VictorJuiz/thesis huggingface dataset:
Investigating the Cardiotoxicity of Proteasome Inhibitors on iPSC Cardiomyocytes
Keywords:  Department of Biology
Biology
induced pluripotent stem cell
cardiomyocytes
proteasome inhibitors
cardiotoxicity
Thesis

Proteasome inhibitors (PIs) are a class of chemotherapeutic drug in treating multiple myeloma (MM), a cancer of plasma cells. PIs are an integral part of MM therapy, but their usage is limited by its cardiovascular side effects. Clinical cases show that patients have suffered from complications such as heart failure and sudden cardiac death after taking these drugs. The mechanism through which PIs cause these effects remains unknown. This study aims to use an iPSC-CM model to recapitulate and investigate the mechanism of cardiotoxicity caused by PIs. First, human iPSC-CMs were treated with three types of PIs (carfilzomib, ixazomib, and bortezomib) at doses and schedules relevant to their clinical usages. Doxorubicin, a well characterized chemotherapeutic known to have cardiac adverse side effects, was used as a positive control. We then conducted a series of in vitro investigations including contractility and viability assays to 1) determine whether iPSC-CMs adequately model clinical features of PI-induced cardiotoxicities and 2) to better understand their effects on the contraction of cardiomyocytes. Results show that the mechanism through which cardiotoxicity occurs is linked to the cells’ inability to contract when treated with moderate to high concentration of the drugs. Cells treated with carfilzomib, bortezomib, and ixazomib respectively showed high, moderate, and low levels of cardiotoxicity. The iPSC-CM model used in this study serves as a high-throughput, efficient approach to screen drugs associated with deaths due to cardiac adverse events in MM patients and will enable future studies to further investigate the effects of PIs on cardiomyocytes.
Investigating the function of a mitochondrial phosphatase, PTPMT1, in Drosophila melanogaster
Keywords: Developmental Biology.

Mitochondria are complex organelles that play a variety of critical roles in the cell beyond the generation of ATP. Reversible phosphorylation, mediated by mitochondrially-localized kinases and phosphatases, is a central mechanism by which mitochondrial activities are regulated and by which tissue-specific mitochondrial functions can be achieved. PTPMT1 is a mitochondrial phosphatase and a vital regulator of mitochondrial activities. Current work points to roles for PTPMT1 in the regulation of mitochondrial respiration, dynamics, and ultrastructure through the modulation of pyruvate importation, the tuning of TCA cycle activity, and the synthesis of cardiolipin. PTPMT1 is ubiquitously expressed; however, little is known about its tissue-specific functions. Additionally, PTPMT1 has predominately been studied in vertebrate models, even though invertebrate models, namely Drosophila melanogaster, provide powerful genetic tools for the dissection of gene function. In Chapter 1 of this dissertation, I review the current knowledge surrounding PTPMT1 in vertebrate models. In Chapter 2, I introduce the Drosophila tracheal system and briefly review what is known concerning the mechanisms of tracheal liquid clearance and gas filling in the fly. In Chapter 3, I use RNA interference and CRISPR-Cas9 technologies to generate and validate Drosophila models of dPTPMT1 depletion. Using these models, I discover a new physiological role for dPTPMT1 in the process of tracheal liquid clearance and gas filling. I further show that gas-filling defects occur following molting in first instars with tracheal-specific knockdown of dPTPMT1 and are accompanied by subsequent activation of innate immune responses. First instars with dPTPMT1-depleted tracheae show mitochondrial clustering prior to failure of liquid clearance and gas filling, suggesting that an initial mitochondrial defect may be responsible for this phenotype. I further ask whether other mutants in the cardiolipin synthesis pathway phenocopy the tracheal phenotypes observed in dPTPMT1-depletion and find that they do not, indicating that dPTPMT1 likely acts through one of its other known substrates or perhaps through a novel substrate to mediate tracheal liquid clearance and gas filling in Drosophila. My work demonstrates a new cell-type specific role for dPTPMT1 in fly tracheal epithelial cells and establishes a new model for the exploration of dPTPMT1's physiological and cellular functions
Investigating the p38 MAPK pathway's role in cell cycle size control
Keywords:  Department of Biology
biology
cell cycle
G1/S transition
Rb
p38 MAPK
retinal pigment epithelium cells
Thesis

Cell size is an important determinant of animal cell function because it sets the rate of biosynthesis and surface transport within the cell. Though there exists a vast range of cell sizes, cells of the same tissue type typically grow to a similar size before progressing through the G1/S checkpoint to duplicate DNA and then divide. Phosphorylation of the retinoblastoma (Rb) tumor suppressor protein has long been known to regulate cell cycle entry at the G1/S checkpoint, and more recently, dilution of Rb by cell growth was proposed as a mechanism through which cell growth triggers the G1/S transition. In addition, it was recently shown that the p38 mitogen-activated protein kinase (MAPK) could also contribute to cell size control with regulation of G1 length. p38 had previously been known to respond to stress stimuli such as cytokines, UV radiation, heat and osmotic shock and involved in cell differentiation, apoptosis, and autophagy. In these studies, p38 inhibited progression through G1 in small cells and not large cells, but not much is known about how it does so. Understanding the mechanism of size control by p38 operates and its relative importance will provide further insight into whether there exists a relationship with the established Rb phosphorylation and Rb dilution G1/S control pathways. Here, by using models of human mammary epithelial cells (HMEC) and retinal pigment epithelium cells (RPE), we observe that size control exists in only select cell lines cultured in vitro. For cell lines exhibiting size control, there is a dose dependent relation to p38 inhibitor drug treatments, confirming a p38 MAPK size control pathway. Furthermore, through analyses of p38 inhibitor and rapamycin drug treatments of wild-type and Rb-knocked out RPE cells, we demonstrate that the role of the p38 MAPK pathway in cell size control does not require Rb. These findings elucidate a crucial mechanism of how cell growth drives cell division that may prove important in further understanding of both development and disease.
Investigating the Role of Nectin-2/DNAM-1 Interactions in the Innate Immune Response to Dengue Virus
Keywords:  Department of Biology
Biology
Immunology
Dengue
Thesis

Dengue virus (DENV) is a mosquito-transmitted virus that infects tens of millions worldwide each year. One currently underexplored behavior of DENV is its ability to regulate the innate immune system. Previous research has shown that DENV activates pathways associated with autophagy, complement, and apoptosis, and can inhibit these pathways in order to prevent antiviral responses. My preliminary investigation showed that monocytes significantly upregulate Nectin-2 following infection with DENV. Nectin-2 is a ligand for DNAX Accessory Molecule-1 (DNAM-1 or CD226), an important activating receptor on natural killer (NK) cells. Due to this upregulation, we hypothesized that Nectin-2 might play a role as an important recognition molecule for NK cells in DENV infection. After a series of optimization experiments, we conducted several tests co-culturing NK cells with DENV-infected target cells, using monoclonal antibodies to inhibit the Nectin-2/DNAM-1 pathway. While our study was terminated prematurely due to the COVID-19 outbreak, we observed evidence that the Nectin- 2/DNAM-1 pathway may have a modest effect on NK directed killing of DENV-infected cells. Further study will reveal whether these results are significant, or whether Nectin-2 may be an indicator of a different biological process.
Investigating the Role of Tomato Plasma Membrane Protein RLK15 in Tomato Defense Responses to Bacterial Infection
Keywords:  Department of Biology
Biology
Pattern-triggered immunity
tomato immunity
pre-invasion defense
post- invasion defense
pattern recognition receptors
reactive oxygen species
salicylic acid
Xanthomonas euvesicatoria
Pseudomonas syringae pathovar tomato
Thesis

Economically important crop plants such as tomato are under constant threat of attack from bacterial pathogens which colonize leaf interior spaces and replicate. Lacking specialized immune cells, plants rely on the innate immunity of their cells to defend against pathogens. In leaves, both stomatal and apoplastic immune responses play key roles in preventing bacterial entry and replication within the tissue. The plasma membrane (PM) is the interface across which these interactions occur. Plant PM proteins termed Pattern Recognition Receptors (PRRs) interact with other PM proteins to activate early intracellular plant defenses following recognition of conserved microbial molecules termed Microbe-Associated Molecular Patterns (MAMPs). Recently, our lab has shown that one PM protein in tomato, Tomato Atypical Receptor-Like Kinase 1 (TARK1), plays a role in positively regulating apoplastic immunity and negatively regulating stomatal immunity. A proteomics screen was performed to identify TARK1 interacting partners and elucidate TARK1’s functions. This led to the identification of a Receptor Like Kinase, designated RLK15, whose function was unknown. To determine if RLK15 played a role in immunity against bacteria, I conducted pre- and post-invasion infection assays with tomato lines with altered RLK15 expression. In pre-invasion assays, I obtained data suggesting that RLK15 may positively regulate stomatal immunity. In post-invasion assays, I found that RLK15 protein accumulates in response to pathogen infection but is not necessary for TARK1 accumulation. I also found that RLK15 nonfunctional mutants demonstrated increased resistance to Xanthomonas strain Xe ∆xopN (deficient in the XopN effector) while RLK15 overexpression lines displayed greater susceptibility to Pseudomonas strain Pst COR- (deficient in production of the virulence factor coronatine). Quantification of reactive oxygen species (ROS) production and salicylic acid (SA) production in response to bacterial elicitation suggests that RLK15 may play a role in the suppression of ROS and SA production during bacterial infection. Taken together, my findings highlight the importance of RLK15 in pre- and post-invasion immune responses to bacteria in tomato.
Investigating the synaptic basis of hippocampal learning and memory
Keywords:  Department of Biology
long-term potentiation
hippocampus
Syntaxin-3
learning
memory
Molecular and Cellular Physiology
biology
Thesis

Memory impairment is a symptom of most neurodegenerative diseases. Yet restoring memory is impossible because we do not understand the molecular correlates underlying it. Currently, the field of neuroscience follows the hypothesis that the biological basis of memory lies in the ability of neural networks to physically change based on experience. Long-term potentiation (LTP), a form of synaptic strengthening that results from strong activation of a synapse, has been discussed as a potential facilitator of memory formation and retrieval. LTP utilizes SNARE complexes containing Syntaxin-3 to increase neurotransmitter trafficking at the postsynaptic membrane, resulting in increased postsynaptic AMPA receptors. LTP has been studied for decades, but a causal link between LTP and memory has been difficult to establish due to the difficulty of deleting LTP without altering other neuronal activities. The present study uses a conditional knockout of Syntaxin-3 in mice to demonstrate that Syntaxin-3 is necessary for LTP and to test the role of LTP in memory. We found that Syntaxin-3 deletion in the hippocampus failed to result in significant differences in performance on memory tasks. Voltage-clamp electrophysiological recordings from hippocampal slices confirmed that LTP was selectively eliminated by the Syntaxin-3 deletion. Evaluation of short-term memory tasks (object location task, novel object recognition task) as well as long-term memory tasks (Barnes maze, fear conditioning) showed that knockout mice and control mice performed similarly without a statistically significant difference. The results of this study indicate that hippocampal LTP may not be required for memory storage and retrieval in these short-term and long-term memory tasks. However, the ability to selectively affect LTP without altering other forms of neuronal activity makes the Syntaxin-3 conditional knockout mouse a key tool for future studies of long-term potentiation.
Investigation of droplet break-up and flow in a concentrated emulsion
Keywords: Mechanical Engineering.

Droplet microfluidics, which uses microdroplets suspended in a carrier oil as individual biochemical reactors, has enabled massive parallelization of reactions with sub-nanoliter reagent consumption per reaction. It has found a wide range of applications, including digital polymerase chain reaction, antibiotic screening, and directed evolution of enzymes. However, unlike solid microwells, droplet-based microreactors are metastable and can undergo break-up into small daughter drops, thereby compromising the accuracy of the assay. Droplets are prone to break-up during the serial interrogation process, in which drops are injected as a monolayer into a taper-shaped channel leading to a constriction for the optical or electronic interrogation of the droplet content one at a time. The onset of droplet breakup sets the upper limit in the throughput of the interrogation process. Few studies have attempted to increase the throughput of the drop interrogation process. Motivated by this gap in knowledge, the work presented in this dissertation had two broad aims: 1) Describe concentrated emulsion flow to gain a fundamental understanding of break-up; 2) Increase drop throughput by reducing drop break-up. To address the first aim, we studied the timescale and special distribution of drop rearrangement events in a low strain-rate regime (Chapter 2, capillary number Ca~10^(-7) to 10^(-2)). In this regime, the interfacial effects dominate the viscous effects. To address the second aim, we developed two strategies to reduce drop break-up and increase drop throughput. Experiments were conducted in high strain-rate regime (Chapter 3-4, Ca> 10^(-2)). In this regime, the viscous effects become important, and emulsions begin to exhibit droplet break-ups. The results presented in this dissertation provide significant guidance to the design of flow control elements in droplet microfluidic devices, improving drop throughput applications, and understanding the effect of microscopic drop behavior on macroscopic emulsion flow behaviors
Investigation of VISTA as a cancer immune checkpoint via structural analysis and therapeutic antibody development
Keywords: Bioengineering.

Immunotherapy has shifted the landscape of cancer treatment with the advent of checkpoint inhibitors, which have become the standard of care for multiple types of cancer. Despite the remarkable success of anti-PD1 and anti-CTLA-4, patient response rates to checkpoint blockade are often only 20-30%, necessitating the need for new therapeutic targets. V-domain Ig Suppressor of T cell activation (VISTA) is an orthogonal checkpoint molecule that dampens the interaction between antigen-presenting cells and T cells to prevent effective anti-tumor responses. In this thesis, I present multiple projects related to the characterization of VISTA as a therapeutic target. First, I report the high-resolution crystal structure of the VISTA extracellular-domain and elucidate an important functional epitope of a clinical VISTA antibody (Chapter 2). I used a genetic and enzymatic de-glycoyslation approach to facilitate crystallization and utilized a display-based sorting strategy to isolate hotspot contact residues. Second, I detail the engineering and characterization of a unique, high affinity, species cross-reactive antibody against VISTA (Chapter 4). Using a combination of immune cell assays, epitope mapping strategies, and syngeneic mouse models, I demonstrate the efficacy of this antibody as a VISTA inhibitor. These efforts highlight a promising candidate for therapeutic development and provide a blueprint for future drug discovery campaigns against this promising new checkpoint target
Investigations of solid oxide-based carbon fuel cells : modeling and experiments
Keywords: Mechanical Engineering.

Solid oxide-based carbon fuel cells (SO-CFCs) provide an efficient energy conversion of carbon into electricity. Furthermore, by keeping the fuel and the air physically separated, a near-capture ready product stream of CO2 is produced. To further our understanding of this promising energy technology, three areas of investigations are presented herein. First, a set of experiments run on CO, H2, and syngas fuel are performed to gain insight into the electrochemical reactions occurring on the anode surface. Current-voltage curves and electrochemical impedance spectra are measured and analyzed, and kinetic parameters are extracted. Second, a previously established comprehensive SO-CFC model is further developed and executed. In particular, the effects of fuel-bound hydrogen on SO-CFC performance is investigated, and design and operational parameters of a commercially viable tubular SO-CFC is explored. Finally, a thermodynamic analysis of sulfur uptake by solid sorbents is performed and visualized using C-H-O ternary diagrams. Sulfur-poisoning is one of the major hurdles for commercialization of the SO-CFC technology. The analysis shows that there is a small window of opportunity at a cell voltage of 0.65-0.7 V where the abundant calcium sorbent is able to bring the H2S and COS mole fractions down to low ppmv levels, enabling the utilization of sulfur-containing carbonaceous fuels in the SO-CFC
Investing thermo-fluidic performance of si-based embedded microchannels-3D manifold cooling system for high power density electronic applications
Keywords: Mechanical Engineering.

High performance and economically viable cooling solutions must be developed to reduce weight and volume, allowing for a wide-spread utilization of hybrid electric vehicles. However, the continued drive for higher device and packaging densities has led to extreme heat fluxes on the order of 1 kW/cm2 that requires aggressive microchannel cooling strategies in order to maintain the device junction temperature ~ 200 C. The traditional embedded microchannel cooling heat sinks suffer from high pressure drop due to small channel dimensions and long flow paths in 2D-plane. Utilizing direct "embedded cooling" strategy in combination with top access 3D-manifold strategy reduces the pressure drop by nearly an order of magnitude. In addition, it provides more temperature uniformity across large area chips and it is less prone to flow instability in two-phase boiling heat transfer. This dissertation presents a series of studies to develop an effective microchannel-based heat exchanger with a fluid router system, the Embedded Microchannels-3D Manifold Cooler (or EMMC). The overall microfabrication efforts for the EMMCs are presented. The configuration of the target EMMC design is introduced and two major fabrication challenges are discussed. For single-phase flow, thermo-fluidic behavior of the EMMC is experimentally examined and validated by a conjugate numerical simulation model. DI water and R-245fa are used as working fluids and the maximum heat transfer rate of 100 kW/m2-K was measured with DI water. Furthermore, the conjugate numerical simulation modeling is heavily used to predict the geometric effect on the thermo-fluidic performance of different EMMCs and used to develop correlations to predict friction factor and Nusselt number of the system. For two-phase flow, forced-convective subcooled boiling is confirmed by the experiments and a systematic trial to calculate exit vapor quality has been made based on a few assumptions. The highly pressurized subcooled boiling delays onset-of-nucleate boiling in the microchannels and this strong condensation effect allows the EMMC to remove higher heat fluxes with low void fraction inside of the channels. The present research motivates further study into flow visualization and different types of boiling heat transfer. The better understanding to the underlying physics of the EMMC will be a key to develop more effective heat exchanger design for high-power density applications
Ion conduction by the picosecond : optical probes and correlations
Keywords: Materials Science and Engineering.

Ionic conductivity is key to electrochemical energy storage in rechargeable batteries, and remains a vital enabler of clean energy. Macroscopically, ion conduction is a random temperature-activated process consisting of rare and incoherent discrete events called hops. In every such event, a mobile ion physically jumps, i.e. undergoes translation, between distinct crystallographic sites in a host lattice. The centrality of fast ion conductors to pressing societal needs demands a mechanistic understanding of ion conduction starting at the timescales of single hops, and including contributions of phonon structure, anharmonicity, and collective phenomena. I have used picosecond-timescale terahertz pump-probe experiments and large-scale molecular dynamics simulations to characterize ionic hopping in a model homologous family of historic fast solid-state ionic conductors, the beta-aluminas, M_1.2 Al_11 O_17.1, with mobile ions M = Na+, K+, Ag+. I have quantified spatio-temporal correlations between hopping events in beta-aluminas using a statistical social-network approach. In all beta-aluminas, each hop of an ion acts as a perturbation to its neighboring mobile ions, and furthermore influences follow-on hops. These correlations extend beyond nearest-neighbor interactions and appear mediated by specific vibrational couplings. Using single-cycle terahertz optical pulses, I have resonantly pumped the hopping attempt frequencies of mobile ions in beta-aluminas. The transient birefringence of the materials following a terahertz pump elucidates the picosecond-timescale dynamics of mobile ions. The statistics of hopping events derived from molecular dynamics explain the unusual liquid-like relaxation in beta-aluminas in addition to the expected vibrational responses. I furthermore experimentally observe signatures of correlated hopping events. In contrast to periodic-perturbation experiments, or to non-resonant pumping, it is the transition state, i.e. an ion caught mid-hop, that is the most sensitive to strong-field terahertz pulses and gives rise to the liquid-like response. To the best of my knowledge, these are the first pump-probe studies of ionic transport in electronically insulating materials, drawing parallels to studies of electronic conduction in photovoltaics. Based on my experimental and computational methods, I discuss the role of local vibrational dynamics and correlated transport in mediating fast ionic conduction and determining the overall activation energy