DNA damage occurs regularly, and although the cell has numerous repair mechanisms to counteract it, some DNA lesions may persist. Many of these lesions can be bypassed by DNA and RNA polymerase, with replicational mutagenesis leading to permanent mutations in the genomic sequence, and transcriptional mutagenesis leading to mutant transcripts that may direct the production of mutant proteins. We hypothesized that transcriptional mutagenesis is a mechanism for initiating adaptive mutagenesis, which occurs in nondividing cells in direct response to a selective pressure, allowing cells to overcome the selection and resume cell division. Our lab has previously demonstrated that DNA damage leading to adaptive mutagenesis in yeast is biased to the template strand relative to transcription, supporting this hypothesis. Unexpectedly, DNA damage leading to replicational mutations in our system is similarly skewed to this strand, although the sequences surrounding these two classes of mutations seems to differ. We hypothesize that sequence context is important for determining whether certain mutation sites are more likely to contribute to replicational or adaptive mutagenesis. To address this hypothesis, our aim was to create Trp5 mutant yeast strains using CRISPR/Cas9 in which the mutation occurred in a sequence context-specific manner, and the reversion of which could be scored for both replicational and adaptive mutagenesis. The CRISPR/Cas9 system required generation of a plasmid containing the target sgRNA (via molecular cloning techniques) as well as a linear repair template with the mutation of interest (via a two-step PCR protocol). Creating both of these components proved challenging, and optimization of the protocols will be required. Once we have all the components for CRISPR/Cas9-mediated mutagenesis, future research involves creating the mutant strains and assessing whether the sequence context affects the mutation frequency with respect to either replicational or adaptive mutagenesis.

In response to various stimuli, proteins undergo changes in shape (conformational changes) that are essential to their function in cell signalling, enzyme activity and many other crucial biological processes. Previous studies have shown that intramolecular distance changes associated with the conformational dynamics of proteins can be probed using transition-metal-ion-fluorescence-resonance-energy-transfer (tmFRET), in which a donor fluorophore transfers light energy to an acceptor metal ion in a steeply distance-dependent manner. tmFRET is both highly sensitive and highly specific: each unique donor-acceptor pair can only measure a limited range of distances reliably. Here, we expand the utility of tmFRET by employing new strategies to create a library of unique donor-acceptor pairs each sensitive to a different range of distances.To showcase the accuracy and precision of our measurements, we use a maltose-binding-protein (MBP) model system, which undergoes a well-studied conformational change upon the binding of maltose. In order to label MBP with Cu(II) ions that act as tmFRET acceptors, we utilize macrocyclic chelator-maleimides, which simultaneously coordinate Cu(II) and covalently modify a cysteine introduced into MBP. Using amber codon suppression, we introduce fluorescent non-canonical amino acids (ncAA’s) into MBP to act as tmFRET donors. Our fluorescence measurements show that tmFRET between the ncAA donor and chelated-Cu(II) acceptor accurately reports MBP’s intramolecular distance changes. We also show that by varying the exact structure of the molecule that chelates Cu(II) we can tune the sensitivity of tmFRET to distances ranging from 0.8-1.8 nanometers. Thus, this chelator-maleimide approach to labelling proteins with tmFRET acceptors could prove to be a powerful tool for researchers studying conformational motions of that scale.

In the US over 100 million people live with diabetes or pre-diabetes. The economic burden of this is approximately $327 billion every year. This study seeks to establish an alternative mode of insulin production using a polyethylene glycol (PEG) transformation of Pleurotus ostreatus. P. ostreatus is a valuable target for genetic transformation due to its lack of endotoxins, rapid growth, and fully sequenced genome. In this study, I transformed P. ostreatus using PEG with a plasmid containing the human insulin gene, a green fluorescent protein (GFP) reporter gene, and a selectable resistance gene. Transformed cells were selected using hygromycin, extracted, and regenerated on growth media. Confocal microscopy confirmed the presence of the GFP and presumably the human insulin gene. An ELISA for insulin and proinsulin will be used in the upcoming months to test for genetic expression, and to determine the efficacy of protein folding in the transgenic fungal cells. This has the potential to not only expand the market for diabetic treatment options, but it initiates a valuable conversation about the importance of diversifying production methods and costs in the treatment of diabetes.

Cancer is an evolutionary process driven by mutagenesis and selection for malignant phenotypes. Correspondingly, mutations that elevate mutation rates, producing a “mutator phenotype,” accelerate tumor formation. Heterozygous mutator alleles affecting the catalytic subunit of DNA polymerase (Pol) epsilon (encoded by the POLE gene) or bi-allelic mutations affecting mismatch repair (MMR) components, such as MSH2, arise frequently in a subset of colorectal and endometrial cancers. In some tumors, mutator alleles from both classes occur together and synergistically cause tremendous genetic instability. A key unanswered question is whether the severe mutation accumulation compromises tumor cell fitness and imposes a selection for “antimutator” phenotypes that allow the tumor to escape extinction. Evidence supporting the selection for antimutator alleles in strong mutator cells have been obtained using budding yeast, whose DNA replication and repair machinery are highly conserved with their mammalian counterparts. Our initial studies in haploid yeast show that mutations in the pol2 mutator alleles represent a major class of antimutator alleles. However, mutator suppression in diploid cells remains understudied. The strongest mutator allele known in human disease is POLE-P286R, which corresponds to pol2-P301R in yeast. We previously evolved diploid pol2-P301R/POL2 msh2Δ/msh2Δ mutator yeast strains to identify candidate antimutators that may arise in diploid cells. We also identified putative human antimutator alleles from The Cancer Genome Atlas database. Here, we test whether these alleles do indeed exert antimutator phenotypes by re-engineering them into diploid yeast. Our findings will provide a direct test of the relevance of antimutators for tumor evolution and define likely antimutator candidates for further study.

Constitutions embody individual rights and government duties, setting higher standards for society and governing institutions. Through legislative actions and judicial interpretation, the interests of the people and the government are constantly in tension and being balanced within the framework of the constitution. Yet critical to the realization of constitutional rights is the actual implementation and real effect of these legal processes. In this project, I raise the question, how are individual rights as outlined in a constitution implemented? To answer, I examine Taiwan's Judicial Yuan, specifically the Constitutional Court and its relationship with the lower courts and legislature, to find if, how, and why the Judicial Yuan, as an entity, impacts fundamental human rights. In particular, I develop an original case law dataset including key constitutional rights cases before the Constitutional Court from 1950 to 2020. Through an in-depth historical case law analysis, I code for whether the Constitutional Court expanded or constrained rights and how the decisions are implemented in lower courts and by the legislature. Preliminary research finds that the Constitutional Court makes rulings that are often incomplete without further action by the lower courts and the legislature. These findings may help answer larger questions of the efficacy of constitutional courts and their ability to bring social change.

 Alzheimer’s disease (AD) is a complex and common neurodegenerative disease in humans. It is diagnosed by the presence of A beta amyloid plaques and tau tangles in the brain during autopsy, decades after neuropathology has developed. Early diagnosis is still not definitive and contributes to the inability to successfully treat the disease at an early stage. Generally, animal models are not reliable for preclinical studies since lower mammals, including laboratory rodents and nonhuman primates, pet dogs and domestic livestock do not naturally develop AD. The reason is based on conformational differences in amyloid precursor and tau proteins. We have recently observed one exception to this list of mammals- the aging pet cat. Formalin fixed paraffin embedded brain sections from ten 16 to18-year-old pet cats obtained from a regional veterinary teaching hospital were stained with antibodies 6E10 and AT8 specific for A beta and phosphorylated tau using standard immunohistochemistry procedures for diagnosing human AD. One cat showed robust tau tangles and moderate amyloid plaques, while a second cat showed moderate tau tangles and mild amyloid plaques. Brains from additional cats need to be examined for AD neuropathology but this preliminary observation suggests old pet cats develop AD. Since pet cats share the same environment as humans, clinical studies to search for ways of early diagnosis and treatment of this naturally occurring disease in cats will have a huge impact on the translational implications for early diagnosis and intervention studies in human AD.

Though renewed efforts in tuberculosis (TB) research have facilitated massive strides towards treating Mycobacterium tuberculosis (M.tb), TB remains a global health problem with an estimated 10 million infections and 1.4 million deaths in 2019. The ability of the pathogen to hide itself inside a granuloma, a mass of immune cells whose precise mechanism of regulation is unknown, prevents both the study of M.tb pathogenesis and the development of drugs or treatments. Current in vivo models have been established to study TB infection using animal models or tissues, limiting its biological relevance as it relates to human disease while current in vitro models lack components of the complex lung microenvironment during infection. We present the creation of a novel microscale infection model, which uses open and suspended microfluidic principles to enable spatial and temporal manipulation of cultures in suspended hydrogel plugs. By stacking together two devices, we demonstrate the ability of a suspended model granuloma consisting of M.bovis BCG (Mycobacterium bovis bacille Calmette-Guérin) and monocyte-derived macrophages to interact with a model vasculature layer consisting of endothelial cells. Analysis of soluble factors for proinflammatory cytokines and characterization of infection-dependent angiogenesis in the vasculature layer are used to verify communication between cultures. In the future, we envision this model expanding to contain multiple immune cell types and to incorporate additional aspects of the lung anatomy to approach a more accurate pathophysiological model as a tool for other researchers.

Local processes in marine ecosystems, including coastal estuaries, modify ocean acidification caused by rising atmospheric CO2. Because ocean acidification poses a threat to shell-forming organisms, it is critical to understand how these processes affect acidification in specific regions. In the Snohomish River estuary, freshwater from river discharge deposits directly into Possession Sound, impacting the salinity and temperature of the area. River discharge in estuaries has been found to be slightly acidic, as well as a source of nutrients that fuel blooms of phytoplankton. Large phytoplankton blooms can lower the pH at depth because of the process of respiration, which releases CO2 and decreases dissolved oxygen levels. My research examines changes in pH with temperature, salinity, chlorophyll, and dissolved oxygen at different depths in Possession Sound, Washington, using data collected from January 2017 through January 2021 with a YSI EXO2 Sonde. I hypothesized that near-surface depths and sites located closer to the river would have lower temperatures and salinities correlating with lower pH. Additionally, lower dissolved oxygen at greater depths would correlate with greater amounts of chlorophyll and a decrease in pH at depth. Depths near the halocline were predicted to have alkaline pH values due to photosynthetic organisms. I analyzed data using Microsoft Excel and R Studio. Results found that with chlorophyll less than ~1.25 RFU, pH was greater than 8.0, while with lower dissolved oxygen, pH was less than 7.75. Temperatures less than 10°C corresponded with more pH values between 7.0 and 7.5, while salinity had no apparent trend. In most seasons, pH appeared to decrease slightly at greater depths. The exception to this was winter, when more acidic pH values were observed at near-surface depths. Overall these results indicate that local processes in the Snohomish River estuary are affecting changes in pH.

In April of 1986, an accident in the Chernobyl Nuclear Power Plant caused a reactor to undergo an uncontrolled chain reaction that resulted in it melting down, and setting fire to the building it was housed in. This disaster spread high levels of radioactivity throughout the surrounding area, which was soon absorbed into the surrounding environment. Nevertheless, the radioactivity in this area has been periodically re-released into the air because of the worsening forest fires that scorch the unmaintained land. This literature review focuses on how the fires spread radioactivity, what effects they have on the surrounding inhabitants, as well as what has and should be done to prevent the further spreading of radioactivity. This review first explores the history of the forest fires and their growing intensity over time, then studies how the radioactivity is absorbed into plants and is biomagnified in animals when moving up the food chain; Next, it explores how it is released into the air by smoke from the fires, and the negative effects exerted on living things, primarily on people. Finally, it looks at what has been done in the past to stop the fires and spread of radioactivity, and what could be done to improve the current situation. As it stands right now, the forest fires are worsening due to climate change. The radioactivity poses a real threat to the surrounding populace, and there is no feasible way of removing the radioactivity from the area. The best solution for this problem appears to be fire prevention through better forest management and mitigating the effects of climate change. However, the radioactivity will persist for millennia within the environment and preventing forests from burning can interfere with their natural ecological processes, so further research into the possible effects of this solution should be performed.

Standard diagnostic tests for common bacterial and viral infections are invasive and uncomfortable, especially for children. Difficulty performing these tests leads to diseases such as strep throat or pneumonia going undiagnosed in children with significant health risks. There is a need for more accessible diagnostics. Our research uses saliva analysis as a diagnostic medium for Streptococcus pyogenes. This presentation depicts a novel saliva collection platform that is designed and engineered to be child-friendly, effective at pathogen collection, and is suitable for home use. We produced prototype devices using 3D printing, computer numerical control (CNC milling), and molding. Human studies were performed to examine usability and inform further design. In addition to engineering the device, we tested coatings and capture of bacteria. Preliminary data finds success in capturing S. pyogenes. Collection of commensal oral bacteria capture is the next step of our human studies. Our expectation is to find success with our device in a human mouth. Once our device and system are established, we plan to expand to include additional diseases. We will be presenting our innovations in the device as well as our preliminary data on the efficient capture of bacteria. We hope this technology will provide diagnostics with a way to provide care to underserved populations as well as help us to better collect data regarding the bacteria and viruses present in previously inaccessible populations. 

Water clusters play a significant role in a variety of processes such as those pertaining to the atmosphere and biological systems, and studies of water clusters have suggested that they could help us learn more about hydrogen bonding. We first must understand the energetics and trends of isolated water molecules in order to better comprehend the spectroscopic properties of water clusters. Afterwards, we can look at water clusters and observe how the energetics and patterns change due to the interactions with other water molecules. We are studying the coupling among vibrations in water molecules and how they are reflected in the spectra. The discrete variable representation (DVR), a method used to solve the Schrödinger equation, was implemented to generate the water spectrum as well as energies and wave functions. The DVR results show that the theoretical intensities are consistent with the experimental results. These results contribute to our goal of analyzing the spectra of more complicated water cluster systems. Diffusion Monte Carlo (DMC) is a different method that allows us to explore larger systems and is used in the analysis of the coupling in assemblies that contain multiple water molecules.

Polymers are commonly seen in our daily lives. Proteins and plastics are both familiar classes of polymeric materials whose utility is heavily relied upon. There are different architectures of polymers, for instance, linear and cyclic, each of which has unique properties. For example, cyclic polymers have a lower viscosity, smaller hydrodynamic volume, and a unique topology as an endless circle. In this project, we are investigating and improving a privileged method to approach cyclic polymers, Ring Expansion-Metathesis Polymerization (REMP), which grows the polymer chain while cyclizing it, using a Ruthenium-based (Ru) system. The goal of the research is to solve the recent major challenge of synthesizing cyclic polymers in a controlled fashion, through systematically modifying the structure of Ru-based initiators. My goal in the research project is to synthesize a precursor ligand, the subsequent initiator, and the monomers(commonly strained alkenes, norbornene). Eventually using the monomers to conduct polymerization reactions and analyze their properties and characteristics with spectroscopic instruments. Preliminary results of this research suggest we can make cyclic polymers that are more evenly distributed in size and weight. Since polymers have played an important role in people’s everyday life, improving the methodology through having better control on making cyclic polymers can make a big contribution to applications in the aspects of biomedicine and energy for our society. For example, cyclic polymers could generate biotherapeutics for the field of medicine; they could also serve as well-behaved and new types of conducting materials for the field of semiconductor.

The island of La Palma is the second youngest in the Canary Island volcanic chain, 475km northwest of Africa, which was created by hot spot magmatism. The earliest activity of each volcano erupted below sea level and was later buried by subaerial eruptions. On the island of La Palma, the seamount, or submarine-building stage of the Taburiente volcano, is uniquely exposed due to uplift and a subsequent giant landslide that exposed the interior of the volcano. It provides access to basalts that record the island’s early magmatic history. I report lead, hafnium, and strontium isotopes of Taburiente’s submarine history in order to infer the mantle origin of the magma source and compare the data to that of the later subaerial volcanic rocks to determine if magma source changes occurred between the two volcanic phases. Each analyzed rock sample was divided in two parts: one was acid-leached to remove possible contamination and alteration caused by hot seawater circulating through rock on the ocean floor and the other was not acid-leached. Both sample sets were then dissolved in strong acids to collect pure Pb, Hf, and Sr aliquots using ion-exchange chemistry and isotopically analyzed using high-precision mass spectrometry. The isotopic ratios of ocean island basalts in general differ greatly from ocean floor basalts. This is the basis for determining if Taburiente’s early volcanic phase is more similar to an ocean floor or an ocean island basalt composition. The data also detail the type of mantle source—whether primordial or recycled rock that was returned from the surface back into the mantle. The leached versus non-leached sample sets quantify how much hydrothermal alteration has occurred within the seamount and if this is a useful technique to apply to future studies of ocean island basalts.

Today, chemical separation processes are a major contributor to the global threat of climate change. Therefore, the development of more energy efficient methods to separate and purify chemical mixtures has become an important field of research. Limitations exist in the current application of metal-organic frameworks (MOFs)- extended, 3D networks formed from metal ions and bridging organic linkers- to fabricate high quality films for gas separation. We were interested in investigating a potential solution to this problem by synthesizing metal–organic cages with well-defined interior cavities and modifiable surface chemistry, which can be engineered to produce materials that bind and store many types of gas molecules. Here, we report a generalizable synthetic route towards lower symmetry cages- an area largely unexplored relative to their symmetrical counterparts- that possess a combination of solution processability, high porosity, and selective gas uptake. To illustrate the robustness of our synthetic route, a series of five metal–organic cages were synthesized and characterized by single crystal X-ray diffraction, gas sorption analysis, MALDI-TOF mass spectrometry, and ¹H NMR digestion experiments. Variable temperature CO₂ gas sorption data show that open binding sites can be generated, and that they remain accessible to molecules even in variants lacking permanent porosity. Our results demonstrate a mixed-ligand platform that supports a wide family of structurally related cages. This opens the door to new types of soft nanoporous materials possessing unique functions and properties, with applications in membrane-based separations, ionic conductivity, and controlled drug release.