Paper-based diagnostics are an exceptionally cost efficient technology for detecting pathogens. In the presence of a rapid spread of diseases to a large number of people, early detection becomes an even more vital process that can mean life or death for individuals infected by such pathogens. This project aims to improve an existing device that performs a multiplexable disposable nucleic acid test in which a lateral flow network is normally horizontal; the problem with the horizontal orientation is that once the paper membranes are saturated fluid will leak out of the membranes which can prevent the device from operating successfully and giving readable results to the user. To avoid this complication without relying on encapsulating the membranes, which can alter the flow physics and introduce an extra material to dispose of at the end of the process, the possibility of running the assay network in the vertical orientation will be evaluated by measuring the success rate of the vertical device in performing the isothermal amplification step in a nucleic assay. The isothermal amplification will be done using an electric heater in an open system which will result in concentrating analytes near the heater and therefore the study will also evaluate the benefits and complications that might arise from heat concentration of analytes in the device and the possibility of using this technique in increasing the sensitivity of the detection process downstream of the heating region.

Currently, disease detection in rural areas of developing countries is hampered by a lack of accurate, convenient and affordable diagnostic tests. The primary purpose of this research is to design and analyze software to diagnose test strips on a mobile device. The tests are paper-based and therefore cheap and appropriate for use in developing counties. Further, the software is simple and intuitive to use and would not require a health worker to have prior training. My role has been to develop an Android application that will allow a user to take a photo of an immunoassay test which then interprets the test results using algorithms run on the mobile device; it then provides a health worker with an objective, automated, and accurate diagnosis at the point of care.

The use of medical ultrasonography as a screening tool has greatly improved the early detection and diagnosis of cancer and disease. However, it has also increased the risks of false positives and unnecessary biopsies that can outweigh the screening benefits. This occurs when the ultrasound finds tumors that do not possess a risk to the patient’s life. As a result, additional screenings are often performed to reduce the percentage of false positives. This is an expensive process that can further increase when different doctors order different ultrasound screenings. In order to improve the design and current usage of Philips ultrasound imaging systems in patient diagnostic procedures, we need to analyze the preexisting patient data regarding the use of ultrasound imaging aystems. To do so, this project aims at building a patient database to store, manage, and classify the data from patients and doctors from the University of Washington department of radiology, regarding the use of ultrasound technology. A posterior statistical analysis of this data will allow us to find the utilization of specific features and modes on the system, and to determine the relationship between the number of false positives and false negatives and potential linkages to settings or image quality. The analysis will be done on small sets of related data, obtained from the system through queries. By comparing the occurrence of false positives and negatives for different settings and parameters, we will be able to determine the best utilization of the ultrasound imaging system. The expected results from this statistical analysis will demonstrate the relation between ultrasound imaging system settings, and the rate of false positives and negatives. This information can then be further used to modify the current usage and design of ultrasound imaging systems.

Prochlorococcus, the most abundant photosynthetic organism in the ocean, has an important impact on our planetary system by providing the basis of many marine food webs, and contributing to approximately 20 percent of our planet’s oxygen. It may play a significant role in other planetary cycles. Through experimentation, we aim to understand how the presence of arsenic affects the distribution of Prochlorococcus, and conversely how Prochlorococcus influences the distribution of arsenic. A cell will try to use arsenate in place of phosphate (because of the similar structure of the molecules), but since arsenate does not function like phosphate, a disproportionate ratio of arsenate to phosphate is harmful. We predicted that MED4, a strain of Prochlorococcus, would die quickly when exposed to one μM of arsenic in the media, because that is more concentrated than what is typically found in seawater. Three control cultures of MED4 were grown in media that contained no phosphorous, and three experimental cultures in media that contained no phosphorous and had a concentration of one μM arsenate. The only phosphorous available was what was contained in the cells. Cell death was quantified by using cells counts from a flow cytometer. The experimental MED4 cell counts decreased immediately and were dead after just four days, indicating sensitivity to arsenic. This was the first in multiple experiments to determine MED4’s tolerance to arsenic exposure. Clearly one μM arsenic is too high for MED4. These results call for future experiments with a lower arsenic to phosphorous ratio, so that we can discover the limits of MED4 survival with regard to the presence of arsenic. We can also compare MED4’s response to those of other strains that possess different combinations of arsenic detoxification genes.

Vibrio parahaemolyticus is the leading causes of seafood-borne bacterial gastroenteritis in the U.S. However, overall lack of understanding of virulence is a major obstacle for predicting and preventing disease. Several outbreaks in 2012-13 in New England were apparently caused by an invasive sequence type 36 strains that appears more virulent than endemic strains. Unlike many other bacteria, V. parahaemolyticus uses quorum sensing (QS) to represses virulence under high cell densities, a form of community communication allowing cells to coordinate gene expression. It has been noted that many pathogenic strains of V. parahaemolyticus historically used in research are quorum blind (QB), leading to increased virulence, and suggesting an intriguing hypothesis that altered QS promotes evolution of more virulent strains. The proposed study investigated the extent to which clinical strains are QB compared to environmental strains, evaluated whether altered QS is likely the result of laboratory handing rather than a natural avenue to enhance virulence, and determined whether highly-virulent ST36 strains are QB. Recently isolated and historic clinical strains and environmental strains were evaluated for their QS characteristics both phenotypically and genetically. Clinical strains were more often QB compared to the environmental strains; however, data (phenotype and genotype) do not suggest that historical clinical strains were more often QB compared to other clinical strains, indicating this is a potential mechanism for evolution of virulence and not artifact of handling. Both recent and historical ST36 strains exhibited normal QS, indicating enhanced virulence is not likely due to quorum blindness. An observation made during the nematode assay shows that a particular strain is more virulent at one time than other. This observation has opened a new field of investigation, where strains locked at two different phenotypes are being compared. This study improves our understanding of the evolution of this pathogen’s virulence, which may help in minimizing human diseases caused by this and other similar pathogens.

The increasing number of antibiotic resistant bacterial strains and the rising number of deaths due to infections by these bacteria underline the need for research into new classes of antibiotics.  The bacterial enzyme DapE has not been targeted by current antibiotics and is an ideal target for the development of new antibiotics.  DapE (N-succinyl-L,L-diaminopimelic acid desuccinylase enzyme) is a promising enzymatic target because it is part of the succinylase biosynthetic pathway that leads to the production of lysine and meso-diaminopimelate (mDap).  Lysine and mDap are vital components of the peptidoglycan cell wall in Gram-negative and Gram-positive bacteria.  Existing classes of potent antibiotics have shown that inhibiting cell wall synthesis has deleterious effects on bacterial cell survival and being able to target both Gram-positive and Gram-negative bacteria will give a DapE inhibitor compound the potential to become a broad spectrum antibiotic.  It has been shown that DapE is essential to bacterial cell growth and proliferation and that the deletion of the gene dapE is lethal to bacteria.  A high throughput screen was performed to test the inhibitory potential of over 30,000 compounds against DapE.  This screen led to two indoline sulfonamide lead structures with promising levels of inhibition of DapE.  The objective of this project is to identify successful methods to synthesize medicinally relevant analogs of the indoline sulfonamide lead compounds, and test their antibiotic activity. An interdisciplinary approach is being used to identify potential inhibitors of DapE.  Molecular docking and molecular dynamics will guide traditional medicinal chemistry in the selection of promising analogs to synthesize.  The current focus is the synthesis of 7-indolinesulfonamides via the intramolecular formation of a cyclic sulfonylurea using chlorosulfonyl isocyanate.  The successful creation of an antibiotic that is effective against currently resistant bacteria will address the medical and bio-defense concerns that arise from antibiotic resistant bacteria.