Bridges to Doctorate Program
Mentor: Dr. Hwan Youn
MOLECULAR BASIS FOR THE UNEXPECTED INVOLVEMENT OF THE CRP F-HELIX IN RNA POLYMERASE RECRUITMENT
The Escherichia coli cAMP receptor protein (CRP) belongs to a family of bacterial transcription factors that play important functions in bacteria such as regulating virulence factors in human pathogens like Listeria monocytogenes and Pseudomonas aeruginosa. CRP, the best-studied member, transcriptionally activates the genes involved in catabolizing various carbon compounds in E. coli. To acquire this transcriptional activation activity, CRP needs both DNA binding and RNA polymerase interaction/recruitment. In CRP, the regions for DNA binding and RNA polymerase interaction are distinct and separable functionally and spatially. The F-helix of CRP is directly responsible for DNA binding and three activating regions (AR1, AR2, and AR3) interact with and recruit RNA polymerase. However, recent data from Dr. Youn’s lab demonstrated that amino acid substitutions at an F-helix residue (Val183) influenced transcriptional activation without apparent changes in DNA binding. This result suggests that the CRP F-helix has a role in positioning activating region(s) indirectly. We hypothesize that the affected activating region is AR1 because it is relatively close to the F-helix. My research goal is to unravel how the F-helix residue (Val183) is functionally connected to AR1. My current project is to make CRP mutants at position 172 in various Val183 mutant background using the method of codon randomization and to select for highly active CRP mutants, in order to test if the interaction between position 172 and 183 amino acids repositions AR1 in affecting RNA polymerase recruitment. Since both residues are non-polar, the combined size of 172 and 183 amino acids is hypothesized to be an important factor for proper CRP function.
Excerpted from Lawson et al. (2004) Current Opinion in Structural Biology
Mentor: Dr. Martin Shapiro
Heart rate variability (HRV) is a measure of the inconsistent gaps between each heartbeat (e.g., 30 milliseconds following a 40-millisecond time interval). HRV has shown to reflect the unconscious control of heart activity through both the parasympathetic and sympathetic nervous systems. The parasympathetic nervous system works quickly to slow down heart rate while the effects of the sympathetic nervous system slowly increase heart rate. For my thesis project, I am investigating the role of HRV as a measure of individual differences in student's decision-making skills under cognitive stress (e.g., test-taking). Previous research has suggested that HRV plays a role in physical, emotional, and cognitive changes. In the present study, I will measure how HRV is affected by a student that is required to adapt to cognitive stress and their ability to make accurate decisions. To assess HRV, I am using Elite HRV wearable devices and comparing them to state-of-the-art Biopac electrocardiogram (ECG) systems. The Elite wearable HRV device will enable the students to monitor resting-state HRV at home. Resting HRV will then be compared to HRV during a delayed matching task and recovery HRV after the task. The delayed matching task will be used to create a stressful event and assess the accuracy of their decision-making skills. I hypothesize that students with higher resting HRV will be able to adapt faster to stress activities and make better decisions than those with low resting HRV.
Mentor: Dr. Chris Miller
META-ANALYSIS OF THE TRIPLE NETWORK MODEL OF DEPRESSION
Primary studies using resting-state functional magnetic resonance imaging (rsfMRI) have reported abnormal functional connectivity in brain regions of the triple network model (i.e., central executive, default mode, and salience networks) of major depressive disorder (MDD). It has been proposed that the aberrant connectivity of these neural networks plays a significant role in the cognitive deficits, emotional dysfunction, and specific symptoms (e.g., anhedonia, rumination, amotivation, and psychomotor retardation) of MDD. The present study aims to advance our understanding of MDD by identifying abnormal patterns of functional connectivity in depressed patients and by examining the relations of these abnormalities to specific clinical symptoms, cognitive deficits, and emotional dysfunctions.
We are conducting the first-ever meta-analysis of the triple network model of depression by quantitatively combining all existing fMRI studies that compared functional connectivity of the central executive, default mode, or salience networks in patients diagnosed with MDD to age-matched healthy controls. These results will be used to evaluate the triple network model of depression and suggest novel neural targets for treatment. They may also be used to increase the efficacy of existing neurostimulation therapies (e.g., transcranial magnetic stimulation) and to inform the development of possible strategies for treatment selection and symptom-targeting in patients with MDD.
Mentor: Dr. Trisha Van Laar
UNTANGLING THE MOLECULAR PATHWAY BETWEEN GLUTATHIONE AND RADICAL OXYGEN SPECIES.
Pseudomonas aeruginosa is a gram negative pathogenic bacteria that can have serious and fatal consequences for patients with cystic fibrosis victims with burn injuries. The bacteria is often acquired from hospital personnel that may have unknowingly served as a vector from previous patients. Unfortunately, the bacteria also displays a natural resistance to antibiotics and has the potential to secrete toxins that result in iron sequestration and the formation of radical oxygen species. As a result, the Van Laar lab has conducted projects to manipulate the pathogenicity of the organism by developing mutant strains of Pseudomonas aeruginosa. Specifically, my project is an extension of previous work published in the Van Laar lab that is interested in untangling the molecular pathway between glutathione and radical oxygen species. Glutathione is a biological antioxidant that is involved with the removal of radical oxygen species. The protein does not operate independently and requires other biological molecules to recycle the antioxidant feature that it provides the cell. In some cases Glutathione could interact with an alcohol molecule and spontaneously interact to form an alcohol glutathione complex that could not remove radical oxygen species. In order to remove glutathione from the alcohol complex it would require a secondary protein called alcohol dehydrogenase to free glutathione. What would be the result of a mutant without alcohol dehydrogenase enzyme? Would Pseudomonas aeruginosa be able to remove radical oxygen species? How would it interact when introduced to alcohol? The answers to these questions are unknown in previous research and the Van Laar lab is interested in providing answers.
Mentor: Dr. Trisha Van Laar
Antimicrobial resistance (AMR) and gene transfer
Probiotics are marketed to humans and domesticated animals globally as providing therapeutic/protective effects against gut microflora imbalances. Probiotics are typically consumed in food or as supplements. Despite the pathogenic characteristics of some Bacillus spp. (B. anthracis, B. cereus), B. subtilis and B. coagulans are deemed “generally recognized as safe” (GRAS) by the FDA and are added to a plethora of probiotic products. Although these species have attained GRAS status, they may still possess genes conferring antimicrobial resistance (AMR) which could be shared among other bacteria. In this study, we aim to determine if probiotic Bacillus are resistant to antibiotics and analyze their potential to transfer AMR genes to potentially pathogenic Bacillus.
Mentor: Dr. Laurent Dejean
Particulate matter (PM) exposure, inflammatory response and reactive oxygen species (ROS).
Fresno is considered one of the most polluted cities in the United States. Previous studies have established a link between particulate matter (PM) exposure and adverse health effects. In our laboratory, we work with Rat alveolar macrophages (NR8383) and study the potential of PM2.5 to induce an inflammatory response catalyzed by a high production of reactive oxygen species (ROS). The chemical composition of PM2.5 is a complex mixture of solid and liquid particles that contains a high degree of variability associated to the time and location of a sample. Components of PM2.5 such as transition metals and quinones are believed to play a major role in PM2.5 cytotoxicity, due to their ability to trigger intracellular ROS production through process such as Fenton reaction or redox cycling. To further investigate PM2.5-associated ROS generation, our team utilized a cell-based fluorescence microplate assay. The fluorophore employed was dichloroflourescein-diacetate (DCFH-DA), which will measure the total intracellular ROS levels triggered by PM2.5 samples and model solutions. A flow cytometry-based assay will be employed to identify subcellular localizations of oxidative stress. This technique will allow for single cell analysis of multiple fluorophores, in which fluorescent dyes MitoSox and DCFH-DA could differentiate mitochondrial and cytosolic ROS. By distinguishing between the separate fluorescent signals, the experiments could provide information about the mechanism to which PM2.5 may contribute to pulmonary dysfunction; and the relative contributions of quinones and transition metals in these processes.
Mentor: Dr. Hwan Youn
Allosteric regulation of CRP (cAMP receptor protein) as a transcriptional activator
CRP (cAMP receptor protein) is a transcriptional activator which regulates hundreds of transcriptional units, it does so by responding to cellular levels of cAMP. CRP has two states active and inactive inside the E. coli cell. Upon binding to cAMP, CRP undergoes an allosteric transition, thereby allowing the protein to bind DNA strongly and specifically. The active conformation of CRP is heavily studied and well-known, but inactive conformation is poorly understood. The inactive structure may have interactions that allow its activity to remain low in the absence of the cAMP. My project aims to identify CRP residues particularly important in the inactive conformation. Putative residues have been selected based on their involvement in charge interactions in the inactive form of CRP but not in the active form. Using site-directed and random mutagenesis I will substitute residues responsible for the charged interactions. I predict that substitution of these residues in the inactive form will increase the activity of CRP in the absence of cAMP by destabilizing the inactive form. Results from these experiments will locate key structural components critical for the inactive CRP form and may reveal the reasons why they are important. Several proteins in this family function as virulence factors in human pathogens like Listeria monocytogenes and Pseudomonas aeruginosa. Once the critical components of regulation are understood, the knowledge can be applied to the homologs in the pathogen in order to develop small-molecule inhibitors of bacterial transcription.
Mentor: Dr. Martin Shapiro
Effect of emotional states on risky economic decision-making.
For my thesis project, I am investigating the effect of emotional states on risky economic decision-making. There is some evidence from our lab that seeing negative images (e.g., a dead body) increases riskiness compared to seeing positive images (e.g., water skiing). In the present study, we are measuring individual emotional reactions and risk-taking behavior. Emotions are being assessed by physiological activity and self-report. Participants complete a gambling task where they solve short memory problems using images; they then have an opportunity to risk points based on perceived accuracy. While playing the game, we monitor three physiological responses, heart rate (ECG), skin conductance (EDA), and facial expression (EMG). We are also using subjective measures of emotion and stress before, during and after the experiment. We hypothesize that those most affected subjectively and physiologically by the images will show the most significant change in their risk-taking behavior.
Mentor: Dr. Alejandro Calderon-Urrea
Identification of the DNA Sequence Responsible for Chalcone Resistance in C. elegans Using EMS-Based Deep Sequencing Mapping.
The project will be focused on identifying the DNA sequence responsible for causing resistance to the organic chalcones 17, 25, and 30. These organic chalcones kill C. elegans at very low concentrations and therefore are excellent nematicidal prospects. This project will help us elucidate the molecular and biochemical mechanisms of action for these chalcones.
Mentor: Dr. Paul Price
How people respond to their implicit racial bias that is inconsistent with their explicit bias.
People exhibit a sample size bias when judging the average of groups. The larger the group, the greater the judged average—even when the actual average is held constant. Sample size bias has been demonstrated for a variety of stimuli and stimulus dimensions. For example, the judged average of a group of numbers increases as the number of numbers increases (Smith & Price, 2010). Similarly, the judged average size of a group of squares increases as the number of squares increases (Price et al., 2014). Recently, Halliday et al. (2016) found the judged average attractiveness of a group of people increases as the number of people in the group increases. In the previous research on attractiveness, however, the stimulus people were at least moderately attractive. But we do not know what will happen when the stimulus people are unattractive. For this reason, we are conducting a study to see whether sample affects judgments of the average attractiveness of groups of unattractive individuals. College student participants will judge the average attractiveness of groups of three, six, and nine people—including groups of men and groups of women—who are either very attractive, moderately attractive, or unattractive. For groups of very and moderately attractive individuals, we expect to see a sample size bias with larger groups being judged to be more attractive on average. For groups of unattractive individuals, larger groups may also be judged to be more attractive on average, which would be consistent with all our previous research on the sample size bias. However, larger groups of unattractive people may instead be judged to be less attractive on average. This result would have interesting implications for understanding why the sample size bias occurs. For example, it would imply that it does not involve a simple “translation” of larger samples to judgments that are higher on the response scale.
Mentor: Dr. Hwan Youn
Comparative analysis of CRP and Clp in DNA binding and transcriptional activation
The CRP/FNR superfamily of transcription factors typically function as transcriptional activators. CRP is a transcription factor, in which the ligand cAMP binds, inducing an a conformational shift of the protein. This change in structure allows for the binding of CRP to the specific DNA binding site. Clp is a CRP homolog, binding to the same DNA binding site.However, Clp does not require the binding of a ligand to assume an active conformation, instead, it sustains an active conformation alone. As to why this occurrence takes place is a current topic of research.
Mentor: Dr. Qiao-Hong Chen
Chemical modifications for anti-prostate cancer agents.
The American Cancer Society estimates over 161,000 new cases of prostate cancer and about 26,730 deaths from this disease will occur in 2017. There are successful treatments for early-stage prostate cancer, but not for advanced, metastatic castration-resistant prostate cancer. There is a huge difference in the incidence of prostate cancer between Western (120 per 100,000 in Northern America) and East countries (less than 10 per 100,000 in Asia). The significantly increased risk of prostate cancer in the first generation of Asian men relocating to the United States suggests a chemopreventive effect of Asian traditional food. Several dietary natural products have been demonstrated to have potential in treating prostate cancer. However, their moderate potency and poor bioavailability have limited their clinical use. My research aims to optimize two dietary natural products, Fisetin and Luteolin, through chemical modifications as anti-prostate cancer agents. The specific aim is to define the in vitro potency of the nitrogen-containing derivatives of fisetin and luteolin.
Mentor: Dr. Laurent Dejean
A Method to Assess Reactive Oxygen Species Production in Alveolar Macrophages Exposed to Particulate Matter
There is a well-established link between atmospheric particulate matter with a diameter smaller than 2.5 nm (PM2.5) and adverse health effects. PM2.5 exposure has been associated with reactive oxygen species (ROS)-linked oxidative stress. For the investigation of PM-associated ROS generation, the dichlorofluorescein-diacetate (DCFH-DA) fluorescent probe is applied in a 96-well microplate format to measure relative ROS levels induced in alveolar macrophages. The objective of this study is to establish a method for the detection of ROS production induced in alveolar macrophages in response to PM2.5 samples collected in the areas of Fresno and Claremont, California.
The macrophage-based ROS detection assay standardized in this study is a part of an integrated approach, which involves the chemical characterization of a unique set of PM2.5 samples and the analysis of these PM2.5 samples in both cell-based and cell-free models. The goal of this project is to analyze a collection of 15 PM2.5 samples with the bioassay and to compare the ROS measurements obtained to the chemical composition of the samples.
Mentor: Dr. Laurent Dejean, Chemistry
The Warburg Effect
There is an imbalance of cell death observed in cancer such as in lymphomas. In other words, there is variation in the anti-apoptotic and pro-apoptotic expression levels. The B-cell lymphoma 2 (Bcl-2) family proteins that regulate cell death, it can have a pro-apoptotic or an anti-apoptotic effect. Current studies show that Bcl-2 is overexpressed in cancer patients. Patients that have high levels of Bcl-2 tend to have a resistance to chemotherapy. Monitoring the expression levels of Bcl-2 in cancer patients is important to provide the most optimal treatment for the patient. The Bcl-2 family protein is localized to the outer membrane of mitochondria. They can be identified by their four domains: BH1, BH2, BH3, and BH4. Members of the Bcl-2 that have an anti-apoptotic response include Bcl-2, Bcl-xL, and Bcl-w. Cancer cells need the energy to survive, their metabolism can be explained by the Warburg effect.
The Warburg effect shows that there is an increase in anaerobic glycolysis vs mitochondrial oxidative phosphorylation. In the 1920s, Otto Warburg observed that cancer cells tend to uptake glucose at high rates compared to normal cells. The consumed glucose is then secreted as lactate instead of being oxidized completely. Tracking the metabolic pathway using the stable isotope Carbon-13 (C-13) will provide a better understanding of cell metabolism. The in vivo labeling of cancer cells (Bcl-2, Bcl-xL) with C-13 glucose will help keep track of the glucose consumption and lactate production. Gas Chromatography-Mass Spectrometry will be used to study changes in the cell metabolic pathway.
Mentor: Dr. Ulrike Muller, Biology
On Biomechanics of Suction Feeding
My research project is about the suction feeding of small organisms. I will build a robotic model of a bladderwort which will aid in studying the suction feeding off that organism. This model will allow us to see the feeding event at a more reasonable time frame, and also how small the gap can get and still have a successful feeding event. To better understand the actual feeding event, I have done high-speed filming of a few feeding events of Utricularia praelonga. Plants in the genus Utricularia are carnivorous plants.
Mentor: Dr. David Lent, Biology
On Short-range and Long-range Visual Navigation of Insects
Wood ants (Formica rufa L.) are minuscule creatures, yet navigate in a very large world. They are among the many ant species that rely chiefly on their sense of vision for navigation in their environment. Studies have shown that wood ants use the skyline panorama to obtain long-range navigation cues for both returning to a previously-identified location, such as a food source, and for returning to their nest upon completion of the trip. For short-range navigation, the chief visual cues to the ant come from images that present a relatively unstable image on its retina. These objects generally are closer to the ant. As such, as the ant travels past such objects, their orientation changes with respect to the ant’s position.
Mentor: Dr. Jason Bush, Biology
Neural Stem Cells and Irradiation
Cranial radiotherapy is often used to treat brain tumors; however, cognitive decline
has been observed as a consequence of this therapy and there are currently no preventative
measures that can be taken to avoid this side effect. Much attention has been centered
on functions that are dependent on the hippocampus since multipotent stem cells are
concentrated here (Gage 2000). Recent studies show a reduction in the number of neurogenic
cells (Monje ML. et al. 2007) and mature neuronal loss in the dentate gyrus portion
of the hippocampus (Raber J. et al. 2004; Fan & Weinstein 2007). It has been demonstrated
that MNPs that received varying levels of irradiation exert acute and chronic levels
of oxidative stress (Limoli CL et al. 2004;2004;2006;2006;2007; Giedzinski E. et al.
2005). I propose to differentiate neural stem cells (NSCs) into neurons, astrocytes,
and oligodendrocytes. I will then irradiate the 3 differentiated cell types in addition
to the undifferentiated cells at varying levels of irradiation. I will then proceed
to evaluate the damage with NMR and various fluorescence microscopy assays.
The purpose of my project is to address a current gap in scientific knowledge pertaining to how multipotent neural precursor (MNPs) cells are affected after receiving varying levels of irradiation. I believe that the use of mitochondrial protectants might help reduce the damage to both NSCs and differentiated cells after irradiation. The objectives are (a) Grow and differentiate NSCs into neurons, oligodendrocytes and astrocytes, (b) Irradiate the 4 cell types without neuroprotectants and evaluate the damage with a variety of fluorescence microscopy assays and NMR and (c) Administer neuroprotectants to the 4 cell types and irradiate them and evaluate the damage in the same way as objective 2.
Mentor: Dr. Mamta Rqwat
On the Biofilm Formation
We are investigating how DTT a reductant and Diamide an oxidation affects biofilm formation on three species of interest, Mycobacterium smegmatis, Candida albicans and Staphylococcus aureus. This study will allow for further understanding of different biofilms and the effects of environmental stresses and provide potential drug targets.
Mentor: Dr. Joy Goto
On the Mechanism of BMAA and its Carbomate Adducts
The environmental neurotoxin beta-Methylamino-L-alanine (BMAA) is considered an etiologic factor in the development of amyotrophic lateral sclerosis (ALS) and ALS-parkinsonism-dementia complex (ALS/PD). The structural moiety of the non-protein BMAA alone is not able to decisively produce neurotoxicity within the neuronal environment. However, in the presence of bicarbonate (HCO3) BMAA undergoes structural alterations to give two different compounds known as the α-carbamate adduct and the β -carbamate adduct. Of the two adducts formed, the β-carbamate adduct contains the greatest structural similarity to the primary excitatory neurotransmitter, glutamate. Both compounds are elongated chains with an acidic carboxyl group at the opposite end. Based on the similarities of both structures and the ability of the glutamate to bind to the ionotropic receptors (iGluRs) it is highly likely that BMAA and its adducts are also able to affect the iGluR as either partial, full, or biased agonist. Considering the onset of ALS/PD does not occur acutely, but rather, over an extended period, the mechanism by which BMAA exhibits its excitotoxicity effects must be through continual neuronal stress in the neuronal milieu. The lack of acute neurotoxicity suggests the dynamic equilibria between BMAA and its respective adducts are variable during periods of metabolic and neuronal stress. Identification of the primary adduct formed, as well as, how it is affected by various conditions such as pH, metals, and regulatory compounds are important in order to identify a possible mode of action for BMAA in the neuronal environment.
Mentor: Dr. Jai-pil Choi
Colorimetric Detection of Alkali and Heavy Metals by Crown Ether Functionalized Au25 Clusters
Gold nanoparticles have been well studied over the past century garnering attention within many disciplines finding wide use in applications such as drug delivery, catalysis, and as chemicals sensors. Gold nanoparticles <3 nm like Au25 is a whole new class of nanoparticles which possess rich optical and electrical properties different from their larger cousins. Since their discovery been under constant investigation. Our work focuses on studying fundamental electronic and optical properties of Au25 functionalized with crown ethers and doped with heavy metals. Crown ethers are well known to chelate cations in solution potentially serving as interactive extensions of Au25. Recent findings have led to an unexpected reaction between Au25 and Hg2+. When mercury is introduced to a solution of Au25, a change in its electronic features are observed, which might suggest alterations within the gold core. Our focus is to study the electrical, optical, and physical properties of crown ether functionalized Au25 and AuxHgy clusters by cyclic voltammetry, UV-Vis spectroscopy, and fluorescence.