High-resolution structures of membrane proteins are necessary for guiding the rational design of novel therapeutics. Membrane proteins make up 30% of our proteome and roughly 60% of all drug targets, yet they only represent ~4% of the structures in the Protein Data Bank. This is because membrane proteins have proven to be extremely difficult to crystallize for traditional X-ray crystallographic studies. Recent advances in single-particle electron cryo-microscopy (CryoEM) technology are beginning to revolutionize the field of membrane protein structural biology by providing near-atomic resolution structural information without the need for protein crystallization. Despite these advances, CryoEM is limited in its ability to characterize small membrane proteins (less than ~200 kDa in size) and dynamic protein-protein interactions. Several important classes of membrane proteins are “too small for CryoEM”, such as the G-Protein Coupled Receptors and the Major Facilitator Superfamily transporters. Moreover, our ability to capture membrane proteins in complex with cytosolic signaling proteins is commonly limited by their inherently short-lived low-affinity interactions. Our research group aims to overcome these hurdles to characterize the regulatory complex formed between the ~100 kDA small membrane water channel, Aquaporin-0 (AQP0) and the calcium signaling protein, Calmodulin (CaM). We intend to enhance the small size of the AQP0 water channel by applying Nanodisc technology, which offers the added benefit of a near native lipid bilayer environment to study the membrane protein. While the system requires further optimization, a preliminary Nanodisc reconstitution has an enhanced mass of ~250 kDa, which is sufficient for high-resolution CryoEM analysis. We are also exploring the use of engineered avidity as a novel approach to enhance the native multi-valent interaction between CaM and AQP0 tetramers. We believe these strategies represent general approaches that may be applied to thousands of other small membrane proteins and transient protein complexes to facilitate their structural characterization. "

Neural injuries (either peripheral or central nervous systems) affect a large number of the human population world-wide and in general are considered irreparable due to the lack of regenerative capacity of the neural tissues. Various neural tissue engineering approaches have been employed to differentiate human neural stem cells (hNSCs) into neurons for the purpose of using them in regenerating the damaged neural tissues. Recently, electrical stimulation was found effective to enhance neuronal differentiation of hNSCs. Light-triggered generation of free electrons on the surface of semiconducting materials is an exciting option to achieve electrical stimulation on hNSCs. However, the band gap of most light-harvesting semiconductors falls in the UV range which is harmful to cells. The main hypothesis of this study is that hNSCs will preferentially differentiate into neurons when cultured on dopamine-modified titanium oxide (TiO2) substrates followed by irradiation by visible light (~ 420nm). We have discovered that TiO2 grown on polydopamine (PDA) coated substrates exhibits an absorption peak in the visible light range. The surface chemistry was confirmed by various surface characterization methods including UV-Vis, SEM, EDS, and XPS. Using this substrate, we investigated the effect of laminin on hNSC adhesion, surface free electron generation on TiO2 by visible light irradiation, and preferential differentiation of hNSCs into neurons. The results from this research open up a new possibility for implantable neural tissue engineering scaffolds that can be activated by light irradiation.

As animals grow, scalar expansion of many types of neurons must be coordinated with animal growth to maintain receptive field coverage and proper connectivity, a process that is not well understood. Resulting from a genetic screen, the Parrish lab has generated and isolated a mutation that results in severe elongation of the ventral nerve cord. This mutation affects a predicted transcription factor of unknown function, senseless-2(sens-2). Although the origin of the defect is unknown, one possible explanation for the defect is that sens-2 regulates growth of the peripheral nerves. In the absence of sens-2 function, the peripheral nerves are unable to elongate properly and, as a result, larval growth leads to greater strain on the nerve cord, which leads to nerve cord elongation. An alternative explanation is that sens-2 is required in all the cells of the nerve cord (neurons and glia) for their growth. Thus, one major aim of this study is to produce antibodies that recognize the sens-2 protein. To accomplish this task, I will generate a plasmid vector for production of sens-2 protein in bacteria, perform a recombinant protein purification and submit the protein for antibody production. Upon receiving the antibody, I will perform a series of dissections accompanied by antibody staining and imaging in order to localize sens-2. A second major aim is to take an unbiased genetic approach to identify factors that interact with sens-2 to regulate nerve cord expansion. If we identify interactions with genes of known functions in growth control or patterning, it will allow us to formulate models to account for sens-2 function in nerve cord elongation. To accomplish this task, I will I will create a series of heterozygous sens-2/deficiency stocks and perform genetic screens to determine which genes in Drosophila interact with sens-2's molecular pathway. 

The blood-brain barrier is a very selective membrane that works to protect the brain from most pathogens; however, this means that it is a difficult therapeutic target for drug targeting. Cutting-edge nanotechnology research is being performed by Dr. Elizabeth Nance on targeted drug delivery mechanisms to combat brain tumors such as glioblastomas, neuromas etc. Nance’s group has found and supported good drug release results with poly-ethylene glycol coating (PEG) for which they hope to perform controlled and targeted release. Currently, there is a lack of molecular level understanding of the drug-polymer interactions that dictate controlled release. Better computational models are needed to understand release kinetics and facilitate rational design of biomaterials to control the release or retention of a particular drug or enzyme. In the present work, molecular dynamics (MD) simulations were used with GROMACS software to study glutamate diffusion in water to validate our method and set a baseline for more advanced systems. Diffusion constants calculated with the Einstein equation based on glutamate diffusion in MD were consistent with experimental values of ∼0.75 μm2/msec. We are currently extending this approach to probe glutamate diffusion in the presence of biocompatible polymers such as PLGA (poly(lactic-co-glycolic acid)) and PEG. Then, we compared and contrasted the diffusion constants to make sure they were on the same order of magnitude as glutamate. We also studied the system kinetics to discover the polymeric matrix that allows the system to get closest to a diffusion-limited rate. We will relay this to Dr. Nance to modify her experiments to lead to improved drug delivery systems that help fight off brain cancers.

Heart failure (HF) is a serious medical epidemic, and the number of those suffering from end-stage, advanced HF continues to rise along with the use of long-term left ventricular assist devices (LVADs) as treatment. Despite improvements in LVAD technology and clinical management, patient response remains significantly heterogeneous with variable predicted outcomes based on measurement of traditional biomarkers. Soluble suppression of tumorigenicity-2 (sST2) is upregulated in HF due to myocyte stretching and fibrosis during disease progression; this protein, as a novel biomarker, may provide insight into pathophysiologic changes that occur following LVAD implantation and potentially help predict patient response. More comprehensive patient selection would reduce the use of unfavorable and expensive surgery, saving money, and lives of those suffering from HF. Thus, we aim to quantify changing levels of plasma sST2 in adult patients who have undergone LVAD implantation. Plasma samples were collected from 100 individuals prior to surgical implantation of a LVAD and at 1-, 3-, and 6-months post-operatively. Levels of sST2 will be measured in all available plasma samples using commercially-available quantitative sandwich monoclonal ELISA kits (Critical Diagnostics, San Diego, CA). Data will be analyzed using repeated measures ANOVA and modeled using growth modeling approaches, as appropriate. We anticipate that plasma sST2 levels will significantly decrease from pre-LVAD to 6-months post-LVAD in this cohort of patients. Moreover, we anticipate discovering differing trajectories of sST2 change that will help differentiate who will do better or worse following LVAD implantation. Our findings may support use of sST2 as a clinically relevant biomarker in HF patients following LVAD implantation. Incorporating sST2 into the model of response to LVAD implantation could lead to improved outcome prediction to LVAD implantation.

 We will use the roundworm Caenorhabditis elegans to study the process of meiosis, which is involved in the formation of gametes (sperm and eggs). Its small size, rapid life cycle, transparency and well-annotated genome allows researchers to track the effects of mutations that disrupt gametogenesis. Our preliminary data demonstrated that a genetic screen for mutations that increase the viability of embryos produced by worms lacking SPO-11, a critical factor regulating meiotic chromosomal inheritance, can identify genes required for the accurate transmission of the genome from one generation to the next. To further test this hypothesis, we will continue this screen to identify additional regulators of meiosis. Our results will be relevant to human genetics, since key regulators of gametogenesis are highly conserved in both worms and vertebrates. Thus, we can learn about the causes of human birth defects by examining worm development. Consequently, these mechanisms will be relevant to understanding gametogenesis in humans and may aide in preventing genetic instability.

Meiosis produces four daughter cells that are genetically distinct. A crucial step of production is the pairing of homologous chromosomes; however, how these chromosomes align by homologous pairing remains a mystery. One contributing factor to alignment is tension within kinetochores. Experimentation with budding yeast reveals that a lack of tension will influence the spindle checkpoint until all sister chromatids are attached to the fiber. In the same manner, we will use budding yeast to investigate homologous interaction as the yeast provides us with an ideal model of recombination rates and life cycles similar to higher eukaryotes. The question of homologous behavior is also presented in DNA double-strand breaks. During prophase, these breaks (DSBs) create interactions between homologs along with inducing repair of homologous recombination. The strand is able to be repaired by the sister chromatids; however, the DSBs prefer interactions with homologous chromatid. How are the homologous chromatids found and used to repair the strand? These types of questions will be better understood with our research focused on how homologous chromosomes align initially during meiosis.

As cancer remains a looming threat to the public, understanding the genetic and biochemical basis of how cancers operate is necessary in order to develop new medicines and treatments. A topic of critical importance when considering the biochemistry of cancers is how specific changes in gene expression control the growth of tumors. I explore this topic by using Drosophila genetics in order to investigate the crosstalk amongst three key growth regulators, RAC-alpha serine/threonine-protein kinase (Akt1), Translationally Controlled Tumor Protein (TCTP), and Yorkie (Yki). The overexpression of Yki in intestinal cells is known to cause intestinal tumors, and our preliminary results indicate that Akt1, TCTP, and Yki interact to control intestinal stem cell proliferation. Nevertheless, signaling crosstalk amongst these three genes is still ambiguous. By using the advantageous genetic toolbox of Drosophila, I created experimental fly lines which allow me to manipulate the expression of each gene or a combination of these genes in Drosophila intestinal stem cells. My goal in this project is to anatomize this signaling crosstalk through monitoring how manipulation of these genes in combinations influence the proliferation of intestinal stem cells using confocal microscopy. Ultimately, we hope to attain a greater understanding of growth in relation to genetic networks that control it.

Drinking water treatment is essential to obtain a healthy regulated source of water that is distributed throughout a community. There are various methods to disinfect water; some have more negative effects toward public health than others. For example, disinfection with chlorine causes disinfection byproducts (DBP) to be produced within treated drinking water. The Environmental Protection Agency (EPA) regulates DBPs because they have the potential to cause cancer and other negative health effects if levels of exposure exceed legal limits. Ultraviolet(UV) light disinfection does not produce these DBPs which is why it is becoming a preferred method for water disinfection. The issue that medium pressure UV disinfection technology faces currently, is the lack of a proven, regulatory accepted method to monitor low wavelengths(LW). These low wavelengths (200-240nm) have been demonstrated in the literature to effectively inactivate regulated viruses, such as adenovirus, that are found in drinking water. The EPA, 2006 Groundwater Rule requires demonstration of 4-log removal/inactivation of human enteric viruses. Being able to take advantage of these LWs could help many communities develop more sustainable performance and compliance. This research is determining if innovative sensors can accurately record LWs that will help prove disinfection. Also, will taking advantage of these LWs result in energy and cost savings providing a more sustainable UV disinfection systems while improving public health protection. The hypothesis is that there will be savings in energy consumption and cost when the UV system operates taking advantage of the LWs. Data was recorded and analyzed from a drinking water treatment plant to determine the effectiveness and reliability of the sensors while demonstrating the potential energy savings. The preliminary results show that when taking full advantage of the LWs the system operates using about 40% less energy which can be correlated with an overall lower operation and maintenance cost.