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2017 Research Showcase
Master's Abstracts
Abstract Title : Invasion Dynamics and Removal Strategies of Rhodactis howesii at Palmyra Atoll
Abstract : Invasive species are known to cause turnovers in dominant biota, often leading to dramatic changes in ecosystem structure and function. Recently in the Central Pacific, parts of the coral reef at Palmyra Atoll have experienced a local benthic domination by the corallimorpharian Rhodactis howesii, endangering the atoll's relatively pristine reef ecosystem. Using a permanent plot time-series spanning 7 years, we track the succession patterns of this invasive species at Palmyra Atoll. Results show increases in R. howesii cover at all sites where it was present. Percent cover of other benthic functional groups indicate that R. howesii invasions were most dramatic at sites with lower percent cover of hard coral and higher cover of turf algae and crustose coralline algae. This suggests that the spread of R. howesii throughout Palmyra can be predicted by benthic composition and potentially topographic structure. The unabated spread of R. howesii has demanded the creation of in situ management practices involving active removal of this highly competitive species on Palmyra's reefs. Given the predictability of R. howesii invasion based on benthic composition, targeted removal and coral transplantation in locations experiencing, or expected to experience R. howesii invasion could potentially halt and prevent further damage to Palmyras reefs.
Advisor : JEAN Y J WANG
Abstract Title : Nuclear ABL Programs Extracellular Vesicles for Transmission of Ionizing Radiation-Induced Bystander Effects
Abstract : Radiation therapy is used to treat many diseases such as cancer. However, untargeted side-effects called bystander effects are observed in patients after being treated with radiation. Previous work from our laboratory has shown that extracellular vesicles derived from irradiated conditioned media from mouse embryonic fibroblasts mediate radiation induced bystander effects by inhibiting clonogenic survival, increase γH2AX, and increase ROS. However, when the protein Abl is mutated so that it no longer can enter the nucleus, the EVs from the irradiated conditioned media of these cells do not cause bystander effects. When nuclear Abl is reconstituted into these mutant cells, the EVs from the irradiated conditioned media of these cells cause bystander effects such as increase γH2AX and increase ROS. This suggests that nuclear Abl is required for extracellular vesicle mediated radiation induced bystander effects. Additionally, there are Abl inhibitors that are FDA approved and might be viable treatments to reduce bystander effects in patients undergoing radiation therapy.
Abstract Title : Molecular Mechanisms of ATF6 Mutations in Photoreceptor Diseases
Abstract : Achromatopsia (ACHM) and Cone-Rod Dystrophy (CORD) are characterized by the absence of cone photoreceptor function, the progressive loss of cone photoreceptors, and for CORD, the progressive loss of rod function. Recently, we found mutations in ATF6, an endoplasmic reticulum (ER) stress regulator gene, in ACHM and CORD patients. Under ER stress conditions, ATF6 migrates from the ER to the Golgi apparatus, where it is cleaved by site-1 and 2 proteases to release its cytosolic bZIP transcriptional activator domain. The bZIP transcriptional activator upregulates chaperones and enzymes necessary for ER protein folding and homeostasis. Here, we functionally characterize the molecular defects of all known ATF6 mutations found in ACHM or CORD families. We performed site-directed mutagenesis to introduce disease ATF6 mutations and expressed the recombinant mutants in HEK293 cells. Primary fibroblasts were collected from two separate ACHM families and 1 CORD family carrying different ATF6 mutations. The ATF6 mutants were assessed for their abilities to traffic from the ER to Golgi, undergo proteolytic cleavage, and transcriptionally upregulate ATF6 downstream target genes. Class 1 ATF6 mutants exhibit impaired ER-to-Golgi trafficking and, therefore, have impaired proteolysis to release the bZIP transcriptional activator domain under ER stress conditions. Class 2 ATF6 mutants produce truncated cytosolic ATF6 fragments that contain functioning bZIP transcriptional activator domains, resulting in ATF6 being able to constitutively activate downstream targets even under normal ER homeostasis conditions. Class 3 ATF6 mutants have an absent or defective bZIP transcriptional activator domain, resulting in the loss of transcriptional activity.
Abstract Title : Transduction of DRG Neurons Following Intrathecal Infusion of AAV9
Abstract : The delivery of AAV9 vectors via the cerebrospinal fluid (CSF) drives strong gene expression throughout the nervous system including the cerebral cortex. We are interested in using this method to deliver Brain-Derived Neurotrophic Factor (BDNF) for the treatment of Alzheimer's Disease (AD). The Tuszynski Lab has previously shown that BDNF provides neuroprotective benefits during early stages of AD. However, expression of BDNF within cells of the dorsal root ganglia (DRG) could potentially have negative side effects, such as hyperallodynia. My project addresses three questions. First, we investigated the level of gene expression in the DRG following intrathecal infusion of AAV9-CAG-eGFP, and found that 26.4% of neurons expressed GFP. Second, 99.5% of transduced cells were observed to be neuronal based on co-localization with CaMKII. Third, there was not a significant difference in transduction between DRGs from cervical and lumbar spine. These results demonstrate that intrathecal infusion of AAV9 drives strong gene expression within both cervical and lumbar DRG neurons, and that the potential for adverse side effects from this transduction should be considered when this technique is used to deliver genes to the brain.
Abstract Title : Potential Role of Siglec-XII in Human Carcinomas
Abstract : Siglecs, or sialic acid binding immunoglobulin-like lectins are signalling molecules expressed in a highly specific manner on cells of hematopoietic origin and are important for regulating the immune response in humans and higher primates. Sialic acids (Sias) are sugars found on the outer ends of vertebrate glycan chains and are recognized by Siglecs. Siglecs are characterized by two extracellular, immunoglobulin-like domains. These are the variable, sia binding V-set domain and the constant C2-set domain. Siglecs also possess a cytoplasmic immunoreceptor tyrosine domain which is either activating or inhibitory. The CD33-related subgroup of Siglecs all possess similar cytoplasmic motifs and are expressed specifically by myeloid cells such as neutrophils, monocytes and macrophages. These CD33-related Siglecs play an important role in lymphocyte activation and regulation of the inflammatory response. One of the CD33-related Siglecs is Siglec-XII, which is undergoing strong negative selection in humans. While all higher primates possess a functional, sia binding Siglec-12, human Siglec-XII is an ortholog of the chimpanzee Siglec-12, having completely lost its ability to bind sialic acids during evolutionary divergence. This loss of binding in Siglec-XII is present in all humans and is caused by mutations in its two V-set domains. In addition, about 60% of humans possess homozygous inactivation of the Siglec-XII gene caused by either a frameshift mutation or a premature stop codon. These mutations result in nonsense mediated decay of the truncated gene product. As a result, only 40% of humans are expected to express Siglec-XII. While Siglec-XII and other CD33-related Siglecs are known to be expressed on cells of hematopoietic origin, Siglec-XII is one of three Siglecs of this subgroup that have been discovered to be expressed on various epithelial tissues as well. In initial screening, Siglec-XII was found to be expressed on both normal prostate and prostate carcinoma tissue. Further investigation was performed with a PC-3, prostate carcinoma, cell line which possessed the homozygous inactivation of Siglec-XII. Restoration of Siglec-XII expression in this cell line induced altered regulation of genes associated with carcinoma progression and also doubled the growth rate of these cells when implanted in nude mice. Here we will compare expression of Siglec-XII in normal epithelial tissue and carcinoma tissue collected from a diverse cohort of individuals to explore the possible role of Siglec-XII in human carcinomas further.
Abstract Title : Insight into the Mechanism of Oximes Against Organophosphate Nerve Agents in Acetylcholinesterase
Abstract : Organophosphates (OP) inhibit the enzyme acetylcholinesterase (AChE) and are effective, biodegradable pesticides when judiciously used. However, certain volatile OP’s have been employed insidiously in terrorism, as evident by the recent Sarin gas attacks in Syria. Current antidotes used to treat OP-poisoning are oximes, which function through nucleophilic attack against the OP, thus "reactivating" the enzyme. To develop efficacious OP antidotes, we focus on the studying the kinetics of oxime-induced reactivation of AChE, in order to understand their mechanism of action against the inhibited enzyme. Studies of oxime catalysis show that the oximate anion is the nucleophile in solution. To investigate whether a similar mechanism occurs in the active center of AChE, we examined the pH dependency and kinetic isotope effect of oxime-induced reactivation on the OP-inhibited AChE. General base catalysis predicts that reactivation rate constants would be expected to increase with pH, since the oximate anion is the attacking species. However oximolysis of the conjugated OP within the active gorge of AChE shows a broad pH dependence with a peak reactivation rate constant at pH values around 7.5. Kinetic isotope studies demonstrate a 50% drop in rate constant, despite the lack of reduced nucelophilicity that arises from in-solution kinetic isotope oximolysis reactions. These findings suggest the possibility of hydrogen bonding networks that allow for the extraction of oxime hydrogen for oximate formation in the active center gorge of AChE.
Abstract Title : Conservation Applications of Stable Isotope Analysis: Can Pups Be Used as Proxies for Adult Females in Dietary Studies on Northern Fur Seals?
Abstract : Stable isotope analysis (SIA) can be used to reconstruct food web dynamics to better understand animal foraging patterns, making it a useful tool for ecologists. SIA determines the ratio of heavy to light isotopes of carbon (13C/12C or δ13C) and nitrogen (15N/14N or δ15N) in a given tissue. These isotopes reflect dietary and habitat choices of the animal in question over the time period represented by the analyzed tissue’s protein turnover. Tissues with fast protein turnover rates (e.g., plasma, liver) reflect isotope values from an animal's recent diet, whereas slower turnover tissues (e.g., muscle, hair, bone) reflect diet and habitat use over longer time periods. However, obtaining tissue samples for analysis from adults (especially large animals that are hard to capture) is difficult compared to their young, and this is the case with otariid pinnipeds. We used northern fur seals (Callorhinus ursinus) as a model species to test the viability of using stable isotope values from pup whiskers as a proxy for data from unrelated adult females in the same population, which has been suggested in previous studies demonstrating pup isotope data accurately reflecting that of their mother’s during gestation and nursing. A longitudinal record of stable isotope values acquired over the near lifetime of an individual are stored in sequential order in the vibrissae, or whisker, of an otariid, thereby generating a time series of isotope values reflecting foraging patterns over time. We collected a single vibrissa from five adult females and five pups, cut them into segments, and analyzed the δ13C and δ15N isotope values for each segment to create a time series of data for each animal. We corrected for differential whisker growth rates between adults and pups, then compared time series for the pups to those of the adults. Preliminary results suggest that pup data can indeed be used as a proxy for adult female data, but further statistical analysis is still needed. An understanding of the relationship between isotope signature patterns of pups and adult females could potentially eliminate the need to sample from adults during pregnancy and nursing, reducing risk to both animals and the human handlers. This would also improve the methodology of stable isotope studies on pinniped populations, increasing their applications for ecological and conservation research.
Abstract Title : Where, when, and why corals grow, shrink, and die: Exploring patterns and natural variations in the spatial distributions and life history of the scleratinian coral genus Pocillopora
Abstract : Coral reefs of the world are currently under a host of stressors due to global threats such as seawater temperature rise and ocean acidification as well as local anthropogenic pressures such as fishing and eutrophication. Thus, there is a dire need for effective monitoring and conservation management efforts in order to protect one of the most diverse and valuable ecosystems on the planet. Such efforts will only prove successful if the scientific community can accurately convey effective conservation strategies to the local populations directly dependent on these local ecosystems. Understanding the life history characteristics of scleratinian corals is a critical component that helps accurately predict the future status of these reefs in a changing world. Yet, with such high species diversity, little is known about the life history characteristics of every specific taxa. Coral reefs are inherently complex systems and such basic understanding of their functioning needs to be examined in absence of local stressors and minimized global impact. Palmyra atoll, one of the most remote and pristine coral reefs in the Central Pacific, provides such a natural laboratory for studying coral reefs without the confounding factors of local human impact. Palmyra serves as a baseline to examine natural variations in spatial distributions and life history characteristics in a pristine reef. We use two dimensional orthoprojection technology (large scale photogrammetry) in a time series fashion to examine four 100m˛ sites in Palmyra. This technology allows us to accurately capture the size, spatial distribution, and fate of over 1000 colonies of the common coral genus Pocillopora from 2013 to 2014. We describe the observed natural variation in the proportions of growth, shrinkage, and mortality in Pocillopora colonies, as well as the different patterns in the spatial distribution of coral colonies as a function of their fate. Our results may aid in more effective management of coral reefs, as well as provide a better prediction of what they will look like in the future.
Abstract Title : Dysfunctional Mitochondria May Promote Fatty Liver Disease and Tumorigenesis
Abstract : Hepatocellular carcinoma (HCC) is the second most deadly cancer worldwide. HCC can result from the progression of fatty liver disease and steatohepatitis to tumorigenesis. Nonalcoholic steatohepatitis (NASH) is becoming an increasing problem in the Western world with increasing occurrence of obesity. NASH is characterized by inflammation and fibrosis, which can advance to cirrhosis and HCC. Our previous study to elucidate the molecular mechanisms of HCC showed that two molecules, Shp2 and Pten, have a synergistic effect to prevent hepatocarcinogenesis. The deletion of Shp2 and Pten led to severely fatty livers and early-onset NASH. Since mitochondria are the major site for fatty acid metabolism, the aim of this study was to explore the role of mitochondria in the development of fatty liver and its progression to HCC using our Shp2 and Pten knockout mouse model. We quantified the mitochondria and examined mitochondrial functions, including respiration, structure, integrity, motility, and gene expression. We show that mitochondrial structure and integrity were compromised in Pten knockout and Shp2/Pten double-knockout mice. Mitochondrial numbers were dramatically decreased in double-knockout mice. We also found that mitochondrial metabolic pathways are dysregulated in Pten knockout mice. These results demonstrate that dysfunctional mitochondria are drivers in the progression of NASH to HCC, and may be a good therapeutic target.
Abstract Title : Visualization of the interaction between Wnt9a and its specific Fzd receptors
Abstract : Wnts are secreted signaling molecules involved in many developmental processes and in maintaining adult tissue homeostasis. Previous work in our laboratory has shown that one particular Wnt ligand is important in hematopoiesis, or the development of blood stem cells. Using a Wnt transcriptional reporter cell line, we have identified two of the fourteen zebrafish Frizzled (Fzd) receptors that our candidate Wnt protein can interact with. Using expression constructs of the Wnt ligand and Fzd receptors fused to different fluorescent proteins or a protein tag, we have developed a strategy to visualize the interaction between Wnt and its Fzd receptors. By exploiting one mechanism by which cells negatively regulate their own signal, cells can internalize the entire ligand-receptor complex. We can then visualize this internatlization by following the fluorescence from these fusion proteins, allowing us to confirm specific interactions between these proteins.
Abstract Title : Microalgae as a Biotechnology Expression Platform: Visualization of Recombinant Protein Expression in the microalga C. reinhardtii with a Self-Cleaving FMDV 2A Peptide
Abstract : Recombinant proteins are widely used for industrial purposes including manufacturing, pharmaceuticals, and cosmetics. Using microalgae as a biomanufacturing platform has recently piqued research interests due to their photosynthetic or heterotrophic capabilities. Another benefit of using microaglae is the native cellular machinery that has the ability to correctly fold and secrete recombinant proteins. Some currently existing obstacles include low heterologous protein expression and poor expression and secretion vectors specific to microalgae. Here we developed a molecular biology tool to overcome some of these challenges by increasing expression and ease of quantification of a secreted recombinant protein. This was achieved by fusing the recombinant protein to a self-cleaving fusion peptide from the Foot and Mouth Disease Virus (FMDV), a secretion peptide, and a GFP fluorescent marker. The ratio of the GFP to protein was established so that by measuring GFP, an accurate quantification can be obtained of the secreted recombinant protein of interest. This transcriptional fusion tool creates a new technique for quantifying recombinant protein in the microalga Chlamydomonas reinhardtii for biotechnology and industrial use.
Abstract Title : Circulating Triglycerides and the Brain's Reward Circuit
Abstract : Little is known about the role that circulating triglycerides have on the brain. While understudied, dietary triglyceride centered neurophysiology holds immense implications regarding metabolic disfunction, diabetes, and obesity - all of which impart strong health and societal burdens. It has been shown that circulating triglycerides in the brain reduce preference for palatable food and motivation to engage in food seeking, both of which are behaviors that are in part determined by mesolimbic function. We suspect that a dietary triglyceride processing enzyme known as lipoprotein lipase (Lpl) is the key sensor involved in triglyceride sensing in the brain, more specifically within the context of reward and motivation. By chronically catheterizing the carotid artery of mice, we are able to directly infuse lipids to the brain via catheter in order to emulate post-meal neurophysiology. Preliminary data reveals that circulating triglycerides upregulate markers of neural activity in the VTA, sub-thalamic nucleus, substantia nigra pars reticulata, pallidum, and cortex when compared to saline infused control mice. We have also employed fluorescent in-situ hybridization in order to delineate the expression patterns of Lpl and dopamine receptors within the mesolimbic circuitry of the brain. We found that Lpl colocalizes with dopaminergic neurons in the ventral tegmental area (VTA), and with striatal d1/d2 dopamine-receptor expressing medium spiny neurons. Considering the expression patterns of Lpl and the effects of brain triglyceride delivery on neural activity as well as behavior, we have further reason to believe that circulating dietary triglycerides are being detected within the mesolimbic circuitry of the brain, and that Lpl is a key factor in this detection. In order gain further insight into the potential role of Lpl as the primary sensor of dietary triglycerides, future experiments will look at the effects of targeted Lpl impairment.
Advisor : JAMES NIEH
Abstract Title : Effects of new insecticide (flupyradifurone) and low quality sucrose on honey bee flight and mortality
Abstract : Insecticides pose a major health concern for honey bee colonies, particularly those involved in crop pollination. Flupyradifurone (FLU) is a new butenolide insecticide effective against pests that are resistant to neonicotinoid insecticides, but the safety profile of FLU for pollinators is not yet well established. We tested the effects of FLU exposure and food quality (sucrose concentration) on European honey bee flight and mortality. Flight performance was measured using an assay of bee flight ability—bees flying in flight mills. Under FLU exposure, bees that consumed low quality sucrose had a higher mortality rate than bees that consumed high quality sucrose. In addition, higher sucrose diet increased flight distance, time and velocity, though FLU exposure did not cause significant changes. Because agricultural insecticide exposure and poor nectar quality can co-occur, we suggest that further studies are necessary to evaluate the true safety of FLU for honey bees that collect nectar and pollen from FLU-treated crops.
Advisor : JACK BUI
Abstract Title : Type I Interferon Regulates Chemoresistance in Breast Cancer Cells
Abstract : Tumor cells are known to acquire resistance to chemotherapy, leading to tumor relapse in patients. Cancer Stem Cells (CSCs) are a specialized subset of tumor cells that show enhanced resistance and have the ability to initiate tumor formation. Induction of CSCs by chemotherapy is a potential mechanism by which tumor cells acquire chemoresistance. Previous research from our lab has shown that interferon beta expression is upregulated in immune-edited progressor cell lines relative to unedited regressor cell lines in the MCA sarcoma model. Progressor sarcoma cell lines had a greater proportion of CSCs (Sca1+ CD90-) which was associated with greater sphere forming capacity in-vitro as well as increased tumor-initiation potential in-vivo. Treatment of breast cancer cell lines with chemotherapy resulted in an increased in interferon beta expression. This study attempts to elucidate the role of tumor-autologous interferon beta in induction of CSC phenotype in response to chemotherapy.
Abstract Title : PHLPP2 Negatively Regulates Phenylepherine (PE)-induced Cardiac Hypertrophy
Abstract : Crucial cellular events such as death, growth, metabolism, proliferation, and hypertrophy are regulated by phosphorylation and dephosphorylation of proteins. PH domain leucine-rich repeat protein phosphatase (PHLPP) is a serine/threonine phosphatase that has been shown to directly dephosphorylate several members of the AGC family of kinases. Removal of PHLPP2 in neonatal rat ventricular myocytes (NRVMs) induces hypertrophic growth and activates fetal gene expression at baseline and potentiates phenylephrine (PE)-induced gene expression 2 fold over siControl. Since removal of PHLPP2 in NRVMs does not affect Akt phosphorylation PHLPP2 must have the ability to repress cellular hypertrophy through regulation of other unknown targets. G-protein coupled receptor kinase 5 (GRK5), an AGC kinase, has been shown to regulate cardiac hypertrophy through de-repression of gene transcription and by directly binding DNA. Upon down-regulation of PHLPP2, stimulation of NRVMs with the hypertrophic agonist PE induced GRK5 accumulation in the nucleus and export of HDAC5. Conversely, overexpression of PHLPP2 blocked PE-induced hypertrophic growth, re-expression of fetal genes and nuclear accumulation of GRK5 in cardiomyocytes. The translocation of GRK5 by PHLPP2 knockdown was dependent on calmodulin binding. Through siRNA studies it was found that GRK5 is necessary for PE-induced hypertrophy following PHLPP2 knockdown. Here we demonstrate for the first time in cardiomyocytes that knockdown of PHLPP2 regulates hypertrophic growth through GRK5. Understanding the signaling pathways affected by PHLPP2 has potential for new therapeutic targets in the treatment of cardiac hypertrophy and failure.