite some early setbacks. Also, I was blessed to work with two secondary mentors, Dr. Kaiqin Chu and Dr. Zach Smith, who were both incredibly helpful and constantly worked with me in the lab. Outside of the lab, getting to meet and interact with the other interns was so much fun! Overall, being an intern at CBST has been one of the most enriching experiences of my entire life. My goals of becoming a doctor and completing research in the medical field have been greatly reinforced, and I now return to my undergraduate studies with more excitement and inspiration than ever before. I have discovered that helping people through research brings me so much joy, and I plan to continue research throughout my life. I am so grateful to everyone at CBST, especially Dr. Ana Corbacho, Dr. Gene Gurkoff, Dr. Marco Molinaro, my mentors, and my fellow interns, for this amazing opportunity!Ana Popovich
The effect of optical tweezers on the cytoskeleton of human immune cells
The study of how the Human Immunodeficiency Virus (HIV) can transfer between cells through virological synapses has been impeded by limitations of the research tools used and reliance on serendipity. Finding active virological synapses at different stages of viral transfer is a challenging and time consuming process, and missing a stage means missing out on many details of the process. A recent approach to improve our yield to observe these events is the use of optical tweezers to bring cells together; however the majority of the work has been done with single point tweezers, and there have been no studies on the effect this might have on the cytoskeleton of the cells. Here we use a spinning disk confocal microscope for 3D live imaging and combine it with multipoint, holographic optical tweezers to trap target cells and move them next to infected cells. This increases the incidence of contacts between cells without harming them, and enables imaging the entire process of synapse formation. As a control for this experiment, Jurkat cells (an immortalized CD4+ T cell line) were transfected with fluorescent tubulin and actin plasmids. Transfection efficiency was checked by direct observation of fluorescence. Titration with different concentrations of G418 antibiotics was performed to determine the optimal concentration that kill all non-transfected cells. Transfected cells were then grown in 6 mg/ml of G418 antibiotics to establish a stable line. Our next step will be to directly evaluate the effect of optical tweezers on actin and tubulin. Because both actin and tubulin are critical to HIV transmission, these experiments are necessary to ensure that the results of future experiments are physiologically relevant and not caused by the tweezers themselves.
Being part of the CBST Summer Internship Program was a wonderful experience. In the first week intensive I participated in many meaningful activities that taught me a lot about myself and other interns. The program also taught me the importance of networking. The CBST retreat allowed me to further develop professional and personal relationships with other interns and members of the scientific community. Working under Dr. Thomas Huser in his research lab was a remarkable experience. I was able to learn many new skills, such as how to use a Deconvolution, Structure Illumination and Spinning Disk microscopes; how to analyze research results using FIJI and Volosity programs; how to stain specific organelles in cells; how to do transfections; and how to do multiple types of cell culture work. My mentors and other lab members were very friendly and helpful. I am thankful for the opportunity to be part of the CBST program.
During postnatal brain development, several genes are upregulted in mice. Among them, Synapse Differentiation Induced Gene 1 (SynDIG1) plays a key role in developing hippocampal neurons. When synapses are being formed, SynDIG1 was found to exist at elevated levels. The opposite is true in mutant mouse lines with defects in synaptic differentiation. This shows that SynDIG1 plays a key role in synapse development. SynDIG1 encodes a novel, postsynaptic transmembrane protein that is a critical regulator of synapse AMPA receptors in cultured hippocampal neurons. Manipulating SynDIG1 levels in cultured neurons is a very powerful tool for understanding its function. This is achieved by transfecting neurons with constructs to overexpress or knock down the protein. However, one major challenge is the very low efficiency of transfection (~1%). Transduction, a virus-mediated DNA transfer, is much more effective at targeting these cells. The aim of the project is to create a viral vector for SynDIG1 knockdown to be able to effectively deliver and express shRNA in neurons to further study SynDIG1’s effects. This lentivirus is created by first constructing a shuttle vector carrying the shRNA sequence. This vector is then co-transfected with packaging plasmids into a cell line to produce virion particles carrying the genetic material coding for the shRNA of interest. Infection of neurons with this virus will result in efficient delivery of the shRNA, which will be processed by the cellular machinery to produce siRNA, resulting in SynDIG1 knockdown.
Thank you CBST and the FASTRAC program for making my 2011 summer phenomenal. The knowledge I have gained by working in a molecular biology lab this summer will really help me as a continuing undergraduate. As an incoming transfer to UC Davis, I feel as though I will have an upper hand in my science classes once I start because I have learned so many new lab techniques, as well as the biology behind those techniques. At first, working in a UC Davis School of Medicine lab was very intimidating. I soon found out that it was not that bad because of the support I received from my mentor as well as others researchers in the lab. Because of CBST/FASTRAC, I was able to move to Davis early and get a feel of what the town is like before I start in the fall. In the few months that I have lived in Davis, I have met so many new people as well as got to interact with my fellow interns on a regular basis. The support that I received from them kept my really motivated this summer. I would like to give special thanks to Dr. Barisone, my primary mentor in the lab, for working side by side with me through everything, as well as Dr. Corbacho for believing in me and allowing me to continue with CBST in the summer. It has been a very rewarding experience.
Development of Liposome-Based Radiometric Probe for Detection of X-ray Enhancement Due to Gold Nanoparticles
Anna Malubay, Zane Starkewolf, Connie Zeng, Ting Guo
Liposomal delivery of chemotherapeutics has been used for nearly 20 years as a standard in cancer treatment. However, recurrence rates in many cancers still remain high due to ineffective drug release from the liposomes. To address this problem, we are developing a controlled-release drug delivery capsule utilizing gold nanoparticle(GNP)-coated liposomes that are activated using a minimal amount of x-ray radiation. This allows the drug to be released from the liposomes only within the tumor cells and will greatly reduce recurrence rates and side effects. The GNP coating enhances the amount of energy deposited by the x-rays and destabilizes the liposome, thus releasing the drug. To detect the enhancement due to GNP coating, we have developed dye-filled liposomes that can be used as radiometric probes. Using these probes, we can then optimize our GNP coating and liposome composition to increase the release of drug. Fluorescent dyes loaded within liposomes can be used in measuring the destruction of fluorescence upon x-ray radiation. Several dyes were used including Cy5, Calcein, and Rhodamine 6G. In liposome control studies, 30% and 60% decreases in the fluorescence of various dyes were observed after 100 and 200 grays of radiation, respectively. Upon completion of the synthesis of the GNP-coated liposomes, the enhancement will be measured. This work will allow for the optimization of the liposome composition as well as the GNP shell that will enable a novel drug release capsule for cancer therapy.
Candice A. Gellner
I want to thank the CBST staff for this great opportunity to be part of the summer
Obtaining access to membrane proteins using cell-free technologies and nanolipoproteins.
We have developed a novel process for the production and assembly of nanolipoprotein particles (NLPs) as a reagent for imaging, drug delivery, immunomodulation and stabilizing membrane protein complexes. Nanolipoproteins (NLPs), also referred to as Nanodisc, represent a novel nano-technology we are exploiting for multiple biotechnology applications. These proteins were first described as components of the human HDL lipoprotein complexes that could be isolated and reconstituted to form NLPs. NLPs are discoidal nanoparticles of 10 – 20 nm that self-assemble around a phospholipid bilayer capable of supporting membrane proteins. This bilayer mimics closely the cell membrane can support small molecules such as dyes and proteins. NLPs present distinct advantages in terms of particle size, monodispersity and consistency: the presence of the circular protein belt constrains the dimensions of the bilayer and ensures that NLP particle size distributions can be monodispersed and consistent between preparations. NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins and may make many more membrane protein related complexes accessible for biophysical and biochemical study. By the simple addition of a few constituents to cell-free extracts, this approach provides a rapid process for the production of functional soluble membrane protein complexes that eliminates the need for cell growth, cell lysis, and subsequent purification, refolding etc. We have demonstrated this process on multiple membrane proteins important for rhodopsin-related proton pumps, drug transporters, host-pathogen interactions, herceptin receptor family and G-coupled protein receptors. Proteins have ranged in size from 10 – 200 kDa, with 2 or more transmembrane domains and have been shown to be biochemically functional. Our approach represents a unique solution to the inherent challenge of generating soluble and functional membrane proteins, facilitating the structural and functional characterization of these critical, yet poorly understood molecules.
As a returning CBST Summer Intern I had previous experience working in the lab and was excited to return. My summer started with the first week intensive in which you get to know the other interns. Another major part of the first week intensive is getting a crash course on what you might encounter in your lab. Although I was a returning student and I had been in lab before I actually had the opportunity to work at a new lab at Lawrence Livermore National Lab (LLNL). The first week intensive was helpful even the second time around because I encountered new people and a different environment. After first week intensive you start “working” in the lab. I have been very lucky over the past summers that I have actually got to start working when I got to the lab, where others had to read articles for two weeks. Either way, working right away or reading articles, is very important to understanding science. As for the weekly seminars I was not required to go because I was a returning intern and since I was in Livermore I was not able to make them. From previous experience the weekly seminars are very helpful and teach you a lot about various things like how to make a resume and make it look appealing. Overall my experience with CBST has been amazing and I am sad I will not get another summer with all of the staff and amazing interns.
Change in Microbial Communities in Solitary Bee Brood Provisions Over Time
Caprice Lee, Ryder Diaz, Neal Williams, Kyria Boundy-Mills, Sharon Strauss
Historical research has shown that bacteria and yeast are associated with bee pollinators in such locations as their brood provisions, nest material, larval bee cadavers, and on the tongues of foraging adult bees. Less understood, however, is if the microbial communities associated with solitary bees change over time or if microbes that associate with solitary bees are consistent throughout the active season for adult bees. To explore this question, brood provisions (pollen nectar masses provided as food resources for developing bees) were examined to determine if both absolute and relative abundance of bacteria and yeast changed over a 5 month period when adult solitary bees are actively provisioning nests. Individual colonies of yeast and bacteria were cultured and isolated from brood provisions of solitary bees from two locations in Northern California. Microbial isolates were amplified by PCR for DNA sequencing of the 16S rDNA region for yeast (primers NL1 and NL4) and bacteria (primers 27f and 152r). We plan to use an Analysis of Variance to compare both the absolute and relative abundance of microbes associated with solitary bees at intervals throughout the season. Microbes may have intergral roles in bee health, fitness and behavior. Expanding our understanding of the microbial communities associated with cavity-nesting bees may provide the foundational information needed to test which microbes may be playing significant roles in pollinator fitness. If microbes affect pollinator behavior and their interactions with plants, this information may reveal potential impacts to plant fitness and community structure.
The CBST Summer Internship has proven to be a truly pivotal experience. There are so many aspects of this internship that have made it immensely beneficial; participating in research, professional development, networking with professors and mentors, making lifelong friendships, becoming familiar with the city of Davis and the UC Davis campus, the list goes on and on! My involvement in the Strauss lab has opened many doors for me. As a transfer student, the disadvantage of only having two years to participate in research (as opposed to four years) makes programs like this summer internship so invaluable. Not only have i begun to understand the research environment, but I have also gained understanding regarding how I want to steer my future and what resources are available to me. I gained so much from workshops, lectures and presentations that this internship provided. My public speaking skills as well as writing and communication in a research setting have been greatly improved. Attending and participating in the Journal Club and attending the biophotonics research conference were also valuable experiences. I feel absolutely prepared for my first quarter at UC Davis and dam excited about making the most of my two years of undergraduate studies. Furthermore, because of this program, there is no question that I will be going to Graduate School. Thank you Ana Corbacho, Gene Gurkoff, Ken Burtis, and Sharon Strauss for giving me the opportunity to truly contribute to science!
Dennis Chang, Vihn Lam, Eduardo Sanchez, Chong-xian Pan and Paul T. Henderson
Department of Internal Medicine, Division of Hematology and Oncology, UC Davis Medical Center, 4501 X Street, Suite 3016, Sacramento, CA 95817
Doxorubicin is an effective chemotherapy drug that kills many types of cancer cells. However, the drug causes unwanted and sometimes fatal side effects like nausea, vomiting, diarrhea, and heart damage. This is due to the doxorubicin affecting not only the cancer cells, but also the normal cells. We are developing a type of targeted therapy that concentrates doxorubicin in a particular type of cancer cells. Studies have shown that a peptide, called PLZ4, has high affinity for human bladder cancer cells and we hypothesize that conjugating Doxorubicin with PLZ4 will increase the effectiveness of treating bladder cancer with Doxorubicin while reducing harmful side effects. We chemically linked doxorubicin to PLZ4 and verified the existence of the product using mass spectrometry. We are now purifying the Doxorubicin-PLZ4 conjugate, scaling up the synthesis and starting experiments with cancer cells to determine if this new molecule can kill bladder cancer cells. This research may lead to a new targeted therapy drug for bladder cancer, but the overall strategy can be applied to many other cancers.
I cannot begin to describe how the CBST summer internship has benefited me. It has been a life changing experience. Just last year I didn’t even know there were such things as summer research internships. I was thinking about how I would ever get a taste of what research is like and now here I am doing research. Although there were some setbacks and frustrating times, I can honestly say I enjoyed doing my research. But what I enjoyed most of all was getting to know everyone in the program and learning from them. My mentor, Paul Henderson, was a great mentor and taught me to take advantage of all the resources in the lab as well as being independent. Ana, Gene, Marco, and all the other CBST staff really supported us through their hard work and passion. And of course my fellow summer interns were awesome. We all came from different backgrounds with different experiences, and brought it together in the same place. We learned a lot from each other and had great times. Thank you N.S.F. Thank you UC Davis. Thank you CBST. And thank you everybody for a life changing summer.
PLP (Pyridoxal-5’-Phosphate) is an active form of vitamin and is an important cofactor in numerous enzymes for amine/amino acid metabolism. For the PLP-Aspertate system, the Schiff base linkage is a vital reaction center to investigate as it dictates many of the photoacidic properties and is a crucial part of the enzyme’s catalytic strategies. It is expected that if the α-carbon-hydrogen bond is energetically weaker, then PLP-Aspertate will be more acidic and exhibit weaker local electron density. Thus, to examine the nature of the α-carbon-hydrogen bond electron density difference maps were calculated using the triplet excited state and singlet excited state of PLP-Aspertate via Gaussian using GaussView. Preliminary findings indicate that irradiation with light increases the enzymatic activity of PLP and other derivatives of pyridoxine.
I am very thankful for being apart of something as amazing as the summer CBST internship program 2011! The experience as a whole was priceless! I have gained so much more than just undergraduate research. I have gained friendships that are amazing, Mentors that I can confide in, memories I can tell others and laugh about with, and best of all I have learned more about myself and what I am able to accomplish. From the First Week Intensive, I knew I was around people who were just like me; thriving for more in the science background and wanting to have a research experience! On that first day I knew I was going to love the internship and everybody who was apart of making CBST happen! It was a great feeling to open up to the many different faces around me and to see how alike we all were no matter what our upbringing was.The CBST summer internship taught me so much, such as: how to read a research article, how not to read a research article, how to communicate with professionals, and more importantly how light can be used to explore biology in so many ways!My research experience in Professor Delmar Larsen’s lab was not your ordinary lab situation. I was able to do computational chemistry calculations through a computer program called Gaussian. It was very interesting! I learned to take steps outside of my comfort zone to get help and answers for questions I had about the program. I am continuing my research in Larsen Lab and hope to contribute great findings towards his research.CBST in one word was AMAZING. I am extremely grateful to be apart of such an awesome opportunity. I was to acknowledge Professor Larsen, and Larsen Lab. I want to especially thank Dr. Ana Corbacho and Dr. Gene Gurkoff, and Dr. Marco Molinaro for making this experience something to cherish. I would also like to thank my fellow interns for making my summer an unforgettable one.
Hiram M. Dominguez
Development of Disulfide Cross-linked Micelles for the Efficient Delivery of Doxorubicin Anti-cancer Drug
Fiorella Candamo, Yuanpei Li, and Kit S. Lam
The use of nanoparticles as chemotherapy drug vehicles is a novel approach towards cancer tissue. In this study, self assembling micelles were used to transport doxorubicin (DOX), an intercalating agent that blocks DNA synthesis and transcription and inhibits topoisomerase type II resulting in DNA disruption that ultimately leads to cancer cell death. Micellar nanocarrier comprised of polymer PEG2k-CA4 has been proven to be a good delivery vehicle for doxorubicin; however, it results in a high hemolytic effect. Following previous work on reversible disulfide cross-linked micelles (DCMs), which have been proven to reduce hemolytic effect, we hypothesized that DOX-loaded micelles comprised of different ratios of two linear-dendritic polymers: PEG5k-Cys4-L8-CA8 which results in disulfide cross-linking upon oxidation of thiol groups and PEG2k-CA4 with no thiol groups for cross-linking would reduce hemolytic activity. We performed a hemolysis assay using our synthesized empty micelles to measure their dissociation into amphiphilic telodendrimer. These telodendrimers disrupt the plasma membranes of red blood cells and result in the release of hemoglobin. The four-hour incubation of red blood cells with our different synthesized micelles showed us that as the concentration of PEG2k-CA4 decreases and the concentration of PEG5k-Cys4-L8-CA8 increases the hemolytic activity decreases. Having as little as 20 percent composition of PEG2k-CA4 polymer still results in considerable hemolytic activity (>5%). On going work focuses on incorporating a cross-linker into the PEG2k-CA4 polymer and loading DOX into micelles made up of this new polymer in order to reduce hemolysis while still benefiting from PEG2k-CA4 efficient DOX loading.
Participating in the CBST summer internship has been one of the most enriching educational experiences. I gained so much knowledge from working in the Lam lab: from cell culturing to synthesizing chemotherapy drug-loaded nanoparticles. Also, I learned how to conduct my own project research and come up with new ideas after reading some scientific articles. At first, I was a little overwhelmed with all the machines and instruments that I had not seen or used before; however, with the help of the other interns who had already done research in the past I started feeling more comfortable. I am extremely grateful for this opportunity because I did not just conduct my own research, but also I was part of a great group of interns that were encouraged to participate in many activities, such as first week intensive seminars, the CBST annual retreat, and journal club. I would like to thank Dr. Ana Corbacho and Dr. Gene Gurkoff for motivating us to participate in journal club because I am sure that the skills that I learned in journal club will help me succeed as a scientist.
Polyvinyl alcohol Nanogels for Efficient Doxorubicin Delivery
Giselle Camarillo, Yuanpei Li, Kit S. Lam
Nanoparticles can prolong the circulation time of conventional anti-cancer drugs and increase the solubility of these drugs. Furthermore, nanoparticles have been shown to efficiently deliver anti-cancer drugs to the tumor site because of the enhanced permeability and retention (EPR) effect. The ideal size of the nanoparticles should be 10-100 nm to take full advantage of the EPR effect. We developed a novel type of nanogel comprising of polyvinyl alcohol (PVA) and cross-linkers to load anti-cancer drugs like doxorubicin (DOX) in order to effectively deliver these drugs to the tumor site. We hypothesized that these nanogels will be stable under physiological conditions and can be triggered to release drug under acidic conditions. We synthesized a series of nanogels with different sizes by varying the size of the PVA as well as the types and ratios of the cross-linkers (two arms, three arms and four arms). The unloaded drugs were removed by running the drug loaded nanogel through centrifugal filter devices. The drug-loaded nanogel on the filters was recovered with PBS. MTS assay will be used to evaluate the cytotoxicity of the blank PVA and drug-loaded nanogel against A549, non-small, cell lung cancer cells. Our data shows that the larger PVA and the negatively charged PVA resulted in higher loading capacity for DOX.
CBST is one of the best ways to get involved with research while gaining a myriad of amazing friends. The first week intensive is the prime opportunity to relish everyone’s diversity and get to better know each person. Everyone in the internship had an inspiring sense of intelligence and dedication that shined through their friendly personalities. The people are only one great aspect of CBST. I worked in Dr. Kit S. Lam’s lab and learned an incredible amount of procedures and laboratory techniques. It was challenging at first since I was unfamiliar with a lab setting but with the help of my mentors and the people in lab, I was always able to manage fairly well. Everyone in the lab was always willing to answer any questions. My project is in its very early stages so a lot of the tests that I ran, I did it several times. It, however, did not get tedious because I was so new to the procedures that it was a learning experience. Overall, the interns and the people in the lab were very helpful and I enjoyed working with both; the lab experience was challenging but rewarding nonetheless.
Morphology is an important indicator of the physiogical state of the cells. Electrical field is known to be able to override other signal in cell migration. However, the effect of the electrical field on cell morphology is still unknown. When cell moves, part of the cell protrudes and part of the cell contracts. An unanswered question is how cell protrudes and contracts in the electrical field. To answer those questions, it is important to develop a method to extract the cell shape and quantify the cell shape. Based on the literature, we successfully developed a procedure to quantify the cell shape and the velocity of cell membrane. The procedure starts with creating binary images. The process of getting binary images is semiautomatic. A Matlab code automatically gets the binary images from normal images. If there is any problem with the binary image, one can use Photoshop Lasso tool to manually get the binary image. Once binary images are created, one can use Celltool program to obtain different quantitative values of the cell shape. Then we used a short custom python code to calculate the velocity of chosen points on the cell membrane. We also wrote a Matlab code to display those quantitative values on graphs and charts.
This internship has been a very good experience for me. In the lab, I could apply what I learned in school to help my lab solving their problems. For instance, I have been successfully developed a protocol to quantify cell shape. Besides working in the lab, we also attended weekly seminar with many interesting and practical topics. In those seminars, I learned many things from CBST alumni such as: how to get the scholarship and how to prepare for graduate schools and professional schools. By talking to the alumni, I know more about what I can do to be successful. I also liked the talk about the entrepreneurship, which was very interesting. From the talk, I got the general idea about how to transfer the result from bench to bed and how important research is. In the middle of the internship, I had an opportunity to go to the CBST annual retreat in Lake Tahoe. In the retreat, I made many new friends and talked to many interesting people. I also had a really good time with other interns in a very beautiful resort. This internship has made my summer very meaningful. I got a chance to work and enjoy the summer at the same time. Thank to the internship, I can work in the really nice lab on a very interesting project. I am going to continue working in the lab in the school year.
THETA RHYTHM STIMULATION RESTORES COGNITIVE ABILITY IN RODENTS FOLLOWING
TRAUMATIC BRAIN INJURY
I could tell you a story for every day that I spent in Davis this summer. But by the time I finished, I’d probably have a book written. The summer of 2010 was filled with amazing experiences inside and outside the lab; I was simply left hungry for more. This year, I was able to return to the UC Davis Medical School Department of Neurological Surgery, where I worked under the mentorship of Dr. Robert F. Berman and Dr. Darrin Lee. My project this summer involved using the Barnes maze paradigm to test the efficacy of a treatment for traumatic brain injury (TBI) using a rat model of TBI. In lab, I practiced previously learned skills and learned new techniques. I also enjoyed seeing the progress made in the lab over the course of the past year. Furthermore, the experience out of lab was equally remarkable; my fellow interns are an amazing group of people. Last but not least, I am inspired by our program mentors, Dr. Corbacho and Dr. Gurkoff. I thank them and the rest of the CBST staff for providing an internship where undergrads can grow as young scientists. This internship is much more that I could have ever asked for, the experience gained and friendships made this summer are priceless.
Interaction of spontaneous retinal activity and semaphorin signaling on stereotyped axon pruning in primary visual cortex
Jeremy Crane, Karl Murray, PhD and Hwai-Jong Cheng, MD, PhD, Center for Neuroscience, University of California, Davis, Davis, CA
Proper functioning of the nervous system is dependent on the establishment of a precise pattern of neuronal connectivity. Functional connectivity can largely be credited to a period of early development when initially formed exuberant neuronal connections are pruned back to eliminate inappropriate contacts. Trimming immature connections promotes a more precise and functional network. Stereotyped axon pruning is a widespread phenomenon that refines neuronal contacts in the neuromuscular junction, as well as the hippocampus and corticospinal tract (CST). Although essential during development, the mechanism of axon pruning remains unresolved. In the visual system, axons originating from layer V pyramidal neurons initially target the spinal cord but are subsequently pruned back to the brainstem. Prior evidence indicates that the semaphorin family of axon guidance molecules, and their cognate neuropilin and plexin receptors, mediate this pruning. In addition, we recently found that pruning of CST axons is dependent on patterned spontaneous activity in the retina. Whether or not these mechanisms operate along the same or separate (independent) signaling pathways has not been addressed. Here, we elucidate the interaction between these pruning mechanisms and their combined effect on remodeling of CST axons. Using mutant mice enucleated at birth and in vivo tracing experiments, we will examine the effect of combined deficits on visual cortex CST pruning. Based on previous work, we predict no difference in pruning deficits when both mechanisms are inactivated compared with previous results using independent inactivation. This would support our hypothesis that these pruning mechanisms operate along the same pathway.
Participating in the CBST Summer Internship program has been the most rewarding, engaging, and memorable experiences of my undergraduate career. Having participated in a summer research program the summer before I became a CBST intern, I can say with confidence that this program is truly a cut above the rest. Coming into the program I didn’t know a single one of my fellow interns, but by the end of the first week intensive I truly felt like I was part of the group and had formed lasting friendships. The first week intensive succeeded in making sure that as interns we became close and would be there to support each other while we tackled our lab work and any research obstacles we might encounter during the length of the program. In no time at all we were enjoying trips to the farmers market and organizing regular social outings outside of our research.
Besides forming lasting friendships with my fellow interns and participating in a plethora of seminars on diversity, resume writing, how to write an abstract, and a phenomenal journal club led by Gene Gurkoff, the highlight of this program was taking on your own research project. Getting paired with a neuroscience lab, I jumped right into my first day learning new techniques and investing myself in my project with a grad student, post doc, and professor to help me along the way. CBST did a phenomenal job pairing me with a lab that not only matched up with my research interests, but also had extremely helpful and insightful members to work with. This unforgettable summer with CBST provided me with priceless career insight, the opportunity to tackle an exciting project of my own, and make enduring friendships!
Developmental progression of electrical activity throughout the anterior-posterior axis of Xenopus laevis spinal cord and skeletal musculature.
Spontaneous Ca2+-mediated electrical activity manifests during nervous system and muscle development and is important for neuronal and muscle cell differentiation. Previous studies have shown that Ca2+ -mediated electrical activity manifests in the Xenopus developing spinal cord from the moment the neural tube closes (22 h post fertilization, hpf) until the embryo hatches (32 hpf). Similarly, developing muscle cells exhibit Ca2+ transients during an embryonic period that correlates with somitogenesis. After hatching, Xenopus larvae develop a tail which allows for effective swimming of the tadpole. Whether the spontaneous embryonic Ca2+-mediated electrical activity is recreated in the most posterior tissues of the larva’s tail remains unknown. We investigated the developmental progression of Ca2+-mediated electrical activity in anterior and tail tissues of the Xenopus tadpole. Anterior and posterior muscle and spinal cord were dissected from stages 33, 37 and 41 (44, 54, 72 hpf, respectively) tadpoles and dissociated cells were loaded with a Ca2+-sensitive dye followed by time-lapse imaging with a confocal microscope. Preliminary results show that Ca2+ transients are evident in cells derived from tail tissues with the highest incidence of active cells at stage 33 decreasing to undetectable levels by stage 41. Cells derived from anterior spinal cord and muscle do not exhibit spontaneous activity. These results indicate that expression of Ca2+-mediated electrical activity in neuronal and muscle cells of the tail is developmentally regulated and suggest that this activity may be relevant for appropriate tail development as well as for the regenerative capacity of these tissues after injury.
A whole new aspect of research that was once limited by textbooks has found its way into my life through this incredible summer research internship. During the first week of the internship, I met with a diverse group of interns, with the same aspirations and mutual passion for science. The first week’s group activities allowed us to get to know each other through joyful and fun activities. There were also empathetic moments during activities that enriched our understanding of each other’s diverse backgrounds and no matter how difficult we seem to find ourselves converged in the same path. The various seminars, presentations from the retreat, journal club throughout the internship better prepared us for presentations, writing CVs and résumés, and especially an insight on various lab projects being worked on throughout different Universities. I built long lasting friendships and memories through get-togethers such as: Journal Club, Farmer’s Market, and most importantly the CBST Squaw Valley retreat. And when it came down to working in laboratories, consumed a plethora of knowledge. The first day of lab, I immediately immersed myself into a textbook of developmental biology. Throughout the summer I worked closely beside my Primary Investigator, Dr. Laura Borodinsky, learning techniques, applying and formulating experiments for my project, eventually I became independent as I continued working on my project. Overall, this summer internship was an invaluable experience. Working in lab everyday did not feel anything like work! I encourage anyone whose passion belongs to science to apply; it is truly an experience you do not want to miss!
The Contribution of Genotoxic Stress to the Formation of Chromosome Bridges in Anaphase
Jesse Garcia and Kenneth B. Kaplan, MCB, UC Davis, Davis CA 95616
Defects in enzymes (e.g., Blooms syndrome helicase (BLM)) that unwind and detangle DNA during replication are associated with genomic instability and human disease. Interestingly, BLM localizes to late segregating chromosome elements called chromosome bridges that are likely to be tangled sister chromatids that arise during replication. However, it is unknown whether genotoxic stresses promote the formation of chromosome bridges or inhibit their resolution during anaphase. To address this question, we propose to challenge budding yeast cells expressing histone H1 fused to GFP with the following compounds: hydroxyurea, an inhibitor of DNA replication; MMS, induces single strand lesions; razoxane, aclurubucin and camptothecin, inhibitors of topoisomerases, enzymes important for the resolution of tangled sister chromatids. We have previously observed that inhibition of DNA replication following HU treatment gives rise to slower to resolve chromosome bridges. Preliminary observations indicate that MMS treatment does not affect chromosome bridge formation or resolution. Further experiments are in progress to further characterize the HU-induced chromosome bridges and to evaluate the effect of other genotoxic stresses on chromosome bridges. The long-term goal is to link the behavior of chromosome bridges to the severity of genomic instability by monitoring DNA double-strand breaks. This may have important implications for cancer onset and therapeutic strategies.
When I began my 2011 summer internship, I had the opportunity to be exposed to the full range of new information, all of which tended to reinforce and solidify my intense interest in biology. I've also had the opportunity to study a number of topics in cell biology and they have been both enjoyable and enlightening, providing me with a new and different perspective on researching. My primary mentor, Dr. Kaplan, and my secondary mentor, Brandon Zipp, have always taken the extra minute to explain theory, concept and process of what needs to happen and my role in the lab. The feeling of utilizing my theoretical background and creativity as a scientist along with strong role models as a backbone is priceless. It was not all work with any play. Activities such as the human knot are always fun especially as an icebreaker to become acquainted with new people. I am highly appreciative of the superb CBST retreat, and my conversations with several of the researchers who helped to clarify their interesting presentations. I know that in addition to the excellent faculty, my fellow summer interns are among the best in the world. I continue to learn that the most diverse individuals can produce the most extraordinary results (i.e. PowerPoint presentations). I’ve had the privilege of continuing to research for two summers and will continue throughout the year. I will never forget the feeling of complete fulfillment after a long day in lab and my mentor says, “Nice, Good job”.
Analysis of Red Blood Cells under Oxygen-deprived Environments
Mentor: James W. Chan1, Rui Liu1
1. NSF Center for Biophotonics Science and Technology, UC Davis, Sacramento, CA, 95817
Raman spectroscopy and microscopy have proven to be powerful label-free analytical techniques for determining the oxygenation level of red blood cells (RBCs). The known conformational differences between normal, sickle and fetal red blood cells (RBCs) indicate that studying them in an initial deoxygenated state using Raman spectroscopy and microscopy at the single cell level may reveal additional insight into the fundamental molecular conformations of different types of hemoglobin. To carry out these experiments in our lab requires additional instrumentation to maintain the cells in an oxygen-deprived environment during Raman analysis. We resolve this by fabricating a chamber that will isolate the cell samples from the outside atmosphere while continuously purging the inside atmosphere with pure nitrogen gas, which is monitored using a flow meter. A slight positive pressure inside (<1 psi) prevents oxygen from leaking inside, guaranteeing that the inside atmosphere will maintain an oxygen free environment throughout the duration of the experiment. We propose to carry out experiments to identify RBC Raman spectra that reflect deoxygenated conditions in a pure nitrogen atmosphere and monitor the deoxygenation process of RBCs in real time when the nitrogen is initially filled in. In addition, we propose to investigate and identify Raman markers that can distinguish between normal, sickle and fetal RBCs under the oxygen-deprived environment.
The CBST summer internship was a fantastic opportunity to gather greater experience, develop more professionally and establish contacts. Under the guidance of my mentors, Dr. James Chan (PI) and graduate student Rui Liu, I was able to put my education as a mechanical engineering major to practical use in the design and fabrication of a nitrogen purge chamber, which would be use for the study of red blood cells using Raman techniques. To be able to develop my skills and expand my knowledge base in biomedical applications was a rewarding experience, and it was a great pleasure to work under two knowledgeable and easygoing mentors. In addition, the CBST retreat provided a clearer outlook on the use of biophotonics in basic research and allowed me to talk to professionals in both industry and research. Not to mention, all my fellow interns were a fantastic collection of intelligent and fun individuals, which made the internship that more enjoyable. I have no doubt that the CBST summer internship was an invaluable experience; as a result, I can now tell others that I have engineered and built an instrument for biomedical applications. Thank you CBST!
Plant-derived Auxins as Inhibitors of Neutrophil Myeloperoxidase: Potential Therapeutic Agents for the Treatment of Cystic Fibrosis
Kim Ngan M Huynh, Sharon Louie, Sean Ott & Jason Eiserich
Department of Internal Medicine, Division of Pulmonary/Critical Care Medicine,
University of California, Davis, CA
Cystic Fibrosis (CF) is a hereditary disease that results in the accumulation of viscous mucus in the lung. As a result, CF patients suffer chronic infections, overwhelming inflammation and oxidative stress that leads to pulmonary dysfunction and ultimately premature death. Chronic inflammation in the CF lung is associated with exuberant recruitment of neutrophils into the CF airway. Neutrophils express the highly abundant hemeoprotein/enzyme myeloperoxidease (MPO) that can cause oxidative injury in the CF airway via the production of reactive oxidants such as hypochlorous acid (HOCl; otherwise known as ‘bleach’). Under pathological conditions the hyperactivation of MPO can cause injury to airway epithelial cells and the levels of this enzyme are positively associated with decreased lung function in CF. We have hypothesized that MPO may be a feasible therapeutic target for the treatment of CF. A survey of natural chemicals possessing an indole moiety reveal plant-based compounds typically termed ‘auxins’ as potential inhibitors of MPO. Accordingly, we have begun to test a number of these indole derivatives as MPO inhibitors such as indole-3-carbinol, indole-3-carboxylic acid, indole-3-acetic acid and indole-3-propionic acid. Spectrophotometric assays have been utilized to assess the ability of these compounds to inhibit MPO. While preliminary results indicate the potential for these compounds to inhibit MPO, more studies are necessary. Successful completion of these studies will define the capacity of these natural products, either through dietary means or in pure form, to be utilized as therapeutic agents to ameliorate oxidative stress and inflammation in CF and related lung diseases.
Being able to participate in the CBST Summer Internship / FASTRACT program is a great opportunity for me. As an intern returning from the CBST Winter program for Community College student, I had one more chance to know more about academia research. However, the summer internship was totally different from the winter one. During the winter, we mostly focused on the techniques and got to work as a group to figure out the solution. For the summer program, we actually participated in the progress of research. Because of the difference, I was excited and nervous at the seam time when I first started my project. For more than two months, I worked in the lab of Dr. Jason Eiserich and was under the mentorship of him as well as Sean Ott. I was assigned the project of screening the possible inhibitor of Myeloperoxidase using Auxins with another undergraduate intern. Both my mentors were really nice and willing to help me anytime. Working in the lab gave me a lot to think about such as my major and my career later. It also helped me narrow down my interest and passion. For me, the highlight of the summer program was the retreat. Through it, I was able to know more about biophotonics and get a chance to meet all these great people. The retreat also strengthened the friendship between interns. I feel grateful for being able to participate in this internship program. I own my thanks to Dr. Corbacho, Dr. Burtis, Dr. Molinaro, Dr. Eiserich and CBST for giving me this occasion.
FUNCTIONALIZED GOLD NANOPARTICLES FOR RADIATION-DIRECTED CHEMOTHERAPY
Larissa Miyachi, Zane Starkewolf, and Ting Guo
Although chemotherapeutic drugs are vital to modern cancer therapy, their systemic toxicity often causes severe side effects and limits practicable dosages, increasing the likelihood of incomplete cancer elimination and subsequent recurrence. Our aim is to overcome this barrier by developing a system where drugs are transported throughout the body by gold nanoparticles (AuNPs) and released only in response to radiation, thus restricting chemotherapeutic activity to cancerous tissues treated with site-directed radiotherapy. To achieve this, drugs are attached to AuNPs using radiation-sensitive linkers that are cleavable by the hydroxyl radicals produced in irradiated solutions. As irradiated AuNPs also generate hydroxyl radicals, it is expected that the AuNPs will, in addition to carrying drugs, further enhance their release under radiation. We are currently using single-stranded DNA as a linker model and the fluorescent dye 6-carboxyfluorescein (6-FAM) as a drug model in studies of radiation-induced linker cleavage and AuNP enhancement. As 6-FAM fluoresces only when detached from AuNPs, the extent of DNA cleavage is determined using fluorometry. When DNA was substituted with a radiation-insensitive linker, the increase in 6-FAM fluorescence after radiation was greatly reduced, confirming that the fluorescence increase observed in previous radiation studies was due to linker cleavage. Studies substituting silica nanoparticles (SiNPs) for AuNPs are currently underway to measure any cleavage enhancement due to AuNP radical generation, as SiNPs do not produce hydroxyl radicals under radiation. Through this ongoing work, we hope to improve the quality of life for cancer patients by providing a platform for more effective chemotherapy.
This summer, I had the pleasure to continue working at the Nanofast Lab where I interned last year and continued to participate afterwards as an undergraduate researcher. Having recently completed my second summer internship with CBST, it is amazing to look back on all that has happened since this program first launched me into scientific research last June. Through the CBST internships, I became established as an undergraduate researcher, acquired vital lab and interpersonal skills, gained key experience in communicating my research, and formed my first friendships as a new arrival in Davis last year. I heartily recommend the CBST Summer Internship to anyone considering a career in research or a similar field requiring initiative and innovation. The experience of becoming an active participant in research is different from completing lab courses, for no pre- or post-lab can fully approximate the sensation of scouring journal articles for a new process or technique and then receiving approval to implement it in the project. In addition to providing opportunities not typically encountered in an academic setting, this internship also provides motivation for further studies, and I am eager to complete my remaining lab courses so that I can gain additional skills and ideas for the project. I wish to thank Dr. Ana Corbacho and all the CBST staff for two amazing summers that will impact my life for years to come as I pursue future graduate studies and my goal of researching the mechanisms or treatment of cancer.
Functional Analysis of Oxygen Carrying Capacity of Normal and Sickle Red Blood Cells using Laser Tweezers Raman Spectroscopy
Mentors: James W. Chan1, Rui Liu1
1. NSF Center for Biophotonics Science and Technology, UC Davis, Sacramento, CA
Optical tweezers have become widely used for the manipulation and analysis of individual
biological cells. A simple and most commonly used configuration is the single beam optical trap in
which a tightly focused laser beam optically immobilizes individual cells within the laser focus. The
perturbative effect of an optical trap operating under such a tight focusing condition on the function
and biochemistry of a live cell is often a concern, with photoinduced damage typically being a primary
focus. Mechanically induced biochemical changes, however, have not been as extensively studied,
even though it is known that optical forces are imposed on a biological cell by a single beam optical
trap that are often strong enough to modify its shape. Herein, we report that a red blood cell (RBC) in a
single beam optical trap transitions from an oxygenated to a partially deoxygenated state with
increasing trapping power using laser tweezers Raman spectroscopy (LTRS). In addition, the
oxygenation states of different types of red blood cells, such as sickle cells and normal adult red blood
cells, reacts differently with the increased optical trapping power ranging from 1.6mW to 20mW,
which provides the rationale for functional analysis of the oxygen carrying capacity of different types of RBCs.
This summer interning at CBST has been a wonderful experience. I would like to thank Dr. Ana Corbacho for organizing such an incredible program, and I would also like to thank my amazing mentors Dr. James Chan and Rui Liu. My mentors was always patient with me, and very willing to explain topics that I didn't understand at all. They were very supportive of me and always guided me on the right track. With this, they have provided a great learning environment for me, pushing me to new heights and making me feel like I'm really part of the team. I cannot thank them enough for everything they have done for me. Now, not only did I have outstanding mentors, but the other graduate students, post-docs and other interns were superb as well. It really felt like a community where we all learned and had an exciting time. I had the opportunity to connect with these people whom I would not get the chance to know if it were not for this summer program. During my stay here, I have learned a tremendous amount of new skills and gained valuable knowledge that I will carry on with me for the rest of my life. I was able to practice my writing and presentation skills under the guise of experienced individuals who gave me helpful feedback so that I may improve myself. Thanks again to all the contributors who have made this summer meaningful!
Personalized chemotherapy by quantification of drug-DNA damage
Department of Internal Medicine, Division of Hematology and Oncology, UC Davis Medical Center, 4501 X Street, Suite 3016, Sacramento, CA 95817
Patients undergoing chemotherapy often do not respond to the treatment, which results in suffering, waste of time that could be spent on better therapies and high cost to the medical system. Currently, there are no proven technologies for predicting which patients will respond to chemotherapy. We are developing a method to measure drug-DNA damage, which has the potential to predict chemoresistance in cancer patients. Our hyposthesis is that low levels of drug-DNA damage will predict drug resistance. Since DNA is the target of many chemotherapy agents, such a test may have broad applicability. My project involves growing cancer cells on petri dishes, isolating the DNA and exposing it to cisplatin, a commonly used chemotherapy agent. The sites of drug-DNA damage will be detected using a proprietary antibody-based technology in collaboration with Life Technologies Inc. Since antibodies recognize specific sites, called epitopes, the antibodies bound to the drug-damaged DNA can be easily measured and quantified. If successful, this work may lead to improved cancer care by enabling personalized cancer therapy.
The journey during the CBST summer internship put a positive impact in my voyage of becoming a researcher. From day one Ana Corbacho and her colleagues put great effort to make my summer experience unforgettable. After preparing us during the first week, learning about safety and what to expect during our time in lab, I was placed in my lab with Dr. Henderson. The first time I meet my mentor it was nerve wrecking, where at first was hard for me to communicate to him because I didn’t want to come across as a simpleton. However, after weeks in the lab and finding out that Dr. Henderson and I share common interest with music and sports, I broke out my shell. Moreover, some of the highlights that made this summer enjoyable were networking with new people, making new lasting friendships, learning the ins and outs of scientific research, and being involved in explorations that will one day make a change in a person life. Not to mention the huge retreat up to Lake Tahoe was great fun. My project was titled Personalized Chemotherapy by Quantification of Drug-DNA Damage, where my mentor and I explore new platforms that doctors in the field can use to predict how cancer patients response to chemotherapy. My involvement was to cell culture cancerous cells, extract their DNA, and expose the DNA to Cisplatin, a common chemo platinum based drug. Overall, being a part of this internship truly convince me in pursuing in the scientific field.
Breaking the detection limit for coherent anti-Stokes Raman (CARS) signals by using doubly resonant coherent anti-Stokes Raman (DR-CARS) signal generation with Acousto-Optical Tunable Filter (AOTF) signal modulation
Iwan Schie, M.A., University of California, Davis, Davis, CA and Thomas Huser, University of California, Davis, davis
Fluorescence microscopy has long been the driving force in the cellular biology field. However, its methodology has had a weakness since it requires exogenic fluorescent probes to select the molecules of interest. Those probes are not photostable and can potentially become toxic or interfere with the cellular environment. In the last decade, some label-free microscopy techniques have been shown to successfully overcome those problems. One of the most promising one is CARS microscopy. It is based on a resonant inelastic scattering between photons and molecules, and provides label-free, chemically specific information from the sample in video rate. However, one of the drawbacks of CARS microscopy is the strong signal dependence on the molecular concentration. We developed a technique that is based on a doubly resonant enhanced signal generation that allows pushing the concentration sensitivity by few orders of magnitude. In this work we show the implementation of a modified signal acquisition setup that theoretically allows increasing the concentration sensitivity to few μM. To overcome the signal detection limit, the double resonant signals are rapidly modulated using an Acousto-Optic Tunable Filter (AOTF),(Gooch&Housego, UK) and detected on a conventional photomultiplier tube (PMT),(Hamamatsu, JP) in conjunction with a Lock-In-detection scheme. The entire signal acquisition setup is controlled via a LabView code.
The value and personal gain from a program such as the CBST internship is really quite immeasurable. From engaging in simple 2-minute conversations with scientists I would never again see to daily teaching from my mentor Iwan Schie, the wisdom I have gained is infinite. Much of what I had learned are skills that will aid me throughout my life whether or not I choose to pursue a career that’s even related to Biophotonics. One especially stressed skill is the ability of an intern to communicate technical information. Whether we communicated through email, powerpoint presentation, computer language, or while creating a scientific abstract, it quickly became evident that the power of knowledge transfer is absolutely mandatory. Oddly enough these normally difficult concepts turned out to be surprisingly easy with the amount of interest I had on the subject and help I had received from fellow colleagues. This brings me to the social aspect of the internship. It was an honor to spend my summer with such a positive group of people all committed to reaching their goals. Since our internship consisted of students from a wide range of backgrounds and personalities, friendships were created effortlessly and I myself will admit that I had gained new perspective literally overnight. I really enjoyed spending time with fellow interns both at work and outside of work and as a result have made long lasting relationships that will far exceed my stay at CBST. Thank you everyone for making this an unforgettable summer!
BIOENGINEERING FLUORESCENT TAG FOR TLL0911 IN THERMOSYNECHOCOCCUS ELONGATUS BP-1
Monica Bower, Mo Kaze, Fiorella Meza-Acevedo, Kristen Beck, Stefanie Yoshizuka, Valerie Metea, Alexandria Magallan, Ling Ou, Nathan Rockwell, Clark Lagarias, and Susan C. Spiller
Thermosynechococcus elongatus is a thermophilic cyanobacterium. T. elongatus was found in the hot springs of Beppu, Japan. Five genes are located on the T. elongatus genome, one of which is tll0911. These five genes show homology with phytochromes from plants, but the chromophore absorbance ranges around 435 nm to 540 nm (blue and green photons). Since T. elongatus cyanobacteriochromes are blue/green photoreversible, they differ from phytochromes which are red/far red photoreversible. The proteins from these genes are called cyanobacteriochromes. When a mutation is introduced into the DNA sequence of the GAF domain (bilin binding pocket of cyanobacteriochromes), the protein changes its shape and forces the linear tetrapyrrole structure (bilin) to become more stationary in the protein’s binding pocket. Our lab takes all of the T. elongatus genomic DNA, amplifies GAF only domains from cyanobacteriochrome genes with PCR, and ligates the PCR product into the pBAD plasmid. We then transform the plasmid into E. coli cells to complete protein expression. Protein purification with chitin beads attaches the chitin binding domain in the Tll0911 protein product that is produced by E. coli. During purification the chitin binding domain is cleaved off leaving only Tll0911 protein. After purification, the protein is analyzed with a spectrophotometer. The description and assessment of the protein is performed by spectral analysis. SDS-PAGE gels are run to determine purity and size. Covalent bonding of the bilin in the protein’s binding pocket is demonstrated by a zinc blot. Finally, the wavelengths of absorption and fluorescence are confirmed with spectrometry.
Being an intern through CBST at Mills College was a great experience! I loved getting to know each of the interns and staff members. Connecting to others who have the same goals as me opened my eyes to a new world of science, particularly biophotonics! Working in Dr. Susan Spiller’s lab began with CBST and will continue throughout my time at Mills. Her interest in my progression of biology was truly amazing. Everyone in my lab made a huge contribution to making my time positive, especially my lab partner. We started projects and finished every single one. I learned different types of lab techniques which were beneficial to any biology lab that I might encounter later in life. I remember how much I learned from the First Week Intensive; the judgment exercises were very helpful to see how I viewed myself and how I view others, which are very important perspectives in science and life. The Retreat at Tahoe was more than I could’ve imagined! I met MORE people who had the same interests as me, and I met some who had already taken the path I want to take in life. I am thankful to be a part of the 2011 Summer Internship group!
A SEISMIC EVALUATION AND BUILDING CODE ASSESSMENT USING THE SAP-2000 COMPUTER SOFTWARE
Rosa Serrano and Sashi Kunnath
Building codes have been established in order for engineers to have a standard set of guidelines to perform a building design. Since earthquakes cannot be predicted, buildings are designed according to the provisions in current codes such as ASCE-7 (American Society of Civil Engineers) and IBC (International Building Code). Damage to structures in recent earthquakes provides the need for evaluations to be done on buildings designed by earlier codes. A building evaluation will provide an assessment of how the structure would perform during earthquakes as well as information on the adequacy of code-based structural design. In this study, a steel moment frame constructed in 1976 was chosen to conduct a seismic evaluation. The six story symmetrical building was designed in Southern California according to the 1973 code. The building was analyzed to determine if the structure would be adequate when compared against the most recent code, ASCE 2007. A dynamic analysis was conducted on the building using the computer software (SAP-2000) to determine the seismic performance of the existing building. Various earthquake accelerations were then applied to the steel building frame to determine the critical earthquake magnitude that would have the most significant effect on the structure. Once a critical earthquake has been selected, the earthquake’s intensity will be increased until the building reaches a failure point. With the obtained results decisions could be made on appropriate seismic retrofitting and on necessary adjustments to the building code.
I could have never predicted what I experienced in the Summer CBST Internship.
NAAG Peptidase Inhibitor Prodrug, PGI-02776, Improves Motor And Cognitive Behavior In Experimental TBI
Rahil Ghiasvand1, Gene Gurkoff, PhD2, Ken Van, MS2 and Bruce Lyeth, PhD2, (1)University of California, Davis, Vacaville, CA, (2)Department of Neurological Surgery, University of California, Davis, Davis, CA
Over 1.7 million people suffer from Traumatic Brain Injury (TBI) annually in the United States. Hypoxia, a secondary injury, is frequently associated with TBI. TBI+hypoxia causes cell death through excitotoxicity, an excessive release of glutamate. Cell death correlates with poor motor and cognitive outcome following injury. N-acetylaspartylglutamate (NAAG), a peptide neurotransmitter, is released with glutamate following injury and activates presynaptic group II metabotropic glutamate receptors inhibiting further glutamate release. Hydrolysis of NAAG into NAA and glutamate by glutamate carboxypeptidase reduces presynaptic inhibition and increases glutamate concentrations resulting in enhanced excitotoxicity. We hypothesize that PGI-02776, a NAAG peptidase inhibitor, will prevent hydrolysis of NAAG, reduce excitotoxicity, and increase motor and cognitive performance in rats following TBI+hypoxia. Using a lateral fluid percussion (LFP) device we induced moderate TBI in rats followed immediately by 30 min of hypoxia. PGI-02776 was administered intraperitoneally immediately after hypoxia. Animals were analyzed on the beam walk and Morris water maze (MWM) to test motor and cognitive behavior respectively. CA2-3 pyramidal cells in the dorsal hippocampus were quantified using stereology. We observed a significant decrease in cell death in TBI+hypoxia rats treated with PGI-02776 and improved performance on the beam walk and MWM when compared to TBI+hypoxia rats treated with saline. In conclusion, PGI-02776 played a vital role in improving motor and cognitive behavior in a rodent model of TBI+hypoxia. Our goal is to translate these data into a clinical trial to determine whether PGI-02776 can reduce the number of people suffering from disabilities related to TBI.
CBST summer internship exceeded my expectations beyond imagination. Not only did I gain extensively through researching at the lab, but my experiences with my fellow inspiring interns and mentors made this summer one of the best. My research involved more than collecting data. I had the chance to analyze, give ideas, and even process the ideas into the research. I wrote an abstract, presented my work, and will now make a poster to present at the SACNAS Conference. The highlight of this summer was feeling like I already had a PhD. Being around doctors, both PhD and MD, I saw what my academia leads to therefore motivating me to work harder. I even had the chance to shadow a neurosurgeon in the operating room. Networking and taking advantage of opportunities is an essential skill I learned. Untangling a human knot, having snowball fights with summer attire while hiking at Lake Tahoe, and going white water rafting were only a few of our adventures. CBST Summer Internship did transform a diversity of students into a group of friends. Developing friends in the science community is a great advantage not only for support, but also for future collaboration. Looking back I am amazed at my gains and am so grateful to be a part of this internship. I highly recommend this internship for those interested in research.
Using COPASI to Simulate the Enhancement of Enzymatic Activity in PLP
Roque Troz, Delmar Larsen
As new ways of data collection arise, there is a need for an increase in the ways in which we can also interpret data. COmplex PAthway SImulator (COPASI) is a computer program designed to simulate and analyze biochemical reactions and their properties. The primary experiment at hand is determining how much light can enhance enzymatic reactions that are not specifically induced by light to begin with. Aspartate aminotransferase (AAT) is an enzyme that can interchange the alpha amino group between glutamate and aspartate. This unique property becomes significant in the metabolism of amino acids. However, AAT can only carry out its designated task in the presence of the cofactor pyridoxal-5'-phosphate (PLP). PLP is a variation of vitamin b6, which is a chromophoric co-factor required for catalyzed activity. Although a chromophore, the enzymatic activity is light independent and thus will proceed without presence of light. PLP can still absorb light, so the next step was to see how exposing PLP to light affected the overall reaction. Initial observations have noted significant increases in enzymatic activity when additionally exposed to light. The COPASI data can serve well as a comparison that will help aid in validating and quantifying how extensively light can enhance these reactions. Although the simulations were not obtained convincingly, there still appears to be promising future ideas in this field.
This summer was another unforgettable experience thanks to CBST. I was fortunate enough to once again have a friend sublet a room to me for the summer in the peaceful Davis community. Additionally, I met many wonderful interns who were from a broad range of different backgrounds. Without my fellow interns along with the CBST staff, I highly doubt the experience would have been as good as it was. The first week consisted of activities which were composed of many ice breakers and allowed many of us get a more personal glimpse into the lives of others and forced us to communicate with people who we might not have ever spoken to otherwise. The seminars along with the networking were invaluable to my personal and professional life. In the lab setting, I overcame an uncomfortable setting by accessing and using all the resources at my disposal, and then some. I had to become fluent in the computer program COPASI; I had never used any computer program of the sort. Fortunately I got in contact with one of the creators of the program and to my astonishment, he was willing to help me become familiar with the program. The annual retreat was arguably the highlight of the summer. We got to enjoy the beautiful landscape of Squaw Valley and also hear the research of some of the brightest minds in the world. Venturing in uncharted territory with the research assignment I was given, meeting dozens of motivated and intelligent students like myself, and the wise words of many experienced individuals has allowed me to grow in confidence, resourcefulness, establish connections and also expanded my horizons in my academic career. Thank you CBST!
Identifying the factors in FBS that inhibit the binding of α2 integrin-transfected K562 cells to One-Bead-One-compound combinatorial libraries containing components of cholic acid or cholesterol
Ruth Lopez, Dr. Wenwu Xiao, and Dr. Kit Lam
The whole cell binding assay is commonly used to identify ligands specifically targeting surface receptors of cancer and stem cells from one-bead-one-compound (OBOC) combinatorial libraries. Several ligands such as LLP2A, LXY1and LXW7 that bind to different tumor cell types have been discovered using this methodology. α2-transfected K562 leukemia cell line has been experimented over time to identify specific ligands against α2 integrin and continues to show negative binding results when screened against several OBOC libraries. Recently, we have performed bead screening against α2-tranfected K562 cells with Dekai and LOR series of libraries with branched hydrophobic components such as cholic acid or cholesterol. Results revealed that cells in old medium, in which cells have been growing for 4-5 days, showed stronger binding to these libraries beads, and cells in fresh media demonstrated no binding at all. Adjustment of the pH of the fresh medium to slightly acidic conditions by adding hydrochloric acid or lactic acid were not able to recover cell binding to the library beads. However, removal of FBS from fresh media illustrated positive binding to the cells. Together, these data suggests that FBS played a role in the binding inhibition of α2-transfected K562 cells to the library. Further inhibition assays using several components in FBS such as low density lipoprotein (HDL), high density lipoprotein (LDL), and serum albumin indicated that serum albumin was responsible for binding inhibition of these cells to the library-beads. We believe serum albumin in FBS binds to the cholic acid or cholesterol moiety of these libraries, thus disrupting the peptide conformation that is necessary for strong binding to specific receptors on the surface of α2-transfected K562 cells. The positive binding peptides identified from the screening may be used to retrieve cells in basal medium with absence of FBS for future experiments.
Being a part of the CBST program has been a once in a lifetime experience. This was my first time experiencing real research in a lab and working towards advancements in Cancer research. The main thing I learned throughout my experience was making sure to remember the reason why I was doing research. It is very easy to get caught up in the details of research, that you often present your findings in such a way that makes sense to only the researcher and fellow colleagues. Being able to translate this information to students and to the public is what was very important to me as a future educator. I thank my researcher Dr. Xiao for teaching me the many different techniques that are used at the Lam lab, Dr. Ana Corbacho for the opportunity to participate in the program, and my fellow interns who made the summer internship program well worth the experience.
The CBST Undergraduate Summer Research Program was by far the best experience of my college career. I had done research in a laboratory at UC Davis last summer, and the opportunity to return to the lab and the project that I was previously assisting in was exciting. The seminars and workshops were greatly beneficial and provided important guidance in not only science, but in other critical fields as well. My critical thinking skills and collaborating skills were greatly improved by this program, and I grew not only as a scientist, but as an individual. I am truly blessed to have met and worked with such amazing individuals this summer and it was very rewarding being able to share this experience with my peers in the program. Through the CBST Research Program, I was able to implement the conceptual knowledge that I had learned in my previous classes to practical applications in the laboratory, which was very rewarding. By being a part of this program, I solidified my desire to pursue a major in the biological sciences department and am motivated to gain even more laboratory experience. In my future, this program will create more opportunities to work in a laboratory when transferring to a university. This is such a unique opportunity for community college students! I am thankful that I was able to have been involved in such an amazing program, and I believe that this experience will aid in my future endeavors at a 4-year institution and in my further academic and social endeavors.
Identifying Pch2 Binding Protein Partners in Saccharomyces cerevisiae
Ryan Solis, Jacob Spector, Sean Burgess
Department of Molecular Cellular Biology, College of Biological Sciences University of California Davis
In eukaryotic organisms, meiosis serves as an essential process in the cell cycle that enables the creation of haploid gametes from diploid cells. This process promotes genetic variation by enabling pairing and crossover recombination among homologous chromosomes to form new allele combinations. Within prophase I of meiosis, the formation of self-induced double stranded breaks (DSBs) occur and result in crossovers between specific pairs of chromosomes. To further continue in the meiotic pathway, the repair of each DSB is critical for the accurate segregation of homologous chromosomes. In budding yeast, Saccharomyces cerevisiae, the Pch2 protein is important for signaling and sensing the accuracy in DSBs repairs. It is speculated that Pch2 works in combination with the pachytene checkpoint to promote an interhomolog bias and can arrest the cell in reaction to chromosomal recombination errors.
It remains unclear what proteins physically interact with Pch2 especially when conducting crossover interference and serving in a dynamic recombination checkpoint. Based on localization to chromosomes in meiosis, it is expected that Pch2 works with a number of protein complexes such as Xrs2. The protein Xrs2 has been identified in a complex responsible for processing DSBs along with Pch2. Though a Yeast Two-Hybrid Screen, we have the potential of discovering more physical Interactors of Pch2. This is possible by carefully isolating transformed yeast cells that enable the transcription of the reporter gene HIS3 when plated on nutrient deficient media. The interacting binding proteins are identified by sequencing the DNA from the plasmids that have been isolated from selected candidates. The sequences are then compared and analyzed along with the yeast genome to identify the interacting proteins. These experiments will be followed by testing the physical interactions of identified proteins using immunoprecipitation and GFP fusions. This approach will allow us to determine localization to chromosomes in the presence or absence of checkpoint activating lesions.
The CBST Summer Internship has been an experience like no other. The opportunity to participate in this program has given me the edge I need to peruse my goals in academia and most importantly, career options. The first few weeks we were given the chance to meet everyone involved in the internship and share useful information that would later prepare us for the research experience ahead. When conducting research, it was quite fulfilling to have the ability to apply all of our previous scientific knowledge in current and potential future discoveries. My most important learning experience was to network effortlessly with various groups to grasp a better understanding of the research conducted at the Burgess lab. Looking back after this internship, I was amazed by the amount of quality work produced by all of us involved and gained an immense amount of knowledge throughout our Formal Research Presentations. Having the opportunity to conduct research under this program has given me a glimpse of potential career options especially life after college. Also it further clarified my understanding of the scientific world. I thank Dr Ana Corbacho, Dr. Gene Gurkoff along with all of the interns. They have made this summer an extremely memorable time that will always be prized.
Development of a chemotaxis assay and identification of chemotactic agents in Giardia intestinalis
Shahane Everett, Anna Wiedmann, Scott Dawson
Department of Microbiology, University of California, Davis
The waterborne flagellated parasite, Giardia intestinalis, causes approximately 280 million intestinal infections world-wide every year. Attachment to the microvilli in the small intestine is believed to cause impairment of absorptive and digestive functions of intestinal cells. There may be a direct correlation between chemotaxis and the mechanism of attachment. Chemotaxis is defined as a cell’s directional change and movement resulting from concentrations gradients of chemoattractants and chemorepellants. This process is uncharacterized in Giardia. By developing a chemotaxis assay for this organism and identifying possible chemoattractants and chemorepellants, we can obtain a better understanding of key molecular processes underlying giardial chemotaxis and assess whether or not chemotaxis plays a role in pathogenicity. To this end, I am developing a technique to observe and characterize chemically guided movements of G. intestinalis to identify chemotactic agents. Several techniques are being tested including (1) agarose plates, which require cells to swim through low concentration agarose towards or away from a test compound, and (2) capillary techniques in which chemotaxis is assessed as a function of number of cells that swim into a syringe or micropipette containing a chemoattractant. Determining the variants Giardia chemotaxes towards will allow us to research the molecular mechanisms and functional causes governing chemotaxis in Giardia intestinalis and possibly characterize the role of chemotaxis in the establishment of giardiasis.
CBST is a program that fulfills every expectation, and more. You are given an opportunity to work in a lab and become part of a research project where learning becomes endless. If that is not rewarding in itself, CBST arranges workshops for you to attend where topics such as building your resume, and future careers are discussed. Lastly, this program organizes a trip to attend a scientific conference with your fellow interns where you can network, have fun with interns, and receive insight on renowned research projects taking place. Being chosen for this internship is certainly an honor. I can sincerely say I have formed life-long relationships with staff and interns at UC Davis and become a stronger student as a result of being a part of CBST internship. Thank you for an amazing opportunity!
Muscle degradation in C2C12 myoblast model upon treatment with dexamethasone
Stephanie Soderberg1, Monica Watson2, David Furlow PhD2
Muscle atrophy is the decrease of muscle size that leads to a decrease in force production, and can affect individuals at every stage of life. Muscle atrophy can be caused by a number of factors including: starvation, immobilization, cancer, and aging. Skeletal muscle is maintained by a balance between protein synthesis and protein degradation, and skeletal muscle in particular is an important reservoir of essential amino acids for the body. It is important that the pathways for muscle atrophy are determined so this balance can be restored and ultimately a new therapeutic target to stop muscle atrophy can be found. Glucocorticoids, a class of hormones, induce muscle atrophy in skeletal muscle by binding to the glucocorticoid receptor (GR), which is a member of the nuclear hormone receptor superfamily. Two genes, MuRF1 and MAFbx, are E3 ubiquitin ligases and are shown to be up-‐regulated with dexamethasone (DEX) treatment. These E3 ligases may target proteins within the sarcomere for degradation, and identifying the dose response effect on sarcomeric structural protein in a Mus Musculus myoblast (C2C12) model will help to determine target genes of MuRF1 or MAFbx. C2C12 myoblasts were differentiated and treated with increasing concentrations of DEX in vitro. We hypothesize that DEX treatment will lead to decreases in protein levels of sarcomeric structural proteins. We also expect to see dose responsive expression of MuRF1 and MAFbx genes with increasing levels of DEX. Further studies should include an in vivo mouse model to study the emergent properties of these pathways.
Yersinia protein kinase A autophosphorylation: Characterization of site-specific mutations
Tasha Barr, Khavong Pha, and Lorena Navarro
University of California, Davis, One Shields Avenue, Davis, CA USA
Pathogenic Yersinia species use the type III secretion system to inject effector proteins into host cells. One of these effectors, the Yersinia protein kinase YpkA, is an essential virulence protein involved in rearrangement of the host actin cytoskeleton and inhibition of phagocytosis. In a mouse infection model, the kinase activity of YpkA was important for Yersinia virulence. Upon translocation, the C-terminal domain of YpkA binds to actin and autophosphorylates on serine residues resulting in increased kinase activity. It has been proposed that actin binding triggers autophosphorylation on Ser90 and Ser95 of YpkA in vitro. Here, we report the identification of 15 additional potential autophosphorylation sites. We generated recombinant GST-tagged YpkA serine to alanine mutants and performed in vitro kinase assays. Our results revealed Ser144Ala, Ser317Ala, and Ser320Ala had decreased autophosphorylation and substrate phosphorylation when compared to wildtype. We previously demonstrated that YpkA inhibits the host Gαq signaling pathway through phosphorylation of Gαq. To gain a better understanding of the serine residues that are critical for YpkA kinase activation in vivo, we performed an immunofluorescence study using HEK cells and GFP-tagged Tubby, a putative transcription factor that translocates to the nucleus upon Gαq activation, as readout for Gαq activation. We have shown that the YpkA mutants (YpkAS90A/S95A, YpkAS144A, YpkAS90A/S95A/S144A, and YpkAS320A) are able to inhibit Gaq signaling similar to wildtype YpkA, suggesting the presence of additional sites in vivo. Identification of the critical autophosphorylation sites will lead to new insights on YpkA kinase activation and its role in Yersinia virulence.
After being able to participate in the CBST 2011 Winter Research Program for Community College students, I already knew this summer would be full of excitement and endless learning opportunities. I spent an amazing twelve weeks working with my Principal Investigator, Dr. Lorena Navarro, and graduate student, Khavong Pha, in the Department of Microbiology at UC Davis. Coming from community college, this was an excellent opportunity to become accustomed to a new campus and a new city prior to the start of the fall quarter. This internship is unlike any other thanks to all the exceptional work that is put in by numerous people. Dr. Ana Corbacho is always an inspiring and outstanding director. The time Dr. Gene Gurkoff spends with the interns is invaluable and he is always incredibly helpful. Dr. Ken Burtis has done a tremendous amount of work for the FASTRAC students ensuring the best transition possible while transferring to a university. The CBST Annual Retreat at Squaw Valley Resort in Lake Tahoe was amazing. Volleyball, swimming, hiking, and campfires were a great chance for the interns to bond and learn from one another. This summer internship far exceeded my expectations. Anyone can find a place at the Center for Biophotonics no matter what major or background. Overall, it has been a phenomenal and rewarding summer becoming a member of the CBST family. Thank you CBST, the Navarro lab, and my fellow interns for making this an unforgettable summer.
Study of photobleaching effect induced by high-repetition rate UV fiber laser on arterial vessel tissue: Application to the development of an intravascular optical diagnostic system
Thong Nguyen1, Yang Sun2, Laura Marcu2,3
University of California Davis, Davis, CA1, Department of Biomedical Engineer, Davis, CA2, NSF Center for Biophotonics Science and Technology, Sacramento, CA3
Atherosclerosis is one of the leading causes of deaths in modern society. The rupture-prone plaque can be characterized by time-resolved fluorescence spectroscopy (TRFS) based on the fluorescence decay characteristics of endogenous fluorophores of arterial wall including collagen, elastin, and lipids. However, the clinical application of TRFS has the limitations of time-consuming data acquisition and complex processing of the results (on the order of tens of seconds). Our novel approach, simultaneous time and wavelength resolved fluorescent spectroscopy (STWRFS), combining multiple bandpass and dichroic filters with optical fibers of different lengths, enables rapid tissue diagnosis (on the order of milliseconds). Due to the fast data acquisition, we are able to translate the STWRFS technique to a near real time intravascular catheter- based system by using an ultrafast fiber laser (355 nm, 1 MHz). Since the laser with high repetition rate is used in such sytem, the photobleaching effect needs to be assessed for different experimental parameters, such as excitation energy, pulse repetition rate and rotational scanning speeds (30 Hz, 15 Hz, and 1 Hz) with the assumption that minimal bleaching would occur at a higher scanning speed. Emission intensity and average lifetime information obtained from 9 porcine aorta specimens will be measured at different linear scanning speeds equivalent to those rotational speeds for intravascular application at several excitation energies and pulse repetition rates. The procedure will be repeated again to statistically analyze the photobleaching effect as well as the fluorescence recovery. Using the results of this experiment, we hope to determine the optimal instrumental parameters for the catheter- based STWRFS system coupling with the ultrafast fiber laser.
During the time working at Dr. Marcu’s lab under the mentorship of Dr. Sun, I was introduced to a cutting-edge technology of biomedical research on development of diagnostic tool for cardiovascular disease. As a person who consider the career in cardiothoracic surgery, this experience was nothing short of amazing. Although working in an engineering lab is a step out of my comfort zone as a chemist, I gained enormously new amounts of knowledge and more importantly, recognized that the answer to a problem sometimes requires the interdisciplinary approach. One of the highlights of the program was the First Intensive week which had many activities such as the outdoor games and group photo taking for interns to get to know each other. The Lake Tahoe Annual Retreat was my first time to attend a meeting of researchers. There were a variety of the seminar topics that provided insights into the world of research and the industry of Biophotonics. I was fortunate to meet with a MD/Ph.D student from UC. Irvine who was shared with me his advice for pursuing medical career. One of the most nerve wrecking, yet enjoyable learning experiences was the Research Symposium. I was nervous at time since I just got to do my experience a few days before the presentation. However, it was fine and I was astounded of the research my peers were doing as well as their great presentation skills. I would recommend the CBST internship to anyone interested in research and/or professional career.
Regulation of oxygen radical formation as a treatment for cell death in a rodent model of pediatric traumatic brain injury
Tomas Tesfasilassie1, Bruce Lyeth, PhD2, Gene Gurkoff, PhD2 and Marike Zwienenberg-Lee, MD3, (1)Neuroscience , Universtify of California, Davis, Davis, (2)Department of Neurological Surgery, Universtify of California, Davis, Davis, CA, (3)Neurological Surgery , Universtify of California, Davis, Davis
Over 1.7 million people suffer from traumatic brain injuries (TBI) each year in the United States, surpassing multiple sclerosis, HIV/AIDS, breast cancer, and spinal cord injuries combined. TBI is the number one cause of death and disability in the pediatric population. One of the many consequences of TBI is cell death which is correlated with deficits in motor and cognitive skills. Cell death can be caused through the formation of free radicals, more specifically reactive oxygen species (ROS). In this study our goal is to characterize the efficiency of selenium, a scavenger drug, in preventing ROS-mediated cell death in a rodent model of pediatric TBI. We hypothesize that administration of selenium after TBI will diminish neuronal cell death and improve behavioral deficits. In order to test whether selenium can decrease cell death we first developed a model of pediatric brain injury that would generate significant cortical and hippocampal cell death as well as cognitive deficits. Using a closed skull cortical impactor we were able to generate a range of TBI severities in pediatric rats as determined by levels of cortical and hippocampal cell death. Subsequent studies will first focus on whether cell death is correlated with behavioral deficits followed by evaluation of selenium as a potential treatment for TBI. If we can demonstrate the efficacy of treatment with ROS scavengers in our rodent model then our ultimate goal is to translate selenium into a therapy to improve outcome in pediatric patients suffering from TBI.
Words can not express the gratitude that I have for this program. From the first week intensive to the last few days of presentations, this program has provided me with priceless knowledge. Coming into the program I was spectacle about research and whether I would like it, but by the end of CBST a certain love of research has bloomed in my heart. Surrounded by intelligent mentors who care about you and hard working interns has been the recipe for one of the best summer of my life. If I could I would rewind time and do this program again and once more after that. I would strongly encourage anyone who is interested in research or even pondering what research truly is to be involved in this program.
Bioengineering a Small, Red Fluorescent Tag For Future Use in HIV Research
Valerie Metea, Rosa Meza-Acevedo, Kristen Beck, Monica Bower, Mo Kaze, Alex Magallan, Stefanie Yoshizuka, Ling Ou, Nathan Rockwell, Clark Lagarias and Susan C. Spiller
Fluorescent tags are a very effective method to label proteins. GFP (green fluorescent protein) is the most widely used fluorescent tag, but as it is green fluorescent, the tags cannot be visualized beyond a certain depth into cells or tissues. Red fluorescent light travels farther, thus scientists can observe a wider range and depth by microscopy. There are red fluorescent tags currently available on the market, but we are searching for a shorter amino acid sequence to lessen the possibility of interference with other processes in living cells. The Spiller Lab is working with the Class II GAF domains of five distinct genes from the T. elongatus genome. My project this summer was to work with one of these genes in particular, tlr1999. To ensure we knew the behavior of the unmutated form of the gene, I expressed and purified samples of the 1999 truncated GAF domain. The next step is to make a point mutation of cysteine to aspartate to prevent photoreversibility, ensuring light absorption and fluorescence in the longer wavelength red spectrum. Our ultimate goal is to bioengineer a small, red fluorescent tag (SmuRF) that can be used to visualize the lymphocyte cytoskeleton interacting with green fluorescent labeled HIV. Visualizing the details of the HIV infection process with advanced microscopy techniques may allow researchers to obtain insight and progress towards a cure.
"The CBST Summer
Internship program was not something I considered participating in when I
initially applied to work in my lab, but I'm very glad I ended up changing my
mind. Through the weekly seminars and talks with different professionals
associated with the program, I began to see the wide field of career options
that scientific research offers. Working with Dr. Spiller in her lab has taught
me many skills necessary for future laboratory research and also has taught me
an appreciation of all the work that goes into scientific progress. Through the
first week intensive and the weeks that followed, I enjoyed spending time with
a great group of people. It has been pretty amazing to spend time with the
other interns, many of whom have future goals similar to mine and to see how
the differences we all learned about in the first week intensive activities
drive us towards those goals."
Role of Sonic hedgehog Signaling during Regeneration of Muscle and Neural Tissue in "Xenopus laevis"
Veronica Hernandez, Laura Borodinsky
Sonic hedgehog (Shh) is a signaling molecule that plays a key role in the embryonic development of the nervous system. This protein is secreted from ventral structures to the developing spinal cord, and regulates the differentiation of ventral spinal phenotypes. The action of Shh on skeletal muscle development remains unclear. Xenopus tadpoles exhibit a remarkable capacity to regenerate the amputated tail offering an ideal model for the study of muscle and spinal cord regeneration. Here we tested the hypothesis that Shh signaling is important for muscle and spinal cord regeneration upon injury. Under anesthesia, we amputated tails from stage 37-39 (53-56 h post fertilization) Xenopus laevis tadpoles and incubated them in control saline or cyclopamine, a Smoothened, Shh co-receptor, antagonist, for 24, 48 and 72 h. Samples were then fixed and processed for whole-mount immunostaining in order to evaluate the extent of muscle and spinal cord regeneration in control and experimental groups. Confocal scanning of stained samples followed by Image J particle analysis reveals that blocking Shh signaling increases the area of regenerated muscle as early as 24 h post amputation. Analysis of the effect of Shh signaling on the regenerating spinal cord is underway. Because blocking Shh signaling leads to faster and more robust regeneration of the muscle, we will test whether enhancing Shh signaling inhibits muscle regeneration by incubating tail-amputated tadpoles with SAG, Smoothened agonist. By better understanding natural muscle and neural tissue regeneration, new therapeutic approaches can be developed to help those with neural and muscle injuries.
Accepting the invitation to be part of UC Davis CBST internship was one of the best decisions I have done in my life. I gained so much valuable knowledge during the 8 weeks in Davis and Sacramento. I not only gained valuable laboratory skills, but I also gained networking skills, and thru this experience I was able to further narrow down the career path I want to follow.
This is the first internship I have done. Coming into a lab setting I was nervous because I didn’t know what to expect. I was fortunate enough to have been assigned to Dr. Laura Borodinsky’s lab. I wasn’t expecting for the primary investigator to be the one that was going to mentor me. Since day one, Dr. Borodinsky, and the rest of the lab members were attentive for any help I may need. Dr. Borodinsky wanted me to have a well-rounded experience with research so she gave me the option of choosing the experiment I wanted to do. The project I decided to do, focused on the role of Sonic hedgehog signaling in the regeneration of skeletal muscle and spinal cord in Xenopus laevis, commonly known as the African clawed frog. In order to do my experiment, I learned how to do whole-mount immunostaining, and how to use a Nikon A1 confocal microscope to do florescence imaging. An important aspect I learned about research is: not everything goes as expected. There are cases when you follow protocol as carefully as possible and the results are not completely off or there are no results at all. In the beginning of this internship I knew I wanted to go into some kind of biology related research. As I completed my experiments and gained more knowledge about the branch of research my mentor was part of, I narrowed down my future career goal to biomedical research.
CBST activities away from the lab were essential to my gain of knowledge. I learned how to read and understand scientific journal articles, how to improve my resume and CV, how to write an abstract, and how the growing field of biophotonics is developing new technologies in the sciences. Dr. Ana Corbacho, Dr. Gene Gurkoff, and Dr. Marco Molinaro were amazing at providing us with these skills. This summer internship was memorable not only because of all the skills and learning I did, but because my fellow interns also made it special.