Student Profiles Archive - Science Poster Day Dean's Prize Winners 2012
Here you will find information about past and present students funded through scholarships administered by the Undergraduate Research Center - Sciences. We are proud of the achievements of our research scholars.
Please click on the program year to get information about the supported students, their mentors and their research projects.
| Ms. Amy Wisdom
Amy Jordan Wisdom
Mentor: Rhonda Renee Voskuhl
Title: Treatment With Estrogen Receptor-Beta Ligand Late in Disease Ameliorates Experimental Autoimmune Encephalomyelitis
Multiple Sclerosis (MS) is an autoimmune disease characterized by inflammation and neurodegeneration. Current MS treatments focus on treating the inflammatory component of the disease and do not directly target neurodegeneration. Estrogen has well-documented neuroprotective effects in many disorders of the CNS, including experimental autoimmune encephalomyelitis (EAE), the most commonly used mouse model of MS. Treatment with an estrogen receptor beta (ERB) ligand has been shown to be effective at ameliorating clinical disease and providing neuroprotection in EAE. However, the protective effects of this ERB ligand have only been demonstrated when administered prior to induction of disease. Therefore, we tested whether ERB ligand treatment could still provide clinical protection and neuroprotection when treatment was initiated after onset of disease. We found that treatment administered both before and after disease induction was effective in ameliorating EAE. In case treatment paradigm, ERB ligand treatment did not affect the peak level of early disease, but promoted recovery during the chronic phase of disease. Our findings suggest that therapeutically administered ERB ligand can successfully treat EAE, which could eventually translate into an effective treatment for MS. Since most adverse effects of high dose estrogen treatment are mediated through ER-alpha, not ERB, novel use of ERB ligands as neuroprotective agents would likely have a very good safety profile.
| Mr. Drake Williams
Drake Winslow Williams
Mentor: Reuben Han-Kyu Kim
Title: Role of IL-36 in Impaired Oral Mucosal Wound Healing and Bisphosphonate-Related Osteonecrosis of the Jaw (BRONJ)
Bisphosphonates (BPs) are potent anti-resorptive agents used to treat bone-associated diseases including osteoporosis and metastatic bone cancer. Reports have indicated that long-term use of BPs can cause BP-related osteonecrosis of the jaw (BRONJ) that occurs at the site of tooth extractions; however, the pathophysiology of BRONJ is poorly understood. In our project, we utilized the BRONJ mouse model, performed a microarray analysis using oral mucosal tissues from the tooth-extracted sites, and found a significant increase in the expression of Interleukin (IL)-36 members (unpublished). In this study, we examined expression and the role of IL-36 alpha both in vivo and in vitro. Immunohistochemical staining was performed against IL-36 alpha on control and BRONJ tissues. Quantitative Real-Time PCR (qRT-PCR) was performed to determine the relative expression of the IL-36 receptor, IL-1Rrp2, and its accessory receptor, IL-1AcP, in four cell types. Finally, the effects of recombinant IL-36 alpha on migration of normal human oral keratinocytes (NHOK) were determined using a migration assay. We found high levels of IL-36 alpha at the oral epithelium in the BRONJ lesions. The expression of IL-1Rrp2 and IL1AcP was high in NHOK compared to other cell types. Finally, we found that IL-36 treatment caused a decrease in the ability of NHOK to migrate. The data suggests that IL-36 is associated with impaired wound healing in BRONJ and that high expression and secretion of IL-36 inhibit migration of keratinocytes in autocrine fashion.
| Ms. Dinali Wijewarnasuriya
Dinali Priasha Wijewarnasuriya
Mentor: Utpal Banerjee
Title: The Role of Cytosolic Calcium in the Maintenance and Differentiation of Hematopoietic Progenitors
In Drosophila melanogaster, the lymph gland is the hematopoietic organ that gives rise to the differentiated myeloid blood cells types that arise from hematopoietic progenitors within the lymph gland. Mechanisms involved in blood progenitor maintenance is an active field of research, and the Drosophila lymph gland serves as a model system to address new signaling pathways or mechanisms involved in progenitors maintenance. Preliminary studies in our lab have suggested that the lymph gland hematopoietic progenitors have high cytosolic calcium compared to the differentiated blood cells. After conducting gain and loss of function experiments on different components of the calcium-signaling pathway, we observed that decreasing cytosolic calcium in the hematopoietic progenitors leads to their differentiation and an loss of the progenitors while enhancing components of the calcium signaling pathway leads to progenitor maintenance. Loss of either nositol trisphosphate receptor or Ryanodine receptor function in the progenitors leads to their differentiation indicating that components of the store operated calcium (SOC) pathway is important for progenitor maintenance. Interestingly, loss of gamma-aminobutyric acid (GABA) receptor function within the progenitors mimics inositol trisphosphate receptor and ryanodine receptor loss-of-function phenotypes. These data obtained by manipulating different components involved in calcium homeostasis suggests a pivotal role for cytosolic calcium in hematopoietic progenitor maintenance.
| Mr. Timothy White
Mentor: Peggy Marie Fong
Title: Spatial and Temporal Variation of Nutrient Limitation and Initial Tissue Nutrient Levels in Tropical Macroalgae
We conducted four macroalgae growth experiments to determine the importance of spatial and temporal variation on the relative strengths of nitrogen (N) and phosphorus (P) limitation along a gradient of nutrient availability. Our study species was Dictyota bartayresina, which was found along the length of Cook’s Bay in Moorea, French Polynesia and often exists in direct competition with coral species. With our temporal growth assay, we demonstrated that the initial tissue nutrient stores of D. bartayresina were temporally dependant by measuring growth rates of samples collected from the same locations on four consecutive days. The temporal N vs P limitation experiment directly supported our hypothesis that the strengths of N and P limitation are highly variable over time. In this experiment, the growth response varied strongly with initial tissue nutrient stores and collection date in all four nutrient treatments (ambient, N, P, or N and P). We confirmed the presence of an initial tissue nutrient gradient in our third experiment, a spatial growth assay. Finally, the spatial N vs P limitation experiment revealed great variability in nutrient limitation between algae collected from different sites that ranged from no limitation to dual limitation. It is important to fully understand algal nutrient limitation if we are to understand how algal growth degrades coral reefs, but these results indicate that this may require a broader investigation than once thought.
| Ms. Lily Vartanyan
Mentor: Sophie X. Deng
Title: Preferential Expression of Potential Limbal Stem Cell Associated Molecules in the Limbal Epithelium
Corneal epithelial stem cells or limbal stem cells (LSCs) are known to reside in the limbus, the border between the cornea and the conjunctiva. Stem cells, in general, have great therapeutic and restorative potential. LSC function at the ocular surface is to replenish the constant turnover of epithelial cells in the corneal epithelium and to maintain the cornea’s integrity and functionality. A deficiency in limbal stem cells can result in ocular discomfort and reduced vision. Putative limbal stem cell markers, such as ABCG2 and ∆Np63a, are preferentially localized to the limbus and expression is absent in the cornea. However, moderate expression is observed in the conjunctiva as well. To successfully isolate LSCs, a definitive cell marker is needed to specifically identify these cells. Microarray analysis identified the overexpression of 143 genes in the limbus over the cornea and conjunctiva. Quantitative real-time PCR results were consistent with these findings. Two of these limbal-specific genes, pituitary homeobox 2 (Pitx2), a transcription factor, and Tenascin C (TNC), an extracellular matrix glycoprotein, were uniquely expressed at the basal and superficial layer in the limbus. Expression was minimal in the cornea for both. Characterizing LSC markers can be used to improve isolation methods, one step in understanding limbal stem cells. The results from these data will provide insights into improving the current treatment for patients with limbal stem cell deficiency by using stem cell therapy.
| Ms. Nany Tu
Nancy Lynn Tu
Mentor: Richard Kent Zimmer
Title: Chemosensory Basis for Keystone Predation
In predator-prey relationships, physiologically requisite biochemical compounds of prey may be exploited and used as honest signals by predators. The extrapallial (EP) fluid excreted by the mantle of the California mussel, Mytilus californianus, contains proteins involved in biomineralization; as these proteins are required for the production of shell, they may serve as the chemosensory signals guiding the primary predators of mussels, seastars, to their preferred prey. Seastar bioassays, comprised of placing the seastar in a tank with prey mimics and tracking its feeding choices, have shown that seastars are as responsive to the extracted and purified EP fluid as a live mussel, indicating that an active recognition cue is found within. After extracting the EP fluid and isolating its components through SDS-PAGE, four component proteins were identified using tandem mass spectrometry: KEYSTONin, paramyosin, heat-shocked protein 70, and an unidentified protein B3. Seastar bioassays have shown a strong feeding preference on KEY-filled faux prey when tested against the control-filled faux prey, thus pointing towards KEYSTONin as being the active cue by which a seastar identifies Mytilus californianus. Seastars are keystone predators and the signals they use can be considered as keystone molecules. Their effects resonate through the intertidal community as seastars reduce competition for space for other sessile organisms by consuming mussels, thereby increasing biodiversity on the rocky wave-swept shores.
| Mr. Vincent Tse
Mentor: Catherine F Clarke
Title: C. elegans fed a Coenzyme Q deficient E. coli diet Live Longer, are Stress Resistant, and Accumulate Less Colonizing Bacteria in their Intestinal Lumen
It has been found that C. elegans fed a diet of GD1, an E. coli bacterium harboring a mutation in the ubiquinone gene ubiG, exhibit an increased life span. This life extending quality of GD1 has been well characterized, but the mechanism is still unknown. In addition, the worms are also more resistant to a variety of stressors including heat and Juglone, a powerful chemical that induces damaging oxidative stress. It was previously believed that this phenomenon was caused by a probiotic health benefit. However through the use of bacteria carrying a GFP expressing plasmid, it has been shown that there is less accumulation of GD1 in the intestinal lumen of worms than those fed the wild type, OP50. We hypothesized that worms fed GD1 have healthier intestinal tracts and are thus more stress resistant than their counterparts fed OP50. After further investigation, GD1 was found to have a fermentative metabolism, are more sensitive to Juglone, and proliferate slower than OP50. These factors combined decrease the ability of GD1 to colonize the intestine. This work can have strong implications in human intestinal tract diseases such as diverticulosis and the impact of microorganisms in our bodies.
| Ms. Mai Tran
Mai Phuong Tran
Mentor: Stephen Smale
Title: Evidence for NF-κB c-Rel Homodimers Binding Cooperatively to IL-12 p40 Promoter to Selectively Regulate Transcription
The transcription factor NF-kB, nuclear factor kappa B, plays an important role in coordinating responses from the innate and the adaptive immune systems. DNA binding is essential in NF-kB dependent transcription. All members of the NF-kB family share partial functional redundancies but differ in specific transcriptional regulation and physiological roles. The NF-kB c-Rel member is important for lymphopoiesis and T-helper cell differentiation. Activation of the pro-inflammatory Interleukin (IL)-12 p40 gene is largely c-Rel dependent. There is evidence that c-Rel homodimers bind cooperatively to the IL-12 p40 promoter, with strong cooperativity at two adjacent NF-kB sites. Cooperative binding was observed only with c-Rel homodimers but not with other NF-kB homodimers or heterodimers. Preliminary electrophoresis mobility shift assay (EMSA) data showed that specific spacing between two NF-kB sites is required for cooperative binding of c-Rel homodimers to the IL-12 p40 promoter. Cooperative binding decreased significantly with a single base-pair insertion/deletion between the two sites and abolished with two- or more base-pair insertion/deletion. Future transfection experiments with luciferase reporter plasmids can be used to test the consequences of spacing changes on transcription activation. Understanding the role of c-Rel cooperative binding can shed light on the c-Rel dependence for IL-12 p40 gene expression and provide insights about a possible mechanism responsible for the specificity mediated by NF-kB dimers.
| Ms. Amy Ton
Amy Nu Ton
Mentor: Luisa M Iruela-Arispe
Title: Progesterone Regulates the Expression of Endothelial-Leukocyte Adhesion Molecules and Inflammatory Cytokines by Endothelial Cells
Many inflammatory diseases, such as cardiovascular disease or autoimmune disorders, have gender specificity underlying their etiology. These gender-related differences may be explained by the varying activity of sex hormones in regulating inflammation. More specifically, progesterone, a female-associated hormone, has been implicated as an anti-inflammatory agent; yet the mechanism of its regulatory action is still unclear. One possibility is that progesterone regulates the trafficking of leukocytes from the blood into the tissue by affecting the activation state of endothelial cells during inflammation. To understand the role of progesterone within endothelial cells, an in vitro model was generated by overexpressing progesterone receptor (PR) in human umbilical vein endothelial cells (HUVECs). PR activated by progesterone treatment of endothelial cells decreased expression of select inflammatory cytokines and leukocyte-endothelial adhesion molecules. Furthermore, progesterone treatment increased NF-kB p65 phosphorylation at serine 536, implicating the inflammatory NF-kB pathway as a possible downstream target of progesterone action. Further understanding of the molecular mechanism by which progesterone regulates leukocyte trafficking will provide greater insight into sex-differential responses to disease.
| Mr. Justin Tiulim
Justin Wayne Wong Tiulim
Mentor: David William Walker
Title: Role of Parkin in Modulating Drosophila Aging
Studies in different organisms have revealed that aging is a complex process associated with a decline in physiological function and increased probability of death. Among other features, aging organisms generally display a decrease in the degradation capabilities of proteins. Consistent with these data, there is an accumulation of protein aggregates in cells that can impair critical cellular functions. The fruit fly Drosophila melanogaster is a well-suited organism to study aging as it is relatively short-lived, mainly composed of post-mitotic cells, has sequenced nuclear and mitochondrial genomes, and multiple genetic tools are available. Since it was already known that Parkin was a gene involved in the regulation of protein degradation and clearing, it was hypothesized that the upregulation of it could produce an increase in lifespan. Using the UAS-GAL4 gene switch, the Parkin gene (a gene involved in ubiquitination) was upregulated during development and adult stages. This overexpression of Parkin during both development and adulthood was shown to increase the lifespan of the flies through survivorship experiments. Furthermore, it was shown through feeding and fecundity assays that long-lived Parkin flies ate regularly and were more fertile than controls. This illustrates that Parkin plays a role in mediating molecular systems, which are necessary for the slowing down of degradation pathways that come with aging.
| Ms. Christine Ryan
Christine Elizabeth Ryan
Mentor: Donald Barry Kohn
Title: Effect of 4-1BB Co-stimulation In CD19-Specific Chimeric Antigen Receptors Targeting B-Cell Malignancies
Chimeric antigen receptors (CARs) are engineered fusion protein receptors combining the specificity of an antibody with the cytotoxic activation capability of a T-cell receptor; a CAR-expressing T-cell is capable of both antigen-specific recognition and targeting. This project involves CD19-specific CARs that target B-cell leukemias and lymphomas. Because engineered T-cells expressing CARs containing only one internal signaling domain have limited persistence and cytotoxic capability, we investigated the effect of 4-1BB co-stimulation. CD19-specific CAR constructs containing part of the T-cell receptor CD3-zeta signaling chain and the 4-1BB co-stimulatory domain were obtained from a collaborator and assessed in primary human T-cells transduced with CAR-encoding lentiviral vectors. The constructs containing the 4-1BB domain were subsequently tested against another set of 4-1BB-lacking CAR constructs in primary human T-cells and in progeny of lentiviral-transduced CD34Title: hematopoietic stem/progenitor cells (HSPCs). The HSPCs underwent normal myeloid differentiation and the progeny exhibited CD19-specific killing capability. In the experiments where higher levels of transduction efficiency were obtained for all CAR constructs tested, the results suggested that 4-1BB co-stimulation enhanced antigen-specific activation of CAR-expressing T-cells, as measured through IFN-gamma ELISA, and CAR-directed specific killing. 4-1BB co-stimulation could thus potentially contribute to more effective CAR-mediated leukemia immunotherapy.
| Mr. Victor Ruiz
Mentor: Kendall N Houk
Title: Conformational Analysis of Bryostatin Derivatives to Elucidate Structure-Affinity Relationships
The bryostatins, partial agonists of the C1 domain in protein kinase C (PKC), are a series of macrocyclic lactones with enormous medicinal potential. This study was designed to investigate the relationship between conformation and activity of several bryostatin derivatives. The first step was to identify all potentially bioactive conformations. This was done using molecular and quantum mechanical approaches in computational chemistry. The second step employed the use of molecular superposition to elucidate possible structural and pharmacophoric elements involved in bryostatin protein-ligand binding. A combination of Merck molecular force field and density functional theory calculations resulted in unique, low-energy conformations beyond the global minimum for each bryostatin derivative. The generation of global minimum structures was especially significant because the majority of these derivatives do not have crystal structures available for analysis. Our superposition results suggest that the in vivo binding conformations of bryostatin are not limited to that described by the crystal structure of bryostatin 1, a key premise for bryostatin design and synthesis. We hypothesize that with conservation of backbone conformation in the recognition domain region, even less stable conformers of bryostatin derivatives can manifest significant binding affinity with PKC.
| Mr. Devin Quinlan
Mentor: Daniel Kamei
Title: A Modified Transferrin-Polyethylenimine Conjugate for the Improved Delivery of Plasmid DNA to Cancer Cells
Gene therapy is an attractive method to treat diseases because it allows for the production of a protein that can alter molecular pathways for a custom-tailored effect. Because these therapeutic agents have a strong negative charge and are unable to cross cell membranes, delivery vehicles, such as polyethylenimine (PEI), have been studied for several years. Additionally, targeting moieties, such as transferrin (Tf), have been conjugated to PEI to target them to cancer cells, since Tf receptors are overexpressed on many types of cancers. This particular strategy, however, may be limited due to the rapid recycling of Tf through the cell, which reduces the probability of these carriers to deliver the therapeutic genes. As a solution, our research group has previously developed a modified Tf that exhibits a higher cellular association. In this poster, we summarize our results in evaluating a system comprised of the modified Tf conjugated to PEI and complexed with plasmid DNA (pDNA). The Tf-PEI conjugate was first purified and characterized prior to the cell studies. Subsequently, using the fluorescent DsRed reporter gene, we obtained data suggesting that the modified Tf-PEI complexes have an increased transfection efficiency compared to the wild-type complexes at a 1:4 Tf:PEI molar ratio on PC3 prostate cancer cells. Due to these initial, promising results, we will further investigate this system as a gene delivery vehicle.
| Mr. Alan Pan
Mentor: Mary A Woo
Title: Clinical Assessment Using Magnetic Resonance Imaging Detects Brain Injury to Regions that Control Memory and Executive Function in Heart Failure
Heart failure (HF) patients exhibit memory and executive function impairments that contribute to HF mortality. Using complex magnetic resonance imaging (MRI) quantitative analysis procedures, structural brain changes have been reported in areas which control these functions (mammillary bodies, hippocampus, and frontal cortex). However, quantitative MRI procedures are not part of standard clinical assessment and it is unknown whether clinical MRI examination can detect changes in these brain structures. Methods: Using standard clinical visual assessment, we examined whole brain regions in 17 HF and 50 controls using high-resolution MR images. Standardized visual clinical scores of mammillary bodies, hippocampus, and the frontal cortex were determined and compared between groups. Results: There were significant differences in right hippocampal size (1.65±0.996 vs. 0.80±0.857; p=0.002) and left frontal cortical atrophy (1.65±0.786 vs. 1.22±0.840; p=0.011) in HF compared to controls. Mammillary body, hippocampal lesions, left hippocampal size, and right frontal cortical atrophy did not show significant differences between the two groups. Conclusions: Clinical examination of routine MR images can detect brain damage in areas controlling memory and executive function in HF patients and may allow clinicians and researchers increased opportunity to evaluate the progression of injury to these brain structures and to more easily identify and evaluate potential treatments for this damage in HF patients.
| Ms. Jessica Ong
Jessica Renee Ong
Mentor: Karen Marie Lyons
Title: Overlapping Functions of the Matricellular Proteins CCN1 and CCN2 during Long Bone Formation
The C (Cyr61) C (CTGF) N(Nov) family of secreted proteins are modular matricellular proteins that are essential for many aspects of development. Our investigation focuses on the first two family members, CCN1/Cyr61 and CCN2/CTGF, which are involved in endochondral bone formation and integrin mediated cell adhesion in vitro, but their roles with integrins in vivo are unclear. CCN1 and CCN2 exhibit a high degree of co-expression within long bones as seen with our preliminary analysis of Ccn1 and Ccn2 enhanced green fluorescent protein (Egfp) mice. In order to study whether CCN1 and CCN2 have overlapping functions in endochondral bone formation, we generated cartilage specific depletions of Ccn1 and Ccn2 in mice to create a comprehensive analysis of genotypes. Our preliminary data illustrate that CCN1 and CCN2 have overlapping functions, since double mutants exhibit a more severe phenotype when compared to single mutants. Upon closer inspection of mutant growth plates, we observed defective chondrocyte proliferation and differentiation. We hypothesize that although CCN1 and CCN2 have overlapping functions in regulating long bone formation, they also have distinct functions from eachother.
| Mr. Takahiro Ohara
Takahiro Erick Ohara
Mentor: Xianjie Yang
Title: Hedgehog Signaling is Activated in Developing Photoreceptor Cells
Photoreceptor cells mediate the first step in vision, capturing light and carrying out phototransduction, ultimately resulting in a neural signal to the brain. These cells are highly patterned and develop from multipotent retinal progenitor cells in the postnatal stage of retinal development. However, the mechanism behind photoreceptor patterning and development is not fully understood. Several in vitro studies have suggested that Hedgehog (Hh) signaling, which is essential for proper embryonic patterning and cell fate specification during development, is involved in photoreceptor birth and differentiation. Here, we investigate the sources and targets of Hh signals during photoreceptor development in the postnatal mouse retina using the Shh-GFP and Gli1-lacZ mouse lines, respectively. Gli1 is a well-known downstream Hh target gene, and its expression serves as a readout of high level Hh signaling. Histological analyses show that developing photoreceptor cells respond strongly to Hh signals secreted from possibly amacrine cells and/or the retinal pigment epithelium. As more cell-based therapies begin to enter the clinical phase, further in vivo studies examining Hh function in the postnatal mouse retina may be useful to understand and treat diseases associated with photoreceptor malfunction and death.
| Ms. Farnoosh Nik-Ahd
Mentor: Carmen Bertoni
Title: Permanent Gene Correction of Muscle Stem Cells in a Mouse Model of Duchenne Muscular Dystrophy
Duchenne muscular dystrophy (DMD) is the most prevalent neuromuscular disorder, affecting 1 in every 3500 males. It is a fatal disease caused by mutations in the dystrophin gene that lead to a complete absence of dystrophin protein throughout the body. Gene correction strategies hold great promise in treating DMD. Our laboratory has pioneered the use of peptide nucleic acid single-stranded oligodeoxynucleotides (PNA-ssODNs) to correct single-point mutations at the genomic level. Here, we show that PNA-ssODNs can target and correct muscle stem cells (SC), a population of cells capable of self-renewing and differentiating into muscle fibers. When transplanted into skeletal muscles, SC that had undergone gene correction were able to engraft and to restore dystrophin expression. Dystrophin was detected in all muscles analyzed both 5 weeks and 6 months after SC transplantation. The number of dystrophin positive fibers was significantly increased 6 months after SC engraftment and protein expression spanned across the longitudinal length of the muscle, demonstrating the long-term stability of this approach. An increase in dystrophin was also detected in response to injury, demonstrating that SC that had undergone gene repair retained their inherent stem cell properties and regenerative potential. Our results demonstrate that gene correction in SC is a valid therapeutic option for the treatment of DMD and offers hope for finding an effective cure to the disease.
| Ms. Eva Ng
Eva S Ng
Mentor: Kent L Hill
Title: The role of Trypanosoma brucei BBSome in flagellar homeostasis and pathogenesis
Trypanosoma brucei is a unicellular mammalian parasite and the causative agent of African sleeping sickness. Its flagellum is required for motility, cell morphogenesis, and host-parasite interactions and houses a wide range of signaling pathways. These pathways rely on proteins that dynamically localize in and out of the flagellum. Recently, it was shown that the BBSome, a protein complex composed of 7 BBS (Bardet-Biedl Syndrome) proteins, mediates flagellar protein trafficking. Loss of the BBSome in other organisms results in disrupted flagellar signaling without generally affecting flagellar biogenesis. The function of the BBSome remains unexplored in T. brucei. We hypothesize that 1) BBS proteins form a complex in T. brucei, 2) are involved in flagellar protein trafficking, and 3) are essential for virulence. To test this, we first performed glycerol gradient sedimentation. BBS proteins co-sediment in a fraction indicative of a complex, possibly the BBSome. We also generated genetic knockout strains to test the role in virulence. Knockout strains were viable in culture with minor growth defects. Mice injected with knockout strains survived longer than those infected with wild-type strains or strains with a similar growth rate before succumbing to infection. These results indicate the presence of the BBSome complex in T. brucei and that loss of BBS proteins attenuates virulence. Thus, the T. brucei BBSome is a promising therapeutic target and will provide us with mechanistic insights into the pathogenesis of this parasite.
| Ms. Naseem Moridzadeh
Mentor: David L Glanzman
Title: Investigation of Zebrafish Startle Reflex Habituation Through Electrical Stimulation of the Trigeminal Neurons.
Zebrafish have been growing as a model organism of interest in the field of developmental biology. Harnessing the ever-expanding repertoire of advantages this organism possesses allows for the investigation of the mechanisms underlying synaptic plasticity. Electrical stimulation of the trigeminal neurons facilitated the investigation of the Mauthner cell, a component of the neuronal circuit critical to the C-start escape response. These neurons are located at the top of the fish’s head and cause the elicitation of the C-start upon stimulation. Repeated stimulation of these neurons led to the establishment of basic habituation. Habituation experiments involving the bilateral and ipsalateral stimulation of the zebrafish trigeminal neurons suggest that habituation of the C-start reflex is pathway specific, rather than a generalized non-specific decrease in the rate of C-start responses. In addition, investigation of the effects of strychnine, a glycine receptor antagonist, on the C-start response to electrical stimulation using a high-speed camera demonstrated significant discrepancies in the responses as compared to untreated fish. Further elucidation of these processes will allow for the establishment and more widespread use of zebrafish as a model for learning and memory.
| Ms. Taylor McCleery
Taylor Leigh Mccleery
Mentor: Daniela F. Cusack
Title: Invasive Tree Species in Tropical Forests Along an Urban-Rural Gradient
Urbanization and anthropogenic activity have major effects on the spread of invasive species in the tropics. Because of human activity, Puerto Rico has only six percent original forest cover and one percent is mature forest with only native species. We surveyed seedling diversity along an urban-rural-remote gradient outside of San Juan, Puerto Rico. Woody species were identified to the species level in three two meter by five meter plots in nine forest stands. We hypothesized that urban expansion and land use change are linked to domination by exotic plant species in understories nearer to the urban center and major roads, indicative of new successional pathways for these forests. Preliminary results show that rural and remote sites had statistically significantly higher species diversity, whereas stem abundance was not significantly different across the watershed. Remote sensing analyses using ENVI revealed that lower Normalized Difference Vegetation Index values indicated more invasive legumes, and lower NDVI values were positively associated with distance to the urban center. Areas closest to the urban center had NDVI values averaging negative 0.05 whereas areas farther away averaged 0.25. Forests more than 6.5 kilometers from the urban center had positive NDVI values. These data suggest a positive effect of urbanization on invasive plant species in the canopy as well as the understory for these Puerto Rican forests, with distance to the urban center playing the strongest role in determining invasive species extent.
| Mr. Alan Avalos
Alan Vinicio Avalos
Mentor: Kenneth Alan Bradley
Title: Host Variation on Murine Chromosome 11 Modifies Inflammatory Response and Resistance to Anthrax
Anthrax is the acute infection caused by the sporulating bacterium B. anthracis. Pathogenesis is associated with the virulence factor, lethal toxin (LT). LT is a bipartite exotoxin composed of protective antigen (PA), which attaches to extracellular host anthrax toxin receptors, and lethal factor (LF), a zinc-dependent metalloproteinase.
PA delivers the catalytic moiety, LF, into the cytosol of the host macrophage. Catalytic activity of LF in the host cytosol activates the Nlrp1b inflammasome, which senses intracellular bacterial signals to initiate host defense pathways. An LT-sensitive Nlrp1b allele is responsible for causing a pro-inflammatory macrophage lysis that results in the release of cytokines in order to recruit phagocytes to the site of infection. Interestingly, a congenic strain of mouse, termed B6.CAST.11M, undergoes a severe early response phenotype that is in part driven by an LT-sensitive Nlrp1b allele. However, The B6.CAST.11M strain has higher resistance to infection by spores than other mice with an LT-sensitive Nlrp1b allele, indicating the influence of additional genetic factors. We hypothesize that these factors may include a recessive-modifier gene on chromosome 11 of the CAST/Ei genome. We have mapped this gene between 66.4 and 74.7 Mbs on chromosome 11. Through backcross-breeding and mapping of chromosome 11 using macrosatellite markers, we are establishing multiple subcongenic lines to identify the modifier gene(s).
| Mr. Rey Martin
Mentor: Sabeeha Merchant
Title: Phenotypic Analysis of the Cadmium-Induced Response in Chlamydomonas reinhardtii
Cadmium (Cd) is an important element to study because it is a toxic heavy metal that accumulates as it moves up the food chain. Understanding how organisms detoxify heavy metals such as Cd by activating detoxifying pathways may provide insight into how these metals can be managed within larger ecosystems. The green alga known as Chlamydomonas serves as a great model organism for this study because it can be grown in a defined medium, it displays phenotypic responses to stress, and its genome has been sequenced. This study aims to identify pathways affected by Cd-exposure by first analyzing the phenotypic response of Cd-exposed cells, and then by analyzing their transcriptome using RNA-Seq. Cd concentrations have been identified to obtain no activity (no Cd), Cd-specific activity (25 uM Cd), and Cd-saturated activity (120 uM Cd) of the detoxification pathways. In a 24 h time course experiment, treatment with 120 uM Cd was found to be toxic to wild-type cells because their growth rate decreases greatly, their ionome is perturbed, their chlorophyll content decreases, and their photosynthetic efficiency is significantly impaired. However, cells treated with 25 uM Cd appear similar to cells not exposed to Cd. These results are necessary to understand and validate transcriptomic analysis of these cells, which is currently underway. A comparison of these responses to Cd in the environment may identify pathways affected by Cd-exposure and may help guide future bioremediation projects focused on Cd-contaminated environments.
| Ms. Kimberly Loo
Kimberly Leticia Loo
Mentor: William Edward Lowry
Title: Manipulating the Developmental Maturity of Pluripotent Stem Cell Derived Neural Progenitor Cells
Human pluripotent stem cells (hPSCs) have the potential to differentiate into many cell types, yet it is not known how similar the process of PSC in vitro development reflects the in vivo process. Recent work from the Lowry lab identified a set of 105 genes whose expression appears to distinguish hPSC progeny from their respective mature tissue derived cells. A number of these genes are expressed in early embryos and fail to be properly turned off in PSC derivatives. These “embryonic” genes include LIN28, DPPA4, and TCF7L1. Furthermore, a second set of genes fails to be properly induced during in vitro differentiation. These “maturity” or “specification” genes include NFIX, HOPX, and ZFP3. In attempts to make PSCs that more accurately reflect their natural postnatal tissue derived counterparts, transfection experiments via electroporation aimed to overexpress NFIX and HOPX in PSC derived neural progenitor cells. Real-time reverse-transcription PCR (qRT-PCR) was used to quantify gene expression levels at the RNA level, while immunofluorescence was used to quantify expression levels at the protein level. Following confirmation of the induction, the affect of the genetic manipulations on the developmental maturity will be determined through gene expression microarrays and in vitro functional assays. Future experiments will also be conducted on additional genes in attempts to bring hPSC derivatives closer to their natural counterparts on a global transcriptome level.
| Mr. Joseph Lee
Joseph Kang Lee
Mentor: Daniela Cusack
Title: Soil Respiration Across an Urban-Remote Tropical Gradient: Variability Among Forest and Invasive Grass Sites
The abandonment of degraded agricultural land in Puerto Rico has led to large-scale forest regeneration in the region; forest recovery occurring contemporaneously with urban growth. As a result of this landscape fragmentation from urbanization, patches of secondary forests now exist within a mosaic of urban infrastructure and may be reacting differently to environmental changes along an urban-remote gradient. In order to explore how these changes may be affecting carbon loss in tropical secondary forest soils recovering from intensive agricultural use, urban and remote secondary forests were studied in the San Juan/Rio Piedras watershed. In this study, measures of soil carbon dioxide (CO2) efflux (collected using a Li-Cor 6400 infrared gas analyzer) were paired to measures of soil temperature and moisture in urban and remote secondary forest and grass sites. The main hypothesis driving this study was that more urban and smaller forest fragments, regardless of cover type, would see higher carbon fluxes relative to larger, more continuous forests as a result of combined “urban” and edge effects. For both cover types, soil respiration was highest in sites closer to the urban center, with steeper declines away from the urban center in forest sites. Average forest and grass soil respiration was 6.3 (plus or minus 0.5) and 7.1 (plus or minus 0.6) micromoles of CO2 per meter squared, respectively. These data suggest that mechanisms driving rates of soil respiration may differ among urban forest and grass areas.
| Mr. Aaron Kwong
Aaron Adrian Kwong
Mentor: Timothy F Lane
Title: Creation of a Versatile Cell Cycle Reporter for In Vivo Analysis of Cell Cycle Progression
Analysis of cell cycle progression is difficult in intact animals due to the need to isolate cells prior to analysis or fix tissues after biopsy. To better study cell cycle progression in vivo, a fluorescent cell-cycle based indicator (Fucci) system was utilized. This system revealed cell cycle phases through the dual expression of two fluorescently labeled cell cycle specific proteins. This system could be very useful for routine cell cycle analyses of various cell lines such as those with perturbations in various cancer regulators. It might also be employed in vivo where direct methods can be employed to visualize cells in live tissue. The Fucci-2A vector, containing both cell cycle genes, was cloned and inserted into a lentiviral backbone. The resulting vector was validated by enzymatic digestion and sequence analysis. With the help of the UCLA vector core, this vector was then packaged into a lentivirus for efficient delivery into target cells. Subsequent infection of various cell lines resulted in successful expression of the fluorescently conjugated proteins as visualized by fluorescence microscopy. A Cre-Lox system was added to the Fucci lentiviral vector to allow for tissue specificity while maintaining strong expression levels with the ubiquitous elongation factor 1a promoter. The resulting lentivirus will be an invaluable tool for in vivo cell cycle analysis that can help identify proteins involved in tumorigenesis.
| Mr. Jonathan Kuo
Jonathan Lan Kuo
Mentor: Craig A Merlic
Title: Pd(II)-Catalyzed Intramolecular Insertions for Polycyclic Ring Synthesis
An intramolecular regioselective and stereoselective Pd(II)-catalyzed coupling of boronate esters with alkene or alkyne insertion has been developed. This methodology provides convenient access to substituted fused or spiro polycyclic ring systems. The reaction involves competing transmetallations and insertions. Furthermore, we have designed substrates which explore the competitive rates of vinyl and aryl transmetallation versus alkene and alkyne insertion.
| Mr. Richard Jin
Mentor: Robert M Prins
Title: Cytotoxic T Cell Functional Responsiveness May Predict Clinical Efficacy of Dendritic Cell Vaccination in Glioblastoma Patients
Tumor immunotherapeutic approaches, in particular dendritic cell (DC) vaccinations, have emerged as innovative strategies in the treatment of glioblastoma multiforme (GBM). Despite their promise, the absence of biomarkers and/or immunological monitoring techniques to assess the clinical efficacy of therapy remains a primary limitation. The purpose of our study is to identify a surrogate for anti-tumor immune responsiveness associated with extended survival from peripheral blood lymphocytes (PBL) in GBM patients undergoing a DC vaccination. We expected to observe a correlation between extended survival and the functional responsiveness of T cells, and/or B cells following DC vaccination. To assess the anti-tumor immune response elicited by therapy, we studied the functional responsiveness of PBL pre- and post-vaccination to immunostimulatory cytokines interferon-gamma and interleukin-2 through the phosphorylation of downstream transcription factors, STAT-1 and STAT-5. Flow cytometric analysis of pSTAT-1 and pSTAT-5 revealed a positive correlation between increased phosphorylation of STAT-5 in cytotoxic T cells and extended survival. Our results suggest monitoring the change in pSTAT-5 populations in peripheral blood cytotoxic T cells before and after DC vaccination may predict the clinical efficacy of a DC vaccination on GBM patients. Furthermore, DC vaccinations may be augmented to specifically enhance cytotoxic T cell functional responsiveness to improve GBM patient survival.
| Ms. Monica Hernandez
Monica Nicole Hernandez
Mentor: Ann M Hirsch
Title: Quorum Sensing is Required for Normal Infection of Nitrogen-Fixing Nodules by Sinorhizobium meliloti and May Regulate the Expression of Many Genes
The phenomenon known as quorum sensing (QS) is a cell-density dependent gene expression. QS plays a role in the symbiotic interaction between the Gram-negative soil bacterium Sinorhizobium meliloti and the host legume Melilotus alba. We inoculated the roots of M. alba with both wild-type and QS mutant strains and then harvested the roots after 7, 14, and 28 days post-inoculation to determine any differences in the nodulation and infection phenotypes. Overall both roots infected with the wild-type and mutant strains produced nodules, but the wild-type strain infected roots produced more partially and fully infected nodules. In contrast roots infected by the mutant strains produced more partially and uninfected nodules. This supports the hypothesis that QS is necessary for the functional bacterial infection of the nodule. Because the QS system could regulate the expression of many genes during the symbiosis, determining when this gene-expression cascade begins is an important part of understanding how bacteria associate with the plants. We are currently making promoter-gusA and promoter-gfp transcriptional fusions to determine when sinI expression occurs during infection.
| Mr. Joseph Hadaya
Joseph Elias Hadaya
Mentor: Aman Mahajan
Title: Left and Right Stellate Ganglia Stimulation Modulates Left Ventricular Function, Interstitial Norepinephrine Levels and Activation Recovery Intervals
The sympathetic nervous system plays a role in sustaining ventricular arrhythmias. Sympathetic innervation of the heart is via the left and right stellate ganglia and is mediated by norepinephrine. Anesthetized pigs underwent a sternotomy to expose the heart and stellate ganglia. Ventricular pressure was recorded with a conductance catheter. Interstitital norepinephrine was sampled with a microdialysis probe on the anterior surface of the heart. Arterial, venous, and coronary sinus blood samples were taken for norepinephrine level analysis. A multi-electrode catheter on the anterior surface of the heart was used to record activation recovery intervals. The right and left stellate were electrically stimulated; data was collected before, during, and after stimulation. Left stellate stimulation resulted in increased pressures with no change in heart rate. Right stellate stimulation resulted in increased pressures with a large change in heart rate. Interstitial and coronary sinus norepinephrine levels increased during stimulation, but arterial and venous levels remained unchanged. Both left and right stimulation shortened activation recovery interval, but right stimulation had no effect on dispersion of repolarization. Both left and right stellate innervate the left ventricle; however, the right stellate exclusively exerts chronotropic control over the heart. This study provides insight into mechanistic control of the left ventricle and documents the value of sympathetic denervation in managing ventricular arrhythmias.
| Ms. Samantha Gebauer
Samantha Kelly Gebauer
Mentor: Axel Schmitt
Title: Eruption and Crystallization Ages for the Breccia Museo Supereruption Deposit
Large volcanic eruptions can impose significant stresses on human populations. The terminal decline of the Neanderthal population of Europe correlates with the eruption of the Campanian Ignimbrite (CI), the largest volcanic eruption in the Mediterranean during the late Pleistocene. Certainty of this contemporaneity hinges on the accuracy of the CI eruption age, which has remained contentious because of inherent limitations in 14C and 40Ar/39Ar dating techniques. Here, we focus on the Breccia Museo (BM) deposit, a complex volcanic breccia in the proximity of the Campi Flegrei caldera (Naples region, Italy) from which the CI deposit erupted. Crystallization and eruption ages for the BM were determined via uranium decay series and (U-Th)/He dating of zircon, respectively. Zircon rims in the BM magma crystallized on average at 49 ka with the eruption age being consistently younger at 41 ka (with analytical uncertainties of approximately 2 ka at 95% confidence for both ages). These results imply that magma presence underneath Campi Flegrei was long-lived, pre-dating the eruption by at least 8000 years. The new BM eruption age overlaps with recalibrated 40Ar/39Ar ages for CI suggesting both deposits formed in the same eruption. The ages refined by this study further substantiate the link between a voluminous eruption of the Campi Flegrei magmatic system and the demise of the Neanderthals.
| Mr. Michael Fice
Michael Peter Fice
Mentor: Kenneth A Dorshkind
Title: Fetal and Adult Waves of B Cell Development Are Differentially Dependent on the PU.1 Transcription Factor
Two distinct B cell populations exist in mice and humans. B-1 cells are associated with innate immune responses, while B-2 cells are part of the adaptive immune system. There is clear evidence that B-1 and B-2 cells are separate lineages that develop from distinct, phenotypically identifiable progenitors. Observations in the literature suggest that B-1 and B-2 development are differentially regulated. In particular, it has been reported that B-1 cells are present in mice with deficient expression of PU.1, a transcription factor known to regulate genes involved in B cell development, but B-2 development is blocked. These results suggest that PU.1 expression is not required for the emergence and maturation of B-1 progenitors. In order to test this hypothesis, we examined B-1 development in PU.1 hypomorphic mice and Mb-1 cre x PU.1 fl/fl mice. The former strain has a severe reduction of PU.1 in all stages of hematopoiesis, while the latter strain lacks PU.1 expression in committed B lineage cells. Here we demonstrate the existence of two waves of B-1 development that are differentially dependent on PU.1. The fetal wave requires PU.1 expression and in its absence no identifiable B-1 progenitors are present. In contrast, B-1 progenitors can emerge in the subsequent neonatal wave of B-1 development in a PU.1 independent manner. These data provide evidence that the transcriptional regulation of B-1 and B-2 development is distinct and provide a basis for the formulation of strategies to manipulate the production of B lineage cells.
| Mr. Benjamin Emert
Mentor: Aldons J Lusis
Title: The Effects of High-Density Lipoprotein on the OxPAPC Response in Human Endothelial Cells
Atherosclerosis is a chronic and progressive inflammatory disorder and the primary cause of cardiovascular diseases (CVDs). The World Health Organization estimates that CVDs kill nearly 20 million people each year, making them the leading cause of death worldwide. Researchers at our lab and others have demonstrated that oxidized 1-palmitoyl-2-arachidonyl-sn-phosphatidylcholine (OxPAPC), a major pro-atherogenic component of minimally-modified low density lipoprotein, alters the expression of over 1000 genes in human aortic endothelial cells in vitro. Conversely, high-density lipoprotein (HDL) has been shown to suppress or even reverse the effects of OxPAPC, thereby protecting the cell from oxidative stress and inflammation. We define this effect as the HDL response. In the hopes of defining the mechanisms mediating this response, we investigated both receptor specific and non-specific modes of action. Using siRNA mediated gene knockdown, we found that the HDL response is independent of the expression of the putative HDL receptor scavenger receptor class B-1. Similarly, we show that cholesterol loading of the cell membrane using cholesterol-cyclodextrin complex is unable to mimic the action of HDL. However, knockdown of krüppel-like factor 2 (KLF2), a transcription factor involved in the inflammatory response of vascular endothelial cells, partially mitigated the HDL response. Taken together, our work identifies a novel role for KLF2 in vascular biology and provides greater insight into the atheroprotective properties of HDL.
| Mr. Jaideep Dudani
Jaideep S Dudani
Mentor: Dino Di Carlo
Title: Rapid Inertial Solution Exchange for Cellular Sample Preparation and Flow Cytometry
Mixing and solution exchange are routine tasks in sample preparation for cell biology. Achieving solution exchange around cells and particles on microfluidic systems would minimize reagent consumption, enable parallelization, automation, and in-line analysis. However, exchanging solution around cells is difficult, as it requires precise control of fluids by way of externally applied forces or precisely fabricated mechanical features. Here, we introduce a strategy for exchange based on passive inertial lift forces. Uniquely, we explore and make use of dominant wall-effect lift that is parallel to the particle rotation direction to achieve controllable cross-stream motion. In this way, particles migrate across laminar streams and enter a new solution without significant disturbance of the interface at rates exceeding 1000 particles per second with sub-millisecond transfer times. We demonstrate the capabilities of rapid inertial solution exchange (RInSE) for preparation of hematological samples and other cellular assays. Further, we characterize improvements to inline flow cytometry after RInSE of excess fluorescent dye and cellular alignment for downstream analysis. Lastly, we demonstrate transfer of cells and particles across multiple streamlines. This approach has broad applications in fields that can benefit from automation as well as areas that require ultrafast solution exchange such as biomarker detection, analysis of low-affinity interactions on cells, kinetic studies, and solid-phase chemistry.
| Mr. Dhaval Dixit
Mentor: Jerome A Zack
Title: Characterization of HIV Infection in CD34Title: Hematopoietic Stem Cell Progenitors Using BLT Mouse
HIV infected patients demonstrate hematopoietic abnormalities mainly related to HIV replication in the bone marrow. Recent studies have suggested that HIV infection does occur in bone marrow derived CD34 cells. Our aim is to understand the biology of HIV infection in the bone marrow using the humanized BLT mouse model. To this end, BLT mice were infected with one of three different HIV strains. Infected mice showed detectable viral loads and loss of CD4 T cells. Bone marrow samples were collected and CD34 cells were purified to assess in vivo HIV infection in the bone marrow. CD34 progenitors were cultured in methylcellulose containing appropriate growth factors to determine whether a single HIV infected progenitor cell can produce multiple HIV infected lineages. We were able to detect the presence of HIV DNA in both the CD34 progenitors as well as their derived colonies. Furthermore, we identified HIV integration sites in both populations, suggesting that infected colonies arise from a single progenitor. Based on our data, infected progenitors can give rise to multiple infected lineages. These results suggest that pluripotent hematopoietic progenitors can be infected by HIV in vivo. This knowledge can be used to target and kill infected hematopoietic progenitors to eradicate one source of latent virus.
| Mr. Jonathan Diep
Mentor: Ren Sun
Title: Monobodies Evolved by mRNA Display as Novel, In Vitro Alternatives to Antibodies
Antibodies have become essential recognition tools for molecular biology research. Allele variation, post-translational modifications, and alternative splicing, however, have made the range of targets in the proteome far exceed the number of commercial antibodies currently available. In vitro selection techniques, such as mRNA display, represent alternative methods for accelerating the generation of antibody mimics called monobodies. We sought to demonstrate in vitro applications for monobodies in immunoassays using monobodies that were evolved by mRNA display to the model targets: the maltose binding protein of Escherichia coli and human immunoglobulin G. Subcloning vectors were designed and constructed for the expression of monobodies either with tags for enzymatic biotinylation by BirA or as alkaline-phosphatase fusions for detection. Monobodies linked to streptavidin-labeled horseradish peroxidase via the C-terminal biotin enabled antibody-free target detection by western blot. In antibody-free enzyme-linked immunosorbent assays (ELISAs), monobodies with alkaline-phosphatase fusions showed a limit of detection of 100 pg. Monobodies, therefore, exhibit comparable performance to commercial antibodies on western blots and ELISAs. This validation of in vitro applications for monobodies evolved by mRNA display suggests that monobodies represent stable, expressible, and economical alternatives to traditional antibodies.
| Mr. Debobrato Das
Mentor: Benjamin M. Wu
Title: Investigation of a Dynamic Biomimetic Apatite Nanoparticle Delivery System for Targeted Non-viral Gene Transfection
Common approaches for gene delivery use nanomedicines that may produce in vivo toxicity and immunogenicity, and are limited in the variety of transportable nucleic acids. This study aims to maximize transfection efficiency and minimize adverse host responses by utilizing biocompatible properties of calcium phosphate (CaP). CaP nanoparticles (NPs) present a unique class of non-viral vectors, which can serve as efficient alternatives for targeted gene delivery. In this project, CaP NPs were constructed from simulated body fluids (SBF), which have historically been used to create apatite coatings that elicit in vivo bioactivity, decreased immunogenicity, and facilitate osteogenic differentiation of adipose stem cells. SBF-CaP NPs co-precipitated with RNAi and pDNA were administered to MC3T3 cells to assess biocompatibility, cellular uptake of CaP-siRNA NPs, and modulation efficiency of intracellular protein and gene expression. As expected, CaP NPs induced consistently less cytotoxicity than traditional transfection reagents such as Lipofectamine. Cells cultured with CaP-GAPDH siRNA NPs demonstrate knockdown of GAPDH activity. Preliminary results indicate comparable up-take efficiencies among miRNA, siRNA, and pDNA sequences using the SBF-CaP, suggesting that this methodology can be applied to deliver a variety of engineered genetic therapeutics. Current efforts focus on the detailed characterization of the NP-RNA/DNA interactions, and cell-NP-RNA/DNA interactions.
| Ms. Madeline Cross
Mentor: Rachelle Hope Watson
Title: Sarcospan Ameliorates Muscle Pathology by Improving Strength in mdx Mice
Sarcospan is a 25kDa component of the dystrophin- and utrophin-glycoprotein complexes that connects the extracellular matrix to the actin cytoskeleton. Mutations in the dystrophin gene cause Duchenne muscular dystrophy and lead to loss of the entire dystrophin-glycoprotein complex. In the mdx mouse model of this disease, the abundance of utrophin-glycoprotein complex is increased around the sarcolemma to partially compensate for the loss of dystrophin. We have previously shown that sarcospan overexpression in dystrophin deficient mdx mice ameliorates the mdx pathology. However, the effect of sarcospan overexpression on muscle contractile function is unknown. In the current report, we investigated the effect of sarcospan on mdx muscle function by electrophysiological stimulation of isolated extensor digitorum longus muscles and monitored muscle mass, myofiber diameter, number, and density. In addition, we performed immunoassays to investigate the effect of sarcospan on other components of the dystrophin- and utrophin-glycoprotein complexes as well as integrin. We report that sarcospan-transgenic mdx muscles were over 40 percent stronger relative to controls and we demonstrate that sarcospan improved expression of the three adhesion complexes around the sarcolemma. This is the first study demonstrating increased force generation in mdx mice overexpressing sarcospan. We conclude that improved attachment of myofibers to the extracellular matrix contributes to the increased muscle strength.
| Ms. Cassandra Coleman
Cassandra Sherean Coleman
Mentor: Anil Bhushan
Title: Identification of Novel Beta Cell Specific Regulatory Elements
Beta cells of the endocrine pancreas are critical regulators of blood glucose homeostasis, which is disrupted during diabetes. The inefficiency of current diabetic treatment has led to interest in cellular reprogramming to generate beta cells from other cell types. Although beta cell fate was originally thought to be determined, recent studies have concluded that cellular identity is plastic. Chromatin modifications at key regulatory elements can enable cellular reprogramming; however, beta cell specific epigenetic elements and their target genes have not yet been identified. To identify these possible beta cell specific regions, we used the Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) method, which has been shown to identify cell type specific clusters of open regulatory elements (COREs) with possible single-gene regulatory function. Preliminary analysis of our data identified potential beta cell specific COREs (Beta COREs) proximal to genes important in the regulation of beta cell identity. To determine the functionality of these elements, Chromatin Immunoprecipitation (ChIP) assays will be performed for H3K4me1 and H3K27ac, which are histone markers indicative of transcriptional enhancers. By analyzing Beta COREs that regulate genes important for beta cell lineage maintenance, we will gain a more thorough understanding of beta cell biology. These elements could be potential targets for cellular reprogramming experiments to convert donor cells, such as liver cells, to beta cells.
| Mr. Kevin Coffey
Kevin Thomas Coffey
Mentor: Axel Schmitt
Title: Determining the Source of Volcanic Ash in Maya Ceramics Using Zircon Crystallization Ages
Volcanic eruptions cause sudden environmental changes, with both negative (inundation with ash, poisoned water) and positive (increased soil fertility) human impacts. Massive volcanic eruptions may have forced Maya population centers to shift from the highlands of Guatemala to the lowlands at the beginning of the Late Classic Period (600-900 A.D.). At the same time, an enigmatic volcanic ash first appeared in lowland pottery. As potential sources of this ash, we have identified 14 volcanic centers of the Central American and Chiapanecan volcanic arcs by considering age, distance, and whole-rock compositions. Correlations between pottery ash and eruptive centers using glass chemistry are inconclusive, largely because firing of the pottery has altered the ash. We are therefore developing the novel tool of using the crystallization ages of zircon, an inert accessory mineral contained within the ash, to correlate pottery ash with its source volcano. Preliminary results indicate that most pottery zircons have ages matching an older zircon population within the approximately 400 A.D. Ilopango TBJ ash, but none correlate with a second, younger population. Additional zircon analyses from pottery and candidate volcanoes will be conducted to establish a robust correlation. This would have implications for the volume and transport direction of the volcanic ash plume and its potential impacts on Mayan societies.
| Ms. Jennifer Chyu
Mentor: William Robb Maclellan
Title: Characterizing the Therapeutic Potential of Induced Pluripotent Stem Cell Derived Cardiovascular Progenitor Cells
Cardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart by their expression of the transcription factor Islet 1 (Isl1). Our lab has recently identified novel cell-surface markers that specifically identify these Isl1Title: multipotent CPCs. We have functionally validated these markers in induced pluripotent stem cell (iPSC)-derived progenitors. The next step in their clinical translation is to show that iPSC-derived CPCs robustly engraft into infarcted myocardium and differentiate into cardiomyocytes to improve heart function and to determine if CPC delivery in a hydrogel cell scaffold can support CPC survival and engraftment in vivo. Fluorescence Activated Cell Sorting (FACS) for the surface markers purify Isl1Title: CPCs from iPSCs. Immunofluorescence imaging and analysis by quantitative real-time PCR were used to validate their progenitor identity. Transplantation of murine iPSC-derived CPCs into species matched infarcted hearts within a hyaluronan-based hydrogel was performed and functional recovery was assessed by echocardiography two and four weeks following induced infarct and intramyocardial cell transplantation. We show here that a combination of cell surface markers specifically label CPCs derived from iPSCs. These progenitors can be expanded in vitro and maintain their cellular identity. Furthermore, CPC transplantation in vivo within a pro-survival hydrogel matrix may serve as a tool for CPC delivery to support cardiac repair following infarction.
| Ms. Ke-Huan Kuo Chow
Ke-Huan Kuo Chow
Mentor: Harold G Martinson
Title: Understanding the Mechanism Behind Coupled Transcription and Pre-mRNA Processing
To understand gene expression in eukaryotic cells, our lab studies the mechanism by which eukaryotic transcription is coupled to mRNA 3’-end processing. Previously, we showed that cutting the mRNA between the RNA polymerase and the poly(A) site with RNase H prevents 3’-end processing. This led us to hypothesize that a stretch of mRNA is required to “tether” the cleavage apparatus to the RNA polymerase. However, since RNase H cutting requires a DNA-RNA hybrid target, it was unclear whether processing was blocked as a result of the cutting, or the binding of an oligo DNA to the RNA. To distinguish between the possibilities, we used MOE oligo that binds to its target without triggering RNase H activity. We showed that processing was not disrupted by this oligo, and that the tether anchors the apparatus to the polymerase. In addition, the need for a tether during apparatus assembly hints at the complexity of the assembly process. Therefore, we investigated the kinetics of the apparatus assembly. Specifically, we examined the recruitment kinetics of two major cleavage factors, CPSF and CstF. Using two chemically modified MOE oligos specific for the factors’ binding sites, we discovered that only the CPSF binding site is required for the recruitment of both factors. We believe that the two factors are recruited to the pre-mRNA together as a complex, while CstF latches on its binding site later. Future efforts will be directed at testing this model, which would provide us with valuable knowledge on eukaryotic gene expression.
| Mr. Stephan Chiu
Stephan Y Chiu
Mentor: Benhur Lee
Title: Novel Nipah Virus Matrix Protein Mutants Reveal Defects in Budding Pathways
Nipah virus (NiV) is an emerging zoonotic virus of the Paramyxoviridae family. It causes fatal acute encephalitis in humans with a mortality rate of 40-92%, resulting in its classification as a Biosafety Level 4 pathogen, the highest level of biosafety containment. The Nipah Matrix (M) protein is essential for the assembly of viral components and subsequent budding from host cells. NiV-M is also capable of independently budding in the absence of other viral components, causing the release of virus-like particles. Previous studies have shown that mutations in critical sites in NiV-M can result in loss of budding function. In this study, fifteen new sites in the Matrix protein that are highly conserved across several genera of the Paramyxoviridae family were identified using phylogenetic analysis. Using site-directed mutagenesis, these sites were independently mutated in order to assay their importance for NiV-M function. Upon transfection into mammalian cells, most NiV-M mutants displayed a moderate to severe degree of budding deficiency. In addition, fluorescence microscopy of NiV-M within transfected cells revealed distinct mutant phenotypes, including exclusion from distinct intracellular holes, and formation of large angular compartments protruding from the cell surface. Further investigation of these NiV-M mutants in the context of other paramyxoviral Matrix proteins may reveal conserved or distinct features in Nipah budding that will facilitate development of novel therapeutic strategies.
| Ms. Vicky Chiang
Mentor: Zhefeng Guo
Title: Structural Study of Abeta42 Oligomers Using Electron Paramagnetic Resonance Spectroscopy
Aggregations of amyloid beta protein fragments in the brain are associated with Alzheimer’s disease (AD), and are believed to play a causal role in AD pathology. In particular, Abeta42, a peptide fragment consisting of 42 residues, appears to form more toxic oligomers and to aggregate faster in comparison to Abeta40, though the two peptides only vary by an additional two residues at the C-terminus of Abeta42. Therefore, understanding the structure of Abeta42 may provide information on AD pathology and lead to therapeutic applications in the long-term. In this experiment, electron paramagnetic resonance (EPR) spectroscopy is used to study at a residue-specific level the structural details of the oligomers of Abeta42, using a fusion protein consisting of GroES and ubiquitin fused at the N-terminus to facilitate solubility. Residue pairs within segment 17-42 of Abeta42 oligomers were mutated and spin-labeled. Inter-residue distances between pairs of spin labels were determined using EPR spectroscopy. These distances were used as structural constraints to model the oligomer structures. These results provide important insights into the structure of Abeta42 oligomers and the mechanism of the oligomerization process involved in Alzheimer’s disease.
| Ms. Andrea Chan
Andrea N Chan
Mentor: Paul Henry Barber
Title: Genetic Analysis of Pseudochromis Color Morphs from the Coral Triangle Leads to the Potential Discovery of a New Species
The Coral Triangle contains the highest levels of marine biodiversity on the planet, suggesting that lineage divergence and speciation events were/are common in this area. Many coral reef fishes display significant intraspecific color pattern variation, which may be indicative of corresponding genetic divergence resulting in speciation due to assortative mating preferences (McMillan et al. 1999). To determine if the various color morphs of Pseudochromis sp. represent distinct lineages, the DNA of 27 individuals from eight regions in the Coral Triangle was extracted and the Pseudochromis ribosomal RNA marker: 16s AR(5’ CGCCTGTTTATCAAAAACAT ‘3) and 16s BR(5’ CCGGTCTGAACTCAGATCACGT ‘3) (Smith and Craig 2007) was amplified. DNA sequences were used to construct the most parsimonious phylogenetic tree under neighbor joining (NJ) equilibrium. There was significant genetic differentiation among the color morphs, suggesting that these lineages are diverging in accordance with color. Thus, there may be little, if any, genetic exchange going on between the color morphs indicating that they are either different species or may be in the process of speciating. Most notably, the genetic distance between two color morphs of P. perspicillatus from Komodo and Morotai was 0.03. This study brings us closer to quantifying the biodiversity of the Coral Triangle, thus increasing our understanding of how new lineages and species are created and enhancing our ability to protect emerging coral reef organisms from extinction.
| Mr. Abraham Botros
Abraham Peter Botros
Mentor: Todd Sigi Hale
Title: Beat-Induced Auditory Brainwave Entrainment
Brainwave entrainment refers in general to any phenomenon that aims to induce synchronization of brainwave frequencies to some repetitive external stimulus. Such entrainment is often aimed at altering mental state or producing positive psychological results and can be accomplished using various auditory methodologies. In particular, a constant “beat” at some desired frequency in the stimulus can cause a sustained frequency-following response, or “entrainment,” of the brain, marked by assimilation of dominant brainwaves to the beat frequency. Previous work has suggested that such auditory beats can induce changes in the physiological functioning of the brain, with associated effects on psychological states and cognitive ability. However, to date, no study has quantitatively compared the distinct methods of auditory entrainment for their respective efficacies in producing such effects. The current study directly examines and compares the three auditory methodologies used to produce entrainment by analyzing their impacts on electroencephalography-measured brain function and on cognition in a small sample of healthy college students. Results from this experiment help to further discern the differences between these methodologies. They also aid in increasing our understanding of human brain function, frequency-following responses, levels of arousal and associated psychological disorders, and cognition, as we continue to strive to understand the elusive mind-body connection as a whole.
| Mr.Jacob Borrajo
Jacob De Riba Borrajo
Mentor: Tatiana Segura
Title: Spatially Controlled Bioactive Signal Incorporation to Guide Stem Cell Fate in Hydrogels
Hydrogels are networks of hydrophilic polymer chains that can be used as tissue culture systems that mimic the natural stem cell niche. Potential applications for hydrogels include expanding and differentiating stem cells in vitro, delivering stem cells in vivo, as well as making tissue constructs. When functionalized with bioactive signals, hydrogel-based artificial niches can be used to guide stem cell fate. This study aims to develop a method to spatially functionalize synthetic hydrogels with bioactive signals via departure of photolabile pendant moieties and enzyme-assisted bioconjugation. Such a hydrogel system would allow for the photopatterning of complex biochemical patterns and gradients, in order to guide complex multicellular tissue formation. In this study, Arg-Gly-Asp (RGD) peptide -a bioactive adhesion motif found in the extracellular matrix (ECM) glycoprotein fibronectin- was used in an effort to enhance cell-ECM interactions and increase mouse mesenchymal stem cell (mMSC) spreading. Indeed, conjugation of bioactive RGD peptides to synthetic hydrogels showed a three-fold increase in mMSC spreading. This mild and biocompatible process allows for bioactive signal incorporation in hydrogels, and future studies will aim to develop a platform to spatially program stem cell fate. Such a hydrogel system could have several applications in tissue engineering, regenerative medicine and stem cell biology as it could present the opportunity to have two-dimensional control of stem cell fate in a three-dimensional culture.
| Mr. Scott Becker
Scott Nicholas Becker
Mentor: Aldons J Lusis
Title: Oxidized Phospholipids Induce the Expression of MicroRNA-21Mentor: and MicroRNA-27aMentor: in Human Aortic Endothelial Cells
There is an established connection between oxidized lipid accumulation in the arterial tunica intima and the development of atherosclerosis, but the precise role of oxidized lipids in the early events of atherosclerosis is not yet fully understood. We suspected that oxidized palmitoyl-arachidonyl-phosphatidylcholine (OxPAPC), occupies a central, microRNA (miRNA)-mediated role in the etiology of atherosclerosis since it is commonly detected in atherosclerotic lesions. Human aortic endothelial cells (HAECs) from three donors were treated on a time course with either 40 ug/mL OxPAPC medium or control medium, and cells were collected at 2, 4, and 8 hours post-OxPAPC treatment. MicroRNA extraction, microRNA stem-loop RT-PCR, and qPCR were performed for each timepoint and treatment in order to quantify microRNA expression. On average, significant induction of miRNA-21Mentor: and miRNA-27aMentor: was observed. Expression of both was upregulated at least twofold, and up to threefold, in OxPAPC-treated cells at 4 and 8 hours post-treatment. Cross-referencing of the miRNA seed sequences with all mRNAs in HAECs suggested potential miRNA-mediated gene silencing of the NF-KB pathway. These findings are crucial because they characterize a major molecular event in early atherosclerosis progression that could be an attractive target for prevention and/or treatment. Future studies will experimentally verify the gene targets of miRNA-21Mentor: and miRNA-27aMentor: in order to better understand their specific roles in HAECs and early atherosclerosis pathology.