2-Cys Prx compõem um grupo de enzimas antioxidantes homodiméricas que atuam na decomposição de hidroperóxidos utilizando uma cisteína reativa (cisteína peroxidásica - CysP). A alta reatividade da CysP é alcançada com o envolvimento de dois aminoácidos vicinais à CysP: uma treonina e uma arginina, que constituem a tríade catalítica. Após a decomposição do hidroperóxido, a CysP forma um dissulfeto intermolecular com um segundo resíduo de cisteína (cisteína de resolução - CysR), o qual é reduzido pela tiorredoxina (Trx). Durante o ciclo redox, estas enzimas sofrem alterações estruturais, mas os mecanismos envolvidos neste processo eram pouco compreendidos. Neste trabalho foi obtida a estrutura cristalográfica de Tsa1 de Saccharomyces cerevisiae, uma 2-Cys Prx. Através de abordagens envolvendo bioquímica e biologia molecular, foi verificada a importância de aminoácidos envolvidos na reatividade e em transições da estrutura terciária e quaternária. Por fim, foram realizados esforços para a determinação da estrutura cristalográfica de mutantes obtidos neste trabalho. / 2-Cys Prx constitute a group of homodimeric antioxidant enzymes that act in the decomposition of hydroperoxides using a reactive cysteine (peroxidase cysteine - CysP). The high reactivity of the CysP is achieved by the participation of two vicinal amino acids: a threonine and an arginine, which constitute the catalytic triad. After the decomposition of hydroperoxide, the CysP forms an intermolecular disulfide with a second cysteine residue (resolving cysteine - CysR), which is reduced by the thioredoxin (Trx). During the redox cycle, these enzymes undergo to changes in the structure, but the molecular mechanisms involved in this process were poorly understood. In this study we have obtained the crystallographic structure of the 2-Cys Prx enzyme Tsa1 from Saccharomyces cerevisiae. By means of biochemical and molecular biology approaches, the importance of amino acids involved in reactivity and structural transitions were determined. Finally, efforts have been performed to the determination of the crystallographic structures of mutant proteins obtained in this study.

Electrospray ionization mass spectrometry (ESIMS) is widely used to characterize neutral and ionic species in solvents. Typically, electrical, thermal, and pneumatic potentials are applied to create electrosprays from which charged ionic species are ejected for downstream analysis by mass spectrometry. Most recently, ESIMS has been exploited to investigate ambient proton transfer reactions at air-water interfaces in real time. We assessed the validity of these experiments via complementary laboratory experiments. Specifically, we characterized the products of two reaction scenarios via ESIMS and proton nuclear magnetic resonance (1H-NMR): (i) emulsions of pH-adjusted water and isoprene (C5H8) that were mechanically agitated, and (ii) electrosprays of pH-adjusted water that were collided with gas-phase isoprene. Our experiments unambiguously demonstrate that, while isoprene does not oligomerize in emulsions, it does undergo protonation and oligomerization in electrosprays, both with and without pH-adjusted water, confirming that C-C bonds form along myriad high-energy pathways during electrospray ionization. We also compared our experimental results with some quantum mechanics simulations of isoprene molecules interacting with hydronium at different hydration levels (gas versus liquid phase). In agreement with our experiments, the kinetic barriers to protonation and oligomerization of isoprene were inaccessible under ambient conditions. Rather, the gas-phase chemistries during electrospray ionization drove the oligomerization of isoprene. Therefore, we consider that ESIMS could induce artifacts in interfacial reactions. These findings warrant a reassessment of previous reports on tracking chemistries under ambient conditions at liquid-vapor interfaces via ESIMS. Further, we took some high-speed images of electrosprays where it was possible to observe the main characteristics of the phenomena, i.e. Taylor cone, charge separation, and Coulomb fission. Finally, we took the freedom to speculate on possible mechanisms that take place during electrospray ionization that affected our system and possibly may influence other common analytical techniques on ESIMS.

<p></p><p>Biomass and shale gas have been proposed as alternate sources of liquid hydrocarbon fuels. Traditional petroleum refining, however, is not capable of directly converting either the highly oxygenated molecular structure of lignocellulosic biomass or the low molecular weight alkanes of shale gas into liquid fuels. In this work, we investigate two processes to generate fuels by upgrading low molecular weight species present in biomass pyrolysis vapors and in shale gas via carbon-carbon coupling reactions of low molecular weight species present in biomass pyrolysis vapors and shale gas. </p> <p>In the first process, fast pyrolysis and hydrodeoxygenation are used to convert woody biomass into hydrocarbons. However, 22% of the carbon in this process forms C₁-C₃ species which are unsuitable for use as liquid fuels. Aldol condensation has been proposed as a means of leveraging carbonyl groups present in the pyrolysis product distribution prior to hydrodeoxygenation in order to couple low molecular weight species such as glycolaldehyde to transform the C₁-C₃ fraction into C₄₊ species. We demonstrate that aldol condensation of fast pyrolysis vapors results in a large (10%) reduction in carbon yield to C₆ species and only a small (5%) reduction in carbon yield to C₁-C₃ species to form C₇₊ products, suggesting that higher molecular weight species undergo significant reaction over the aldol condensation catalyst. We demonstrate a pathway by which levoglucosan can be converted into levoglucosenone, which then forms C₇₊ species through self-aldol condensation and condensation with light oxygenates. </p> <p>In the second process, light olefins in shale gas, consisting primarily of ethane and propane, are dehydrogenated and oligomerized into higher molecular weight species. Ni cation sites exchanged onto microporous materials catalyze ethene oligomerization to butenes and heavier oligomers, but also undergo rapid deactivation. The use of mesoporous supports has been reported in the literature to alleviate deactivation in regimes of high ethene pressures and low temperatures that cause capillary condensation of ethene within mesoporous voids. Here, we reproduce prior literature findings on mesoporous Ni-MCM-41 and report that, in sharp contrast, reaction conditions that nominally correspond to ethene capillary condensation in microporous Ni-Beta or Ni-FAU zeolites do not mitigate deactivation, likely because confinement within microporous voids restricts the formation of condensed phases of ethene <a>that are effective at solvating and desorbing heavier intermediates that are precursors to deactivation</a>. Deactivation rates are found to transition from a first-order to a second-order dependence on Ni site density in Ni-FAU zeolites with increasing ethene pressure, suggesting a transition in the dominant deactivation mechanism involving a single Ni site to one involving two Ni sites, reminiscent of the effects of increasing H₂ pressure on changing the kinetic order of deactivation in our prior work on Ni-Beta zeolites.</p><br><p></p>

Evidence has accumulated that the voltage dependent anion channel (VDAC), located on the outer membrane of mitochondria, plays a central role in apoptosis. The involvement of VDAC oligomerization in apoptosis has been suggested in various studies. However, it still remains unknown how exactly VDAC supra-molecular assembly can be regulated in the membrane. Previous studies suggested the possible influence of various proteins on the formation of VDAC oligomers, but the important issue of the VDAC oligomeric state regulation by lipids has not been studied so far. Nevertheless, the effect of lipids on the oligomerization of several membrane proteins has been mentioned in the literature and in general, protein-lipid interactions are under extensive investigation. In the present work, I addressed the influence of lipids on VDAC oligomerization experimentally by reconstituting the fluorescently labelled VDAC in giant unilamellar vesicles (GUVs)—a chemically well defined, cell-free minimal model system. Fluorescence cross-correlation spectroscopy was performed to determine the oligomeric state of VDAC. I investigated the effect of important for apoptosis anionic lipids, phosphatidylglycerol and cardiolipin, on VDAC oligomerization. I demonstrated that phosphatidylglycerol significantly enhances VDAC oligomerization in the membrane, whereas cardiolipin disrupts VDAC oligomers. These results suggest that up- or down- regulation of these lipids in mitochondria during apoptosis can tune VDAC oligomerization in the membrane. Thus, this study sheds light on the role played by the above-mentioned lipids in the regulation of VDAC oligomerization during apoptosis and provides additional information on the molecular mechanisms of the programmed cell death. Another objective of this work was to investigate the partitioning of VDAC into liquid disordered or liquid ordered lipid phases. The existence of lipid domains or the lipid rafts in mitochondria and VDAC enrichment in these rafts is still under debate. Additionally, mitochondrial VDAC was recently found in the plasma membrane. The role of this VDAC is not known, however, it was shown to be located in caveolae (specialized lipid rafts) and play an important role in neuronal apotosis and Alzheimer’s disease. Therefore, VDAC partitioning to the lipid rafts is an interesting question for investigation. The possibility to reconstitute VDAC into minimal model systems–GUVs with phase separation, allowed to reveal the preferential partitioning of VDAC into liquid disordered lipid domain, which suggests either non-raft localization of VDAC or the requirement of the other factors for the recruitment of VDAC into lipid rafts.

<p>Michelle N. Ingle. M.S., Purdue University, December 2019. Computational Modeling of Transforming Growth Factor-β2 Receptor Complex Assembly. Major Professor: David M. Umulis.</p> <p> </p> <p> Transforming growth factor (TGF)-β1, TGF-β2, and TGF-β3 are secreted signaling proteins that play an essential role in tissue development, immune response, and physiological homeostasis. TGF-β ligands signal through a tetrameric complex made up of two type I receptors (TβRI) and two type II receptors (TβRII). Dysregulation of TGF-β signaling has been linked to uncontrolled cell proliferation and cancer metastasis. An accurate understanding of TGF-β’s receptor complex assembly pathway may allow for pharmacological intervention and/or preservation of proper TGF-β signaling.</p> <p> Amongst the ligand types, TGF-β1 and TGF-β3 are efficient signalers, presumably by strong binding to both type I and II receptors. However, TGF-β2 has a very weak affinity for TβRII and requires an additional membrane-bound protein called betaglycan (BG) to achieve similar levels of downstream signaling. While computational modeling has been performed on the signaling pathway of the TGF-β system, to date no computational modeling has aimed to decipher BG’s role in the potentiation of TGF-β2 signal. To determine the role of BG in selectively facilitating signaling by TGF-β2, we developed computational models with different assumptions based on the levels of cooperativity between receptor subtypes and types of BG behavior (No Receptor Recruitment model, Single-stage Receptor Recruitment model, and Two-stage Receptor Recruitment model). </p> <p> With each of the receptor recruitment models we hypothesized that BG uses two domains to successfully enhance TGF-β2 signaling. This model was first proposed in Villarreal et al., 2016 and is further investigated in this work using a two-step computational approach. First, a root mean square error (RMSE) calculation was performed between our computational models with no BG present and published experimental signaling data in cell lines with no BG present. Lower RMSE values indicate the simulated data is more representative of experimental signaling behavior when no BG is present. The second round of model validation was performed by adding BG into the simulations and comparing its behavior to experimentally determined and hypothesized behaviors of BG. </p> <p> In summary, the simulations indicate there may be more cooperative receptor recruitment present in the system then stated in literature. Furthermore, it appears that BG binding to TGF-β2 ligand through two domains provides an effective transfer mechanism that can be tuned to control differential signaling between TGF-β ligand subtypes. Experiments were then suggested in order to support or refute one of the models offered in this thesis. For the purpose of uncovering how BG enhances TGF-β2 signaling, the computational work performed in this thesis highlights the areas where researchers should focus their experimental efforts and provides a baseline model for further computational work in the TGF-β system.</p>

Cleavage and polyadenylation are two necessary messenger RNA (mRNA) maturation steps for gene expression. The Cleavage and Polyadenylation Specificity Factor (CPSF) complex, which recognizes the AAUAAA polyadenylation signal and executes the cleavage reaction, is indispensable for these two processes. In this thesis, I describe my study of the regulation and functions of two non-canonical isoforms of the CPSF subunit WDR33. In addition, I provide detailed analyses on our current knowledge of CPSF subunits’ functions and their influences on a diverse collection of biological processes and conditions. In Chapter1, I provide a general introduction to cleavage and polyadenylation, WDR33, innate immune response via molecular pattern recognition, and the cGAS-STING pathway. Chapter 2 presents my original research on non-canonical WDR33 isoforms, termed WDR33v2 (V2) and WDR33v3 (V3). I determined that their mRNAs are produced by alternative polyadenylation. Both V2 and V3 proteins lack multiple WD repeats, but they can interact with and stabilize each other. This is a novel mode of protein-protein interaction, which I termed WD repeat complementation (WDRC). Unexpectedly, I found that even though V2 and V3 are isoforms of a polyadenylation factor, they are not themselves polyadenylation factors. Regulated by the NF-κB pathway, they are interestingly immune factors involved in the cGAS-STING pathway that induces immune responses against cytosolic double-stranded DNA. V2 decreases STING disulfide oligomerization and suppresses STING-mediated interferon β induction, but facilitates STING-mediated autophagy. Binding of V3 to V2 via WDRC prevents V2’s regulation of STING, suggesting that V3 is a V2 inhibitor. My findings thus further our understanding of STING-mediated immune responses. More broadly, these findings also demonstrate that isoforms produced by alternative mRNA processing can be functionally unrelated. In light of the versatility of the WDR33 gene, I performed a literature review in Chapter 3 on both the canonical and non-canonical functions of CPSF. I first summarize the general functions of CPSF subunits. Subsequently, I discuss their involvements in a variety of biological processes and conditions. This discussion reveals that different processes involve different CPSF subunits. Although CPSF is responsible for only two simple biochemical reactions, it has profound influences on cellular homeostasis. Together, my thesis studies reveal new insights into the molecular mechanism of the cGAS-STING pathway, underscore the importance of alternative mRNA processing, and provide the latest analyses of the functional significances of CPSF.

Biomass-derived hydrocarbons that include gasoline, diesel, and jet fuel will help replace finite fossil fuel hydrocarbons of the same range. This study showed that temperature could be controlled in a scaled-up reactor system using three types of syngas. The CO conversion, selectivity and amount of product created from each type of syngas were examined. Clean syngas composed of 40% H2, 20% CO, 12% CO2, 2% CH4, and 26 % N2 was used to test ideal stoichiometric molar values. Clean syngas composed of 19% H2, 20% CO, 12% CO2, 2% CH4, and 47 % N2 was used to test an ideal contaminateree synthesis gas situation to mimic our particular downdraft gasifier. Gasifier wood syngas composed of 19% H2, 20% CO, 12% CO2, 2% CH4, 46 % N2, and 1% O2 was used in this study to determine the feasibility of using gasified biomass syngas to produce gasolinerange hydrocarbons.

Many zeolites are deployed in various industrial processes owing to their robust catalytic performance and hydrothermal stability. Reactions in zeolites are catalyzed via framework aluminum. The Si/Al ratio is a metric that describes the relative aluminum content in zeolites. However, several researchers noted that the proximity of aluminum in the framework could impact the catalyst output [1–3]. In this work, the influence of paired acid sites is examined in ZSM-22. The 1-dimensional nature of ZSM-22 allows for direct assessment of aluminum proximity without the influence of channel intersection. Theoretical investigations via static density functional theory (DFT) optimization calculations on isolated and paired BAS in ZSM-22 revealed a potential increase in deprotonation potential energy (DPE), indicating a weaker acid with closer aluminum sites. One specific paired model, however, suggested stronger acid behavior, likely due to unfavorable proton-proton interactions influenced by proximity and orientation. Additionally, ammonia adsorption calculations inferred improved adsorption by isolated models, possibly due to unfavorable ammonium-proton interactions in the paired models. Reaction state calculations of ethylene and propylene oligomerization suggested enhanced stabilization of reactant molecules in paired sites. The synthesis of ZSM-22 showed sensitivity to precursor ratios and conditions, but pure samples were successfully achieved through iterative optimization. Catalytic testing of ethylene oligomerization with these samples, classified by their Si/Al ratios and unique fractions of paired acid sites, showed a correlation between higher fractions of paired BAS and increased catalytic activity and selectivity. Samples with higher fractions of paired BAS displayed a higher activity and selectivity for heavier hydrocarbons, explained by the enhanced adsorption capacity of paired BAS for larger reactant molecules, prompting further oligomerization and enhanced catalytic activity. Our findings demonstrate the impact of BAS proximity in dictating the activity and selectivity in ZSM-22 and provide valuable insights for designing more efficient industrial zeolite-based catalysts.

The aim is to produce aliphatic liquid hydrocarbons using heterogeneous ethylene oligomerization. Thiscould potentially produce renewable synthetic fuels. Heterogeneous catalysis has some advantages overhomogeneous catalysis regarding some sustainability aspects. To achieve this, a setup was built using a heatedfixed bed reactor with an in-situ has chromatography to study conversion and gaseous products, and ex-situGC as well as NMR for analyzing liquid products. Ethylene was oligomerized on a commercial ZSM-5 zeoliteunder varying temperature conditions and feed gas dilution with hydrogen or helium. The gas and liquidproducts were analyzed and evaluated. Additionally, the ZSM-5 was studied at different silica to alumina ratios. The thesis discusses how conversion, liquid yield and selectivity of gas products using GC together withanalysis of liquid products using H-NMR can be used as a simple and quick evaluation. The liquid product isevaluated by the distribution of olefinic and aromatic hydrocarbon species using the hydrogen signal area inthe characteristic chemical shifts of olefinic and aromatic hydrogen. At 250-400oC, 6 bar of ethylene, with andwithout feed dilution, and WHSV of 204 h-1, conversion was consistently above 95% for the diluted 400oCruns. Though the liquid yield fell to around 6%, compared to the best yield at 18% for the pure 300oC run.Diluting the feed had a positive effect on increasing olefinic hydrogen signal while decreasing aromatichydrogen signal. The difference between diluting with H2 or helium had a surprisingly small effect. Decreasingthe Si/Al ratio had no significant effect on performance, while increasing the Si/Al ratio made the zeolite loseits catalytic ability. With a pure ethylene feed the lowest aromatic hydrogen signal was found at 350oC, whilethe olefinic signal did not vary too much with temperature. With diluted feed the higher temperature did leadto a lower olefinic hydrogen signal and higher aromatic hydrogen signal.

No description available.