Many genes are associated with microcephaly, a condition characterized by a small head size. Through exome-sequencing, we identified de novo missense mutations in an actin polymerization gene, ARPC4, in four individuals with microcephaly, mild developmental delay, and mild intellectual disability. ARPC4 is involved in actin filament formation. Actin is an essential component of the cell’s cytoskeleton that gives the cell its structure and aids in cell movement and division. The goal of this research project is to understand the molecular mechanisms that lead to the disease phenotype observed in these patients. I began by using CADD, a measurement used to predict the deleteriousness of single nucleotide variants. The mutations identified in the patients were located in highly conserved loci, indicating they might be pathogenic. To provide further evidence of the pathogenicity of the ARPC4 variants, we were interested in determining the functional effect these variants have on actin polymerization – specifically, the role of ARPC4 in this mechanism. I established fibroblast cell lines from two patients with the same ARPC4 mutation. I performed immunofluorescence staining for actin to quantify the amount of actin present in the patients and control cell lines. Preliminary data from this experiment suggests that there is greater abundance of actin filaments in the control sample compared to the patients’ cells. To observe the effect of decreased actin abundance on cell migration, I executed a scratch migration assay. The results from this study elucidate the impact of ARPC4 in actin polymerization, and establish actin deficiency as a clinically recognizable cause of microcephaly.

Human T-Cell Leukemia Virus type 1 (HTLV-I), a retrovirus that currently 10 to 20 million people are infected with, has been shown to be associated with the Adult T-cell Leukemia (ATLL) and HTLV-associated myelopathy-tropical spastic paraparesis (HAM/TSP). 1 After an extended period of time, 3-5% of those infected with HTLV-1 will develop ATLL or HAM/TSP. The viral Tax protein is thought to have involvement in the development of these diseases. The Tax protein inhibits telomerase and topoisomerase-I which in turn inhibits the process of DNA repair. Tax prevents apoptosis and does not allow cells to enter the G0 phase of mitosis. The oncogenic properties of Tax are correlated to the interaction of Tax with host cellular proteins which are strongly influenced by the large amount of Tax protein made from the integrated provirus. We are using viral promoter magnetic pull-downs to investigate HTLV viral-host interactions involving minimal -306bp viral promoter containing vCRE enhancer sites and Tax-recruited host proteins. Experiments have used electrophoresis to determine whether or not the magnetic beads bind the promoter DNA. Absolute quantitation is necessary for consistency and reproducibility in analysis of proteins suspected of recruitment to the promoter.

Tuberculosis (TB) kills more than one million people annually. Bacteria of the Mycobacterium genus, including M. tuberculosis, build a complex cell wall containing mycolic acids. This cell wall is difficult to penetrate, so specialized antibiotics are needed. Even with newly developed drugs, bacteria adapt quickly and exhibit resistance at an alarmingly rapid pace. One adaptation that allows the community to survive is production of biofilms. Formation of biofilm is one of many quorum sensing behaviors known in pathogenic mycobacteria. This additional layer surrounds a microenvironment where bacteria can thrive with a very low concentration of antibiotic. Thus, one alternative method to treat TB is to control biofilm formation. In this research, a set of marine bacterial strains, including several bacteria that exhibited swarming behaviors and several from the same environmental samples that did not, were cultured, extracted, and analyzed by 1H NMR and LC-MS as well as in newly-developed biofilm and growth inhibition assays. Initial results showed one group of bacteria produced an organic compound that induced biofilm production in mycobacteria. This was an unexpected result. One representative strain producing a strong biofilm inducer was grown on large scale (10L) then extracted using progressively less polar eluents on a reversed-phase SPE column. The biofilm-inducing fraction was then separated using flask column chromatography. One major component was analyzed spectroscopically using 1D and 2D NMR techniques along with Mass Spectrometry. This compound could result in a strategy to interfere with biofilm formation in mycobacteria, thus making antibiotics more effective.

Glutaredoxins (GRXs) are small oxidoreductase enzymes that can reduce disulfide bonds in target proteins. The genome of the model plant Arabidopsis thaliana has more than 30 GRX genes, but the biological function of most of these GRXs is unknown. We previously found that a small group of Arabidopsis GRX genes is specifically activated by nitrate, a common source of nitrogen in the soil. In order to better characterize the function of one of these nitrate-regulated GRXs, AtGRX660, we generated transgenic Arabidopsis plants that continuously overexpress the AtGRX660 gene. We isolated RNA from 12 independent transgenic lines and quantified AtGRX660 mRNA levels via real-time reverse transcriptase PCR. Three elite lines displaying >100-fold increase in basal AtGRX660 transcript levels were selected for further analysis. AtGRX660-overexpression lines and wild-type plants were grown on soil in a controlled environment growth chamber for characterization of shoot phenotypes, and on vertically-orientated plates of plant growth media for characterization of root phenotypes. All AtGRX660-overexpression lines displayed a dwarf shoot phenotype, with significant reductions in shoot biomass, total leaf area, and silique length compared to wild-type plants. In addition, root system architecture was highly altered. While primary root growth was normal in the transgenic plant lines, lateral roots were almost completely absent. Phase contrast microscopy demonstrated that lateral root primordia develop in the transgenic lines, but these primordia do not elongate and emerge from the primary root. Overall, these results suggest that AtGRX660 acts a negative regulator of shoot organ development and inhibits lateral root elongation. Our findings could have agricultural relevance in plant drought tolerance, since AtGRX660 differentially affects primary root system growth (root system depth) and lateral root system growth (root system breadth).

Herpes simplex virus (HSV) infects mucosal epithelial cells and can cause recurrent, painful ulcers. HSV is not curable and establishes lifelong latency in host neuronal ganglia. Herpes affects more than 400 million people globally, with an increased risk of genital ulcer disease, HIV acquisition, and transmission of HSV-2 to partners or neonates. Persons with altered T cell immunity have been reported to have prolonged and more frequent lesions, suggesting that the adaptive immune responses are associated with HSV clearance. Additionally, there is evidence of innate cell and T lymphocyte recruitment to the infection site to help clear the lesion. While there is extensive evidence on the role of T cells in these lesion infiltrates, little is known about B cell activation and antibody response during HSV reactivation. The study of B cells and antibodies can be used in developing a HSV vaccine since all licensed vaccines induce robust antibody responses. Antibodies block virus entry and mediate antibody-dependent cell-mediated cytotoxicity (ADCC). That is why a better understanding of local antibody responses against HSV-2 is needed to develop a successful vaccine. My project examines whether HSV antibody-producing B cells are found in areas where HSV-2 reactivates and the potential role that these antibody-producing cells play in host clearance of the virus. To examine this question, ddPCR using B-cell-specific probes were used to identify different B-cell subtypes responding to the HSV infection. Results to date have shown high variability with few emergent patterns. In the future, additional samples will be examined using new protocols for better cDNA synthesis, and more positive and negative controls to increase the accuracy of the data.

Stem cells are known for their ability to self-renew and differentiate into specialized cells. A key factor that drives self-renewal and differentiation is the specific localization of cell-fate determinants inside the cell cytoplasm. In this work, we focused on a protein called Numb, commonly known to inhibit the Notch signaling pathway responsible for the regulation cell-fate. We sought to understand the correlation between cell spreading, morphology and Numb localization. We studied human adipose-derived stem cells (HADSCs) in two conditions: 1) Cells fully spread after 24 hours and 2) Cells that were actively spreading or retracting. In each condition we analyzed the distribution of Numb fluorescence expression inside the cell cytoplasm. We found that in cells that were allowed to fully spread, Numb was highly localized only on cells with pseudopodia (“arm-like” cytoplasmic protrusions). Numb was evenly distributed when cells were fully spread, had a large area and circular morphology. In the second condition, cells that were actively spreading or retracting were small and circular in morphology. Interestingly, these cells had a high fluorescence expression of Numb specifically localized at the cell edges. These results seem to indicate that Numb localization was found in areas of membrane activity, such as pseudopodia and spreading/retracting cells. This could be due to the fact that Numb has been found to participate in integrin turn-over (cell adhesion). Based on these results our next step will be to modify membrane activity and cell adhesion and observe the effects on Numb localization and stem cell differentiation. Regulating stem cell fate via protein localization will allow us to enhance the production of differentiated cells for regenerative medicine applications.

The ability of an organism to dissociate environmental cues for either safe or threatening situations is key for survival. Generalized fear is an adaptation in which behavioral responses for threatening stimuli are produced to non-threatening cues. In mice, discriminative or generalized fear responses are modeled using a fear conditioning paradigm of two shock intensities, 0.3 mA or 0.5 mA. Mice trained at 0.3 mA intensities could discriminate between safe and threatening cues, yet mice trained at a 0.5 mA intensities displayed generalized fear behavioral responses. Previously, we had identified that dopaminergic neural activity was critical for the ability to discriminate between cues when shocked at 0.3 mA. Dopamine neurons express Kv7.3 potassium channel subunits, which modulate neural activity. I hypothesized that Kv7.3 subunits on dopamine neurons would be critical to threat discrimination and mice that had undergone mutagenesis of Kv7.3 would show generalized fear discrimination. My approach used transgenic mice expressing Cre-recombinase and viral-mediated gene delivery of cre-inducible CRISPR-Cas9 plasmids targeted for the specific mutagenesis of Kv7.3. Mice underwent a three day paradigm known as fear conditioning. Baseline freezing behavior was assessed by playing two tones, A and B. This was followed by fear conditioning trials where at the end of tone A, mice received a shock of either 0.3 mA or 0.5 mA (CS+) and at the end of tone B, mice received no shock (CS-). On the third day, mice underwent a probe trial to assess final ability to discriminate between threatening and non-threatening cues. These findings could provide insights into the mechanisms underlying neurological disorders and serve as a guide for future pharmacological interventions.

Over 350 million people are affected by major depression on a global scale according to the World Health Organization with an increasing economic impact of over $210 billion annually with a growth of 21.5% since 2005. The ability to switch behaviors or strategies when faced with chronic stress is an important determinant to the susceptibility and longevity of depression within people, also referred to as cognitive flexibility. The objective of this proposed study is to analyze the effects of the endogenous cannabinoid (ECB) system on cognitive flexibility through the lateral habenula. Cannula implant surgeries will be conducted on rats to input antagonist or agonist drugs into the brain. Behavioral tests will be conducted on rats to examine whether these manipulations will promote or deter cognitive flexibility.