Research Abstracts

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Diane Aguilar

Faculty Mentor: Dr. Karin Kram

CSU Dominguez Hills

Generating Point Mutations in Escherichia coli to Study Bacterial Evolution

Understanding the evolutionary mechanisms of how bacteria adapt to stressful conditions can help us understand how bacteria become resistant to antibiotics or become more virulent pathogens. The bacterial life cycle consists of lag, log, stationary, and death phase. After the death phase there is a phase known as long-term stationary phase (LTSP). LTSP is not well studied, but is more analogous to the natural environment than normal laboratory conditions. In LTSP the cells are in a stressful environment due to low nutrient availability, high pH, and other stressful factors. Cells can survive for many months or even years in LTSP in the lab due to adaptive mutations which help the cells survive these stressful conditions. Two genes, cytR and sspA, are frequently mutated in these long-term cultures. Because mutations in these genes are frequently observed in cultures that have experienced LTSP, we hypothesize that they may play a role in the cell’s survival. Our goal is to recreate the point mutations identified in cells adapted to LTSP and determine how these mutations affect the fitness of the cell. We will use many molecular techniques such as the CRISPR/Cas9 system, Circular Polymerase Extension Cloning (CPEC), transformation, and recombineering to recreate these point mutations. We will confirm the mutation by DNA sequencing. We will then compete our transformed cells with wild-type cells to determine if the mutations are advantageous to the cell’s survival in long term-stationary phase.

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Nataly Arias

Faculty Mentor: Dr. Shehla Pervin

Charles Drew University

Reduction in Mammary Cancer Stem Cell Population Contributes to Slow Growing MDA-MB-468 Xenographs

Despite breast cancer being the second leading cause of cancer deaths in women, little is understood about the genetic and epigenetic factors that play a role in its development. Far less is understood about a distinct cancer type diagnosed in African-American (AA) women: triple negative breast cancer (TNBC), which is noted to have low incidence, but high mortality rate. TNBC lacks estrogen , progesterone, and HER2 receptors. These aggressive tumors have high mitotic rate, angiogenesis, upregulation of macrophage infiltration, and mammary cancer stem cells (MCSC). Our lab examined xenographs from MDA-MB-468, HCC70 and HCC1806 AA TNBC cell lines to understand the mechanisms that promotes their aggressive tumor growth. We found HCC70 and HCC1806 cell lines had a higher growth rate and developed large tumors within a few weeks. In contrast, MDA-MB-468 cell line developed smaller tumors that showed small growth for 3-6 months. This study aimed at understanding the mechanisms that contribute to the slow development of xenographs from MDA-MB-468. Immunoblot and quantitative polymerase chain reaction techniques (qPCR) were performed to examine the differences in expression of cancer stem cell markers and cytokines in the three aforementioned cell line xenographs over different time frames. Our study found that ALDH, a cancer stem cell as well as macrophage markers, that plays a key role in tumor development, was downregulated in the MDA-MB-468 xenographs. Understanding the pathways that intrinsically downregulates cancer stem cell population could help us better understand mechanisms that could inhibit tumor growth and could lead to effective therapeutic interventions.

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Albert Barrios

Faculty Mentor: Dr. Shehla Pervin

Charles Drew University

Increased Expression of DUSP9 Increases Embryonic Stem Cell Population to Promote Breast Center

Breast cancer is influenced by mammary cancer stem cells (MCSCs) that promote its initiation and progression. MCSC population is heterogeneous, where a subset has embryonic stem cell (ESC) characteristics that contribute to aggressive tumor characteristics. Our lab found a correlation between reduced pERK½ levels and upregulation of DUSP9 in African American Triple Negative (AATN) breast tumors. Tumor cells and host microenvironment increased DUSP9 expression in MCSCs and ESCs. We further investigated the significance of DUSP9 expression in ESC/MCSC populations in AATN breast cancer cells.

We examined expressions of DUSP9, pERK½, and ESC markers (Oct¾ and Sox2) from breast cancer cells and its mammospheres by immunoblot analysis and quantitative Polymerase Chain Reaction. We observed effects of adipocytes and Oncostatin M on DUSP9 and ESC expressions. We reduced DUSP9 expression by ShRNA as well as pharmacological inhibition of all phosphatases by treatment with sodium orthovanadate, a phosphatase inhibitor. We performed soft agar assay to examine the importance of DUSP9 in ESCs.

We observed that adipocytes and Oncostatin M increased DUSP9 and ESC markers expression. Mammospheres from breast cancer cell lines showed upregulation of DUSP9 and MCSC/ESC markers, which coincided with reduced levels of pERK½. Our study suggests that increased DUSP9 expression contributes to the survival of MCSC/ESC populations in aggressive AATN breast tumors. The downregualtion of DUSP9 with ShRNA reduced the expression levels of DUSP9 and ESCs. Inhibition of DUSP9 with pharmacological inhibitor, sodium orthovanadate, caused increase in pERK½ and reduced SOX2 expression. This data indicate that DUSP9 could be target to reduce breast cancer initiation and progression.

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Natalya Cardona

Faculty Mentor: Dr. Sonal Singhal

CSU Dominguez Hills

The impact of climate heterogeneity in niche evolution and diversification of Encelia in Western North America.

The Encelia genus is a notable example. The species in this genus evolved within the last five million years and rapidly radiated throughout North American deserts to inhabit a spectrum of niches. Ecological niche modelling (ENM) uses geographic and environmental information to characterize the niche of a species.These models can then be used to study niche evolution and species diversification. Here, we use ENMs to study the potential factors driving niche evolution of Encelia in Western North America (the deserts of California & Mexico). In particular, we test if closely-related species have similar or divergent niches. This will allow us to understand the impact of climate heterogeneity in rapid species radiation. More generally, our work will characterize the effects of climate change in evolution.

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Jose Garfias

Faculty Mentor: Dr. Kenneth Rodriguez

CSU Dominguez Hills

Theoretical Study of 3FC-TZN and its Host-Guest interaction in the Determination of Stable Electroactive Materials

Reliable theoretical H NMR, HOMO, and LUMO is computed for the molecular recognition (Host-Guest) and its role on redox activity, the 2, 4, 6-Tris(p-ferrocenylanilino)-1,3,5-triazine (3FC-Tzn) with cucurbit[n]uril that has been synthesized experimentally. The Density Functional Theory (DFT) of B3LYP/6-31G* method is used to optimize each molecule and then optimize both structures together using the DFT calculations with the M062X/6-31G* method in solvent of DMSO. These results are used to compare the computational calculations with the experimentally synthesized Host-Guest. These theoretically calculated values are within a 5% relative error when compared to the experimentally chemical shift. The 3FC-Tzn is a star shaped molecule with three repeating units of p-Ferrocenylaniline groups connected by central triazine core. The 3FC-Tzn is used as a guest to form a Host-Guest complex with cucurbit[n]uril. The cucurbit[n]uril is a macrocyclic molecule and acts as host to form an intercalated Host-Guest complex with neutral and cationic molecule. In artificial system, redox active compounds have been used in batteries, sensors, and solar cells. Such redox active compounds have been known to play an important role in nature and systems that are artificial. Many enzymes regulate their activities through redox process naturally. It has been reported several redox active compounds in the literature. Among them, Ferrocene has attracted more attention because of its low oxidation potential as well as its reversible one electron transfer process. Ferrocene is an organometallic complex with an iron (II) that is sandwiched between two stacked cyclopentadienyl rings. Having been synthesized from commercially available Ferrocene and cyanuric chloride that was characterized by different spectroscopic techniques, such as NMR, Mass Spec, IR, and UV-Vis and compared to reliable computational modeling.

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Melissa Guardado

Faculty Mentor: Dr. Fang Wang

CSU Dominguez Hills

Zebrafish: A Model To Study Skin Disease

Zebrafish are small freshwater fish that are used as model organisms for biomedical research. These fish have high similarity to human at a molecular level, and are also inexpensive to maintain. Gene expression can be detected and manipulated in their embryos and their transparency allows scientists to observe the development process easily. Zebrafish have been very useful to for modeling heritable human diseases, including various skin diseases. Different disease models can be created by altering zebrafish genome or its gene expression.

In my lab, I work with zebrafish and look at the interactions between the skin cells and neuron cells and see how they can affect the sense of touch in the zebrafish. I am specifically learning a procedure called in situ hybridization, which is a technique used to detect specific mRNA within individual cells to provide insight into gene expression. This procedure requires many steps and it is a three day procedure that we use to know if it confirms gene expression in skin cells or other tissue in these stages: 20 ss, 52 hpf, and/or 72 hpf. In the future, I hope to further enhance my skills and knowledge to be able to go more in depth to make better treatment for these skin diseases in humans.

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Jenna Knight

Faculty Mentor: Dr. Matthew Wright

LA BioMed

Effect of Nicotine and High Fat Diet on Breast Cancer

Traumatic brain injury (TBI) can affect neurocognitive performance. We examined the relationship between Hispanics and Caucasians with and without traumatic brain injuries with regards to attention and processing speeds on the Symbol Digit Modalities Test (SDMT). The sample (N=84) consisted of 32 healthy participants (N=9 Hispanic; N=23 Caucasian) and 52 TBI patients (N=14 Hispanic; N=38 Caucasian). Groups were separated by ethnic identity either Hispanic and Caucasian. TBI patients were tested 6 months post-injury and 12 months post-injury. The SDMT test consisted of both oral and written portions. All participants passed validity testing. 3x2 ANOVAs were run to examine the relationship between Hispanic/Caucasian and TBI on SDMT performance. Group effects (control and TBI groups) were found for both SDMT written and SDMT oral performances. As expected caucasians outperformed the Hispanic groups for controls and 6 month TBI groups on the SDMT oral, and the 6 month TBI Caucasians outperformed other groups for the SDMT written. Surprisingly, Hispanics in the 12 month TBI group outperformed the other two groups in the SDMT oral, and both the 12 month TBI and control groups outperformed the corresponding Caucasian groups

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Erik Martinez

Faculty Mentor: Dr. Karin Kram

CSUDH

Identifying genes that play a role in long-term survival of Escherichia coli

Escherichia coli can adapt to live in an environment without any additional nutrients for long periods of time. When E. coli grows in an environment where nutrients are scarce, they enter long term stationary phase (LTSP), which mimics a natural environment. In order to identify nonessential genes that play a role in survival during LTSP, we competed cells in the KEIO collection and wild type (WT) E. coli cells. The KEIO collection is a set of E. coli strains, each with a single-gene deletion of a nonessential gene. The KEIO collection represents all non-essential genes – with a total of 3985 strains. We performed competitions during 10 days of incubation, into LTSP. We observed cell growth after one, five, and ten days of incubation. After completing the entire collection, we identified 102 gene deletions that affected the cell’s ability to compete with WT. 29 strains with deletions had an advantage over WT cells, whereas the remaining 72 strains had a disadvantage when competed against the WT. Moving forward, we will confirm the phenotype of mutant strains to ensure that no additional factors were influencing the competition. We will then determine why these genes play a role in survival into LTSP by determining their function in long-term cultures.

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Jocelyn Molina

Faculty Mentor: Dr. Jorge Espinoza-Derout

Charles Drew University

Electronic Cigarettes Modulate the NAD+/PARP1/SIRT1 Axis in the ApoE knockout model

Electronic cigarette or electronic nicotine delivery systems (ENDS) have become an exceptionally popular alternative to conventional nicotine cigarettes. It is known that the use of tobacco can lead to DNA damage and mitochondrial dysfunction. A group of mice were exposed to saline, e-cigarettes without nicotine [e-cigarette (0%)] and e-cigarette (2.4%) nicotine [e-cigarette (2.4%)] for 12 weeks. Western blot analysis showed that mice treated with e-cigarette had increased PARP1 activity associated with reduced levels of SIRT1. Also, a decrease of NAD+/NADH levels was observed. These results demonstrate the adverse effects of e-cigarettes exposure leading to NAD+ deficiency which suggests a mechanistic link between e-cigarettes exposure-induced DNA damage and mitochondrial dysfunction.

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Alondra Pena

Faculty Mentor: Dr. Kamrul Hasan

Charles Drew University

Electronic Cigarettes Modulate the NAD+/PARP1/SIRT1 Axis in the ApoE knockout model

Electronic cigarette or electronic nicotine delivery systems (ENDS) have become an exceptionally popular alternative to conventional nicotine cigarettes. It is known that the use of tobacco can lead to DNA damage and mitochondrial dysfunction. A group of mice were exposed to saline, e-cigarettes without nicotine [e-cigarette (0%)] and e-cigarette (2.4%) nicotine [e-cigarette (2.4%)] for 12 weeks. Western blot analysis showed that mice treated with e-cigarette had increased PARP1 activity associated with reduced levels of SIRT1. Also, a decrease of NAD+/NADH levels was observed. These results demonstrate the adverse effects of e-cigarettes exposure leading to NAD+ deficiency which suggests a mechanistic link between e-cigarettes exposure-induced DNA damage and mitochondrial dysfunction.

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Jason Williams

Faculty Mentor: Dr. Patrick Still

CSU Dominguez Hills

NMR Methods in the Elucidation of Quinone Alkaloids from Mostuea brunonis with Activity Against U87 Brain Glioma Cells

Mostuea brunonis is a shrub or climbing liane, with white flowers that may be pink or lilac with an orange to red throat, which is distributed primarily in sub-Saharan Africa. An extract of Mostuea brunonis was obtained from the NCI Active Repository Program and tested for cytotoxic activity against U87 glioblastoma cancer cells, using a sulforhodamine B colorimetric assay. A chromatographic sub-fraction of M. brunonis containing the quinolone alkaloid, pumiloside (1), showed a U87 glioblastoma cancer cell survival rate of 25.7%. Adjacent chromatographic fractions revealed the presence of a m/z = 511 [M+H]+ signal in the crude LC-MS spectrum. Purification of this signal employed a binary solvent gradient method using 20:80 to 50:50 (ACN-H2O) over 10 minutes using a Waters X-Bridge C-18 column. Preliminary inspection of the m/z 511 signal showed the compound to be an analogue of 1 fully saturated at position C18/19. Further analysis of the 1H NMR spectra also showed additional resonances at 2.6ppm and 5.9ppm suggesting a new analogue of 1. Further dereplication of the structure and 2D NMR spectra is being done to fully elucidate the compound. The results obtained here are support for structurally novel compounds from M. brunonis that have potential applications toward the treatment of refractory brain glioblastoma (GBM) cancers.

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