Alzheimer’s disease (AD) is a progressive neurodegenerative disease that affects millions of people. Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive stimulation typically used in psychiatric conditions such as depression and anxiety. rTMS works by using an electric current to generate a transient magnetic field, depolarizing neurons in a target region and creating lasting changes in brain connectivity via synaptic plasticity. Patients with AD show disruptions in the Default Mode Network (DMN), a network of brain regions typically active during rest and crucial for memory consolidation. We hypothesize that strengthening the DMN through rTMS targeted at the left Brodmann 8AV region, selected for being an easily accessible node of the DMN, will improve memory in AD patients. To test this hypothesis, we are conducting a single-blind, single-arm, randomized cross-over trial of rTMS on early-stage AD patients over a 12 week period with week 1 where we scan for the 8AV region via MRI, during week 3 and 8 being the placebo or treatment week. We measure our primary outcome of the participants’ speed of forgetting —a novel index of memory function—through an individualized, adaptive memory test. To eliminate potential confounding variables, we also measure depression and anxiety symptoms during the 1st, 8th and 12th week of the study. Additionally, functional MRI scans will be analyzed for potential structural or functional differences caused by treatment. Preliminary results from our initial participants have shown promising improvements, and we are hopeful that similar outcomes will be observed in the remaining participants. Successful results would provide a novel target for AD treatment using rTMS, and support further investigation of rTMS as a viable treatment option.

Dilated Cardiomyopathy (DCM) is a heart disease characterized by the thinning and dilation of the heart's walls, which leads to a decrease in blood pumping ability and can progress to heart failure. Many genetic mutations, primarily in components of the sarcomere, have been implicated in causing DCM. One such mutation is the beta myosin heavy chain mutation E525K. This project aims to understand the molecular mechanism by which E525K leads to disease progression using stem cell-derived cardiomyocytes and a unique multi-level approach to characterizing contractility and cell morphology. Here we show that the E525K mutation can lead to both hyper- and hypocontractility depending on the scale of the analysis. We found that in isolated cells, E525K mutants experienced a 65% decrease in sarcomere shortening and that in engineered heart tissues, the max force produced by tissues with mutant cells was 39% lower than WT tissues. Meanwhile, in isolated myofibrils with the mutation peak force was increased by 45% when stimulated with pCa 4.0 calcium. Morphological analysis showed that mutant cells on average have fewer, smaller, less organized sarcomeres than WT cells. This demonstrates that E525K myosin, though linked to DCM, which is associated with hypocontractility, exhibits both hyper- and hypocontractile effects. Cardiomyopathy affects an estimated 1 in 500 people worldwide and is a major cause of heart failure. Novel pharmacological treatments that activate or inhibit myosin are becoming available; however, cardiomyopathy’s genetic nature complicates treatment. Our findings highlight that the underlying mechanisms causing cardiomyopathy can vary greatly even between patients showing similar disease phenotypes. From a clinical perspective, this complicates what medications should be used, demonstrating that a deeper understanding of the underlying mechanism by which cardiomyopathy-related mutations cause disease is imperative to diagnose and treat patients optimally.

As Cannabis use is becoming more widespread there is growing concern regarding the respiratory exposures of employees working in indoor cannabis processing facilities. Employees in these occupational settings are frequently exposed to volatile organic compounds (VOCs), particulate matter (PM), other respiratory irritants, and allergic sensitizers. These exposures are linked to work related illness and disease, such as occupational asthma. Notably, a fatality, in 2022, in a Cannabis worker due to occupational asthma highlights the urgent need for improved exposure controls. Cannabis processing workers experience prolonged and frequent exposure via inhalation with little knowledge on the respiratory hazards of this work. This study aims to evaluate the efficacy of a local exhaust ventilation (LEV) system to reduce exposure to airborne hazards during automated joint filling. Automated joint filling is a common process in Cannabis production facilities, using mechanized equipment pre-ground material is dispensed into pre-rolled cones. This method is preferred in the field as it increases both consistency and efficiency. Over a ~2-hour sampling period across eight batches of pre-rolled joints, we conducted gravimetric sampling for inhalable PM using two inhalable aerosol samplers (IOMs) positioned at the workbench and in the breathing zone. VOC exposure was assessed using thermal desorption tubes and photoionization detectors (PIDs), while continuous respirable PM concentrations were measured using a Nanozen DustCount monitor. Testing air concentration for PM and VOCs with and without the LEV mechanism is being conducted to determine its effectiveness at reducing exposure. We hypothesize that this may be an effective solution, as the LEV has controlled these agents significantly in other similar workplace settings. As this field grows due to recent state by state legalization of Cannabis, these findings hold great impact for workplace safety regulation and solutions. Additional research should be gathered on long-term exposure effects and preventive mechanisms.

Dense-core vesicles are membrane-bound structures that carry neuromodulators such as insulin, dopamine, and serotonin. The peptides within dense-core vesicles are initially larger precursor proteins that undergo enzymatic processing to achieve their functional forms. During the defecation motor program in Caenorhabditis elegans, dense-core vesicles released from the intestine harbor neuropeptides that trigger neurons which activate enteric muscles, promoting the act of defecation. Failure of certain proneuropeptides to mature into neuropeptides results in decreased frequency of defecations. CPD-1, a conserved transmembrane carboxypeptidase, is a poorly understood processing enzyme that affects the defecation motor program. I built on our knowledge of EGL-21, another carboxypeptidase known to process neuropeptides and peptide hormones, to better understand CPD-1’s function. I show here that these two carboxypeptidases, EGL-21 and CPD-1, process neuropeptides necessary for successful defecation patterns. Mutants lacking egl-21 had decreased defecation frequency while worms lacking both egl-21 and cpd-1 had an even lower defecation frequency. Additionally, my results show that CPD-1 is expressed in intestinal cells and can compensate for EGL-21’s function. Finally, I am conducting experiments to determine whether one of CPD-1’s targets is NLP-40, an important neuro-like peptide released from the intestine that regulates defecation. These results contribute to our broader knowledge of peptide processing in dense-core vesicles.

The locus coeruleus (LC) is a major neuromodulator source with widespread projections to distinct functional targets that influence arousal, anxiety, learning, and other behavioral states. Our lab has previously shown LC excitation triggers the release of norepinephrine (NE) into the basolateral amygdala (BLA). Recent studies suggest LC terminal stimulation may release DA into the dorsal hippocampus (dCA1) enhancing novelty-associated spatial learning. Our recent data show LC stimulation evokes DA release. Previously, release across regions, paradigms, and behaviors typically associated with LC have not been characterized, due to difficulty in separating DA from NE using traditional sensing methods. Due to this, the relationship between the LC and other DA systems remains unclear. To understand the mechanisms by which the LC may release DA independently of the ventral tegmental area (VTA), a major DA source, we have employed optogenetic stimulation to evoke release from neuron terminals and quantify the release dynamics of NE and DA. We used fluorescent biosensors to detect NE and DA, captured by a fiber optic cable and amplified to observe the relative dynamics of DA release. These sensors have tuned affinity and selectivity for NE and DA and use fluorescence as a proxy for neuromodulator release. In this project, we aim to elucidate how and under what conditions the LC is releasing DA across regions with different functions during aversive and appetitive behaviors. These data will enhance our understanding of the LC neuromodulator signaling that can become maladaptive and afflict anxiety, addiction, and more, and also demonstrate that the release of DA from the LC is dependent on the behavior induced.

A physiological response to acute stress, called anxiety, is thought to be an adaptive feature that allows us to adjust our behavior to better approach the situation causing stress. However, in anxiety disorders this response is maladaptive, leading to excessive anxiety. A key neural circuit is the projection from the locus coeruleus (LC) to the basolateral amygdala (BLA), and activation of this circuit produces anxiety-like behavior. However, little is known about how this alters the activity of BLA neurons. My Mary Gates research project seeks to utilize machine learning to understand how neuromodulatory input from the LC to the BLA alters the correlated activity of BLA neurons and their encoding of anxiety-like behavior. Mice expressing the excitatory opsin ChrimsonR in the LC and the calcium indicator GCaMP6s in the BLA received tonic (5hz) stimulation of LC terminals within the BLA through a GRIN lens to mimic stress-like release of norepinephrine into the BLA. LC terminals were stimulated while recording individual BLA neuron activity during a conflict-based test of anxiety-like behavior, the Elevated Zero Maze (EZM). To evaluate the correlated activity of BLA neurons as a function of stimulation, I used caGraph, a Python package that utilizes graph theory approaches to test the correlation of neurons from calcium imaging data. I investigated how stimulation affects graph theory communities (densely connected clusters) and clustering coefficients (strength of clustering) and found that stimulation causes an increase in the clustering of BLA neurons. To test the functional consequence of these ensemble shifts, I am using classification algorithms to assess the population encoding of the BLA neurons. I expect that stimulation of the LC terminals will increase the encoding of anxiety-like behavior. The findings of this project deepen our understanding of how the LC-BLA circuit mediates anxiety-like behavior, and may uncover novel treatment strategies.

Neuronal and endocrine cells store and secrete molecular cargos like neurotransmitters and metabolic hormones through the regulated secretory pathway. Dense-core vesicles (DCVs) originate from the trans-Golgi network and undergo a maturation process involving peptide processing and cargo sorting before being stimulated to release their cargos outside the cell. Dysregulation of this process leads to a wide range of neurological and metabolic disorders; yet the molecular mechanisms underpinning it remain poorly understood. Vesicular traffic are largely coordinated by the Rab family of GTPase proteins.  Previous work identified the conserved proteins TBC8 and RUND1 as regulators of DCV maturation in Caenorhabditis elegans; and both proteins bind active GTP-bound RAB2. TBC8 is the putative RAB2 GTPase activating protein (GAP) which promotes conversion of GTP-RAB2 into GDP-RAB2, thereby inactivating the Rab. RUND1 interacts with both active GTP-RAB2 and TBC8, yet its precise function remains unknown. This study aims to characterize the biochemical function of TBC8 and RUND1 in regulating RAB2 activity. Using purified proteins, we demonstrate that TBC8 greatly promotes RAB2 GTP hydrolysis, indicating it is the bona fide RAB2 GAP. Additionally, we show that RUND1 strongly inhibits TBC8-stimulated RAB2 GTP hydrolysis, suggesting RUND1 may compete with TBC8 for RAB2 binding. Given this interplay between RUND1 and TBC8 in binding RAB2, we hypothesize that RAB2 exhibits exclusively pairwise interactions with its partners. To test this, we will use mass photometry to study whether RUND1 and TBC8 can bind RAB2 simultaneously or if one complex is preferentially formed. Based on our current findings, we propose a model where TBC8 promotes RAB2 inactivation by stimulating GTP hydrolysis and RUND1 blocks RAB2 inactivation by TBC8, prolonging the activate state of RAB2.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus known to cause Kaposi sarcoma (KS), a cancer of the soft tissues, and several other diseases. KSHV has two distinct replication cycles: a latent and lytic cycle. During latent infection, only a small section of the viral genome, the KSHV latency-associated region (KLAR), is expressed. Spindle cells, the main proliferating cell type in KS tumors, are thought to be of endothelial origin and are primarily latently infected. Due to lowered viral gene expression during latency, these cells have few viral factors to target therapeutically. Cellular factors required for infected cell survival, like altered metabolic pathways, are potential therapeutic targets for latently infected cells. One metabolic pathway altered by latent infection is fatty acid synthesis (FAS). My research focuses on understanding how KSHV infection induces FAS in cells. Since previous research has shown that expression of KLAR is sufficient to increase lipid droplet formation, a measure of FAS induction, I hypothesize that expression of one of the four genes present in KLAR is likely what upregulates this metabolic pathway. To test this hypothesis, I infected telomerase-immortalized microvascular endothelial (TIME) cells with lentivirus containing a viral plasmid overexpressing one of the four KLAR genes. I then measured lipid droplet formation across each transduced cell population using a flow cytometer. This project is still in progress; however, if the increase in lipid droplet production in transduced cells is similar to the increase observed in cells latently infected with wild-type KSHV, then I will conclude that the over-expressed gene was sufficient to up-regulate FAS. Identifying which viral gene induces FAS in infected cells will provide a new direction for future mechanistic studies and aid in identifying potential therapeutic targets for KSHV-associated diseases.

Hypertrophic Cardiomyopathy is the most common form of hereditary heart disease affecting ~1:500 individuals, characterized by progressive thickening of the left ventricular wall. The first mutation linked to this disease was the heterozygous R403Q mutation in human beta-myosin heavy chain (β-MHC). Conflicting reports of contractile kinetics between human myectomy samples vs transgenic mouse and rabbit models motivated us to study the molecular mechanisms of altered contraction in a CRISPR/Cas9 gene edited human inducible pluripotent stem cell line. Following differentiation to cardiomyocytes (hiPSC-CMs) and maturation in culture, we isolated sub-cellular contractile organelles called myofibrils. Myofibril contractile kinetics from this line had slowed force development and cross-bridge detachment, with reduced maximal force compared to the WT line. hiPSC-CMs were cast into fibrin matrices to form three-dimensional, engineered heart tissue (EHT) for measures of twitch force and contractile kinetics. At 1Hz stimulation, heterozygous mutation EHT’s exhibited a hypercontractile phenotype compared to WT EHTs, with slowed relaxation kinetics. Since the penetrance of our heterozygous R403Q hiPSC-CMs is unknown, we are now studying a homozygous iPSC-CM line where 100% of the β-MHC is mutated. This will allow us to assess the direct contribution of the mutation to the disease contractile phenotype. We will repeat the myofibril and EHT measures of contractile properties and perform stopped flow kinetics analysis on isolated myosin to determine ATP turnover and ATP hydrolysis product release rates. This will provide molecular mechanistic insight of the contractile abnormalities, allowing development of therapeutic interventions that specifically target the mechanisms that alter contractile function. 

Most of our chemicals come from petroleum, a nonrenewable resource and a significant source of pollution. Purple non-sulfur bacteria (PNSB) also produce some of these chemicals from one-carbon (C1) feedstocks, however, genetic engineering toolkits are underdeveloped for these organisms. The ability to integrate heterologous genes is a crucial component of genetic engineering toolkits, enabling stable and precise gene expression. Despite their metabolic versatility, PNSB lack well-characterized genomic integration sites, limiting advanced strain engineering efforts. Here, we identify and characterize genomic integration sites in Rhodobacter sphaeroides 2.4.1 that can serve as stable integration loci for heterologous gene expression. Using RNA-Seq transcriptomic data, we identified intergenic regions with minimal transcriptional activity, ensuring that insertions into these regions would not disrupt native gene function. Seven candidate integration sites were selected across the genome, spanning both chromosomes and plasmids. Two-step allelic exchange was used to integrate “landing pads” for Serine Recombinase-Assisted Genome Engineering (SAGE), a site-specific recombination system, into candidate sites. Our next step is to use the SAGE system to integrate fluorescent reporters into these sites to assess positional effects on gene expression. These seven integration sites serve as a testbed, allowing us to validate the workflow for integration into a broader range of genomic locations. Our findings will provide a resource for engineering R. sphaeroides and expand the genetic toolkit for PNSB, facilitating their use in synthetic biology and bioproduction applications.

Heart disease is linked to one in every five deaths in the United States, indicating the need for more research on causes and possible treatments. Dilated cardiomyopathy (DCM) is an inherited cardiomyopathy characterized by a decrease in contractile function, which often leads to heart failure and death. DCM is a disease of progressive remodeling and is often not diagnosed until later in life once a patient becomes symptomatic and the progression of disease is difficult to discern. By understanding the specific molecular etiology of DCM, treatments can be specialized and better prevent progression to end-stage heart failure. One protein associated with DCM is the motor protein myosin. In the Regnier Lab, we study the dilated cardiomyopathy-associated myosin mutation R369Q to better understand the molecular mechanisms leading to DCM. To explore this, I used Human Induced Pluripotent Stem Cells (hiPSCs) that have been CRISPR-edited to contain the mutation, differentiated into cardiomyocytes and cultured on patterned surfaces to promote maturation and alignment of the contractile organelles called myofibrils. I electrically paced and live-imaged these cells to capture single-cell contraction to compare differences in sarcomere shortening and quantify cell-level effects of the R369Q mutation. I also analyzed flat glass imaging to measure cell area and potential differences in sarcomere alignment. My goal is to better understand how the structural and functional differences on a single cell level fit into the context of a DCM mutation's effects in the heart. Ultimately this will allow a better understanding of the R369Q mutation’s pathogenic effects and give better insight into how possible future treatments can potentially combat the effects of DCM.

Mechanistic target of rapamycin (mTOR) functions in a protein complex with raptor to control protein synthesis in eukaryotes. A reduction of function mutation in C. elegans raptor is resistant to hypoxic death. This mutation, a missense at amino acid 1033 in the daf-15 gene, is interesting because the mutation site is conserved in all mammals, suggesting that this work could shed light on hypoxic injury mechanisms in humans. The Crowder lab has discovered that a mutation called tm11331 in a gene involved in purine metabolism blocks the hypoxia resistance of the raptor mutation. We hypothesized that the tm11331 mutation restores normal protein synthesis to the raptor mutant and therefore restores hypoxic sensitivity. For my project, I examined this hypothesis by measuring nucleolus size as an indirect measurement of protein synthesis. Four strains were used in this assay: unmutated (wild-type) worms, worms with the raptor mutation, worms with the tm11331 mutation, and worms with both raptor and tm11331 mutations. From previous experiments, we know that raptor mutants have smaller nucleoli than wild-type worms, indicating that protein synthesis rates are lowered in mutated worms. We would therefore expect that protein synthesis rates and nucleolus size would be restored in worms made hypoxia sensitive by the addition of tm11331. For this assay, all strains contained a fluorescent protein that labelled the nucleoli, allowing me to image nucleoli under fluorescence. I processed each image and measured average nucleolus size in worms from each strain. Our data shows that the tm11331 mutation increased nucleolus size in strains both with and without raptor mutation. In fact, the combination of tm11331 and the raptor mutation was not significantly different from wild type. Thus, our data supports the hypothesis that the tm11331 mutation restores hypoxic sensitivity by normalizing protein synthesis.

Strokes and heart attacks caused by a lack of oxygen, called hypoxia, are among the most prevalent form of debilitating diseases in the United States. Hypoxia has been shown to cause hypoxia-induced-fragmentation of the mitochondria altering their size, shape, and distribution (known as the mitochondrial dynamics). However, to what extent these dynamics are involved in hypoxic cell death remains unestablished. The Crowder lab through a C. elegans mutagenesis screen discovered a reduction-of-function mutation in rapTOR that confers strong hypoxia resistance. rapTOR functions in a complex with mTORC1 to control cellular metabolism including mitochondrial function. We decided to investigate whether the hypoxia resistance of the rapTOR mutant is from alterations of mitochondrial dynamics in response to hypoxic injury. To measure the mitochondrial dynamics, I visualized the mitochondria with an outer membrane fluorescent protein, in wild type and mutant worms with and without hypoxic exposure. I analyzed the images blinded to their genotype and hypoxic condition and scored mitochondria as primarily fragmented or tubular, which served as a surrogate for detecting changes in mitochondrial dynamics. For a more quantitative analysis, I utilized image processing MATLAB code and determined differences in images using principal component analysis. My analysis showed hypoxia induces small, rounded mitochondria in C. elegans resembling mitochondrial fission. I found the mitochondria in the rapTOR mutant displayed decreased hypoxia-induced-fragmentation after hypoxia. Then when I combined the rapTOR mutant with a hyperfragmented mitochondria mutant it showed fragmented mitochondria with and without hypoxic exposure. However, the double mutant is also hypoxia resistant, which is not consistent with our hypothesis that mitochondrial fragmentation drives hypoxic cell death. Therefore, we reject our hypothesis and conclude that rapTOR is hypoxia resistant from a mechanism distinct from that controlling mitochondrial fission.

Gynogenesis is an asexual reproduction strategy where sperm is necessary for fertilization, but the resultant offspring have no paternal DNA and two maternal sets of chromosomes. This strange reproductive strategy has never been observed before in nematodes (round worms), until a few years ago when a previous student at Ailion Lab observed the phenomenon when investigating the hybrid offspring of two species of Caenorhabditis roundworms; C.Becei and C. Nouraguensis. On their own, neither of these species exhibit asexual reproduction. Furthermore, C. Nouraguensis females normally produce haploid eggs, but when cross bred with C. Becei, they began to produce almost only diploid eggs. It is known that asexuality has arisen from previously sexually reproducing species, but the exact mechanisms of this evolution are unknown. This research project uses CRISPR techniques to attach fluorescent proteins to key structures involved in meiosis, which can then be imaged to reveal any irregularities which could explain the production of diploid eggs instead of haploid. The main goal is to understand the cellular mechanisms which facilitate such a dramatic change in reproductive strategy. 

Bacteria are constantly under dynamic environmental pressures and must promptly respond to survive. Bacterial general stress responses (GSRs) allow adaptation to perceived environmental changes via two-component and phosphorelay systems. The pathogenic alphaproteobacteria Bartonella quintana uses the body louse as a vector for infecting its target host, humans. It must adapt to two disparate environments, the human bloodstream and the gut of the body louse. Upon niche transfer, B. quintana is able to activate its GSR via a partner-switching mechanism involving an elegant molecular dance between alternative sigma factor RpoE, anti-sigma factor NepR, and anti-anti-sigma factor PhyR. The switching transfers NepR away from RpoE to PhyR, which activates gene transcription. Published works have revealed a molecular mechanism for sequestration via formation of a 1:1 dimer triggered by post-translational modification (PTM). However, a protein data bank (PDB) crystal structure (4QIC) shows a 2:2 tetramer, although it has not been observed in solution. We utilized size exclusion chromatography, multi-angle light scattering (MALS), small-angle X-ray scattering (SAXS), and protein modeling under various buffer conditions to identify conditions favorable for tetramer formation. MALS was chosen to determine the precise molecular weight of our chromatogram peaks, while SAXS was chosen to compare specific chromatogram peak scattering curves to PDB crystal structures and provide an overall shape for relevant peaks. Strikingly, our results revealed the tetramer forms in the absence of phosphorylation in solution, and the dimer is the dominant species under PTM favorable conditions. These results are loosely consistent with the literature but indicate the complexity of the alphaproteobacteria GSR is not fully understood. A possible explanation for the tetramer is that it maintains stress-related transcription despite the absence of a PTM.

Amyotrophic lateral sclerosis (ALS) is a progressive disease affecting 5000 people currently in the United States that is due to the degeneration of motor neurons, leading to muscle weakness, paralysis, respiratory failure, and ultimately death. To date, there has been extensive research investigating the underlying cause of the neurodegeneration that occurs in ALS, as well as attempts at targeted therapeutic interventions. CNM-Au8 is an investigational drug employing active gold (Au) nanocrystals designed to support neuronal survival by enhancing cellular energy production and reducing oxidative stress. The results of two randomized controlled phase 2 studies, the Healey Multiplatform Study and RESCUE-ALS, have suggested possible benefits from this therapy in both delaying disease progression, stabilizing respiratory function, and improving survival. The University of Washington (UW) is also a site for the Second Intermediate Expanded Access Protocol (EAP), allowing patients ineligible for the trials to receive the medication. The EAP follows an open-label, multi-center design, with all participants receiving daily oral doses of CNM-Au8. Participants undergo regular assessments every 12 weeks in person or via remote telehealth visits, allowing flexibility based on disease progression and external factors such as COVID-19 infection. The study tracks disease progression using measures such as the ALS Functional Rating Scale-Revised (ALSFRS-R) and slow vital capacity (SVC). ALSFRS-R is a questionnaire that evaluates a patient’s ability to perform daily activities, including speech, swallowing, mobility, and breathing. SVC is a measure of respiratory function crucial in monitoring ALS progression. 

Heart disease remains the leading cause of death in the United States, with the limited regenerative capacity of cardiac tissue resulting in long-term functional deficits following injury or defects. There is a critical need to develop physiologically relevant engineered heart tissues (EHTs) for disease modeling, drug discovery, and even cardiac surgery. Extrusion-based bioprinting offers a promising approach to generate EHTs with high spatial precision using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, most extrusion-based bioprinting methods rely on hydrogel-rich bioinks to achieve desirable rheological properties, often leading to low cell densities that limit tissue functionality. Here, we show that the cell’s properties can be leveraged to form high cell density bioinks with suitable rheological properties, without the need for excessive hydrogel content. Using these boinks, we bioprinted cardiac tissues (400 M cells/mL) around flexible polydimethylsiloxane (PDMS) posts (2mm diameter) to assess contractile force output and electrophysiological characteristics. The printed cells began spontaneously beating after two days, maintained high viability (>80%), and formed mechanically robust tissues with strong structural integrity. These findings highlight the feasibility of high cell-density bioprinting for cardiac tissue engineering and provide a foundation for future work aimed at generating complex, functional EHTs with high cell-density and spatial precision.

Kaposi’s sarcoma (KS) is a cancer caused by Kaposi’s sarcoma-associated herpesvirus (KSHV). While most KS tumor cells are latently infected, where KSHV is inactive, all current treatments for herpesviruses target lytic infection. The Lagunoff lab has shown that latent KSHV infection, similarly to cancer cells, induces the Warburg effect, in which glycolysis is used as an energy source rather than oxidative phosphorylation. Inhibition of lactate dehydrogenase (LDH), an enzyme that catalyzes the last step of glycolysis, increases cell death specifically in latently infected cells. This indicated that the KSHV-induced upregulation of glycolysis was necessary for the survival of these cells; however, it is unknown how KSHV induces this requirement. The goal of my proposal is to determine the viral mechanism for the induction of the Warburg effect in latently infected cells. During latent infection, only the KSHV-latency-associated-region (KLAR) of the viral genome is expressed. KLAR encodes 4 genes: vFLIP, vCyc, LANA, the kaposins, and a cluster of 12 microRNAs. I hypothesized that one of the genes or miRNAs is necessary and/or sufficient to induce the requirement for glycolysis in latently infected cells. To test for necessity, I am using KSHV recombinant viruses that have a deletion in vFLIP, vCyc, the kaposins, or the entire miRNA locus to infect endothelial cells. To test sufficiency, our lab has created lentiviral vectors that contain one of the KLAR genes or the miRNA locus to overexpress these genes in endothelial cells. I anticipate that vCyc and/or the miRNA locus might exhibit necessity/sufficiency, since prior studies have identified these as important for the regulation of other metabolic pathways. Understanding KSHV’s alteration of specific metabolic pathways in latently infected endothelial cells provides novel therapeutic targets for the inhibition of latent KSHV infection and ultimately KS tumors.

G proteins play a vital role in regulating neuronal activity by acting as key intermediaries that relay extracellular signals inside the cell, triggering a cascade of further signaling events that impact cellular function. This signaling can modulate the activity of ion channels in the neuronal membrane, which control membrane excitability by opening or closing in response to signals, thereby affecting the cell's electrical potential. We are studying the signal transduction pathway that acts downstream of the heterotrimeric G protein Gq to regulate the NCA cation channel in Caenorhabditis elegans. My project focuses on characterizing an unidentified mutant yak133, which has a distinct phenotype defined by deep body bends, also referred to as "loopy." This phenotype suggests that yak133 could be connected to Gq signaling, as activating the Gq pathway leads to a loopy phenotype. The goal of my project is to identify the gene affected by yak133 and understand how it functions to modulate the NCA channel. I narrowed down a list of candidate genes from whole genome sequencing of yak133 by performing a genetic cross to deficiency strains that lack a specific segment of DNA. I then carried out a forward genetic screen and identified a new recessive mutant, yak193, which appears to affect the same gene. I am currently preparing this strain for genome sequencing, and by analyzing both mutants, I expect to identify the gene affected by yak133 and yak193, as they should share mutations in one gene in common. This work will provide relevant insights into the molecular mechanisms regulating neuronal activity and how disruptions in this pathway affect motor and behavioral function. Since many of the genes in C. elegans are conserved in humans, these findings could have broader implications, potentially advancing our understanding of human neuronal function and related disorders. 

Genetic mutations in Caenorhabditis elegans (C. elegans) worms can be studied to understand disruptions in pathways relevant to those in humans, due to ortholog between worm genes and human counterparts. These mutations can manifest as an unmotivated phenotype where the worm displays decreased motivation to move. To explore this phenotype, we performed a series of crosses on a strain of mutated worms to map and identify which gene the mutation is on and to gain a better understanding of the underlying reasons behind the unmotivated phenotype. Our work thus far has led to the potential uncovering of a new gene correlating with this phenotype that has never been associated together before. The worm mutation named yak187 was first generated through random mutagenesis. I performed crosses between yak187 worms and various other strains that each contained a fluorescent marker on a different chromosome. Results yielded little correlations between yak187 and any of the chromosomes we tried. We continued crossing with more strains that contained markers near the ends of chromosomes of suspect and eventually narrowed our highest probable linkage to the right arm of the X chromosome. There are no mutants with this phenotype known in this region yet so our next steps are to sequence the whole genome to pinpoint the location. Furthermore, we have reason to believe that this mutation impacts the dense core vesicle (DCV) pathway impacting neuropeptide release. This pathway is important for regulating body functions, development, and emotions. Disruptions to DCV processes can result in diminished abilities for organisms to operate correctly, resulting in similar consequences as those seen in the mutated worms. The overall pathway involving the production and maturation of DCVs and the secretion of neuropeptides is similar to that in humans, making the study of this system in C. elegans further more exciting.

The primary objective of this study is to evaluate the safety and effectiveness of the study drug Mevidalen, in alleviating symptoms in individuals with mild to moderate Alzheimer's disease dementia. Mevidalen is a selective positive allosteric modulator of the dopamine D1 receptor. The efficacy of this drug is being assessed by examining the patient's cognitive function, daily activities, sleep patterns, Alzheimer disease progression, physical activity levels, and overall stress. I am conducting patient appointments to collect relevant data for the statistical analysis of the study drugs efficacy and safety. Patients are between the ages of 60-80 years old, and are experiencing mild to moderate memory loss. Cognitive function tests including MMSE to gauge the patients working memory, and C-SSRS to monitor mental health throughout the course of this trial. Vital signs and ECG's are measured multiple times during each appointment to track the patient's overall health. Patients are either assigned and titrated to a placebo, low dose study drug, or moderate dose study drug. This is a double blind study, so both the researchers and the patients are blinded to the drug assignment. Over the course of 14 weeks, the patient is monitored by a neurologist at periodic visits, and via an Ax6 wristwatch device that measures sleep patterns. The hope is that this drug is effective, and will soon become a FDA approved therapy for Alzheimer disease dementia, to alleviate memory loss symptoms from patients around the world.

In recent years, soft end-effector prototypes for agricultural harvesting applications have seen a rise in research and development from numerous sources. Soft robot manipulators in agriculture are necessary because of delicate produce requiring a wide area of force application to reduce bruising, as opposed to small points of contact through rigid gripper materials. Novel designs for delicate and clustered fruits and berries such as blackberries, strawberries, and blueberries are of highest demand. This is because of their small size, fragility, and the narrow windows of fruit harvest due to ripeness. These limitations for berries and vine plants necessitate the use of manual labor as opposed to assisted labor for harvesting other fruits and vegetables like apples and pears, and full harvest automation of other fruits and grains like corn and wheat. As novel proof-of-concept designs describe solutions to these limitations, sensing mechanisms for control loop compensation such as visual and tactile are required to control the parameters required when harvesting fruits. These parameters of surface roughness, overall ripeness, blemishes, etc. require thorough and precise sensing capabilities to reduce fruit waste and resulting costs. The purpose of this paper is to discuss the state of novel agricultural end-effector prototypes for harvesting non-automated produce. This review describes the materials and methods of actuation for end-effectors of small, difficult to automate, and/or delicate agricultural needs with focus on sensing methods, variability and scalability to differently sized produce, and cost-effectiveness. End-effector design prototype and case study research papers are used to produce conclusions through analyzing qualitative data and subjective results. Design improvements, future considerations, and gaps in research are covered to aid the advancement of the most promising prospective designs and potential innovation.