Due to the rise of pathogen genetic knowledge and implementation methods of diagnoses pathways for patients, an increase in access to speedy and efficacious therapies is needed. The development of inexpensive, high-performance point-of-care (POC) tests does not only improve healthcare in low resource settings, but also moves the diagnoses out of hospitals and into homes and primary care offices. Paper-based microfluidic devices help both identify and screen for pathogens by allowing for more rapid care and treatment. The multiplexed, autonomous, disposable nucleic acid amplification test devices fabricated in the Yager Laboratory performs sequential functions - sample preparation, nucleic acid amplification, and lateral flow detection – for target identification. My project aims to bridge the last two segments into a single unit by introducing a method of inhibitory competition that permits for measurable, fluorescent-based analysis of isothermal DNA amplification from dry reagents in porous media. To prove this concept, a simplified experimental model of a multi-region, paper-based nucleic acid amplification test was designed, and new interference amplification mixtures developed. The isothermal strand displacement amplification (iSDA) technique utilized in these devices incorporates a fluorescent hybridization probe that allows for the detection of the increase in amplicons using an optical detection method. By incorporating a dilution of comparable, secondary strand of DNA as an internal control, a competition for reagents is established which creates different chemical sensitivities in the amplification zones. As a result, target DNA from the pathogen is amplified against a competitive threshold in a range. In the long run, fluorescence imaging of all regions can yield a quantitative measure of the concentration of pathogens in a sample. Overall, this project tests the fundamental groundwork for a new family of optical-based, quantitative devices by testing the use of fluorescent detection for POC tests, and investigating the principle of inhibitory competition.

 Neurodegeneration from proteostatic stress spreads from cell to cell. However, the cellular pathways that contribute to this spreading are poorly understood because there is no simple genetic model established to study this. We can use Caenorhabditis Elegans as the model system because the nematode secretes the ECVs outside into the extracellular environment. I have found that transgenic nematodes (Caenorhabditis elegans) that express human versions of the toxic proteins characteristic of Alzheimer's disease (Amyloid beta) are transported by extracellular vesicles (ECVs). Therefore, I am now developing this novel model system for studying extracellular vesicles signaling and its role in spreading age-related neurodegeneration. The powerful genetics of C. elegans will allow me to study the cellular pathways that contribute to that spreading of these toxic molecules. By harvesting the nematodes from growth plates and centrifuging the worm pellet in buffer, we are able to separate the worms from the raw wash, which contains the ECVs. The raw wash is then run through a size exclusion column to separate the ECVs, and the different size samples are either run through a BCA Protein Assay to determine amount of protein in each sample based on an absorbance reading, or run through an SDS-PAGE Protein Gel and treated with antibodies to isolate the aβ protein. My preliminary results show that the aβ protein is in fact located within a specific range of samples from the size exclusion column. The ability to isolate the extracellular vesicles and locate the source of aβ protein and its proliferation should have a significant impact on the future of Alzheimer’s therapy. If we know more about the exact location and behavior of the deleterious protein, we can apply the knowledge to studies on human cerebral spinal fluid and develop drugs that target this specific pathway in humans.

Global climate change causes weather patterns to be increasingly unpredictable, which has a major impact on plant productivity. Humans are dependent on the agricultural economy and loss of crop productivity would be devastating. Due to their sessile nature, plants are unable to escape environmental stresses and must be able to acclimate to rapidly changing conditions. Arabidopsis thaliana is an important plant model organism, which has around 30,000 genes (about as many as humans). Arabidopsis genes have been identified, but not fully characterized, which is why it is the optimal model organism to work with. There are also many genomic tools available in the reference organism Arabidopsis. One such tool is RNA interference. Using small RNAs, we knockdown an accumulation of gene products and screen to find which genes play important roles in that particular function – drought resistance, for example. In our Arabidopsis RNAi screen, we chose to use trans-acting small-interfering RNAs (tasiRNAs) for multiple reasons. TasiRNAs have a lower chance of off-target transcript silencing than other siRNAs. They are naturally occurring in plants and have a low chance of transgene silencing. Lastly, multiple tasiRNAs can be incorporated into the library at different positions at varying concentrations. Our RNAi screen includes tasiRNAs that target every gene in the Arabidopsis genome, which allows for easy identification of causative changes and the ability to screen for multiple phenotypes with one library. These screens allow us to identify essential genes and other complex interacting gene systems important to plants under various environmental stresses. In the future, this technology can be developed on a bigger scale with crops.

According to the World Health Organization, more than 100 million people annually drop into poverty because of health care related costs. Point-of-care (POC) diagnostic testing works to alleviate this issue. Point-of-care diagnostics is testing that delivers cheap, accurate, and timely diagnosis in the hospital, the field, the office, or the home. In this way, POC testing can provide vital health information in a way that reduces the burden of health care globally. The lab of Dr. Paul Yager has developed a POC diagnostic device known as the multiplexed autonomous disposable nucleic acid amplification test, or MAD NAAT, which returns results in less than one hour. This device uses a two-dimensional paper network (2DPN) to automate the fluidic steps of a nucleic acid-based diagnostic assay, which detects the presence of one or more pathogens (e.g. MRSA, RSV) in a patient’s test sample. The downstream region of the 2DPN uses nitrocellulose strips to support a visual, colorimetric detection system. The detection system produces a series of visible red lines on the nitrocellulose strip that indicate the result of the test and associated controls. My research is focused on the optimization of the detection system, specifically: increasing the visible intensity of these red lines without increasing device cost. I have investigated the effects of varying the geometry of the 2DPN through the detection region. The goal of this research is to obtain the most intense test line signal with the least amount of expensive reagents, at the lowest possible pathogen count, in the least amount of time. I have also used MATLAB to implement patient sample analyzations for the MAD NAAT device. These efforts support further development of an affordable POC device, which will improve patient health outcomes in communities that lack proper healthcare.

 Cyclic diadenosine monophosphate (c-di-AMP) is an essential nucleotide for bacterial virulence. C-di-AMP has been extensively studied in Listeria monocytogenes (Lm), but is also produced by other pathogens including E. coli, C. trachomatis, and M. tuberculosis. Although c-di-AMP is a signaling molecule in bacteria, host cells detect it as a pathogen-associated molecular pattern. During infection, the host receptor STING senses c-di-AMP and stimulates expression of interferon-beta, an anti-viral molecule. To date, STING is the only known sensor of c-di-AMP, and its promotion of an anti-viral versus anti-bacterial response has been somewhat perplexing. However, our lab has recently discovered another host c-di-AMP sensor, the murine aldo-keto reductase1C13 (AKR1C13) or “RECON”, that induces an alternative, anti-bacterial cellular response to infection. Based on the importance of bacterial clearance and the ubiquity of AKRs across species, we began to investigate whether other murine and human AKRs respond similarly to c-di-AMP. Sequence alignment of AKR genes and data on tissue-specific expression of these genes returned six candidates: the murine AKR1C12 as a potential paralogue, and human AKRs 1C1, 1C2, 1C3, and 1C4 as potential homologues. Cloning techniques allowed me to insert these genes into an E. coli expression vector that I used to purify recombinant protein via affinity chromatography. The binding affinity with c-di-AMP of each protein was tested with radio-ligand binding assays to determine dissociation constants (K_d) and we additionally used enzyme activity assays to quantify inhibition of protein activity. AKRs that bind c-di-AMP will be further characterized in vivo in mammalian cell lines in which expression has been knocked down via lentiviral transduction. Further work in cell lines may elucidate novel pathways that are activated downstream of c-di-AMP detection. Understanding the pathways that naturally clear infections is important to potential medical advances in bacterial infection management and prevention.

The efficient production of cellular energy via the mitochondrial electron transport chain is necessary for the growth, health, and lifespan of all organisms. However, the genetic pathways that impact the regulation of energy production are still largely undefined. This is largely because the electron transport chain is so essential for life that genetic mutations which disrupt its functioning are lethal, preventing their study. The small roundworm Caenorhabditis elegans has been a useful model because its powerful genetics have allowed researchers to perform forward screens to identify mutations to electron transport chain proteins that can significantly reduce ATP production but are not severe enough to kill the animal. Preceding studies in my lab (Dr. Kaeberlein, Pathology) have identified how mutations that on the ISP-1 molecule itself can restore more normal functioning. My research focuses on identifying genes other than ISP-1 that can also functionally compensate for the reduction in ATP production efficiency. I am currently identifying the genetic loci of three mutations that suppress the loss of ATP production through single nucleotide polymorphism (SNP) gene mapping, and whole genome sequencing. These process require genetic crosses and high-throughput PCR and restriction digest protocol. Results from this experiment would give the first insights into the cellular pathways that can impact energy production.

Expansion Microscopy (ExM) is a revolutionary type of fluorescence microscopy that enables super-resolution on conventional microscopes of fixed specimens that have been chemically incorporated into a hydrogel polymer matrix, enzymatically digested, and physically expanded. The original ExM approach utilized special tri-functional antibodies containing a DNA-dye-linker complex to tether the fluorescent dyes to the expandable polymer and yielded an isotropic expansion of ~4x in all directions and a lateral spatial resolution of ~70nm. Recently, we have created a simplified ExM methodology, using conventional fluorescently labeled antibodies or fluorescent proteins, and a variety of commonly available reagents yielding increased fluorescence retention for cultured cells and tissue specimens. Additionally, we have extended this new approach of ExM to Drosophila larvae and brain tissue using an optimized solution of enzymes to digest the robust outer surface of larvae (cuticle) and allow for uniform expansion of the polymer. These simplified methods for expansion microscopy will enable super-resolution microscopy on a wide range of biological samples without the need for specialized reagents or equipment.