Browsing by Subject "Mechanism"
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Item Open Access Genomic Analysis of Cancer Heterogeneity and Oncogenic Mechanisms(2014) Jiang, XiaoleiThe development of cancer is a process by which an accumulation of genetic changes leads to uncontrolled replication of cells. Since the process of mutation is random, the set of alterations that occur and accumulate during tumorigenesis in one individual is different from that of another. These genetic differences drive tumor heterogeneity. One of the first technologies used to explore genome-wide heterogeneity was the microarray, which can be used to measure the expression of tens of thousands of genes. By exploring differences in expression of not just single genes, but groups of genes that may be altered in one set of tumors compared to another, researchers were able to classify subtypes of cancer that had relevance in disease aggressiveness, treatment, and prognosis. Furthermore, by looking at genome-wide patterns of expression, it is possible to identify specific oncogenic pathways that are activated and critical in driving tumor cell survival, growth, or metastasis. My research utilizes the patterns of expression derived from microarray analyses to study tumor heterogeneity, particularly in response to targeted cancer therapy, and mechanisms of cell death following oncogenic deregulation.
One of the cancer types that has been explored through expression array analysis is B-cell lymphoma. Human aggressive B-cell non-Hodgkin lymphomas (NHL) encompass the continuum between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), and display considerable clinical and biologic heterogeneity, most notably related to therapy response. We previously showed that lymphomas arising in the E&mu-Myc transgenic mouse are heterogeneous, mirroring genomic differences between BL and DLBCL. Given the clinical heterogeneity in NHL and the need to develop strategies to match therapeutics with discrete forms of disease, we investigated the extent to which genomic variation in the E&mu-Myc model predicts response to therapy. We used genomic analyses to classify E&mu-Myc lymphomas, link E&mu-Myc lymphomas with NHL subtypes, and identify lymphomas with predicted resistance to conventional and NF-&kappaB targeted therapies. Experimental evaluation of these predictions links genomic profiles with distinct outcomes to conventional and targeted therapies in the E&mu-Myc model, and establishes a framework to test novel targeted therapies or combination therapies in specific genomically-defined lymphoma subgroups. In turn, this will rationally inform the design of new treatment options for aggressive human NHL.
The second aspect of my thesis looks at the mechanisms of apoptosis following oncogene deregulation. The Rb-E2F pathway is a critical oncogenic pathway that is frequently mutated in cancers. Alterations in the pathway affect genome-wide expression in the cell, which in turn lead to deregulation of the cell cycle. The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors that cooperate with E2F to mediate the specificity of E2F function. In this work, we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.
Taken together, these studies highlight the importance and power of analyzing genome-wide patterns of expression in investigating cancer heterogeneity, its ability to help predict treatment response, and its role in discovering the mechanisms behind the consequences of gene deregulation.
Item Open Access Mechanisms of Silver Nanoparticle Toxicity in Laboratory Suspensions and Complex Environmental Media in Caenorhabditis Elegans(2014) Yang, XinyuThe rapidly increasing use of silver nanoparticles (Ag NPs) in consumer products and medical applications has raised ecological and human health concerns. Significant progress has been made in understanding the toxicity of silver nanoparticles (Ag NPs) under carefully controlled laboratory conditions. The goals of this dissertation were to investigate the mechanism of Ag NP toxicity under both laboratory conditions and environmental backgrounds, using Caenorhabditis elegans (C. elegans) as a model system. A key question for addressing these concerns is whether Ag NP toxicity is mechanistically unique to nanoparticulate silver or if it is a result of the release of silver ions. Ag NPs are produced in a large variety of monomer sizes and coatings, and since their physicochemical behavior depends on the media composition, it is important to understand how these variables modulate toxicity.
In order to test the hypothesis of a particle-specific effect, multiple techniques were used, including analytical chemistry, pharmacological rescue, and genetic analysis. Results suggested that dissolution was important for all tested Ag NPs and oxidative stress (a particle-specific effect) was important only for some Ag NPs, especially the citrate-coated Ag NPs (CIT-Ag NPs). The hypothesis of the particle-specific effect was further tested by investigating the cellular uptake and damage co-localization upon exposures to CIT-Ag NPs. I found that Ag NPs crossed all layers, including the pharynx, gut, and also embryos through trans-generational transfer. Sites of damage were examined through transmission electron microscopy (TEM), and CIT-Ag NPs showed a more severe and deeper level of damage compared to ionic Ag. In addition, pharmacological inhibitors in parallel with genetic mutants (deficient in both endocytosis and lysosomal function) were used to explore the impact of those pathways on Ag NP uptake and associated toxicity. I found that endocytosis was important for CIT-Ag NP uptake and toxicity. Most intriguingly, one of the lysosomal deficient mutants was much more sensitive than wild type to reproductive inhibition after exposure to CIT-Ag NPs but not ionic Ag, constituting a clear nanoparticle-specific toxic effect.
These laboratory mechanistic studies, however, cannot be directly extrapolated to complicated environmental conditions, including variable amounts of natural organic matter (NOM), different temperatures and salinities, surface sulfidation, etc. My general hypothesis was that complex environmental medium would reduce Ag NP toxicity. In support of this, the environmental conditions present in mesocosms resulted in a loss of toxicity one week after dosing/spiking. In laboratory studies, I found that that increasing temperature and salinity tended to increase Ag NP toxicity, while sulfidation reduced Ag NP toxicity, acting as a &ldquonatural antidote&rdquo. I studied two types of NOM, Suwannee River and Pony Lake fulvic acids (SRFA and PLFA respectively). PLFA rescued toxicity more effectively than SRFA. Therefore, CIT-Ag NP-NOM interactions were explored in depth using PLFA. Using hyperspectral dark field microscopy, I was able to detect the formation of Ag NP-PLFA complexes and the limited tissue uptake of Ag NPs (with and without PLFA). Consistent with the reduced acute toxicity of Ag NPs by PLFA, I also found a rescue effect of PLFA on Ag NP-induced ultrastructural damage.
In conclusion, Ag NP toxicity resulted largely from dissolution and in some cases also from a particle-specific effect. However, Ag NP toxicity was strongly altered by environmental matrices. Continued in depth elucidation of Ag NP behavior, cellular uptake pathways and trafficking, and their interactions with other environmental factors will be invaluable in predicting, designing, and remediating the potential/existing environmental implications of silver-related nanotechnology.
Item Open Access Mechanistic Analysis of Gold(I) Catalysis through Generation and Direct Observation of Reactive Intermediate Analogues(2019) Kim, NanaCationic gold carbene complexes have attracted significant attention, being postulated as intermediates in a range of gold-catalyzed transformations. Regardless of the remarkable progress in the gold (I) catalysis, our fundamental understanding on the key intermediate species and the subsequent reactivity, and mechanistic insight is deficient. This is mainly due to the lack of proper model system with sufficient reactivity, as the majority of known gold carbene complexes are heteroatom stabilized or sterically hindered, and because of a dearth of direct intermediate observations in catalytic systems. Lewis acid mediated leaving group abstraction from a neutral gold precursor provides a convenient method for the generation of rare examples of reactive gold carbene species in high yield and purity, addressing the issue with isolation of such transient species as well as allowing in situ spectroscopic analysis. Subsequent trapping experiment with nucleophiles provides kinetic information about relevant catalytic transformations, and the -ionization strategy is further extended toward generation of transient -cationic propyl gold species for studying gold to alkene carbene transfer reaction.
Item Open Access Mindcraft: a Dynamical Systems Theory of Cognition(2014) Barack, DavidThis dissertation develops a theory of cognition, driven by recent developments in the electrophysiological investigation of the neuronal mechanisms that support adaptive behavior. In the first chapter, I situate the theory in the conceptual landscape of the philosophy of mind, distinguishing componential from systemic dynamical theories of cognition. In the second chapter, I analyze two case studies from electrophysiological cognitive neuroscience, arguing that cognitive neuroscientists are beginning to uncover the dynamical components of cognition. Drawing on the recent literature on mechanisms and scientific explanation, I propose a revised definition of a mechanism that accommodates these dynamical mechanisms, as well as making room for their implementation by physical mechanisms. In the third chapter, I argue that the investigation of a particular class of intelligent behavior begins with the construction of a formal model of the processing problem for that behavior, where this model is distinct from the physical device and the functions performed by the device's components. In the third chapter, I argue that the component dynamical mechanisms of cognitive systems are distinct from though implemented by physical mechanisms. These dynamical mechanisms are described by sets of differential equations, possess a set of organized components and activities that execute the formal models of processing, and are implemented by the physical machinery of the cognitive system, such as the brain. After I argue that these multiple interacting dynamical mechanisms are the components of cognition, defending this componentiality claim against several objections, I define the implementation relation that holds between dynamical and physical mechanisms. I next discuss the grounds for inferring the existence of dynamical mechanisms that are type distinct from physical mechanisms, their implementing substrate. In the fourth chapter, I argue that these dynamical mechanisms are reused: they can execute different formal models and be implemented by different physical substrates. I define this concept of reuse, situating it in the debate on theories of reuse, and illustrate how dynamical mechanisms are reused in cognitive systems.
Item Open Access Model Studies of Proposed Intermediates in Homogeneous Gold(I) Catalysis(2012) Brown, Timothy JustinThe ability of gold(I) complexes to function as catalysts for myriad organic transformations has led to a dramatic increase in their utilization. Among the homogeneous reactions catalyzed by gold(I), carbon-carbon and carbon-heteroatom bond forming processes are of particular interest for the fields of organic synthesis and pharmaceutical development. Discussed herein are gold(I)-catalyzed methods for the intra- and intermolecular functionalization of alkenes, alkynes, and allenes with nitrogen- and oxygen-based nucleophiles leading to new C‒X bonds (X = N, O).
Approximately 26 cationic gold π-alkene complexes, containing either IPr [IPr = 1,3-bis(2,6-diisopropylphenyl)imidazol-2-ylidene] or P(t-Bu)2o-biphenyl ancillary ligands, were isolated or generated and six complexes were analyzed by X-ray crystallography. Spectroscopy, X-ray crystallography, and alkene binding studies are in accord with a gold−(π-alkene) interaction dominated by σ-donation from the alkene to gold. Kinetic analyses of degenerate isobutylene exchange in both the IPr and phosphine systems are consistent with associative pathways for isobutylene exchange involving cationic bis(alkene) intermediates.
Reaction of a 1:1 mixture of (L)AuCl [L = P(t-Bu)2o-biphenyl or IPr] and AgSbF6 with internal alkynes led to isolation of the corresponding cationic, two-coordinate gold π-alkyne complexes in ≥90% yield. Equilibrium binding studies show that the binding affinities of alkynes to gold(I) are strongly affected by the electron density of the alkyne and to a lesser extent on the steric bulk of the alkyne. Treatment of a suspension of (IPr)AuCl and AgSbF6 with terminal arylacetylenes led to the formation of thermally unstable gold π-alkyne complexes of the form [(IPr)Au(eta2-HC≡CAr)] SbF6 in ≥86 ± 5% yield, which were characterized by spectroscopy without isolation. Warming these complexes to 0 °C led to C(sp)-H bond cleavage and formation of dinuclear gold(I) σ,π-acetylide complexes of the form {[(IPr)Au]2(eta1,eta2-C≡CAr)} SbF6, three of which were isolated in 99% yield and one of which was characterized by X-ray crystallography.
A family of 7 cationic gold(I) π-allene complexes were isolated and fully characterized in solution, and in three cases by X-ray crystallography. Degenerate intermolecular allene exchange kinetic studies for three of the allene complexes are in accord with a two-term rate law of the form rate = k1[complex] + k2[complex][allene] with with Gibb's free energy barriers of 17.4 - 18.8 kcal mol-1 (1) and 15.2 - 17.6 kcal mol-1 (2). Variable temperature NMR analysis of these complexes established facile intramolecular -face exchange through 1-allene intermediates or transition states with barriers of 8.9 - 10.9 kcal mol-1 for phosphine and 9.5 - 12.2 kcal mol-1 for IPr complexes.
Mechanistic investigation of gold(I)-catalyzed intramolecular allene hydroalkoxylation established a mechanism involving rapid and reversible C-O bond formation followed by turnover-limiting protodeauration from a mono(gold) vinyl complex. This on-cycle pathway competes with catalyst aggregation and formation of an off-cycle bis(gold) vinyl complex.
Item Open Access Molecular Mechanism of Zipper Interacting Protein Kinase(2011) Chambers, Jenica AnnmarieDiseases caused by smooth muscle dysfunction such as hypertension and asthma are major public health concerns, a better understanding of the signaling pathways that regulate smooth muscle contraction could identify new drug targets. The opposing effects of two enzymes; calcium/calmodulin regulated myosin light chain kinase (MLCK) and smooth muscle myosin phosphatase (SMPP-1M) determine the amount of force generated by smooth muscle. The calcium-independent signaling mediated by myosin phosphatase is regulated by several kinases which include zipper interacting protein kinase (ZIPK). Our laboratory has shown that ZIPK is able to phosphorylate and inhibit SMPP-1M which results in increased smooth muscle contraction. Additional studies demonstrated that ZIPK is also regulated by phosphorylation. The goal of this study is to identify kinases in the context of smooth muscle that regulate ZIPK and to define the events required for ZIPK activation.
A proteomic approach which employed ATP-affinity chromatography coupled with mass spectrometry isolated discreet kinase activities towards ZIPK, these activities were attributed to integrin-linked kinase (ILK) and Rho kinase 1 (ROCK1). ILK phosphorylates ZIPK at Thr180 while ROCK1 phosphorylates ZIPK at Thr265 and Thr299.
Additionally the ATP-affinity media used for kinase enrichment in the proteomic screen was used as a tool to measure ZIPK activation. Pre-incubating ZIPK with ROCK before the assay resulted in increased binding which suggests phosphorylation of ZIPK by ROCK is activating. Increasing the substrate concentration in the assay resulted in increased ZIPK binding, this result was only observed when the assay was performed with the full-length protein. Phosphorylation of residues in the kinase domain along with substrate binding relieves inhibition and results in kinase activation.
Finally fluorescence microscopy along with targeted mutations of ZIPK was used to determine the mechanism of cellular transport. This was done to address the difference in cellular localization between human and murine cells. The localization of human ZIPK is dictated by nuclear localization sequence 2 (NLS2) and the phosphorylation state of Thr299; the mechanism is not shared by the murine form of ZIPK.
Completion of this work has provided additional information about the signaling interactions that take place in smooth muscle; the results suggest that ZIPK is a convergence point for multiple signaling pathways that lead to SMPP-1 inhibition and subsequently smooth muscle contraction. This study also contributes significantly to our knowledge of the molecular dynamics that lead to active full length ZIPK. Future research that employs animal modeling as a tool to investigate ZIPK will be informed by the experiments that address the cellular localization of ZIPK.
Item Open Access Nitrogenase Electron Tunneling Pathways Analysis: From the 4Fe-4S Cluster to the P-cluster(2018) Zhai, YujiaThe biological reduction of N2 to NH3 catalyzed by molybdenum nitrogenase requires eight steps to finish a completed catalysis cycle. This reaction cycle is associated with ATP-driven electron transfer (ET) from the Fe protein to the MoFe protein, and part of ET is experimentally confirmed to be ‘conformationally gated’. Although the overall sequence of ET in nitrogenase has been studied for decades, the nature of coupling between ET pathways and nucleotides binding/protein-protein docking is still unclear, especially from theoretical aspects. Here, we have utilized submicrosecond classical molecular dynamics simulations to allow the ADP-bound and ATP-bound nitrogenases to simulate their conformations in real biological systems. Then the Pathways plugin implemented by Balabin et al was employed to calculate the ET coupling and visualize the ET pathways between the F-cluster and the P-cluster in nitrogenase. The comparison of the ET couplings (the F-cluster to the P-cluster) we calculated and the edge-to-edge distance between the ET donor and acceptor suggests that the coupling pathways grow in strength more that that would be expected from simple distance changes. This result additionally indicates the electron of Fe protein is protected prior to the ATP binding and the protein-protein docking, using pathway switching effects.
Item Open Access Potential consequences of adverse lifestyle factors on decision-making as modeled by the Drosophila melanogaster egg-laying process(2023-04-14) Camacho, SabrinaStudies have shown that lifestyle factors including impaired gut microbiome health, advanced maternal age, and a diet high in sugar may negatively impact cognitive functioning, but their effects on decision-making have not been thoroughly examined. This study aimed to describe the effects of these three factors on decision-making as well as to determine whether the mechanism behind these effects is metabolic or sensory. This was assessed using Drosophila melanogaster egg-laying chamber assays in which Drosophila were given two choices of substrate on which to lay their eggs: sucrose vs. plain or sucrose vs. sucrose. It was found that neither a reduced gut microbiome nor advanced maternal age influenced decision-making. A high-sugar diet resulted in increased sucrose preference. Neither a metabolic nor a peripheral sensory mechanism explained this phenotype, for ingesting just the nutritious element of sucrose nor just peripheral sensing of the sweet element of sucrose was sufficient to increase sucrose preference. An internal sensory mechanism using Gr43A neurons partially accounted for this phenotype, for the lack of internal sensor activity prevented the unfavorable assessment of sweetness, increasing the perceived value of sucrose. It can be concluded that a diet surpassing healthy sugar levels caused adverse changes in decision-making through a combination of metabolic and sensory mechanisms. This study fills the gap in research about whether lifestyle factors affect decision-making in humans and in Drosophila. The results of this study can be a motivator for people to adopt healthier diets and monitor their sugar intake.Item Open Access Understanding Cognition(2015) Steenbergen, Gordon J.Cognitive neuroscience is an interdisciplinary enterprise aimed at explaining cognition and behavior. It appears to be succeeding. What accounts for this apparent explanatory success? According to one prominent philosophical thesis, cognitive neuroscience explains by discovering and describing mechanisms. This "mechanist thesis" is open to at least two interpretations: a strong metaphysical thesis that Carl Craver and David Kaplan defend, and a weaker methodological thesis that William Bechtel defends. I argue that the metaphysical thesis is false and that the methodological thesis is too weak to account for the explanatory promise of cognitive neuroscience. My argument draws support from a representative example of research in this field, namely, the neuroscience of decision-making. The example shows that cognitive neuroscience explains in a variety of ways and that the discovery of mechanisms functions primarily as a way of marshaling evidence in support of the models of cognition that are its principle unit of explanatory significance.
The inadequacy of the mechanist program is symptomatic of an implausible but prominent view of scientific understanding. On this view, scientific understanding consists in an accurate and complete description of certain "objective" explanatory relations, that is, relations that hold independently of facts about human psychology. I trace this view to Carl Hempel's logical empiricist reconceptualization of scientific understanding, which then gets extended in Wesley Salmon's causal-mechanistic approach. I argue that the twin objectivist ideals of accuracy and completeness are neither ends we actually value nor ends we ought to value where scientific understanding is concerned.
The case against objectivism motivates psychologism about understanding, the view that understanding depends on human psychology. I propose and defend a normative psychologistic framework for investigating the nature of understanding in the mind sciences along three empirically-informed dimensions: 1) What are the ends of understanding? 2) What is the nature of the cognitive strategy that we deploy to achieve those ends; and 3) Under what conditions is our deployment of this strategy effective toward achieving those ends? To articulate and defend this view, I build on the work of Elliot Sober to develop a taxonomy of psychologisms about understanding. Epistemological psychologism, a species of naturalism, is the view that justifying claims about understanding requires appealing to what scientists actually do when they seek understanding. Metaphysical psychologism is the view that the truth-makers for claims about understanding include facts about human psychology. I defend both views against objections.
Item Open Access Zinc Oxide Nanostructures: Synthesis, Doping and Growth Mechanism(2013) Cho, JinhyunOver the past decade, the study of zinc oxide (ZnO) II-VI semiconducting nanostructures has been a burgeoning research area because of this material's unique electrical and optical properties. Despite the promise of its characteristics for numerous applications, usage of ZnO in the fabrication of nanoscale devices on a commercial scale remains a challenge because of our lack of knowledge of the underlying physics and chemistry of nanostructures. Sustainable progress in nanowire manufacturing techniques requires that we first undertake basic studies to address these poorly understood underlying concepts before we embark on applied engineering. If these fundamental studies prove successful, then characterization, fabrication, and large-scale integration of nanostructures that use ZnO could be applied to a range of engineering fields. This doctoral dissertation is primarily concerned with the synthesis and doping required for the creation of novel ZnO nanostructures and the growth mechanisms of such structures. Numerous studies have been made of various kinds of ZnO nanostructures. However, no studies have been reported of systematic theoretical modeling that uses both density functional theory and as-synthesized nanostructures to explain the growth mechanisms involved in these devices. First, sulfur-doped ZnO nanostars, synthesized through a hydrothermal method, will be discussed. This section uses ab initio simulations in discussing the synthesis of novel ZnO nanostructures and their proposed growth mechanisms. Moreover, this discussion also addresses the optical properties of ZnO structures that cause sulfur doping to enhance their emission of green light. The next section introduces a novel synthetic methodology to reliably produce well-aligned vertical ZnO nanowire arrays on amorphous substrates. Vertical alignment of nanowires significantly improves the performance of devices like LEDs and solar cells. Because these vertically aligned arrays have historically been made using sapphire substrates that hinder their commercialization, substantial effort has been invested in using ZnO nanocrystal seeds to grow vertically aligned ZnO nanowires on silicon substrates. Well-known synthetic methods, such as zinc acetate dissolved in methanol or zinc acetate combined with sodium hydroxide (or potassium hydroxide), have typically been used in pursuit of this goal without a detailed understanding of the mechanisms of seed creation. The consequence of this lack of knowledge has been inconsistent reproducibility in growing vertically aligned nanowires on silicon substrates. This discussion includes the details of mechanisms that explain the why and how of creation of vertical/misoriented ZnO nanocrystal seeds on silicon substrates. In addition, a preferential c-axis-oriented ZnO nanocrystal seed has been successfully synthesized using a solution composed of ammonium hydroxide (NH4OH) and zinc acetate (Zn(O2CCH3)2). Lastly, the synthesis of sea urchin-like microstructures known as ZnO sea urchins will be introduced. Among the various kinds ZnO structures, the ZnO sea urchin is a integrated structure composed of a 3-D microsphere and 1-D nanowires. Dye-sensitized solar cells (DSSCs) made of ZnO sea urchins have shown a higher power conversion efficiency than planar nanowires. This is because ZnO sea urchins have a higher surface area per unit of volume than planar nanowire arrays. This larger surface area allows larger amounts of dye to access the semiconducting nanowires. We have synthesized the sea urchin structures composed of ZnOxPy microspheres, a mixed of zinc phosphide (Zn3P2) and ZnO phase, encapsulated in an array of ZnO nanowires. Synthesis of these interesting structures was achieved without resorting to the prefabricated 3-D microsphere templates that other groups used in previous studies. This new approach to the synthesis of ZnO sea urchin structures was accomplished by simply adding Zn3P2 powder to the C (graphite) and ZnO source powders in a chemical vapor transport method. The ZnO sea urchin's material properties and growth mechanism will be characterized and discussed in detail.