Browsing by Author "David F. Ollis, Committee Member"

Now showing 1 - 12 of 12

Biochemical and biophysical characterization of compartmentalizing proteases from the hyperthermophilic microorganism Pyrococcus furiosus
(2004-06-10) Chang, Lara Samofal; Steven W. Peretti, Committee Member; David F. Ollis, Committee Member; Todd R. Klaenhammer, Committee Member; Robert M. Kelly, Committee Chair
Proteases catalyze the cleavage of peptide bonds in peptides, polypeptides, and proteins using a hydrolysis reaction. From a biological standpoint, these enzymes are critical for cellular survival, particularly in removal of denatured proteins during stress events or of proteins that have completed their functions. Various proteases play distinct roles in the degradation of proteins, including proteinases that break down proteins and peptidases that break down the resulting oligopeptide products to single residues. The hyperthermophilic versions of proteases are useful for several reasons: they are easier to study because of their relative structural simplicity and, compared to their mesophilic counterparts, they are more stable in harsh conditions such as high heat. The focus of this study was on the biochemical and biophysical characteristics of two multi-subunit compartmentalizing proteases from the hyperthermophilic archaeon Pyrococcus furiosus (T[subscript opt]=100°C). The first protease was an oligopeptidase, PfpI (Pyrococcus furiosus protease I), and the second was a proteinase, called the proteasome. Both proteases are ubiquitous in all domains of life. However, they are theorized to have distinctly different roles within P. furiosus. The proteasome may be one of the primary proteinases, with access to its active sites tightly controlled by ATPase regulators that appear to be dependent on cellular environment. In contrast, the role of PfpI may be degradation of the smaller peptides that result from proteasome and other proteinase action. PfpI is a homo-multimer of 18.8-kDa subunits that assemble into hexameric rings. These rings then stack to form dodocamers and higher forms, with three active sites buried in hindered positions within each ring. Trimer, hexamer, and dodecamer forms were purified separately, with the dodecamer at least three-fold more specifically active than the smaller forms. It was also found that PfpI was only able to cleave oligopeptides up to 17 residues, preferring aromatic residues at the P₁ position. As the substrate length was increased, the cleavage by PfpI became less specific and confined to the C- and N-termini. The precise role of PfpI in P. furiosus still remains to be determined, with a particular need for studies of recombinantly expressed versions. The 20S proteasome, along with a theorized ATP-dependent regulator PAN (proteasome-activating nucleotidase), was investigated from several angles. Both enzymes, including native and recombinant forms, were tested for biochemical and biophysical characteristics as isolated structures and in combination. In particular, the PAN ATPase activity was tested primarily to observe its effects on different forms of the proteasome. Furthermore, both were subjected to targeted cDNA microarray experiments during heat shock of native P. furiosus. The P. furiosus proteasome was the first archaeal form investigated that contains two forms of the beta subunit instead of one. Subsequently, one of the primary focuses of the study was to elucidate the roles of the two (48% identical) beta subunits. Distinct differences in activity, stability, and level of ATPase-based stimulation were observed for the various proteasome forms. These differences were based on the presence or absence of one of the three subunits and the assembly temperature. The beta-2 subunit appeared to be the catalytic center for proteinase activity, while the beta-1 subunit played a stabilizing role. PAN was able to stimulate the native form of the proteasome during degradation of polypeptides but inhibited the native heat-shocked form in the same reactions. It was concluded that PAN, which is highly up-regulated during heat shock, may stimulate the native proteasome form, while the heat-shocked proteasome (containing higher levels of beta-1) may associate with a different set of regulating proteins.
Biochemical, Biophysical and Biotechnological Studies of Class II Xylose Isomerases from Hyperthermophilic Thermotoga Species
(2005-10-15) Epting, Kevin Lee; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member; Amy M. Grunden, Committee Member; Robert M. Kelly, Committee Chair
Xylose isomerase (XI) (D-xylose ketol isomerase, EC 5.3.1.5) is used to convert D-glucose to D-fructose in the production of high fructose corn syrup (HFCS). Here, the biochemical and biophysical properties of xylose isomerases from hyperthermophilic Thermotoga species are examined with regard to their potential for HFCS production at elevated temperatures. The effects of divalent metal cations on structural thermostability and inactivation kinetics of class II XIs from two mesophilic, one thermophilic, and one hyperthermophilic bacteria were examined. The three less thermophilic XIs were stabilized in the presence of Co²⁺ and Mn (and Mg²⁺ to a lesser extent), while the melting temperature of TNXI (T[subscript m]~100 degrees C) showed little significant variation. TNXI's kinetic inactivation was non-first order for all metal cases, and was modeled as a two-step sequential process. Unlike other class II enzymes examined, metals are required for TNXI activity but are not essential for structural thermostability. To determine if xylose isomerases from Thermotoga maritima (TMXI) and Thermotoga neapolitana (TNXI) could be utilized in HFCS production, the enzymes were compared with a commercial class I enzyme from Streptomyces murinus (SMXI) (Sweetzyme T™). While the soluble enzymes exhibited bi-phasic inactivation, the immobilized enzymes were characterized by a first order decay rate. A simple mathematical model was developed which utilizes the soluble enzyme kinetic data and immobilized inactivation rates to calculate productivities as a basis to compare enzymes under different process conditions. The extended N-terminus of class II XIs makes them attractive targets for attaching a carbohydrate-binding domain (CBD) for immobilization. Modifying the length of the N-terminal amino acid insert demonstrated that approximately half of the insert (to about residue 19) could be deleted while retaining activity; removing larger sections or the entire N-terminus caused the enzyme to misfold. A fusion protein (TNXI-CBD) with a thermostable CBD cloned from a hyperthermophilic chitinase (Pyrococcus furiosus 1233) attached to TNXI's N-terminus was created. The ability of the fusion protein to immobilize the enzyme to chitin beads was examined.
Charge Defects in Low Temperature Silicon Nitride/Silicon Interfaces for Application in Computational Clothing and Electronic Textiles
(2002-07-26) Park, Kie Jin; Gregory Parsons, Committee Chair; George W. Roberts, Committee Member; David F. Ollis, Committee Member
The purpose of this research has been to 1) explore materials prepared using plasma enhanced chemical vapor deposition (PECVD) for amorphous silicon thin film transistors (TFTs) fabricated on large area flexible polyimide substrates, and 2) develop new concepts to make smart fabrics for computational clothing using the flexible TFTs. For item 1), silicon nitride films, as gate dielectric of TFTs, were deposited using various processing gases having different NH3/SiH4 gas ratio with constant temperature and various temperature at constant processing gas ratio. It was shown that as NH3/SH4 ratio increases, NH/SiH ratio increases. Apparent leakage current decreased but the flat band voltage was shifted with increasing NH/SiH in the films. It was proposed that the decrease in apparent leakage current with increasing NH/SiH ratio was related to charge screening effect as well as film improved insulating quality. The interface charge and bulk charge densities for SiNx films with different processing gas composition have been calculated. The interface charge density increases with increasing NH/SiH causing flatband shift and increasing total charge density. It was believed that the interface charge is generated by stress build up at Si/SiNx interface and increases with NH/SiH ratio. We found that as substrate temperature increases the NH/SiH ratio remains constant, the apparent leakage current increases and the flat band voltage shifts. Because the net total charge is compensated as substrate temperature changes, charge screening effects were believed to be less important than effects of composition change under the conditions studied. Interface charge density increased also with temperature. This was consistent with flat band voltage shift with temperature. For item 2), amorphous silicon TFTs were formed successfully on large area polyimide substrate using back channel inverted staggered structure and novel masks design. Linear and saturation mobility are 0.026 and 0.059 cm2/V•s, respectively. To address TFTs into clothing, conductive thread contact was investigated. Finally, using novel method, we attempted to form an electronic NOR gate using thin film transistors on polyimide, woven directly into a cotton fabric.
Crosstalk Between Phosphoinositide 3-Kinase and Extracellular Signal-regulated Kinase Pathways in Platelet-derived Growth Factor Receptor-mediated Signaling
(2005-11-25) Wang, Chun-Chao; David F. Ollis, Committee Member; Robert M. Kelly, Committee Member; Jason M. Haugh, Committee Chair
Cells control their behavior by maintaining a balance between signal transduction pathways. The disruption of this balance has been implicated in the evolution of normal cells into cancer cells. People expected to inhibit or activate a specific pathway to achieve the cure of cancer. However, signaling pathways are usually woven together through so-called cross-talk interaction and it is almost impossible to design a drug specifically targeting a pathway. Thus, the fundamental understanding of the intracellular signaling processes is important on medicine and biotechnology. Platelet-derived growth factor (PDGF) is a potent stimulator of cell growth and motility. PDGF-induced phosphoinositide 3-kinase (PI 3-kinase)/Akt and Ras/extracellular signal-regulated kinase (Erk) pathways are thought to play central roles in the regulation of cell survival and proliferation. Crosstalk between PI 3-kinase/Akt and Ras/Erk pathways has provided important hints for how PDGF receptor activation may control cellular function. The apparently contradictory findings among recently studies suggest that the cross-talk mechanisms are not fully understood. In this study, we aim to systematically analyze this signaling network, comprised of Ras/Erk and PI 3-kinase/Akt pathways, stimulated by PDGF. Molecular level control over the signaling network, through genetic and pharmacological intervention, will allow these complex regulatory interaction to be isolated. We assessed the phosphorylation of Akt and Erk as the outputs of PI 3-kinase/Akt and Ras/Erk pathways respectively, and detected DNA synthesis and cell numbers to analyze the effect of these two pathways on the resulting life and death decisions. Accumulating evidence has demonstrated that differences in the strength of stimulus, and the duration and magnitude of activity of signal molecules, determine signaling specificity. We offer a quantitative approach — crosstalk titration — to analyze the effect of our intervention strategies on the signaling network. Our results demonstrated that Ras signals are required for full activation of PI 3-kinase/Akt, consistent with the idea that the PDGF receptors and Ras-GTP modulate PI 3-kinase activity in a cooperative fashion. The relevance of PI 3-kinase for Erk activation is complicated. We observed that PI 3-kinase may play multiple role in regulation of the Erk pathway, both positive and negative, dependent on the magnitude of PDGF and the strength and duration of signaling molecular. The extent to which PI 3-kinase activity influences Ras/Erk signaling is still uncertain. Additionally, we found that PDGF-induced DNA synthesis involved the functions of Ras, PI 3-kinase, and MEK. The relevance of Akt on PDGF-induced DNA synthesis is not fully understood, leading the questions open for further investigation.
Functional Genomic, Microbiological and Biochemical Characterization of Plant Biomass Deconstruction by the Extrememly Thermophilic Bacterium Caldicellulosiruptor saccharolyticus
(2009-11-16) VanFossen, Amy; Jason M. Haugh, Committee Member; Robert M. Kelly, Committee Chair; David F. Ollis, Committee Member; Amy M. Grunden, Committee Member
Functional genomics analysis of metal mobilization by the extremely thermoacidophilic archaeon Metallosphaera sedula
(2010-04-20) Auernik, Kathryne Sherlock; Jason M. Haugh, Committee Member; David F. Ollis, Committee Member; Amy M. Grunden, Committee Member; Robert M. Kelly, Committee Chair
AUERNIK, KATHRYNE SHERLOCK. Functional genomics analysis of metal mobilization by the extremely thermoacidophilic archaeon Metallosphaera sedula. (Under the direction of Dr. Robert Kelly.) Biomining processes recovering base, strategic and precious metals have predominantly utilized mesophilic bacteria, but relatively low yields have impacted wider application of this biotechnology. However, the use of high temperature microorganisms offers great potential to increase metal mobilization rates. Metallosphaera sedula (Mse) is an extremely thermoacidophilic archaeon with bioleaching capabilities, although little is known about the physiology of this microorganism. To better characterize Mse, its genome was sequenced and a whole genome oligonucleotide microarray was constructed for transcriptional response analysis. The physiological and bioenergetic complexities of Mse bioleaching were studied focusing on iron oxidation, sulfur oxidation, and growth modes (heterotrophy, autotrophy, and mixotrophy). The transcriptomes corresponding to each of these elements were examined for clues to the mechanisms by which Mse oxidizes inorganic energy sources (i.e. metal sulfides) and fixes CO2. Quinol/terminal oxidases important for maintaining intracellular pH and contributing to ATP generation via proton pumping were stimulated by different energy sources. The soxABCDDâ€™L genome locus (Msed_0285-Msed_0291) was stimulated in the presence of reduced inorganic sulfur compounds (RISCs) and H2, while the soxNL-CbsABA cluster (Msed_0500-Msed_0504) was induced by Fe(II). Two similar copies of the SoxB/CoxI-like cytochrome oxidase subunit, foxAAâ€™ (Msed_0484/Msed_0485) were implicated in fox cluster oxidation of Fe(II), as well as other energy sources. The doxBCE locus (Msed_2030-Msed2032) did not respond uniformly to either Fe(II) or RISCs, but was up-regulated in the presence of chalcopyrite (CuFeS2). A similar response was also observed for a putative rusticyanin (Msed_0966, rus), thiosulfate: quinone oxidoreductase (Msed_0363/Msed_0364, doxDA), and a putative sulfide:quinone oxidoreductase (Msed_1039, sqr), all three of which are candidates to serve as primary electron acceptors from inorganic substrates. Putative proteins implicated in the generation of reducing equivalents were identified (Dms/Sre-like reductase and Hdr-like reductases). Mixotrophy in Mse was defined as a strong preference for organic carbon combined with concomitant use of multiple inorganic (and organic) energy sources, if available. This growth mode was observed during CuFeS2 bioleaching, with organic carbon most likely obtained via recycling of lysed cell material.
Identification, Characterization, and Physiologic Analysis of Proteolytic Enzymes in Hyperthermophilic Organisms
(2008-12-06) Michel, Joshua Klaus; Robert M. Kelly, Committee Chair; Todd R. Klaenhammer, Committee Member; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member
Capable of growth at or above 80°C, hyperthermophilic organisms encode a myriad of proteolytic enzymes, including a number of homo- and hetero-multimeric complexes. These large hyperthermophilic proteases are often comprised of fewer distinct subunits compared to the less thermophilic bacterial and archaeal homologs; thus they provide an attractive model system for study. Whole genome transcriptional response analysis was used to survey both previously characterized and putative proteases in the hyperthermophilic archaea Pyrococcus furiosus and Sulfolobus solfataricus and hyperthermophilic bacterium Thermotoga maritima. The proteolytic transcriptional response of these three organisms demonstrated a complex synergistic relationship between the ATP-dependent proteases (responsible for initial degradation of proteins) and the ATP-independent proteases that liberate free amino acids from smaller peptides. Additionally, all three proteolytic systems showed up-regulation of protease genes involved in the degradation of misfolded and regulatory proteins during cellular stress response to changes in environmental pH and temperature. To a lesser extent, the ATP-dependent proteases (e.g. Clp) were also involved in the response of T. maritima to increased levels of extracellular acetate; this was accompanied by decreased transcription of metabolic genes and entry into stationary-phase. Thermal stress conditions also affected expression and multi-subunit composition in the P. furiosus proteosome, yielding a more thermostable complex. The P. furiosus genome encodes three proteasome component proteins: one α (PF1571) and two β proteins (β1-PF1404; β2-PF0159), as well as an ATPase (PF0115), referred to as Proteasome-Activating Nucleosidase (PAN). Proteosome assembly and characteristics were found to be highly dependent on the environmental growth conditions. Increased growth temperature (shift from 90 to 105°C) resulted in a 2-fold up-regulation of β1 mRNA within five minutes, suggesting a specific role during thermal stress. Consistent with this data, two-dimensional SDS PAGE revealed that incorporation of the β1 protein relative to β2 into the 20S proteasome (or core particle, CP) increased with increasing temperature for both native and recombinant versions. The recombinant form of PFα+PFβ1+PFβ2 CP assembled at 105°C was found to be more thermostable and have different catalytic rates and substrate specificities, when compared with a recombinant form of PFα+PFβ1+PFβ2 assembled at 90°C or the PFα+PFβ2 version assembled at either 90°C or 105°C. These results indicate that the β1 subunit in the P. furiosus 20S proteasome plays a thermostabilizing role in archaeal proteasome function during thermal stress when polypeptide turnover is essential to cell survival. In contrast to P. furiosus, the hyperthermophilic archaeon Archaeoglobus fulgidus produces a 20S proteasome comprised of two distinct subunits, α (AF0490) and β (AF0481). Combination of A. fulgidus &alpha and P. furiosus β1 and/or β2 yielded hybrid proteasome CPs that display characteristics different then the wild-type enzymes. Notably, A. fulgidus α was found to preferentially assemble with P. furiosus β1, even in the presence of AFα. The A. fulgidus recombinant proteasome exhibited comparable biochemical properties to the P. furiosus complex (α+β2 or &alpha+β1+β2), albeit with a reduced optimal temperature. However, the recombinant A. fulgidus 20S proteasome and hybrid CPs were not substrate-inhibited as was the case for the recombinant P. furiosus 20S proteasome. Taken together, these results demonstrate that proteasomes can be constructed with subunits from different hyperthermophiles, and that subunit composition influences biochemical and biophysical properties. The fact that hybrid inter-generic versions can be created in vitro also suggests that CPs in particular archaea may have arisen from common sources. Furthermore, the ability to interchange subunits and alter composition of the proteasome suggests that this system may provide a useful platform for designing proteases with unique activities or specific biophysical properties required for any biotechnological application.
Intercellular Communication in Hyperthermophilic Microorganisms
(2006-11-28) Johnson, Matthew Robert; David F. Ollis, Committee Member; Jason M. Haugh, Committee Member; James W. Brown, Committee Member; Robert M. Kelly, Committee Chair
In the microbial world it is becoming apparent that many syntrophic, symbiotic and competitive interactions that occur within and between species are driven largely by a form of cell-to-cell communication known as quorum sensing, in which communication within and between species occurs through the use of highly specific signal molecules. In this work, evidence is presented through functional-genomics approaches that cell-to-cell signaling is a phenomenon not limited to mesophilic bacteria. In Thermotoga maritima, a hyperthermophilic bacterium, a previously uncharacterized small peptide (TM0504) that is very highly expressed under syntrophic growth conditions was found to have quorum sensing properties, inducing the cell-density dependent expression of glycosyltransferases to form exopolysaccharides. Exopolysaccharide production enabled the close association of T. maritima to the hyperthermophilic methanogen Methanococcus jannaschii, underlying the synthrophic transfer of hydrogen between species. Upon further examination, it was found that distinct life cycles exist within this syntrophic relationship, with rapid growth and aggregation in the co-culture followed by detachment of the two species in stationary phase. This process is postulated to be driven by an unknown quorum sensing system, allowing the detachment and spread of these organisms into new growth environments. In addition, evidence was provided that showed that Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally near 100°C, both produces and responds to a recognizable form of AI-2, a furanosyl borate diester and known universal autoinducer of quorum sensing in mesophilic bacteria. As P. furiosus and all other members of the Archaea lack the LuxS enzyme involved in AI-2 biosynthesis in mesophilic bacteria, an alternative pathway must be involved. Purification of native AI-2 biosynthetic enzymes from P. furiosus crude cell extracts using a biological reporter assay allowed for the isolation of fractionated cell-free extracts that could convert adenosine to a species that triggered quorum sensing in a reporter strain of Vibrio harveyi. Through the use of the available genome sequence, it was proposed the production pathway for AI-2 involves the phosphorylation of ribose from adenosine through the activity of a eukaryotic-like MTA-phosphorylase (PF0016). In fact, the recombinantly produced MTA phosphorylase could complement fractionated P. furiosus biomass to produce enhanced levels of AI-2 activity from adenosine at 90°C. Other components of the pathway are under investigation, but likely includes a ribose phosphoisomerase (PF1258) to produce phosphorylated ribulose, which can be non-enzymatically converted to (4S)-4,5-dihydroxy-2,3-pentanedione (DPD). A potentially unique contribution of thermal energy in the conversion is proposed as this step is significantly accelerated at hyperthermophilic temperatures over rates observed at mesophilic temperatures, suggesting temperature may have had a role in directing the evolution of cell-to-cell signaling systems. Overall, these results suggest quorum sensing phenomena occurs in hyperthermophilic microorganisms, where it likely plays an important role in regulating intra- and inter-species interactions and defining microbial phenotypes.
Leading Edge Dynamics during Spreading and Migration
(2008-10-20) Krajcovic, Matej; Balaji Rao, Committee Member; David F. Ollis, Committee Member; Jason M. Haugh, Committee Chair
Cell migration is an essential part of wound healing and cancer metastasis. Cells interpret adhesive and soluble stimuli from the environment into directed movement. During cell migration, cells protrude in the direction of motion, forming a leading edge, and retract at the trailing edge. The signaling events at the leading edge are crucial for efficient migration. Cells bind to extracellular matrix (ECM) via integrin receptors. Phosphoinositide 3 kinase (PI3K) is activated in fibroblasts during spreading on fibronectin and poly-lysine. Blocking of integrin receptors by Î²1-integrin antibody inhibits adhesion on fibronectin but not on poly-lysine. PI3K activity is not affected by integrin blocking on poly-lysine. Active Rac, a Rho GTPase, stimulates formation of lamellipodia at the leading edge. We used fluorescent probes for Rac and PI3K in conjunction with total internal fluorescence microscopy (TIRF) to examine spatial distribution of Rac and PI3K. In randomly migrating fibroblasts, Rac and PI3K are co-localized most times in the cell protrusions. The mechanisms governing the formation and disassembly of integrin-ECM adhesion complexes at the protruding leading edge are central to the understanding of cell migration. We have developed a simplified mathematical model that incorporates adhesion and protrusion dynamics mediated by Rac/Pak (p21-activated kinase) signaling at the leading edge. Nascent adhesions either disassemble (turn over) or mature into stable focal adhesions. Stable adhesions can be involved in two distinct pathways of protrusion inhibition. We performed a bifurcation analysis with respect to density of ECM and existence of two possible steady states has been shown. A stochastic version of the model employing the Gillepsie algorithm was developed to model the effect of diffusion in a linear geometry. At certain conditions, multiple states of activation are present within the leading edge.
Life Cycle Assessment of Chemical Processes and Products
(2007-10-28) Li, Yong; Ruben G. Carbonell, Committee Member; Perry L. Grady, Committee Member; Christine S. Grant, Committee Co-Chair; David F. Ollis, Committee Member; Michael R. Overcash, Committee Co-Chair
In this study, environmental assessment of carbon dioxide application in soybean oil and bitumen production has been investigated using life cycle approach. In soybean oil production, the initial life cycle comparison finds that the lab scale CO2 system is not as good in life cycle impacts as the hexane system. However, reasonable engineering improvements of typical scale-up practices will make the CO2 technology better than hexane and eliminate the hexane emissions. Utilization of membrane techniques to separate the small molecular CO2 from the soybean oil hydrocarbon appears to be a much better R&D direction for development. In bitumen production, we find extraction step consumes more energy than other steps in the life cycle of bitumen production. Hot water extraction requires more energy than carbon dioxide extraction because of the heating demand of large water flow in the process. Furthermore, Three carpet products: polyvinyl chloride (PVC) backed tile, styrene butadiene latex (SBL) backed broadloom, and polyurethane (PU) backed broadloom, are studied and compared using a life cycle approach. We find that the supply chains of carpet products play very important roles in the life cycle environmental performance of carpet products. Nylon fibers, as the economically significant component in carpet products, are also environmentally significant in the life cycles of carpet products. Our results show that SBL broadloom has the least global warming impact, HH cancer impact, and HH noncancer impact.
Physiological, Biochemical and Biotechnological Characterization of Glycoside Hydrolases from the Hyperthermophilic Bacterium
(2003-11-19) Chhabra, Swapnil R; Robert M. Kelly, Committee Chair; David F. Ollis, Committee Member; Denns T. Brown, Committee Member; Saad A. Khan, Committee Member
The genome sequence of Thermotoga maritima MSB8, encodes for the highest number of glycoside hydrolase genes amongst hyperthermophilic Bacterial and Archaeal genome sequences reported to date. The ability of T. maritima to utilize the polysaccharides galactomannan and CM cellulose as carbon sources can be attributed at least in part due to the presence of the genes cel5A (TM1751), man5 (TM1227) and cel74 (TM0305). The encoded proteins Tm Man5 and Tm Cel74 are extracellular marked by the presence of N-terminal signal peptides whereas Tm Cel5A is intracellular. Biochemical properties of recombinant versions of Tm Man5 and Tm Cel74, expressed in Escherichia coli, correlated well with predictions made by sequence comparisons. Thus, Tm Man5 was found to be a strict -mannanase while Tm Cel74 was found to be a strict endoglucanase. In contrast, although Tm Cel5A shows sequence similarity to an endoglucanase, its biochemical characteristics point to dual substrate specificity such that Tm Cel5A was found to hydrolyze both -mannan and -glucan polysaccharides. Glu-137 (proton donor) and Glu-253 (nucleophile) were found to be the catalytic residues in Tm Cel5A while Glu-329 was the catalytic nucleophile in Tm Man5. A mutation of these residues in each protein resulted in a complete loss of hydrolytic activity. Currently, Tm Cel74 is the only endoglucanase in Family 74 of glycoside hydrolases that lacks the presence of a cellulose-binding module at its C- terminus. Fusion of a binding module to the C-terminus of Tm Cel74 allowed the chimeric protein to bind and hydrolyze ii microcrystalline cellulose. Gene expression profiles of cel5A and man5 using Northern hybridizations and cDNA microarrays suggested co-regulation during growth on mannose and -1,4 mannan polysaccharides. Overall expression levels of cel74 were several fold lower than the other extracellular endoglucanase gene cel12A (TM1524) during growth on -1,4 glucan polysaccharides. Global gene expression analysis using a targeted cDNA microarray indicated the presence of tight regulatory mechanisms for glycoside hydrolase expression in T. maritima during growth on different carbon sources. Mixed model data analysis revealed co-regulation of genes within potential operons as well as sets of spatially distant gene strings with similar expression profiles, suggesting the presence of regulons in the T. maritima genome. This information in conjunction with biochemical characteristics of encoded proteins, was used to predict pathways for polysaccharide uptake and utilization in T. maritima. The research presented in this work provides a framework for future studies using full genome microarrays of T. maritima and other hyperthermophiles for the identification of glycoside hydrolases with novel sequences and substrate specificities.
Reactions of High-k Gate Dielectrics: Studies in Hafnium, Zirconium, Yttrium, and Lanthanum-based Dielectrics and in-situ Infrared Results for Hafnium Dioxide Atomic Layer Deposition
(2006-05-08) Kelly, Michael Jason; David F. Ollis, Committee Member; Gregory N. Parsons, Committee Chair; H. Henry Lamb, Committee Member; Veena Misra, Committee Member
According to the International Technology Roadmap for Semiconductors (2004) integrating a high dielectric constant (high-k) material into the gate stack will be necessary within the next two years (i.e., by 2007) to maintain the rate of scaling that has come to characterize the microelectronics industry. This work presents results for Y-, Zr-, Hf-, and La-based high-k gate dielectrics prepared by ex-situ oxidation of sputtered thin metal films and for HfO2 prepared by atomic layer deposition (ALD). The kinetics of substrate consumption during formation of yttrium silicate thin films were studied. We find results consistent with high-k dielectric formation by a two-step process in which yttrium metal reacts with the silicon substrate to form a metal silicide which is then oxidized to form the yttrium silicate dielectric. In other experiments, we show flatband voltage shifts of -0.2 and 0.95V in devices containing Zr-based dielectrics formed by oxidation of 8Å of Zr metal on Si at 600°C in N2O for 15 and 300s, respectively. Silicon oxidized in the same environment does not show this shift. The fixed charge scales with EOT for these films and is consistent with charge generation due to disruption of the SiO2 network by metal ions. Zr-based dielectrics exhibit this effect more strongly than Hf-based dielectrics. We show that La-based dielectrics absorb atmospheric H2O and CO2, and that reactions between these materials and deposited silicon electrodes are accelerated when H2O or other OH species are present at the interface. We show that the electrical properties of gate stacks having Ru and RuO2 electrodes in contact with PVD Y-silicate are more stable during thermal anneal than similar gate stacks having PVD ZrO2 or CVD Al2O3 dielectrics. For this work, we configured a Fourier transform infrared spectrometer for in-situ attenuated total reflection measurements and investigated ALD deposition of HfO2. We report the direct reaction of tetrakis(diethylamino) hafnium (TDEAHf) with SiH groups on HF-last Si. Island growth of HfO2 occurs, and SiH features are still present and shrinking after 200 cycles. To the best of our knowledge, these are the first in-situ FTIR results presented for atomic layer deposition using TDEAHf/H2O chemistry.