Dartmouth Institute for Health Policy and Clinical Practice
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The Role of Polo-like Kinase in Polarizing Germline Determinants in the C. elegans Zygote
Get PDFAsymmetric cell divisions are essential processes across diverse organisms that give rise to two daughter cells with different cell fates, allowing to the generation of cell diversity. During these divisions, the establishment and maintenance of cell polarity is required to control the segregation of cell fate determinants along a polarity axis. Moreover, cell polarity must be coordinated with the cell cycle to ensure the precise spatiotemporal distribution of cell determinants.
In the C. elegans one-cell embryo, PAR proteins establish anterior-posterior polarity that directs the asymmetric segregation of cytoplasmic cell fate regulators. Following symmetry breaking, anterior PAR proteins (PAR-3, PAR-6, PKC-3) and posterior PAR proteins (PAR-1, PAR-2) define distinct cortical domains. These cortical PAR domains guide the polarization of downstream cytoplasmic factors such as MEX-5, MEX-1, PIE-1, and POS-1 by locally regulating their mobility. As a result, these factors become enriched in either the anterior or posterior cytoplasm, establishing cellular asymmetries required for the cell fate specification following the first division.
Han et al. (2018) demonstrated that during the asymmetric division of the C. elegans zygote, PLK-1, in complex with MEX-5/6, phosphorylates POS-1 to inhibit its retention in the anterior cytoplasm, thereby promoting its segregation to the posterior. This PLK-1 phosphorylation-dependent mechanism is crucial for the proper asymmetric segregation of POS-1. In this dissertation, I use quantitative live-cell imaging techniques, biochemical approaches and CRISPR-Cas9 based genome editing to understand the role of PLK-1 in regulating segregation of other cytoplasmic cell fate regulators during asymmetric divisions of the early C. elegans embryo. In Chapter 2, I show that PLK-1 phosphorylates MEX-1 to inhibit its retention in the anterior cytoplasm. While PLK-1 phosphorylation is essential for POS-1 function but contributes to MEX-1 function only at an elevated temperature. I also show that the disordered proteins MEG-1/2 are specifically involved in regulating the segregation of MEX-1.
In Chapter 3, I show that PIE-1 can also serve as a substrate of PLK-1. Preliminary data suggest that PLK-1-mediated phosphorylation regulates both cytoplasmic and centrosomal PIE-1 asymmetry during the early divisions of the C. elegans embryo. Loss of PIE-1 asymmetry on the centrosomes results in abnormal enrichment of nuclear PIE-1 in a somatic blastomere and leads to increased sterility. Taken together, my dissertation work provides molecular insights into the role of PLK-1 phosphorylation in regulating the polarization of cell fate determinants during the asymmetric division of the zygote
Framework Material-Based Chemiresistive Arrays for the Detection and Differentiation of Toxic Gases
No full textConductive framework materials, namely metal─organic frameworks (MOFs) and covalent organic frameworks (COFs), are uniquely poised to serve as gaseous chemiresistive sensors. This dissertation systematically investigates the structure–property−performance relationships within these materials for the low-power, sensitive, and selective detection of toxic gases of biological and toxicological importance.
Chapter 1 highlights conductive framework materials as a burgeoning area of research for application in electronically transduced devices as chemiresistors. We present a logical compilation of work using novel framework materials design and synthesis to build highly tunable, processable, and versatile chemiresistors for the detection and differentiation of gases.
Chapter 2 introduces the use of hexahydroxytriphenylene-based MOFs for gas detection. The structure–property−performance relationships are investigated through comparative sensing, in conjunction with spectroscopic investigations. Mechanistic details are put forth to rationalize the incredible ability for the array to selectively detect and differentiate various mixtures of gases during dual analyte synchronous exposure.
Chapter 3 details the synthesis of novel phthalocyanine-based COFs for the detection and differentiation of gasotransmitters and ammonia for improved gas sensitivity compared to previous materials. We detail the fabrication of robust COF devices and their performance as chemiresistive sensors in dry and humid environments of nitrogen and air. This work improves upon the field in terms of sensor array performance, while also fundamentally probing these materials to deconvolute the mechanisms of material−analyte interactions.
Chapter 4 details the development and implementation of an educational experiment for students consisting of the synthesis and activation of a cyclodextrin MOF for passive CO2 detection and uptake. The experiment provides students with a practical and accessible introduction to fundamental chemical concepts and the principles of green chemistry.
The appendix puts forth preliminary data detailing the effect of metal center location and identity within a bimetallic MOF on the chemiresistive properties. Precise control over atomic composition and morphology is used to deconvolute chemiresistive properties.
Finally, a concluding chapter discusses the findings and implications of the dissertation work in the broader context of the field, summarizing the fundamental insights
Advancing novel contrast agents for applications in fluorescence-guided neurosurgery using fluorescence cryotomography
Get PDFFluorescence guided surgery (FGS) is an emerging surgical technique that aims to help surgeons identify tissue types to assist in surgical decision making - ideally providing “cut-by-color” guidance. A primary application for this approach is the identification of tumor tissue to achieve more accurate and complete tumor removal while simultaneously minimizing damage to surrounding normal tissue. The cornerstone of tumor visualization for FGS is the administration of fluorescent contrast agents, or in some cases metabolic precursors, designed to accumulate specifically in tumor tissue, and a wide range of agents that use different targeting mechanisms are currently under investigation. Although these fluorescent contrast agents often provide elevated tumor contrast, many require long incubation times, which complicate their clinical use and/or exhibit imperfect tumor specificity which may confound the surgeon’s ability to accurately distinguish cancerous from healthy tissue.
This project aims to improve FGS techniques for tumor resection by advancing new fluorescent contrast agents with more favorable properties. These properties include a short administration-to-imaging time, which eases the use of these agents in the clinic, and significant improvements in diagnostic performance. To achieve these aims, we used whole-body fluorescence cryotomography with co-registered contrast-enhanced MRI and histopathology to evaluate a panel of candidate fluorescent contrast agents. Specifically, we focused on applications in neurosurgical fluorescence guidance and identified a lead candidate agent which displayed rapid, diagnostically accurate and persistent contrast that closely correlated to contrast-enhanced MRI and ground-truth tumor as early as 10 minutes after administration. The lead candidate agent was also directly compared with two clinically established fluorescent contrast agents—each requiring administration schedules of 3 to 24 hours—in both small and large animal models, including an inducible glioma model in genetically modified pigs. In these studies, the candidate agent either outperformed or displayed equivalent performance metrics while offering a significantly shorter administration-to-imaging time. The fluorescence cryotomography system is further leveraged in this thesis to investigate region of interest impact on fluorescent contrast agent performance metrics, the delivery of genetic medicines, metastatic spread of tumor models and biodistribution of fluorescent reporters
Neutrophil Inflammatory Death Regulation by Bacterial Type III Secreted Effectors
No full textYersinia and Pseudomonas use a T3SS to secrete effectors into host cells that inhibit phagocytosis and modulate immune responses, such as inflammasome activation. Inflammasomes promote the activation of the proinflammatory protease caspase-1. The activation of an inflammasome sensor in response to a signal or ligand initiates the assembly of the inflammasome leading to activation of caspase-1 which cleaves GSDMD and pro-inflammatory cytokines IL-1β and IL-18 into their mature forms. As neutrophils are recruited in large quantities to sites of infection, they are excellent candidates to drive inflammasome dependent responses. Neutrophils have been shown to produce multiple inflammasomes, like pyrin and NLRC4. For this study, I developed a primed neutrophil infection model to study the role of effectors in regulating inflammasome activity during infection. This model was validated by infection with Y. pseudotuberculosis and shows a requirement for the pyrin inflammasome for secretion of IL-1β and pyroptosis in response to ∆yopM. I also showed that pyrin is dispensable for inflammasome activation during infection with P. aeruginosa. Using a mouse model of infection, I show that the expression of pyrin in myeloid cells promotes protection against infection with Y. pseudotuberculosis ∆yopM or yopJC172A∆yopM, and this prevents the formation of microcolonies in the spleen. Finally, using an ex vivo model I investigated the role of ExoS and the hypersecretion of ExoS from P. aeruginosa on neutrophil cell death pathways. Using a combination of ExoS mutants and CF clinical strains, as well as various mouse lines containing defects in inflammasome components, I show that the activation of the NLRC4 inflammasome in neutrophils is inhibited by ExoS. Inhibition of NLRC4 by ExoS promotes necrosis and this is enhanced when ExoS is hypersecreted. Additionally, I show that ExoS ADPRT activity promotes this inhibition, and infection using a mutant lacking ExoS ADPRT activity activates NLRC4-dependent IL-1βsecretion and pyroptosis. Regulation of inflammasomes by bacterial effectors in macrophages have been well documented, however these interactions in neutrophils have been less well studied. Here, I extend our understanding of neutrophil inflammasomes and the role of effectors in regulating cell death responses to infection with Y. pseudotuberculosis or P. aeruginosa
From Trauma to Treatment: Chronic Neurobehavioral Consequences and Emerging Treatments
No full textSevere trauma exposure related to traumatic brain injury (TBI) or psychiatric disorders like post-traumatic stress disorder (PTSD) leads to neurobehavioral and physiological impairments that reduce the quality of life of individuals and remain difficult to treat with current therapeutic options. Further, these conditions are highly comorbid, notably in PTSD with blast-traumatic brain injury (bTBI) commonly experienced by military members via explosive forces, leading to complex and heterogenous outcomes. To address this, preclinical models have been developed to recapitulate aspects of TBI and PTSD. This thesis utilizes two such models in rats, bTBI and the single prolonged stress (SPS) model for PTSD, to elucidate chronic consequences and to evaluate emerging interventions. Chapter 1 demonstrates that a model of bTBI produces behavioral dysfunction at chronic time points across domains (i.e., anxiety and depressive-like, motivational, and fear phenotypes) and corresponding changes in brain structure and function, revealed through neural oscillations and neuroimaging. Chapter 2 evaluates the efficacy of 3,4-methylenedioxymethamphetamine (MDMA) to reduce expression of cued fear behavior in bTBI rats. This revealed that MDMA as an adjunct to re-exposure of conditioned trauma, akin to exposure therapy, had efficacy for reducing fear behavior and resulted in acute and persisting oscillatory changes related to MDMA. In Chapter 3, the SPS model was used to investigate potential stress hormonal changes and anxiety-like and social behaviors related to PTSD, with and without cue re-exposure. These behavioral and physiological impairments were not revealed at the time points tested, showing this commonly reported protocol may need modification to serve as a robust and intervenable model for PTSD. Collectively, these multimodal studies advance our understanding of trauma exposure on chronic neurobehavioral outcomes and serve to guide emerging interventions for bTBI and PTSD
Advances Towards a Robotic Management Vehicle Suited to Nurse Row Crops to More Efficient Outcomes
Get PDFEnhancing agricultural production while reducing input costs remains a central challenge in modern row-crop management. Recent advances in computation, imagery, and sensors are enabling more efficient practices across various agricultural domains, and automation technologies are increasingly available to manage tasks central to perennial crop development. Automation in row-crop agriculture, by contrast, lags behind. This thesis explores utilizing small, unmanned ground vehicles to transform row cropping through the implementation of unconventional, in-season management strategies. The first focus of this work considers improvements to nitrogen fertilization using small, autonomous vehicles. An agronomy experiment in corn assessed the effects of gradually applying nitrogen fertilizer over time, with the goal of evaluating how this approach impacts both yield and nitrogen input requirements. Alongside parametric analyses, this study demonstrates the feasibility of using micro-UGVs to manage fertilization despite payload limitations. To unlock these precision practices, autonomous operations under-the-canopy must be improved substantially. Navigating densely planted row-cropping environments with cameras or ranging sensors is challenging due to sensor occlusion, lighting variability, and the difficulty of distinguishing flexible surfaces like leaves from rigid obstacles like stalks. A novel tactile-based perception system comprising a mechanical feeler sensor and supporting algorithms was engineered to detect nearby obstacles like rigid cornstalks while filtering out flexible features like weeds and leaves. Then, through simple kinematic relationships, the system was used to accurately determine the position of these obstacles, such that a blind robot can traverse the messy environment. Through simulation and real-world testing, the system demonstrated effective navigation in complex agricultural iii conditions, overcoming challenges like row curvature, planting gaps, dense weeds, and canopy variability—without relying on vision or ranging sensors. Additionally, mobility is crucial to navigating row crops; tight row spacing constrains vehicles and limits their ability to recover from immobilizing conditions. A second prototype vehicle is presented with innovative mechanical systems that restore mobility in response to incipient immobilization and extricate the vehicle from challenging terrain. The results of this thesis establish novel models and methods to overcome visual impairment and immobilization for agricultural robots, enabling new, unconventional forms of precision agriculture for row crops
Biochemical and molecular analysis of Anr-dependent microoxic fitness factors in Pseudomonas aeruginosa
Get PDFPseudomonas aeruginosa is a prominent opportunistic pathogen that is often found in microoxic environments like sites of infection and colony biofilms. P. aeruginosa can grow at oxygen (O2) at concentrations as little as 3 μM and also grows anaerobically by generating energy using nitrate as an alternate electron acceptor. An important way P. aeruginosa adapts to low O2 is through Anr, a transcription factor that upregulates many genes important for microoxic and anoxic fitness.
Specifically, Anr regulates mhr which encodes a hemerythrin protein (Mhr) that binds O2 with affinities relevant to microoxia and is also necessary for full fitness of P. aeruginosa in colony biofilms. In this thesis, we present data that suggest Mhr is important for growth in colony biofilms, but not in planktonic cultures. Mhr has an 11-amino acid C- terminal extension not found in all hemerythrins that we show is necessary for membrane localization which is required for the Mhr contribution to fitness. Interestingly, Mhr has an epistatic relationship with the membrane-bound, high-affinity cbb3 oxidases that allow P. aeruginosa to respire O2 at low concentrations. Data presented in this thesis show that the presence of the cbb3 oxidases was also important for Mhr membrane localization. Together, these data suggest that the cbb3 oxidases promote Mhr membrane localization and contribute to the Mhr-dependent microoxic fitness phenotype.
Anr and the transcriptional regulator Dnr, also regulate denitrification, a process that allows P. aeruginosa to utilize nitrate as an alternative electron acceptor and ultimately transforms nitrate to nitrogen gas using a series of membrane-bound reductase enzymes. While denitrification is best described in the context of anaerobic growth, P. aeruginosa also performs denitrification in oxic conditions as well. The data in this thesis expand upon the contribution of oxic denitrification to P. aeruginosa fitness in commonly-used laboratory media like lysogeny broth (LB) and artificial sputum media. Our data show that Dnr and Dnr-regulated norB are important for P. aeruginosa fitness only in conditions where nitrate was being consumed. The work presented in this thesis presents important information about Anr-regulated factors that contribute to P. aeruginosa fitness in microoxic environments
Optimal Hypergraph Connectivity with CUT Queries
Get PDFFinding connected components in undirected hypergraphs—hypergraph connectivity—is a fundamental problem in computer science. It can be framed as a special case of Symmetric Submodular Function Minimization (SSFM), where the objective is to determine if the non-trivial minimizer is zero. This thesis develops an optimal algorithm for hypergraph connectivity within the \CUT query model, where an algorithm probes a subset of vertices to learn the weight of the hyperedges ``cut by that partition.
Our approach is constructive, culminating in an optimal algorithm for the general problem by first developing the necessary tools for two foundational subproblems. The main contributions of this thesis are: \begin{itemize} \item \textbf{Spanning Forest in Weighted graphs:} An optimal (up to a constant factor), polynomial-time, zero-error algorithm to reconstruct a maximal spanning forest in weighted undirected graphs using \CUT queries. \item \textbf{Partition Learning:} An optimal deterministic algorithm for learning a hypermatching—a problem we term ``partition learning —using \RANK queries. \item \textbf{Hypergraph Connectivity:} Leveraging these preceding results, we present an optimal, polynomial-time, zero-error algorithm to find the connected components of capacitated hypergraphs using \CUT queries. \end{itemize} Collectively, these results establish a complete and optimal framework for solving hypergraph connectivity in the \CUT query model
