Current data on CP violation in B_d -> eta' K_S and B_d -> phi K_S, taken literally, suggest new physics contributions in b -> s transitions. Despite a claim to the contrary, we point out that right-handed operators with a single weak phase can account for both deviations thanks to the two-fold ambiguity in the extraction of the weak phase from the corresponding CP-asymmetry. This observation is welcome since large mixing in the right-handed sector is favored by many GUT models and frameworks which address the flavor puzzle. There are also interesting correlations with the B_s system which provide a way to test this scenario in the near future.

Even without grand unification, proton decay can be a powerful probe of physics at the highest energy scales. Supersymmetric theories with conserved R-parity contain Planck-suppressed dimension 5 operators that give important contributions to nucleon decay. These operators are likely controlled by flavor physics, which means current and near future proton decay experiments might yield clues about the fermion mass spectrum. I present a thorough analysis of nucleon partial lifetimes in supersymmetric one-flavon Froggatt-Nielsen models with a single U(1)_X family symmetry which is responsible for the fermionic mass spectrum as well as forbidding R-parity violating interactions. Many of the models naturally lead to nucleon decay near present limits without any reference to grand unification.

Every year infectious diseases are among the top major causes of death worldwide. Infectious diseases disproportionately impact resource-poor areas. While most developed countries have the ability to screen and quickly detect infectious diseases in humans, animals, and plants, developing countries lack this ability. In such areas, access to medical treatment and testing facilities is severely limited. Additionally, developing countries are more densely populated, which makes infectious disease surveillance exceedingly more difficult. As a result of this, infectious diseases tend to spread more quickly and outbreaks tend to occur more often in developing countries. Additionally, the increasing rate of antimicrobial resistant infections and emergence of novel infectious diseases increase the likelihood of global pandemic occurrence.

It is evident that developing novel devices for detection of infectious diseases would be greatly beneficial to global health. It is important to note that developed countries would benefit equally from such a device. Pandemics tend to burden the economy and put strain on resources even in developed countries. The impact of such a device is determined by a set of criteria put forth by the World Health Organization (WHO). Based on the ASSURED criteria, an ideal device for point-of-care (POC) diagnostics must be affordable, sensitive, specific; and yet it must also be user-friendly, rapid and robust, equipment free, and deliverable to end-users. Additionally, this device must be easily adaptable for detection of novel pathogens. The nucleic acid amplification test (NAAT) meets many of these criteria. However, NAAT’s current workflow involves many user liquid handling steps as well as a thermocycler.

This thesis aims to address these issues by first automating isothermal nucleic acid amplification, followed by coupling the amplification step with the lateral-flow immunoassay (LFA), which is a paper-based diagnostic device. Thus, taking advantage of the affordability and POC features of the LFA. The aim of these efforts is to work toward the ultimate goal of developing a device that receives the patient sample, then carries out all the steps in NAAT in an automated manner and produces results with just a push of a single button.

Point-of-care (POC) diagnostics are easy-to-use, low-cost devices that can rapidly detect a biomarker of interest without the need for highly trained personnel or expensive equipment. In resource-limited settings, POC testing is often the only viable option for patients, which highlights the importance of developing sensitive technologies for early disease detection and monitoring of treatment methods. Hydrogels are flexible 3D structures made with crosslinked hydrophilic components that absorb large amounts of water. These systems are sensitive to different chemical or physical stimuli, and the magnitude of their response is usually directly proportional to the magnitude of the applied external stimuli. In the diagnostics field, hydrogels are mostly being used for enzyme detection and immunodiagnostics. For these applications, the biomarker acts as a signal that triggers a volume, color, or state change of the hydrogel, an example of the latter being a gel-sol transition or vice versa. This technology often requires the chemical modification of at least one of the main hydrogel components so that the system is specific towards the molecule of interest. Accordingly, these devices need to be manufactured in a laboratory setting with complex equipment and often expensive reagents, and they also require personnel with extensive knowledge and experience to conduct the assay and its analysis. Thus, it would be desirable to design a hydrogel system that is responsive to the presence of the biomarker of interest without the need for chemical modification of the reagents to design a robust, POC-friendly device that can be manufactured in all parts of the world. To develop this system, we focused on calcium alginate dextran hydrogels beads for the detection of abnormal concentrations of ions, such as bicarbonate and phosphate, in patient samples. This system can be synthesized by a simple ionic crosslinking method based solely on the complexation of the oppositely charged species, i.e., alginate and calcium, which comprise the gel matrix. For disease detection, we took advantage of a calcium depletion mechanism based on the reaction of the biomarkers of interest with the divalent calcium cation in the calcium alginate gel matrix. The timeframe for hydrogel degradation was therefore correlated with the sample’s ion concentration, making our POC device semi-quantitative. To our knowledge, this is the first demonstration of the use of nonionic polymers to improve the morphology of calcium alginate hydrogel beads using a simple processing method that involves minimal labor, equipment, and reagents. The simplified bead synthesis protocol combined with a user-friendly device that we developed allows for the rapid detection of high serum bicarbonate or urine phosphate levels at the POC.

Providing effective healthcare to underrepresented populations is a major challenge worldwide due to environmental, economic, and social disparities. Both developing countries and low income rural/urban areas in the US tend to have reduced healthcare access which limits the frequency and quality of medical screenings as well as treatment options. This has led to an increased effort in the development of point-of-care devices which emphasize, immediate and onsite results. One device that has the potential to fulfill these requirements is the lateral-flow immunoassay (LFA). While the LFA has been successful in the form of the over-the-counter pregnancy test, it still does have some disadvantages that limit its ability to fully replace sophisticated laboratory-based assays. Throughout this thesis, I will be discussing the development of cutting-edge technologies to address the two major disadvantages of the LFA which are its poor quantitative characteristics and low sensitivity.Cerebrospinal fluid (CSF) leaks are a common complication of numerous procedures in Otolaryngology. These leaks are relatively difficult to diagnose and the current laboratory-based tests to do so have a turn-around time that is unacceptable for rapid clinical decision making. We addressed this problem by developing the first rapid diagnostic test kit for the detection of CSF leaks. In Chapter 2, we describe the development of a barcode-style LFA for the semi-quantitative detection of beta-trace protein, which is present in CSF at high concentrations. This work was expanded upon in Chapter 3, where we created a complete, standalone rapid diagnostic test kit composed of our barcode-style LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Clinical studies demonstrated excellent predictive capabilities of this kit in distinguishing patient samples containing CSF from those that did not. In some cases, a more quantitative readout than what the barcode-style LFA can achieve is required. In Chapter 4, we developed a new technique to introduce an even more quantitative readout to the LFA without the need of electronic readers. This technology was applied to the therapeutic drug monitoring of the cardiac glycoside digoxin. Digoxin capture and binding occurs on a modified LFA test strip which utilizes platinum nanozyme probes that possess catalase-like activity. This test strip is combined with a reaction that controls the anisotropic etching of gold nanorods to produce a wide range of visible color hues dependent on the initial digoxin concentration in the sample which allowed for naked-eye quantification. We then shifted to the development of technologies to improve the detection limit and sensitivity of the LFA. Our lab previously discovered the phenomenon of an aqueous two-phase system (ATPS) separating on paper, which allowed for the seamless integration of biomarker preconcentration and detection on the LFA. In Chapter 5, we have extended the functionality of an ATPS separating on paper to automate the sequential delivery of signal enhancement reagents in addition to concentrating biomarkers. The timing of reagent delivery was controlled by changing the initial composition of the ATPS and a mathematical model was developed to help predict the ATPS flow behavior. We applied this technology to automate biomarker preconcentration and nanozyme signal enhancement on the LFA, resulting in a 30-fold improvement in detection limit over the conventional LFA when detecting Escherichia coli, all while maintaining a single application step. In late 2019, a new virus known as SARS-CoV-2 began to sweep across the globe, leading to the COVID-19 pandemic. The development of highly sensitive and easily deployable rapid diagnostic tests is a vital aspect of managing this pandemic. In Chapter 6, we discuss ongoing efforts to develop a highly sensitive, nanozyme signal enhanced LFA for the rapid detection of the SARS-CoV-2 nucleocapsid protein. A 3D-printed casing was designed to store the test strip and all reagents in a way that maintains user friendly operation by eliminating liquid handling steps. Besides developing highly sensitive diagnostics, the ability to deploy these tests quickly and in a highly scalable manner is crucial for being able to manage a pandemic. The work described in Chapter 7 highlights the integration of novel fibronectin-based antibody mimetics, called monobodies, into the LFA for the detection of the nucleocapsid protein of SARS-CoV-2. These monobodies are advantageous over monoclonal antibodies in that they are rapidly developed and produced recombinantly in Escherichia coli which is more affordable and scalable than monoclonal antibody production.

Ethological studies not only provide insights on how and why animals perform extraordinary behaviors, but they also provide an opportunity to understand how animals are disturbed by human activities in the Anthropocene. Ecotourism, a popular recreational activity, is a worldwide growing business that can be used as a tool for biodiversity conservation. However, a lack of fundamental knowledge and effective management plans for an ecotourism site may lead to an increase in anthropogenic disturbances and ultimately degrade a site’s biodiversity. In Ubon Ratchathani, Thailand, locals discovered a unique migration of freshwater shrimp known as “Parading Behavior”, an unusual type of upstream migration in which hundreds of thousands of shrimp climb out of water and walk on land to the headwater. This behavior has captured public attention and Shrimp Watching tourism has been promoted as a must-see event at Ubon Ratchathani. While striking, this behavior is poorly understood. Preliminary evidence suggests that tourists might disturb the shrimp population during migrating. Unfortunately, we know very little about the biology of, and anthropogenic threats to, the shrimp. Therefore, my dissertation aims to: (1) understand proximate and ultimate causes of the parading behavior, (2) investigate the effect of light from tourists on parading shrimp, and (3) evaluate perceptions toward the parading shrimp from locals, stakeholders, and tourists. I use the results from these behavioral studies together with an understanding of tourists, locals, and stakeholders' attitudes toward the parading shrimp to mitigate the anthropogenic disturbances and design an effective sustainable ecotourism management plan.

Infectious diseases are a significant problem, accounting for 1 in every 4 deaths worldwide. The field of bioengineering is constantly innovating and advancing diagnostic technology; however, more often than not, these innovations are accessible only to communities with privileged resources. This has led to a growing focus on point-of-care (POC) diagnostics. Due to their ease of use, speed, and low cost, POC diagnostics can effectively test patients in resource-poor settings. One of the most well known POC technologies is the lateral-flow immunoassay (LFA). Most easily recognized for its use in pregnancy tests, LFA is a paper-based diagnostic that produces visually interpreted results using a colorimetric indicator decorated with antibodies specific to the target. Recently, we have seen rapid advancement in the fields of paper fluidics and paper diagnostics, which can have a tremendous impact on the future of LFA technology. In light of this, we focused our work in 3 distinct directions, which involves development of quantitative experimental methodologies, mathematical modeling, and improving the ease-of-use of the advanced LFA technology.

Development of new paper-based devices would benefit significantly from being able to quantitatively assess the effects of engineering the device on important LFA parameters. For example, it would be useful to know the effects of manipulating the gold nanoprobes and test strips on the forward (kf,s) and reverse (kr,s) rate constants for the probe binding to and dissociating from the test line, respectively. We discuss our novel approach for determining these rate constants and the volumetric flow rate by using mathematical modeling and radioactive iodine-125 (125I). Moreover, we demonstrate how radioactivity and paper strips can also be used to determine the volume of fluid in and before the test line, the concentration of gold nanoprobes, and the number of antibodies per gold nanoprobe.

As the field of paper-based diagnostics continues to rapidly expand, it becomes more important to incorporate modeling into their design. A model can be used to determine the effects of LFA parameters on desired outputs, such as the amount of probe bound to the test line. Such predictions become increasingly important as systems become more complicated, and the effects of changing different operating conditions become less intuitive due to the many physical, chemical, and biological processes that are simultaneously occurring. Moreover, a mathematical model allows the engineer to quantitatively predict the influence of well-defined changes in certain parameters. We have derived a simple model that could be used in combination with our novel estimation methods for LFA parameters to predict the amount of probe binding to the test line, an important performance indicator of LFA. We will discuss the derivation of the model, and demonstrate the model’s ability to predict empirical results.

Our lab’s recent innovations have improved LFA sensitivity by utilizing aqueous two-phase systems (ATPSs) to thermodynamically concentrate the target molecule prior to detection. In the third direction, we describe a diagnostic design that dehydrates the ATPS components within the paper, creating a non-dilutive, one-step diagnostic process. We investigate the importance of ATPS component dehydration order and demonstrate an improvement in the limit of detection for Chlamydia trachomatis LFA using our dehydrated ATPS diagnostic design.