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2014 Research Showcase
Master's Abstracts
Abstract Title : How do seasonal changes in food web dynamics and climate affect carbon dioxide flux from San Diego reservoirs?
Abstract : Many lakes around the world emit carbon dioxide (CO2) to the atmosphere, but there is little information on whether man-made water reservoirs release or absorb CO2 emissions. It is essential to determine if reservoirs sequester or release CO2 to the atmosphere so we can determine the impact of water impoundment on global carbon cycling. We sampled three reservoirs throughout San Diego, CA, all of which receive most of their water from the Colorado River. We sampled each reservoir weekly for one year to measure seasonal variation in the abundances of bacteria, pelagic fungi, phytoplankton, and zooplankton, as well as abiotic factors such as water chemistry (pH, nutrients, conductivity, ions, dissolved organic carbon [DOC], and partial pressure of carbon dioxide [pCO2]). We found that San Diego reservoirs are most often undersaturated with CO2 and vary seasonally in both different trophic level abundances and pCO2. We found an increase in bacteria biomass and pCO2 concentration during the winter, yet the increases were not correlated with DOC concentrations. These patterns suggest that the increases resulted from factors other than the input of terrestrial organic matter from the Colorado River or primary production from within the reservoirs. The data indicate that San Diego reservoirs function mainly as sinks of carbon from the atmosphere. Our data illustrate how ecological processes and species interactions vary seasonally to determine the sequestration or emission of atmospheric carbon in semi-arid reservoirs.
Abstract Title : 5alpha-Androstane-3alpha,17beta-diol (3alpha-diol) regulates amino PTHrP in human non-small cell lung carcinomas
Abstract : Lung cancer has one of the highest death rates of all human cancers with non-small cell lung carcinomas (NSCLC) making up the majority of diagnoses. NSCLC tumor products include parathyroid hormone-related protein (PTHrP), an oncoprotein known to cause hypercalcemia and other cancer side effects. The full length PTHrP protein is capable of being cleaved into several daughter peptides, including the portion resembling parathyroid hormone, amino PTHrP 1-34. The amino PTHrP peptide is a negative prognostic indicator; patients have shorter survival if their tumors express higher levels of it or its cognate receptor, PTH1R. Amino PTHrP expression has been discovered to be less frequent in tumors of male NSCLC patients compared to females, but the reasons are unknown. It was our hypothesis that androgens negatively regulated amino PTHrP, explaining the lower expression in males. To test this, we treated BEN human squamous lung carcinoma cells, both with and without transfected androgen receptor (AR), with exogenous testosterone, dihydrotestosterone, and R1881, then measured amino PTHrP 1-34 by ELISA. None of the three androgens significantly affected BEN cell amino PTHrP. However, 5α-Androstane-3α,17β-diol (3α-diol), a metabolite of dihydrotestosterone that is produced robustly by lung cancer cells, caused significant time- and dose-dependent decreases in amino PTHrP levels. These effects were independent of ectopic overexpression of AR and were enhanced when enzymes regulating 3α-diol levels were inhibited. These results show that 3α-diol is capable of lowering the expression of amino PTHrP 1-34 by AR independent pathways in NSCLC.
Abstract Title : The modulation of IL-27 signaling during antiviral immunity.
Abstract : Interleukin-27 is a pleiotropic cytokine important in antiviral immunity. Previous research from our lab showed immune response defects to LCMV viral infection in IL27R-/- mice including decreased type I interferon, reduced antibody production, and loss of viral control. To assess the temporal and therapeutic importance of IL-27, we treated mice with IL-27 or blocked its receptor and monitored host responses to chronic LCMV infection. Neither IL-27 administration nor IL-27R blockade altered the immune responses to LCMV. In contrast, studies with IL27R-/-IL6R-/- mice recapitulated the IL27R-/- immune defects. Taken together, our work demonstrates an important role for IL-27 in viral control.
Abstract Title : The Role of Extracellular Matrix Protein CTGF in Disease Progression of Blast Crisis Chronic Myelogenous Leukemia
Abstract : Chronic Myeloid Leukemia (CML) is a progressive hematopoietic malignancy where expression of the oncogenic fusion protein BCR-ABL in leukemia stem cells (LSCs) prevents the proper differentiation of myeloid progenitor populations, leading to accumulation of undifferentiated blasts. Current treatments target BCR-ABL with tyrosine kinase inhibitors (TKIs); though effective, it fails to eradicate the bone marrow niche-residing LSCs responsible for patient relapse or progression of CML to its terminal stage, Blast Crisis (BC). Previous studies in our laboratory have shown that these malignant LSCs exhibit stem-like behaviors such as quiescence, self-renewal, and induction of pro-survival gene expression through alternative splicing of BCL2 family members. Similar to previous literature, BC CML LSCs co-cultured on stroma are resistant to pharmacological inhibition compared to culturing the cells alone, indicating a role of the extracellular matrix (ECM) in promoting LSC survival. We performed RNA-seq and qPCR of Lin-CD34+CD38+ progenitor cells in CML patient samples and found a significant increase in BC CML versus chronic phase (CP) CML expression of Connective Tissue Growth Factor (CTGF), an ECM protein that aids in cell adhesion and has been shown to predict therapeutic resistance in cancers such as acute lymphoblastic leukemia (ALL). After sub-cloning the gene into an overexpression construct, we introduced CTGF into the BC CML cell line, K562, through lentiviral transduction. Analysis of cell lysates by qPCR revealed a significant overexpression of human CTGF, validating that the lentiviral construct was functional. Interestingly, K562 CTGF cells exhibited a significant increase in pro-survival BCL2 family genes. In line with this, K562 CTGF cells cultured alone or co-cultured with stroma exhibited robust expansion of cell numbers compared to K562 cells transduced with control vector. Future studies will focus on the roles for CTGF in normal hematopoietic stem cells and CP CML cells with particular regard to cell number, differentiation capacity, and ability to engraft into normal hematopoietic niches in vivo in RAG2-/-γc-/- and NSG-S mice. Delineating the effect of CTGF and the ECM in the transition of CP to BC CML could reveal the mechanisms that LSCs utilize to escape current forms of treatment and shed new light on its pathology, allowing for the development of new therapeutic strategies.
Abstract Title : Functional Studies of a Novel Mutation in NTRK1
Abstract : My research is on the functional ramifications of two novel SNPs in the NTRK1 gene. We will be running western blots to analyze the effects of these mutations on TrkA phosphorylation and interactions with its protein partners. Experiments will be carried out in HEK-293T cells that will be transiently transfected with recombinant NTRK1 protein that contains a HaloTag moiety. Cells will then be treated under various conditions that influence receptor activation. We will then use the Tag to isolate the mutant proteins and examine their phosphorylation state and co-factor binding.
Advisor : KIM E. BARRETT
Abstract Title : Precipitation of Behavioral Defects Due to Inflammation in the DSS Mouse Model of Colitis
Abstract : Anxiety, depression, and altered memory are increasingly being associated with intestinal diseases, including inflammatory bowel disease (IBD). Understanding the link between these behavioral changes and IBD may provide important clinical relevance since concomitant mood disorders often increase a patient?s risk of requiring surgery and developing secondary functional gastrointestinal diseases (FGIDs). In the current study, anxiety-like behavior, as assessed by the light/dark box test, and non-spatial memory, as assessed by the novel object test, were determined at the peak and following the resolution of inflammation using the dextran sodium sulfate (DSS) mouse model of acute colitis. DSS was administered for 5 days via drinking water followed by either 3 or 9 days of normal drinking water for comparing behavior during active versus resolved inflammation, respectively. Mice were weighed throughout the study, colon lengths were measured at the time of sacrifice, and histological analysis was conducted in order to assess the degree of colonic disease. In addition, the composition of the gut microbiota was characterized using qPCR of DNA extracted from fecal pellets. Mice at 8 days post-DSS demonstrated impairments in non-spatial memory and anxiety-like behavior compared to controls. These behavioral defects did not persist following resolution of intestinal inflammation and were normalized by 14 days post-DSS. Furthermore, shifts in the composition of the gut microbiota were evident at the peak of intestinal inflammation ? notably as decreases in lactobacilli and segmented filamentous bacteria (SFB) ? which were also reversed by the time of resolution. Taken together, our findings support that in a murine model of IBD, changes in mood and behavior are present during acute inflammation in the presence of shifts in the composition of the gut microbiota.
Abstract Title : Invariants of center-surround interactions
Abstract : Sensitivity to visual stimuli depends on their spatial and temporal context. Effects of temporal context (as in motion adaptation) that appear to be contradictory in narrow samples of stimuli follow a simple invariant pattern when viewed broadly. Thus, local gains and losses of sensitivity caused by motion adaptation added up to a global shift of the spatiotemporal contrast sensitivity function (CSF; Kelly, 1979) when studied over the full domain of CSF (Gepshtein, Lesmes & Albright, 2013). Similarly contradictory results are commonly observed in studies of spatial contextual modulation (?center-surround interactions?) in narrow samples of stimuli. We asked whether a simpler pattern of sensitivity changes can be discovered in center-surround interactions when they are studied broadly, across the domain of CSF. We measured contrast sensitivity using direction discrimination in drifting Gabor patches with or without surrounding gratings. Center stimuli were sampled from a constant-speed ?slice? of CSF (i.e., at constant ratio of temporal to spatial frequencies). When the surround was present, its spatiotemporal frequency and contrast were fixed within an experiment, its orientations were either collinear or orthogonal to the center, and its motion direction was either same or opposite to the center. The results varied across observers when center and surround moved in the same direction, possibly due to individual effects of motion capture. But the results were invariant across observers for all other conditions.
Abstract Title : Depletion of Ovarian Cancer Initiating Cells by a Monoclonal Antibody Against ROR1
Abstract : ROR1 is a type I orphan-receptor tyrosine-kinase-like surface protein that is expressed during embryogenesis and by various cancer cells, but not on normal adult tissues. Its expression is associated with less-well-differentiated tumors with high metastatic potential (Zhang et al Am J Pathol, 2012, PMCID 3509760; Cui et al Cancer Res, 2013, PMCID 3832210). We evaluated the expression of ROR1 in human ovarian cancers and examined the activity of newly developed anti-ROR1 monoclonal antibodies (mAbs, e.g. UC-961) found to have activity against ROR1-expressing breast cancer cell lines and B-cell leukemias. We examined primary human ovarian cancer cells engrafted into immune-deficient NOD/scid mice (PDX) for human ROR1 expression and for phenotypic and functional characteristics of cancer-stem cells (CSC). In each tumor specimen, we observed different proportions of ROR1-expressing cells. Expression of ROR1 associated with markers of CSC, such as aldehyde dehydrogenase 1 (ALDH1). Moreover, the ROR1+ cells from any given PDX sample had a greater capacity to form spheroids in vitro and to engraft immune-deficient mice than did ROR1-negative cells isolated from the same tumor population. Treatment of ovarian cancer cells with the anti-ROR1 mAb UC-961, but not control IgG, reproducibly impaired their ability to form spheroids, and to engraft into immune-deficient NOD/scid mice. In subsequent studies, we isolated ALDH1+ and ALDH1- cells from the same PDX samples. As noted in prior studies (Ginestier et al Cell Stem Cell, 2007, PMCID 2423808), ALDH1+ cells engrafted more effectively than ALDH1- cells. However, treatment of ovarian cancer cells with the UC-961 mAb abrogated the difference in the capacity of ALDH1+ versus ALDH1- cells to engraft NOD/scid mice. Analysis of the few tumors that did develop in mice engrafted with UC-961-treated, but not control IgG-treated, tumor cells revealed that, compared to the original PDX tumor sample, these tumors had lower proportions of ALDH1+ cells and reduced expression of the polycomb ring finger oncogene, BMI1, and changed the expression level of markers associated with the epithelial-mesenchymal transition (e.g. E-Cadherin and vimentin). Collectively, these studies indicate that expression of ROR1 is associated with CSC and that anti-ROR1 mAbs such as UC-961 can interfere with CSC function and self-renewal, suggesting that such biologics may be useful in the treatment of patients with ovarian cancer.
Advisor : WILLIS LI
Abstract Title : Screen for heterochromatin promoting drugs in drosophila melanogaster
Abstract : It was previously found that the JAK/STAT pathway directly plays a role in heterochromatin formation, a form of chromatin that is tightly compact and is essential for gene silencing, chromosome organization and preservation of genome integrity. Antagonizing heterochromatin formation induces tumor genesis in Drosophila melanogaster hematopoietic tumor model. Observing this, we intend to screen a small set of molecule compounds for their ability to promote heterochromatin formation in hopes of discovering a potential anti cancer drug. Using Drosophila, we initially screen for drugs by observing the eye color phenotype of flies with the DX1 gene. Eye color change in these flies upon treatment of the drug should correlate to change in heterochromatin formation. We found 12 significant compounds in which ≥20% of DX1 male populations had significant eye color change (scale 3 or lower).Five out of thirteen of these drugs are alkylating agents, which may indicate that this somehow plays a role in heterochromatin formation.
Abstract : Type 2 innate lymphoid cells (ILC2) are a new population of innate lymphocytes lacking expression of most lineage markers. These ILC2 lack antigen specificity and are instead activated by release of IL-33 and IL-25, two epithelial derived cytokines. Similar to type 2 T helper cells, ILC2 produce IL-5 and IL-13, two cytokines shown to be important in the development and pathology of asthma. Additionally, the cysteinyl leukotrienes are important inflammatory mediators and potent bronchoconstrictors derived from arachidonic acid. 5-lipoxygenase initially converts arachidonic acid into leukotriene A4 (LTA4), LTC4 synthase subsequently generates the cysteinyl leukotrienes, LTC4, LTD4, and LTE4. Expression of the cysteinyl leukotriene receptor 1 and 2 (CysLT1R and CysLT2R) on ILC2 was shown both by flow cytometric analysis and PCR on sorted ILC2. After exogenous leukotriene C4, D4, and E4 was administered intranasally, ILC2 were shown to proliferate and produce IL-4, IL-5, and IL-13 as measured by intracellular flow cytometric cytokine staining. We show here that the cysteinyl leukotrienes play not only an important role as potential bronchoconstrictors and mediators of inflammation, but also as regulators and activators of type 2 innate lymphoid cells.
Abstract Title : Dephosphorylation of tau in lactating rats subjected to acute restraint stress
Abstract : Chronic stress has been consistently implicated as a risk factor for the development of neurodegenerative diseases like Alzheimer?s disease (AD). Hyperphosphorylated tangles of tau are a hallmark of AD pathology, and while the specific mechanisms that link stress and AD vulnerability have not been fully elucidated, our lab has shown that acute and repeated restraint stress leads to an increase in hippocampal tau phosphorylation (tau-P) in rodents, a process that is regulated by the two known corticotropin-releasing factor receptors (CRFR1, CRFR2). A naturally occurring attenuation of stress response is present in female rats during the last part of pregnancy and throughout lactation. In line with this, the hippocampus of a lactating female is more resistant to the effects of a neurotoxin and the ratio of hippocampal Tau-P/Tau is modified by pregnancy and lactation. To test the hypothesis that the decreased sensitivity to stress seen during lactation can modulate stress-induced tau-P, cohorts of virgin, lactating (14-19 days postpartum), or weaned (21 days postpartum) female rats were subjected to either 30 minutes of restraint stress or no stress (control) and sacrificed either 20 minutes or 24 hours after the episode. Western blot was used to analyze differences at two, well characterized N and C-terminal AD-relevant tau-P epitopes (AT8 and PHF-1), as well as relevant kinases. Lactating rats sacrificed 20 minutes after the stress episode exhibited a significant decrease in tau-P compared to lactating control rats as well as virgin rats subjected to the same stress treatment. We found that lactating rats exposed to stress and sacrificed 24 hours after the episode showed a significant increase in tau-P compared to the lactating stressed rats sacrificed at 20 minutes, and returned to levels similar to lactating control rats. This suggests a steep, yet transient stress-induced dephosphorylation of tau in lactating rats.
Abstract Title : Characterization of ubiquitin like modifier ISG15 in t(8;21) leukemia development
Abstract : Acute myeloid leukemia (AML) is a genetically heterogeneous disease with very poor prognosis. The 8;21 chromosomal translocation is the most common genetic abnormality in AML, breaking at 8q22 and 21q22.3, generating the AML1-ETO fusion protein. This translocation occurs in 15% of all cases of AML and 40-50% of the M2 subtype. An alternatively spliced isoform of AML1-ETO, containing an extra exon 9a of the ETO gene known as AML1-ETO9a (AE9a), leads to rapid development of leukemia in mouse models. Interestingly, microarray data indicated upregulation of ubiquitin activating enzyme E1 like (UbE1L) gene is upregulated in AE9a leukemia cells. UbE1L is a critical enzyme for protein modification by a ubiquitin like small protein ISG15. This protein modification is also called ISGylation. In the current study, I examined the possible contribution of increased UbE1L expression in AE9a induced leukemia development using both in vitro cell culture and in vivo UbE1L and ISG15 knockout (KO) mouse models. UbE1L selectively transfers its substrate, a free interferon stimulated gene-15 encoded protein ISG15, to the UbcH8 E2 conjugation enzyme. With the help of ISG15, E3 ligases, they catalyze protein ISGylation. ISGylation, strongly induced upon interferon treatment, is known to play a role in innate immunity. The UbE1L KO mice have increased basal and inducible levels of free ISG15 and lack conjugated ISG15. The ISG15 KO mice lack both free and ISG15 conjugates. In vitro colony forming unit (CFU) assays using AE9a transduced UbE1L KO bone marrow cells showed decreased colony formation. More analyses indicated the reduced proliferation, differentiation and self-renewal of UbE1L deficient hematopoietic progenitor cells. AE9a transduced ISG15 KO bone marrow cells showed similar colony morphology to wild type AE9a cells. These results suggest that free ISG15 is responsible for the proliferative ability of these colonies. Using undenatured and denatured ISG15 treatment of AE9a expressing cells confirmed effect of UbE1L on the colony formation of hematopoietic progenitor cells. A currently ongoing retroviral mediated bone marrow transplantation (BMT) study is used to verify the role of UbE1L and free ISG15 in leukemia development.
Abstract Title : DNA Barcoding: Cryptic Species in San Diego's Intertidal Polychaete Community
Abstract : The sandy intertidal areas of San Diego are host to a diverse array of polychaetes about which little is known. As beach restoration projects threaten the decline of sandy intertidal communities, we face the possibility of losing undiscovered biodiversity. Thanks to DNA Barcoding, a simple and easy way exists to identify unique species and quantify biodiversity in the sandy intertidal. The COI region of the mtDNA was sequenced for 80 polychaetes collected from nine beaches around San Diego. A phylogenetic analysis showed 9 distinct clades separated by high genetic divergence, suggesting nine different species. Of these nine species, three were previously identified as Scolelepis squamata (can't italicize?) due to their nearly identical morphology. However, these three species showed a mean 19.6% divergence after sequencing, indicating a cryptic species complex. This discovery suggests the possibility of further classification issues and unrealized biodiversity in these understudied communities.
Abstract Title : Interferon-β Selectively Inhibits Interleukin-2 (IL-2) Production via cAMP Responsive Element Modulator (CREM)-mediated Chromatin Remodeling
Abstract : Interferon beta (IFNβ) is one of the most recognized and approved therapeutic options for the treatment of multiple sclerosis (MS). However, the mechanism by which the drug works is still unclear. Interleukin-2 (IL-2), a cytokine produced in activated T cells, plays an important role in mediating autoimmunity. Here we demonstrated that IFNβ selectively inhibits the production of IL-2 upon T cell receptor (TCR) stimulation. Furthermore, IFNβ alters histone modification of the IL-2 promoter through the recruitment of histone deacetylases (HDACs) by the interferon induced transcriptional suppressor cAMP responsive element modulator (CREM). These findings can help elucidate how IFNβ mediates T cell responses in MS.