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1
Drawert, Brian J. "Spatial Stochastic Simulation of Biochemical Systems." Thesis, University of California, Santa Barbara, 2013. http://pqdtopen.proquest.com/#viewpdf?dispub=3559784.
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Recent advances in biology have shown that proteins and genes often interact probabilistically. The resulting effects that arise from these stochastic dynamics differ significantly than traditional deterministic formulations, and have biologically significant ramifications. This has led to the development of computational models of the discrete stochastic biochemical pathways found in living organisms. These include spatial stochastic models, where the physical extent of the domain plays an important role; analogous to traditional partial differential equations.
Simulation of spatial stochastic models is a computationally intensive task. We have developed a new algorithm, the Diffusive Finite State Projection (DFSP) method for the efficient and accurate simulation of stochastic spatially inhomogeneous biochemical systems. DFSP makes use of a novel formulation of Finite State Projection (FSP) to simulate diffusion, while reactions are handled by the Stochastic Simulation Algorithm (SSA). Further, we adapt DFSP to three dimensional, unstructured, tetrahedral meshes in inclusion in the mature and widely usable systems biology modeling software URDME, enabling simulation of the complex geometries found in biological systems. Additionally, we extend DFSP with adaptive error control and a highly efficient parallel implementation for the graphics processing units (GPU).
In an effort to understand biological processes that exhibit stochastic dynamics, we have developed a spatial stochastic model of cellular polarization. Specifically we investigate the ability of yeast cells to sense a spatial gradient of mating pheromone and respond by forming a projection in the direction of the mating partner. Our results demonstrates that higher levels of stochastic noise results in increased robustness, giving support to a cellular model where noise and spatial heterogeneity combine to achieve robust biological function. This also highlights the importance of spatial stochastic modeling to reproduce experimental observations.
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Barb, Jessica Gaus. "Biochemical, Genetic, and Cytogenetic Studies of Stokesia laevis (Stokes Aster)." NCSU, 2007. http://www.lib.ncsu.edu/theses/available/etd-11302007-145604/.
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Stokesia laevis (J. Hill) Greene is a herbaceous perennial with blue, lavender, violet, albescent, pale yellow or pale pink flowers. All cultivars are diploid (2n=2x=14) except for ?Omega Skyrocket, a tetraploid (2n=4x=28) cultivar selected from a wild population. Anthocyanidin and copigment aglycones extracted from floral tissue were characterized using high-performance liquid chromatography. Results indicated that blue, lavender, violet and albescent flowers contained petunidin, though albescent flowers contained a substantially smaller amount. Pale pink flowers were found to contain only cyanidin. Anthocyanidins and carotenoids were not present in pale yellow flowers. All flowers contained the flavone luteolin. Genetic analyses suggested that at least three genes (A, P, Y) each with two alleles control flower color: A permits normal synthesis of anthocyanins and other flavonoids, a reduces synthesis and/or prevents the accumulation of anthocyanins and other flavonoids; Y permits normal synthesis of anthocyanins, y completely blocks synthesis of anthocyanins; P produces petunidin, p produces cyanidin. All three genes are completely dominant, and yy is epistatic to A and P. We provide a model for flavonoid biosynthesis in Stokesia. Study of karyotypes and meiotic behavior of diploid cultivars and ?Omega Skyrocket? suggest that ?Omega Skyrocket? is an autotetraploid form of Stokesia. The karyotype of ?Omega Skyrocket? was almost indistinguishable from the average diploid karyotype. Meiotic pairing in diploids was normal (i.e. 100% bivalents); no meiotic irregularities such as laggards/bridges were observed and disjunction was equal (7:7). Meiotic pairing in ?Omega Skyrocket? demonstrated a high frequency (60%) of quadrivalent formation, though later stages of meiosis were regular with balanced disjunction (14:14). Meiosis in synthetic autotetraploids and triploids from crosses of diploid cultivars × ?Omega Skyrocket? included univalents, bivalents, trivalents, quadrivalents, 5-valents; abnormalities (i.e. laggards, unequal and/or premature disjunction, chromosome bridging, chromosome stickiness) were observed. Nuclear 2C DNA content for diploids and synthetic tetraploids was 20.3 pg and 39.9 pg. Nuclear 2C DNA content for ?Omega Skyrocket? was 37.3 pg (i.e. 8.2% less than twice the 2C DNA content of diploids), indicating that genomic downsizing has likely occurred in this cultivar. Differences in chromosome symmetry between the diploid and tetraploid karyotypes and the reduction in nuclear DNA content observed in ?Omega Skyrocket? both suggest that some divergence has occurred between ?Omega Skyrocket? and its diploid progenitor. A crossability study was conducted to determine the ploidy level and the frequency of progeny produced by interploid and intraploid crosses of Stokesia. A high percentage (70%) of progeny were aneuploids (i.e. 2x-1 to 6x+3) with the total percentage of aneuploids ranging from 92% to 94% in 2x × 3x, 3x × 2x, 3x × 3x, 3x × 4x and 4x × 3x crosses. Progeny (94%) from 2x × 2x crosses were diploids, and progeny (81%) from 2x × 4x and 4x × 2x crosses were triploids and 3x±1 aneuploids. Progeny from crosses of synthetic tetraploids were mostly tetraploids (16%) and tetraploid aneuploids (69%). Unreduced gamete production was estimated to be 0.7% to 1.4%. Reciprocal crosses of identical 2x and 4x parents produced viable progeny, demonstrating that a triploid block is not present in this species. Triploid fertility was higher than expected; crosses using triploids produced seed 38% of the time with an average seed set of ~2 seeds/inflorescence. Fertility of synthetic tetraploids was reduced relative to their progenitor diploids; pollen viability was reduced by 36% and the percentage of inflorescences producing seed and average seed set/inflorescence were reduced by ~50%. Pollen size was positively correlated with ploidy level (i.e. DNA content).
3
Edwards, Lorraine Katy. "Biochemical characterization of mammalian high mobility group protein A2." FIU Digital Commons, 2006. http://digitalcommons.fiu.edu/etd/3118.
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The high mobility group protein HMGA2 is an architectural transcription factor, which is expressed during embryogenesis. Aberrant expression causes benign and malignant tumor formation. The protein possesses three "AT hook" domains and an acidic Cterminal. HMGA2 is natively unstructured, however it forms a homodimer. In this study site-directed mutagenesis was used to create single methionine mutants, HMGA2Q37M, HMGA2I71M and HMGA2Q85M. These mutants were cross-linked using EDC and then cleaved using CNBr to determine which domains are involved in homodimer formation. Our results indicate that the second "AT hook" domain may interact with the C-terminal. We then labeled a peptide containing the C-terminal (CTP) with tetramethylrhodamine-5- maleimide (TRM). We found that the CTP-TMR binds to HMGA2Α95-108, which lacks the C-terminal. These results suggest that the C-terminal is required for homodimer formation. The techniques used within this study can be applied to forensics and with further research HMGA2 may have a forensic application.
4
Mistry, Dharmit. "Mechanistic studies of some chemical and biochemical reactions." Thesis, University of Huddersfield, 2014. http://eprints.hud.ac.uk/id/eprint/23444/.
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Three aspects of chemical and biochemical reactions were investigated. 1. The relative reactivities of pyrophosphate (phosphorus(V)) and pyro-di-H-phosphonate (phosphorus(III)) and its derivatives have been analysed at various pHs. The hydrolysis rate of pyro-di-H-phosphonate (PP(III)) was found to be higher than pyrophosphate at all pHs. Using ITC and NMR, pyrophosphate showed metal-ion complexing abilities whereas pyro-di-H-phosphonate showed weak or no complexing to metal-ions, although the rate of hydrolysis at pH 7 slightly increased compared to the spontaneous hydrolysis of PP(III). The enzymatic hydrolysis of pyrophosphate, which is thought to occur via MgPP(V)2-, occurs efficiently and is close to being diffusion controlled. Pyro-di-H-phosphonate on the other hand does not act as a substrate or as an inhibitor of pyrophosphatase. 2. Dichloromethane (DCM) is an alkylating agent for pyridine, producing methylene bis-pyridinium dication (MDP) upon refluxing the solution. The kinetics and mechanism of hydrolysis of methylene bis-pyridinium dication have been studied. Below pH 7 MDP is extremely stable and hydrolysis is first-order in hydroxide-ion. Above pH 9 an unusual intermediate is formed on hydrolysis which has a chromophore at 366 nm in water and its formation is second-order in hydroxide-ion. The carbon acidity of the central methylene group was also investigated kinetically using H/D exchange and the pKa was surprisingly high at 21.2 at 25oC (I = 1.0 M). 3. Isothermal titration calorimetry (ITC) is a technique mainly used by biochemists to obtain a range of physical and thermodynamic properties of a reaction. Analysing the data can become difficult when investigating complex reactions involving more than one step, for instance metal-ions binding to an enzyme. In this work models have been developed to simulate sequential reactions. These were used to simulate experimental ITC data for metal-ions: Zn2+, Co2+ and Cd2+ complexing to the active sites of BcII, a metallo β-lactamase responsible for antibiotic resistance, providing additional information on the mechanism by which this enzyme acts to deactivate β-lactam antibiotics. The simulations suggest that BcII has two very similar binding affinities to metal-ions which are filled sequentially.
5
Hart, Jaynee E. "Biochemical and genetic approaches to modulate phototropin photoreceptor sensitivity." Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/30991/.
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The necessity of light for plants to sustain their autotrophic lifestyle has made the optimization of growth to maximize light capture a crucial strategy for survival in light-limiting environments. Increases in light capture can be achieved through alterations in plant architecture, such as modifications to leaf position and stem length. Responses to the light environment are mediated by a network of photoreceptor proteins, which sense specific wavelengths of light and respond to light excitation by initiating signaling. Higher plants respond to red and far-red light through the phytochrome family, blue light through cryptochromes, the zeitlupe family, and phototropins, and UV-B light through the UV RESISTANCE LOCUS 8 photoreceptor. Of these photoreceptor proteins, the phototropins (phots) are perhaps the most closely tied to photosynthetic efficiency. Higher plant phots, phot1 and phot2, mediate leaf expansion to maximize the surface area available for light capture as well as control movement and positioning responses, such as petiole inclination, movement towards more favorable light conditions through phototropism, and, at a cellular level, chloroplast movement. Furthering the role of phots in optimizing responses upstream of photosynthesis, phot1 and phot2 also control stomatal opening in response to blue light, allowing the uptake of carbon dioxide into the leaf for fixation into sugars. In general, these responses are redundantly coordinated by both phot1 and phot2, with phot1 acting as the primary sensor due to its greater sensitivity. Because of the profound effect phots have on photosynthetic competence, the studies presented here examine phot1 with the goal of understanding the physiological role of phot1 sensitivity in plants and explore the possibility that enhancing phot1 sensitivity could increase plant growth. Phots consist of two N-terminal light sensing LOV (Light, Oxygen or Voltage) domains, LOV1 and LOV2, coupled to a serine/threonine kinase domain at the C-terminus. Each of the LOV domains bind a flavin mononucleotide (FMN) chromophore that allows these domains to perceive blue light. In darkness, FMN is non-covalently bound within each of the LOV domains, which repress the activity of the kinase domain. When FMN is excited by blue light, a covalent bond is formed between a conserved cysteine residue present within each LOV domain and FMN. LOV2 specifically is coupled to the kinase domain through two alpha helices, Jα and A’α, which become disordered following the formation of the covalent photoadduct. The unfolding of these alpha helices relieves repression of the kinase domain, initiating signaling. The onset of phot1 signaling is characterized by phot1 autophosphorylation and the dephosphorylation of the phot1 signaling partner NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3). Over time, the covalent photoadduct decays and phot1 returns to its inactive dark state, completing the photocycle. The chemistry of the phot1 photocycle in vitro is understood in detail, but its downstream signaling following activation remains relatively elusive, with only a handful of signaling partners and phosphorylation substrates identified. For the sensitivity of phot1 to be thoroughly explored, how the phot1 photocycle affects plant growth as well as how phot1 activity is modulated by signaling partners needed to be addressed. Therefore, a biochemical approach was used to introduce mutations within LOV2 to slow its dark reversion to prolong signaling and investigate how this modulates phot1 sensitivity in vitro and in planta, and, secondly, a genetic strategy was employed to uncover whether any signaling processes can modulate phot1 sensitivity in plants. Compared to other photoreceptors that receive blue light through LOV domains, dark reversion of phot1 following a light stimulus is relatively fast, with the lit state lasting only approximately 15 minutes, while other LOV domains remain activated for many hours. To generate slow photocycle mutants of phot1, previous characterizations of slow photocycling LOV domains were exploited to engineer the phot1 photocycle to have a slower dark reversion by introducing mutations into LOV2. To study the photocycle in vitro, the phot1 light-sensing module consisting of the LOV1 and LOV2 domains (LOV1+LOV2) was heterologously expressed and purified from E. coli and the photocycle was measured spectrophotometrically. Using this approach, 13 LOV2 variants were generated and examined to identify slow photocycle mutants. Three mutations in LOV2, N476L, V478I, and L558I, were found to slow the LOV1+LOV2 photocycle in vitro. Following identification, these mutations were introduced into full-length phot1 expressed heterologously in insect cells to verify the autophosphorylation activity of each mutant. Following the characterization of the candidate slow photocycle mutants in vitro, each phot1 photocycle mutant was examined in planta in a phot1phot2 double mutant background to see whether possession of a slow photocycle increased phot1 sensitivity. Of the three candidate mutations, V478I and L558I were verified as possessing a slow dark reversion through the phosphorylation status of NPH3. NPH3 is dephosphorylated in a phot1-dependent manner following light treatment; it was found that in the presence of wild-type phot1, the phosphorylated form of NPH3 is recovered around one hour following a return to darkness after phot1 stimulation by blue light. By contrast, the dephosphorylated state of NPH3 was sustained in phot1-V478I and -L558I for a substantially longer period of time, consistent with a slow phot1 photocycle and prolonged phot1 activation in these mutants. Surprisingly, it was found that these mutants were less sensitive than wild-type phot1 for phototropism in response to low intensity light treatments. Furthermore, biomass accumulation was not increased in the phot1-L558I mutant under growth conditions consisting of very low light. While the photocycle mutants did not exhibit increased sensitivity or growth in response to continuous light treatments, evidence from collaborators indicated that phot1-L558I is more efficient than wild-type phot1 for the chloroplast accumulation response following brief pulses of blue light. While the role of the phot1 photocycle under continuous irradiation remained unclear, this enhanced chloroplast accumulation response implies that the phot1 photocycle is important for its sensitivity to brief irradiations. Unlike phot1, further work with phot2 later indicated that introducing a slow photocycle mutation to phot2 LOV2 can significantly increase growth in a phot1phot2 mutant background under continuous low light. To investigate other factors that may affect phot1 sensitivity, a genetic screen was undertaken in an attempt to identify suppressors of phot1 activity. The LOV2Kinase (L2K) transgenic line, which expresses a truncated version of phot1 in a phot1phot2 double mutant background, was previously found to be unable to respond to low-intensity blue light, though it can mediate phot1 responses when the light intensity is increased. Because L2K possesses this conditional phenotype, random mutations were introduced into the genome of L2K-expressing plants and a screen was established to identify mutants that were able to respond to low-intensity light with the hypothesis that those mutations could lie within suppressors of phot1 activity, allowing L2K to signal under circumstances where it ordinarily could not. Using this approach, three independent candidate suppressor mutants were identified that had increased sensitivity for the petiole positioning response under low light. One suppressor mutant was identified as a novel allele of the phytochrome B red light receptor, the second is likely to be a mutant of a transcription factor, and the identity of the third candidate suppressor is still not known, though it overexpressed the L2K protein. These candidate suppressors may represent novel modulators of phot1 activity and possible mechanisms for how these candidate suppressors may act on phot1 activity are discussed. In summary, both the biochemical and genetic approaches yielded mutants with increased sensitivity for phot1-mediated responses, enabling a more detailed understanding of how phot1 sensitivity influences its activity and plant growth.This lays the groundwork for extending the increased sensitivity observed in response to pulses in the photocycle mutants to responses other phot1-mediated responses, and for integrating new models of suppression of phot1 activity into our framework for phot1 activation and signaling.
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Khartabil, Rana. "User-centered design and evaluation of a dynamic biochemical pathway visualization tool." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/26944.
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Information visualization is a field of computer science that deals with the computerized visualization of complex information in a form that is easier for human beings to comprehend. Information visualization has applications in many domains, including business, science, and medicine. Visualization of biochemical pathways, as graphs of nodes representing biochemical entities and arcs representing the relationships between entities, is one such application.
This thesis begins by reviewing work that has been done on the usability of information visualization techniques, and in particular these that apply to biochemical pathways. Then, the thesis presents three different usability evaluation techniques that are used to gather information about existing biochemical pathway visualization tools. These are (1) conducting videotaped evaluation sessions of existing biochemical visualization tools, (2) collecting questionnaires, and (3) conducting a brainstorming session. The results from these studies are used to define the requirements, design, and build a biochemical pathway visualization tool, taking into account conclusions drawn from both literature and user studies. The tool is then tested and compared to existing tools.
Results show that the developed tool has more relevant features to biochemical pathway visualization than existing tools, accomplishes certain tasks faster than other tools, and is intuitive and easy to use. In addition, positive feedback from users is documented.
At the end of the thesis, we make some generalizations to the area of information visualization and we then present areas for further research.
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Woo, Sung Sik Ph D. Massachusetts Institute of Technology. "Fast simulation of stochastic biochemical reaction networks on cytomorphic chips." Thesis, Massachusetts Institute of Technology, 2016. http://hdl.handle.net/1721.1/107292.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2016.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 169-181).
The large-scale simulation of biochemical reaction networks in cells is important in pathway discovery in medicine, in analyzing complex cell function in systems biology, and in the design of synthetic biological circuits in living cells. However, cells can undergo many trillions of reactions over just an hour with multi-scale interacting feedback loops that manifest complex dynamics; their pathways exhibit non-modular behavior or loading; they exhibit high levels of stochasticity (noise) that require ex- pensive Gillespie algorithms and random-number generation for accurate simulations; and, they routinely operate with nonlinear statics and dynamics. Hence, such simulations are extremely computationally intensive and have remained an important bottleneck in computational biology over decades. By exploiting common mathematical laws between electronics and chemistry, this thesis demonstrates that digitally programmable analog integrated-circuit 'cytomorphic' chips can efficiently run stochastic simulations of complex molecular reaction networks in cells. In a proof-of-concept demonstration, we show that 0.35 [mu]m BiC- MOS cytomorphic gene and protein chips that interact via molecular data packets with FPGAs (Field Programmable Gate Arrays) to simulate networks involving up to 1,400 biochemical reactions can achieve a 700x speedup over COPASI, an efficient bio- chemical network simulator. They can also achieve a 30,000x speedup over MATLAB. The cytomorphic chips operate over five orders of magnitude of input concentration; they enable low-copy-number stochastic simulations by amplifying analog thermal noise that is consistent with Gillespie simulations; they represent non-modular load- ing effects and complex dynamics; and, they simulate zeroth, first, and second-order linear and nonlinear gene-protein networks with arbitrary parameters and network connectivity that can be flexibly digitally programmed. We demonstrate successful stochastic simulation of a p53 cancer pathway and glycolytic oscillations that are consistent with results obtained from conventional digital computer simulations, which are based on experimental data. We show that unlike conventional digital solutions, an increase in network scale or molecular population size does not compromise the simulation speed and accuracy of our completely parallel cytomorphic system. Thus, commonly used circuit improvements to future chips in our digital-to-analog converters, noise generators, and biasing circuits can enable further orders of magnitude of speedup, estimated to be a million fold for large-scale networks.
by Sung Sik Woo.
Ph. D.
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Pérez, Verona Isabel Cristina. "Approaches for the exact reduction of large-scale biochemical models." Thesis, IMT Alti Studi Lucca, 2020. http://e-theses.imtlucca.it/303/1/P%C3%A9rezVerona_phdthesis.pdf.
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Mathematical models are a fundamental tool used in many
branches of science and engineering to gain insights into the
dynamics of systems. In Systems Biology [122], these models
facilitate the analysis of complex biochemical networks that
describe molecular interactions at different level in living organisms.
In the last years, several efforts have been dedicated
to the characterization of biological systems [98, 127, 128, 165,
209], specific interaction mechanisms [10, 15, 44, 45] and biological
phenomena such as oscillations and bistability [80,
119, 147, 175]. As result, the use of mathematical models of
biological processes have shown to be an useful asset in the
development of several medical applications including drug
design and target therapy [55, 110, 179, 197].
There are two main problems to solve when working with
these types of models. The first one is the typically large computational
cost required for their analysis, which is giving in
part to the large number of configurations in which components
such as proteins, or genes are present in the living cell.
The second problem is related to the difficulty in calibrating
large models, since they are generally associated with many
parameters whose experimental estimation in living cells is
notoriously difficult to make.
In this dissertation we explore several approaches for the reduction
of biological systems, paying special attention to the
interpretability of the aggregated system. In general, we are
interested in techniques that produce an exact reduction, i.e.,
algorithms that given an input chemical network, produce a
smaller network (consisting of fewer species and reactions)
that preserves the output dynamics of interest to the modeler,
e.g. [137, 201].
We present a framework for the automatic analysis of largescale
quantitative repositories of biological models, with special
support for models written in the well-known SBML [103]
specifications. For networks with stochastic dynamics, we
provide equivalences at the level of the Markov chain that
can be applied to several models including biological networks
and epidemic processes on complex networks. In addition,
we approach the simplification of biochemical models
by focusing on their steady-state behaviour [2], a stable condition
attained by the biological system when the influence
of the initial condition can be disregarded. Here, we discuss
a method for the computation of equilibrium points, with the
guarantee of yielding the unique equilibrium of the ODE under
defined graph-theoretical conditions.
Overall, this dissertation provides a detailed analysis of techniques
for the reduction of quantitative models of biochemical
reaction networks, together with the interpretation of the
biological and functional characteristics of the reductions obtained
over a wide collection of case studies from the literature.
Interestingly, the inspection of the obtained reductions
has revealed reducible motifs, i.e., structures embedded in the
structure of the network that are often compressed in the models
analyzed. These findings provide the intuition that these
techniques can abstract beyond the behavior of the system to
capture structural/functional segments of the network that
can be simplified with no (or little) effect in the dynamics of
the model.
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Santra, Tapesh. "Evolutionarily stable and fragile modules of yeast biochemical network." Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2644/.
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Gene and protein interaction networks have evolved to precisely specify cell fates and functions. Here, we analyse whether the architecture of these networks affects evolvability. We find evidence to suggest that in yeast these networks are mainly acyclic, and that evolutionary changes in these parts do not affect their global dynamic properties. In contrast, feedback loops strongly influence dynamic behaviour and are often evolutionarily conserved. Feedback loops are often found to reside in a clustered manner by means of coupling and nesting with each other in the molecular interaction network of yeast. In these clusters some feedback mechanisms are biologically vital for the operation of the module and some provide auxiliary functional assistance. We find that the biologically vital feedback mechanisms are highly conserved in both transcription regulation and protein interaction network of yeast. In particular, long feedback loops and oscillating modules in protein interaction networks are found to be biologically vital and hence highly conserved. These data suggest that biochemical networks evolve differentially depending on their structure with acyclic parts being permissive to evolution while cyclic parts tend to be conserved.
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Royle, Christopher. "Physiological and biochemical responses to frequent milking in dairy cows." Thesis, University of Nottingham, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385209.
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Irvine, Darrell J. (Darrell John) 1973. "Spatially controlled presentation of biochemical ligands on biomaterial surfaces using comb polymers." Thesis, Massachusetts Institute of Technology, 2000. http://hdl.handle.net/1721.1/109636.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2000.Vita.
Includes bibliographical references (p. 243-257).
by Darrell J. Irvine.
Ph.D.
12
Widdowson, Philip. "Biochemical and biophysical characterisation of Anopheles gambiae NADPH-cytochrome P450 reductase." Thesis, University of Liverpool, 2010. http://livrepository.liverpool.ac.uk/1496/.
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As the principal vector for the transmission of the Plasmodium falciparum parasite, and hence the spread of malaria in Sub-Saharan Africa, Anopheles gambiae is a globally significant species of mosquito. Over recent years, the efficacy of established insecticides has waned and there is a constant need for novel effective compounds. Cytochrome P450 reductase (CPR) is a diflavoprotein known to have a central role in phase I metabolism of xenobiotic compounds and, in mosquitoes, this involves the detoxification of insecticides. Due to an inherent lack of understanding regarding the mechanisms of action of A. gambiae CPR, the selective inhibition of this enzyme is a previously untried approach. This project aims to biochemically characterise A. gambiae CPR in direct comparison to the human enzyme. It was found that A. gambiae CPR was deficient in bound FMN, and to a lesser extent FAD, relative to human CPR with 20 % less FMN bound to the purified mosquito protein. Following the dissection of A. gambiae CPR into its constituent FMN- and FAD-binding domains, and using Isothermal Titration Calorimetry (ITC), a 4-fold decreased was observed for the binding affinity for FMN in the A. gambiae FMN-binding domain as compared to the equivalent human protein. The redox potential of the oxidised/semiquinone transition of the A. gambiae FMN-binding domain was -92 mV, much more negative than the published value for human FMN-binding domain. These data suggest a clear difference between these enzymes in the binding strength of FMN and its propensity to accept electrons. The binding characteristics of NADPH nucleotides were probed in some detail. Comparison of the binding of NAD+ and NADP+ revealed a strong bias for the phosphate containing NADP+. In addition, the position of the phosphate was important as 3’-AMP bound very poorly whilst 2’-AMP bound more strongly. 2’, 5’-ADP binding highlighted the importance of additional stabilising interactions involving the 5’-phosphate. Comparison of 2’, 5’-ADP and NADP+ binding confirmed that the 2’-phosphate interaction was the principal site for NADPH recognition and provided the majority of the binding energy for this interaction. A. gambiae CPR was shown to bind NADPH nucleotide analogues 2’-AMP, 2’, 5’-ADP and NADP+ much less strongly than the human enzyme highlighting a potentially significant difference in coenzyme binding. Binding affinities for the nucleotide ligand to intact CPR and the isolated FAD domain showed that the FAD-binding site is fully contained within the FAD-binding domain However, differences in the thermodynamic parameters between the intact enzyme and the isolated FAD-binding domain suggest that, although not directly involved in NADPH binding, the presence of the FMN binding domain had an effect on the overall binding energetics. Despite an apparent difference between A. gambiae and human CPR in flavin incorporation and NADPH binding affinity, it was interesting that the activity of cytochrome c reduction of both enzymes was similar. The measured Km with respect to NADPH corroborated the ITC data by suggesting a stronger interaction of the coenzyme with human CPR compared to A. gambiae CPR. There was an approximate 2-fold increase in potassium ferricyanide reduction with the isolated A. gambiae FAD-binding domain compared to the intact enzyme with the presence of the FMN-binding domain again seemingly imparting an effect of events involving the FAD-binding domain. In order to fully understand and rationalise all of the data, a comprehensive structural determination of A. gambiae CPR is required. With this in mind, isotopic labelling and subsequent biophysical analysis was carried out on the intact CPR and its FMN- and FAD-binding domains. Successful labelling was achieved for all samples, including the deuteration of the intact CPR and FAD-binding domain However, the greatest success involved the FMN-binding domain with sufficient triple resonance spectra collected for backbone assignments. Although this success could not be matched for the intact CPR and FAD-binding domain, the work has provided a solid base for more a comprehensive study in the future.
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Hocking, Damien. "Object-oriented simulation of chemical and biochemical processes /." Title page and contents only, 1997. http://web4.library.adelaide.edu.au/theses/09PH/09phh6847.pdf.
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Klejnot, Marta. "Structural and biochemical insights into members of the kinesin and ubiquitin ligase families." Thesis, University of Glasgow, 2013. http://theses.gla.ac.uk/5108/.
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The thesis focuses on characterisation of two different protein families, kinesins and ubiquitin ligases, that are involved in different biological processes. Kinesins constitute a superfamily of microtubule-based motor proteins, fulfilling important cellular functions, ranging from intracellular transport to cell division. They also play a role in primary cilia and Hedgehog signalling. Ubiquitin ligases are enzymes that catalyse the transfer of ubiquitin to the lysine residues of the substrate. Marking protein substrates with ubiquitin alters their functions and fates. With nearly 700 different ubiquitin ligases in humans, they control a vast array of cellular processes. The first part of this thesis summarises results relating to kinesins. Protocols for creation of a panel of kinesin motor domains, a useful tool for anti-mitotic inhibitors specificity testing are presented. Furthermore, biochemical, kinetic and structural features of a poorly described kinesin motor protein - Kif15 are reported. Kif15's motor domain structure is described and compared to Eg5's catalytic core. Moreover, the influence of Eg5 inhibitors on Kif15 ATPase activity is investigated. Scouting for small molecules targeting Kif15 is also performed. Kif15 interaction with microtubules in various nucleotide-bound states is characterised. The possibility of a secondary microtubule binding site in the tail of Kif15 is examined. The binding of Kif15 with partner proteins is also investigated. Additionally the high-resolution structure of the human Kif7 motor domain is presented, providing the first step towards structural characterisation of this Kinesin-4 family member. The second part of this thesis concerns a ubiquitin ligase, Trim28. Trim28 was first reported as a transcription corepressor, working by recruiting proteins that drive the heterochromatin state, whilst its mechanism of action as a ubiquitin ligase remains elusive. Attempts to crystallise and determine the three-dimensional structure of Trim28 are described. Additionally, Trim28 functioning as an E3 ubiquitin ligase, and its interactions with E2s, KRAB domains of various zinc finger proteins and with the Mage-C2 protein are investigated. The results provide the foundation for future studies on Trim28.
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Mayer, Matthias. "A structural and biochemical comparison of wild-type and recombinant propanediol utilising bacterial microcompartments." Thesis, University of Kent, 2016. https://kar.kent.ac.uk/54756/.
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Bacterial microcompartments (BMCs) likely represent the largest protein complex found in bacterial cells (estimated 18,000 subunits). BMCs are associated with specific metabolic processes such as carbon fixation in the case of carboxysomes or carbon utilisation in the case of the 1,2-propanediol utilisation metabolosome. With all BMCs there is still much to learn about the structure and function of these supramolecular assemblies. In this project the structure and function of the Pdu BMC has been studied through a combination of recombinant DNA technology, proteomics, fluorescence microscopy, transmission electron microscopy (TEM) and atomic force microscopy (AFM). Initially, wild type Citrobacter freundii (C. freundii) Pdu BMCs were compared to recombinant BMCs that had been produced in Escherichia coli (E. coli). These were found to have very similar profiles in terms of BMC shell composition, size (120-130 nm on average) and shape. However, recombinant empty BMCs (eBMCs), generated by the coexpression of the genes for just the shell proteins, were found to be distinctly different in that they incorporated more of the first shell protein of the operon, PduA, and were also significantly smaller (55-80 nm on average) than the complete BMCs. This work provides the first recorded application of AFM for the study of BMCs and revealed that the eBMCs were stiffer and less flexible than complete BMCs and it is unclear if differences in shell protein composition or the lack of the internal scaffold is causing the reported biophysical differences. One key property of the BMCs is their ability to absorb substrate and cofactor molecules to allow the internalised metabolic pathway to operate. Adenosylcobalamin (Ado-B12) is required as a coenzyme for the diol dehydratase conversion of 1,2-propanediol (1,2-PD), but the uptake of cobalamin into the BMC was not shown. By feeding cells exogenous cobalamin, it was possible to demonstrate that cobalamin is specifically partitioned from the cytoplasm into the BMC. Moreover the synthesis of fluorescently labelled versions of cobalamin allowed this partitioning to be followed ex-vivo with purified BMCs.
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Laine, Marjo L. "Functional, biochemical and structural analyses of Plasmodium falciparum pyruvate dehydrogenase complex." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5043/.
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The apicomplexan parasite Plasmodium is the causative agent of the devastating tropical disease, malaria. The World Health Organisation reported that in 2010 there were an estimated 219 million malaria cases and about 660,000 deaths. Sub-Saharan Africa is the worst affected, with 80% of malaria deaths and 90% of cases occurring in this area of the world. P. falciparum causes the most severe form of the illness and accounts for the majority of malaria deaths. Parasite resistance to antimalarials including the most effective drug, artemisinin, is becoming an increasing problem, thus research into new drug targets is vital. The pyruvate dehydrogenase complex (PDC) is one of the alpha-ketoacid dehydrogenase complexes, which are involved in energy and amino acid metabolism. The PDC catalyses the transfer of the acetyl group from pyruvate to coenzyme A (CoA) to form acetyl-CoA. The complex comprises three enzymes; pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and dihydrolipoamide dehydrogenase (E3). E2 has a multi-domain structure where the C-terminal catalytic domains (CD) of several E2 subunits interact to form the core of the complex. This core is an icosahedral 60-mer in mammals, plants and Gram-positive bacteria and an octahedral 24-mer in Gram-negative bacteria. The N- terminal part of E2 consists of the sub-unit binding domain (SBD) and 1 to 3 lipoyl- domains (LD) creating the ‘swinging arm’ of the PDC, which facilitates substrate channelling. E1 (a heterotetramer) and E3 (a homodimer) bind to the SBD to form the functional PDC. In humans, PDC converts pyruvate to acetyl-CoA, which leads into the citric acid cycle located in the mitochondrion. P. falciparum PDC, however, is found only in the apicoplast and produces acetyl-CoA for fatty acid biosynthesis. Plasmodium PDC has recently been shown to be important for parasite progression from the asymptomatic liver stage to the symptomatic erythrocytic stage. Thus, inhibiting PDC could prevent development of malaria. This study focuses on the role of the PDC in P. falciparum blood stages and the identification and characterisation of structural and biochemical differences between parasite and human PDC that may be ultimately exploited for drug or vaccine development. I have optimised the expression and purification of soluble recombinant mature-length P. falciparum (Pf) E2 (His-rPfE2m), truncated PfE2 consisting of the SBD and CD (His- rPfE2bc) and mature-length apicoplast PfE3 (His-rPfaE3) to obtain mg amounts of protein for biochemical and structural analyses. Each of the recombinant proteins was catalytically active. Analytical ultracentrifugation (AUC) showed that His-rPfE2m forms the typical trimer building blocks required for the large E2 core formation. Sedimentation velocity (SV) experiments showed a main species with a sedimentation coefficient of 6.6 ± 0.1 S, which corresponded to a species consistent with the PfE2 trimer size observed in sedimentation equilibrium (SE) studies. However, no 24-mer or 60-mer core species were detected in SV experiments. SE analyses did show some larger molecular mass species (> 1.5 MDa), however, whether these represent a PfE2 core complex was inconclusive due to interference by aggregated protein in the sample. My conclusion from these data is that PfE2 may form a large core structure but that this is very unstable compared with E2 multimers from other organisms, where the core complex is readily formed and maintained. Similar results were obtained from SV and SE analyses of His-rPfE2bc; only trimers are present. Small angle X-ray scattering (SAXS) was used to further analyse the trimer structure. The solution structure obtained for His-rPfE2bc revealed the linker between the SBD and the CD is extended and partially flexible. This conformation, which would allow SBD interaction with E1 and E3, is supported by cryo-electron microscopy images obtained by others for E2 from other organisms. AUC on His-rPfaE3 showed a main species with a sedimentation coefficient of 6.1 ± 0.1 S and SE analyses of the protein confirmed it to be a dimer as expected. These findings were further corroborated with the solution structure obtained using SAXS. Deletion of the PfE2 gene from P. falciparum was unsuccessful, as the correct gene locus was not targeted. This could be due to the gene being required for parasite survival in the blood stages. However, it could also be due to the difficulty of P. falciparum gene manipulation and hence, further attempts to delete PfE2 using different approaches will be required. The PfaE3 gene was successfully deleted, thus as shown in previous studies with murine malaria species, the gene is not essential in the human malaria parasite, P. falciparum. The PfaE3 deletion mutants did not show increased susceptibility to oxidative stressors compared with the wild type strain, suggesting that PfaE3 does not play a role in defence against oxidative stress. However, the mutants were less susceptible to triclosan, an inhibitor of the fatty acid biosynthesis enzyme FabI. In addition, the mutant parasites maintained synchronicity over more than four replication cycles as opposed to WT parasites, which gradually lost synchronicity over this period of in vitro culture. Further work will need to be carried out to fully characterise the role of PfaE3 in P. falciparum.
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Bray, James William. "Quantification of training load, neuromuscular fatigue, biochemical and endocrine responses to fast bowling in cricket." Thesis, University of Hull, 2017. http://hydra.hull.ac.uk/resources/hull:16447.
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Recent professionalization, the emergence of, and ever-increasing popularity of limited overs cricket, have resulted in traditional playing schedules evolving and expanding. Consequently, players now compete for much of the year, experiencing periods of condensed fixtures. To meet these increased demands, the aforementioned contribute to effecting team performance and player health. Thus, the prevalence of injuries, especially amongst fast bowlers, has been shown and attributed to rises in competition workloads. Therefore, the main aim of this thesis was to explore the application of micro-electro-mechanical systems (MEMS) to quantify the training load of fast bowlers. Furthermore, I sought to assess relationships between both internal and external training load variables and proposed markers of fatigue and recovery. The first preliminary descriptive research study (Chapter 4) aimed to prospectively quantify fast bowling workloads during a typical season of professional domestic county cricket (April – September). Data were collected from fixture scorebooks, with descriptive bowling workloads determined by calculating frequencies of overs and deliveries bowled. This was further calculated dependant on both bowler classification (opening [O-B; n = 2] or support [S-B; n = 6]) and competition format (multiday [MD], One-day [OD] or Twenty20 [T20]), respectively. Significant differences were found in total number of overs (296.1 overs; 95% CI 37.8 to 554.4; P = 0.03) and deliveries (1764.8 balls; 95% CI 183.0 to 3346.7; P = 0.03) bowled between O-B and S-B, respectively. Multiday cricket was the only format where, significant differences between bowlers were found; total number of overs (289.9 overs; 95% CI 88.2 to 491.6; P = 0.01) and deliveries (1739.3 balls; 95% CI 529.3 to 2949.3; P = 0.01) bowled. The aim of experimental study one (Chapter 5) was to assess the between-match and within-match between-over variability of external training load measures during T20 cricket competition. MEMS data were collected from eight fast bowlers in 17 matches of domestic T20 competition, spanning two seasons. MEMS variables were categorised into total distance (TD), low- (≤ 14.4 km.h-1) and high- (≥ 14.4 km.h-1) speed running distance, total sprint distance (≥18 km.h-1), number of sprint efforts and PlayerLoadTM ([PL] arbitrary units; AU). Data were log-transformed to provide the coefficient of variation (CV; expressed as percentages). The between-match variability was greatest in high-speed running distance (32.9% CV), total sprint distance (49.0% CV) and number of sprint efforts (48.0% CV). Similarly, within-match between-over high-speed running distance (12.8% CV), total sprint distance (17.1% CV) and number of sprint efforts (12.3% CV) elicited the greatest variability, yet, this was markedly reduced compared to between-match observations. However, TD and PL were found to be relatively stable measures of external training load (range; 5.5–13.3% CV), both between-match and within-match between-over. Experimental study two (Chapter 6) investigated short-term neuromuscular fatigue (NMF) of fast bowlers and relationships to match performance during a typical season of professional academy OD limited overs cricket. Baseline measures of lower body NMF were assessed via flight time (ms) from a countermovement jump (CMJ). These measures were repeated every morning of competition; NMF was additionally assessed within 30-min after the cessation of the bowling innings (CMJ-FIRST or CMJ-SECOND). MEMS data were collected from six fast bowlers, with supplementary descriptive fast bowling workloads classifications (LOW, MODERATE and HIGH). There were significant reductions in flight time pre to post bowling innings (Δ 19 ms; P = 0.008). Moreover, similar reductions in flight time were found in LOW – MODERATE (Δ 30 ms; P = 0.03) and LOW – HIGH bowling workload groups (Δ 43 ms; P = 0.003), respectively. Finally, experimental study three (Chapter 7) investigated neuromuscular, biochemical and endocrine markers of fatigue after four spells of simulated fast bowling. Eleven fast bowlers completed differing spells of simulated fast bowling based on the Cricket Australia-Australian Institute of Sport (CA-AIS) fast bowling skills test. NMF were assessed via flight-time from a CMJ; pre (-0.5-h) and post (+0.5 and +24-h) simulation, with blood (Creatine kinase; CK) and saliva (Cortisol; sCort) samples collected in parallel. During each simulated fast bowling trial (4-, 6-, RANDOM- & 10-overs), internal (heart rate exertion index [HREI]) and external (PL) training load was quantified using MEMS. There were small, significant reductions in CMJ flight time pre to post (Δ 21 ms; P < 0.01) and pre to 24-h post (Δ 8 ms; P = 0.001) simulation, respectively. Overs bowled appeared to significantly affect NMF for up to 24-h post simulation. Furthermore, changes in CK were found to best correlate with estimated TD (r = 0.48; P = 0.002) rating of perceived exertion (RPE r = 0.47; P = 0.002) session-RPE (r = 0.48; P = 0.002), HREI (r = 0.45; P = 0.003) and PL (r = 0.41; P = 0.009) 24-h post simulation, respectively. The findings of this thesis demonstrate that during limited overs cricket, high-speed locomotive activity is highly variable amongst fast bowlers. Furthermore, fast bowlers are shown to experience short-term NMF, which appears to be magnified based on descriptive fast bowling workload characteristics. Collectively, these findings have importance for practitioners, who seek to facilitate performance by informed training prescription based on replicating match and training demands.
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Liu, Weizhi. "Microcapsule internalization by cells in vitro caused by physical and biochemical stimuli." Thesis, Queen Mary, University of London, 2014. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8551.
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There is a growing interest in micro sized vehicles with the function of storing, targeting and controlled releasing of substances during the past few decades. However, delivering the desired drugs inside micro containers to living cells is a particular challenging topic of material science. Microcapsules made of polyelectrolyte multilayers exhibit low- or non-toxicity, appropriate mechanical stability, variable degradation and can incorporate remotely addressable release mechanisms in responding to stimuli and external triggering, making them well suitable for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. PSS/PAH) or natural (i.e. DS/PArg) polyelectrolyte and cells, with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by human umbilical vein endothelial cells (HUVECs). Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional CLSM images reveal that microcapsules functionalized with neuraminidase can be internalized by endothelial cells, whereas ones without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 hours. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5, however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelium barrier to cross membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by cleaving the glycocalyx in their close proximity with minimum disruption of the glycocalyx layer, therefore they have high potential to act as drug delivery carriers to pass through the endothelium barrier of blood vessels into the surrounding tissue.
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SINGH, SHERJANG. "ELECTROCHEMICAL PROPERTIES OF SiC-C MICROELECTRODE SYSTEM FOR NEURONAL AND BIOCHEMICAL SENSING." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1123783498.
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Riaz, Muhammad Qasim. "Influence of Biochemical Environments on Surface Fatigue of Additive Manufactured Ti-6Al-4V." Youngstown State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1485534165584066.
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Vadrevu, Suryakiran. "Biochemical investigation of phosphodiesterase type IV post-translational modification, cellular localisation and interaction with associated binding proteins." Thesis, University of Glasgow, 2008. http://theses.gla.ac.uk/219/.
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cAMP is a secondary messenger that is involved in a variety of signalling pathways through its effectors including EPAC, PKA and ion channels. cAMP signalling regulates processes such as memory, muscle contraction and inflammatory responses. PDE enzymes offer a mechanism to negatively regulate elevated cAMP levels elicited by activators of adenylyl cyclase. Studies have shown that cAMP signalling is compartmentalised through binding of PDEs to A-kinase anchoring proteins (AKAPs) that scaffold PKA regulatory subunits. In this study post-translational-modification of PDE4 isoforms is investigated. SUMOylation is a relatively newly identified post-translational modification that is known to regulate the structure and function of its substrates. PDE4 isoforms of the PDE4A and 4D subfamilies are SUMOylated by an E3 ligase, PIASy. SUMOylation alters the rolipram sensitivity and potentiates the PKA mediated activation of the isoforms whilst it confers protection from ERK-mediated inhibition of PDE4 activity. SUMOylation alters the association of PDE4 isoforms with binding partners like β-Arrestin, AKAP18 δ and UBC9. Rolipram is an archetypal PDE4 specific inhibitor. In this study it is shown that in cells expressing a GFP tagged form of PDE4A4 undergoes redistribution into accretion foci upon chronic treatment with rolipram. Data suggests that foci formation requires protein turnover and is regulated by signalling pathways such as PI3 kinase pathway, p38 MAP kinase pathway and PKC pathways. Further, the Immunomodulatory drug Thalidomide® also inhibits foci formation. PDE4 isoforms have isoforms specific N-terminal regions, which play a crucial role in sub-cellular localisation and protein-protein interactions. It is shown here that PDE4D5 interacts with a novel RhoGAP called ARHGAP21 which has been previously reported to bind β-arrestins. This interaction is independent of GAP activity of ARHGAP as well as PDE4 activity. Previous reports have indicated a role of β-Arrestin, PDE4 and ARHGAP21 in regulation of actin cytoskeleton dynamics. Hence complex β-Arrestin-PDE4-ARHGAP21 may play a crucial role in regulating actin dynamics.
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Geldenhuys, Greta. "Egyptian goose (Alopochen aegyptiacus) : sensory, biochemical and physical meat quality as affected by gender, diet and ageing." Thesis, Stellenbosch : Stellenbosch University, 2014. http://hdl.handle.net/10019.1/95871.
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Thesis (PhD(Food Sc))--Stellenbosch University, 2014.ENGLISH ABSTRACT: In Southern Africa, the hunting of wildfowl species has increased considerably in the past few years. Crop farmers incur major financial losses due to the feeding activities of Egyptian geese (Alopochen aegyptiacus); consequently a large number of geese are hunted in an attempt to reduce the damage caused. With the current absence of scientific information baseline research investigating the meat quality of this species is essential. The sensory profile of Egyptian goose meat was found to be very distinct in relation to the characteristics of other well-known fowl species. It has very strong game aroma and flavour attributes with a prominent metallic aftertaste. The intense aroma and flavour notes were linked to the substantially higher iron content, as well as the high overall polyunsaturated fatty acid content as revealed by chemical profiling. The trained sensory panel also found the meat to be very tough (high shear force) compared to the other species. To identify the factors which may affect the overall consistency of the meat quality, the influence of three main effects namely; season (grain vs. non-grain diet), gender and portion was investigated. This revealed that season had the largest effect and harvesting periods should therefore be considered. The main issue is the higher intramuscular fat (IMF) content in winter (July), as well as the substantial difference in the fatty acid profiles of the two seasons. The forage vs. grain based diets during certain periods of the year leads to variation in the content of key fatty acids in the meat i.e. oleic acid, linoleic acid and α-linolenic acid. In winter, the meat had a characteristic, prominent game and metallic aroma while the summer (November) profile was governed by “sweet-oily-duck” and beef-like sensory notes. The fatty acid differences also result in variation between the omega 6 to omega 3 ratios of the seasons; the portions from winter are within the recommendations (ratio<5) and those from summer not. Regarding gender, the females had a lower carcass yield but higher IMF content. The female breast portion was also more tender (lower shear force). In attempting to elucidate the toughness of the meat, possible causes have been proposed. The pH decline in the pectoralis muscle occurs quite rapidly and it is possible that the high rigor temperature (>20 °C) may contribute to the increased toughness. Regardless of the proteolytic enzyme activity during the rigor period, the meat is still tough at 36 h post mortem and the proteolytic contribution may be overshadowed by the background toughness, i.e. the connective tissue content and fibre structure. The latter was confirmed when the breast portions were aged for 14 days and no change (decline) in the shear force values was observed even though myofibrillar degradation did occur (during ageing). Given the lack of a decline in the shear force, the aging of Egyptian goose meat as a means of improving the overall toughness cannot be proposed without further research. The study in its entirety provides substantial proof that the commercial utilisation of Egyptian goose meat is feasible.
AFRIKAANSE OPSOMMING: Die jag van wildsvoël spesies in Suider-Afrika het aansienlik toegeneem in die laaste paar jaar. Graanboere lei jaarliks groot finansiële verliese as gevolg van die voedingsgedrag van kolganse (Alopochen aegyptiacus). Gevolglik word ‘n groot hoeveelheid ganse elke jaar geoes om sodoende die skade te verminder. Met die huidige tekort aan wetenskaplike inligting is grondslag navorsing rakende die vleiskwaliteit van hierdie wildsvoël spesie noodsaaklik. Die sensoriese profiel van kolgansvleis is baie uniek in vergelyking met die vleis van ander bekende voël spesies. Dit word gekenmerk deur die sterk wildagtige aroma en geur tesame met ‘n baie prominente metaal nasmaak. Hierdie intense aroma en geur hou verband met ‘n baie hoër yster-inhoud, asook ‘n hoë poli-onversadigde vetsuur profiel soos uitgewys deur die chemiese karakteriserings studie. Die opgeleide sensoriese proepaneel het ook die vleis van hierdie voël spesie beskou as baie taai in vergelyking met die vleis van ander spesies. Ten einde te bepaal watter faktore die algehele variasie in vleiskwaliteit sal beïnvloed, is drie hoofeffekte naamlik, seisoen (graan teenoor nie-graan dieet), geslag en porsie ondersoek. Die verskeie studies het uitgewys dat seisoen die grootste invloed het en daarom sal die periodes waarin ganse geoes word in ag geneem moet word. Die hoër intramuskulêre vetinhoud in winter (Julie), asook die aansienlike verskil in die vetsuur profiele van die twee seisoene is die vernaamste verskille. Die weiding (hoofsaaklik gras) teenoor graan diëte wat gevolg word in sekere dele van die jaar lei tot variasie in die inhoud van belangrike vetsure (oliensuur, linoleïensuur en α-linoleensuur) in die vleis. In die winter het die vleis die kenmerkende en prominente wild geur en metaalagtige nasmaak getoon terwyl die profiel in die somer (November) hoofsaaklik bestaan het uit “soet-olierige-eend” en beesvleis geure. Die vetsuur verskille lei ook tot ‘n verskil in die omega-6 tot omega-3 verhouding van die seisoene; in die winter is die porsies binne die aanbevole voedingsvereistes (<5) terwyl die somer porsies nie aan hierdie vereistes voldoen het nie. Rakende die invloed van geslag, het die vroulike voëls ‘n laer karkas massa getoon tesame met ‘n laer intramuskulêre vetinhoud. Die borsie van die vroulike kolganse was ook sagter. In ‘n poging om die taaiheid van kolgansvleis te verklaar, is ‘n paar moontlike oorsake voorgestel. Die na-doodse pH daling in die borsspier vind redelik snel plaas, daarbenewens is dit ook moontlik dat die hoë temperature (>20 °C) waartydens rigor mortis plaasvind, kan bydrae tot die taaiheid. Ongeag die werking van die proteolitiese ensieme tydens die rigor periode was die vleis steeds taai 36 uur na dood, daarom was die proteolitiese bydrae moontlik oorskadu deur die agtergrond-taaiheid, dit wil sê die bindweefsel inhoud en vesel struktuur. Laasgenoemde is bevestig toe die borsspiere verouder is vir ‘n tydperk van 14 dae. Geen verandering (afname) in die instrumentele taaiheid is waargeneem nie, selfs al het miofibrillêre afbraak plaasgevind. Aangesien daar geen afname in instrumentele taaiheid opgemerk is nie kan veroudering van kolgansvleis, met die doel om die taaiheid te verbeter, nie aanbeveel word voor verdere navorsing nie. In geheel voorsien hierdie studie beduidende motivering rakende die moontlike kommersiële aanwending van kolgansvleis.
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Young, David. "Spectroscopic, structural, mutational and biochemical characterisation of quinol-dependent nitric oxide reductase of Neisseria meningitidis." Thesis, University of Liverpool, 2015. http://livrepository.liverpool.ac.uk/2034441/.
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Quinol-dependent nitric oxide reductase (qNOR) is a homodimeric, membrane-bound metalloenzyme with the catalytic function of reducing two molecules of nitric oxide to nitrous oxide. In bacteria, qNOR can serve either as a respiratory enzyme, or in pathogens as a defence mechanism against cytotoxic levels of nitric oxide radicals generated by the host immune response. One such pathogen is Neisseria meningitidis, a cause of meningitis and septicaemia worldwide. Various biochemical, biophysical, and computational techniques have been applied in study of the molecular mechanism underlying the reduction of nitric oxide by nitric oxide reductase, yet clearly defined mechanics of the enzymatic reaction have yet to be concluded. Understanding of the enzymatic mechanism requires a high-resolution crystal structure of the enzyme to provide a basis for computational calculations and directed biochemical/biophysical analysis. A crystal structure of the qNOR of Geobacillus stearothermophilus was solved to a resolution of 2.5 Å, but significantly in a catalytically inert form unsuitable for reliable characterisation of the active site. By this structure, molecular dynamics studies of potential solvent channels to the active site has led to the identification of a hydrophilic pathway from the cytoplasm for transfer of protons required for catalytic turnover. This would be the first nitric oxide reductase identified to take chemical protons from the cytoplasm instead of the periplasm (as occurs in the isofunctional cytochrome c-dependent nitric oxide reductase), a striking feature in respiratory terms as this membrane separation of proton/electron uptake would infer an electrogenic property to qNOR as yet unobserved in nitric oxide reductases. This thesis presents efforts to characterise the qNOR of Neisseria meningitidis, a highly active reductase that, from preliminary analyses, appears promising in its potential to retain a catalytically active form for crystallographic studies. This qNOR was heterologously over-expressed in a bacterial host and purified into a detergent micelle suspension. Spectroscopic techniques were employed to elucidate the electronic state of the active site of qNOR, and amperometric assays were used to study the nature of the nitric oxide reductase activity. N. meningitidis qNOR was found to have nitric oxide reductase activity significantly greater than that of other qNORs and largely independent to pH changes, and displayed Raman characteristics distinct from other qNORs. Crystallography of qNOR yielded X-ray diffraction to ~4.5 Å, and a crystal structure solved to a resolution of 5 Å. From this, significant structural homology to the high-resolution G. stearothermophilus qNOR crystal structure could be observed. Based on solvent networks identified in the crystal structure of G. stearothermophilus, a cytoplasmic pathway for chemical protons to the active site has been proposed. Research described in this thesis involves site-directed mutagenesis of residues believed to play a role in proton transfer. It was found that none of the mutant variants tested significantly affected the nitric oxide reductase capability of qNOR.
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Dyer, Scott Douglas. "Evaluation of the efficacy of the stress protein response as a biochemical water quality biomonitoring method." Thesis, University of North Texas, 1991. https://digital.library.unt.edu/ark:/67531/metadc798352/.
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The stress protein response (SPR) is a conserved and ubiquitous mechanism that enables cells to tolerate a wide variety of environmental insults. This response involves the preferential synthesis of an array of proteins with different molecular weights. These proteins perform a variety of functions, such as protein folding, multimeric protein assembly, steroid receptor binding, and heme catabolism.
To evaluate the potential use of the SPR as a biomonitoring tool, a stepwise plan was utilized that proceeded through various physical and chemical laboratory exposures and culminated with a field validation study. The goals of the laboratory exposures were threefold: (1) determine the time required for induction of the SPR; (2) determine the dose-responsiveness of the SPR; and (3) compare the increased syntheses and accumulations of stress proteins to classical toxicological endpoints (i.e. percent mortality, LC50, LC1, etc).
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Vandongen, Robert. "[Biochemical and epidemiological basis of hypertension] : published works submitted to the University of Adelaide for the degree of Doctor of Science /." Title page and summary only, 1987. http://web4.library.adelaide.edu.au/theses/09SD/09sdv246.pdf.
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Baeza, Rita. "Comparison of technologies to control the physiological, biochemical and nutritional changes of fresh cut fruit." Manhattan, Kan. : Kansas State University, 2007. http://hdl.handle.net/2097/494.
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Lauer, Nathan T. "Physiological and Biochemical Responses of Bald Cypress to Salt Stress." UNF Digital Commons, 2013. http://digitalcommons.unf.edu/etd/454.
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Bald Cypress (Taxodium distichum (L.) Rich.) is native to freshwater wetlands of Florida. The vitality of cypress within coastal freshwater wetlands is threatened by saltwater intrusion. Biomarkers to detect sub-lethal salinity stress were developed using a controlled greenhouse study. Cypress saplings maintained at elevated salinities of 4 and 8‰ exhibited a decrease in maximum quantum yield (MQY) and an increase in non-photochemical quenching (NPQ). Cypress leaves exhibited an increase in Na+, H2O2, and free proline content compared to plants maintained in freshwater. These biomarkers were used to detect salinity stress within a population of cypress associated with the lower St. Johns River where saltwater intrusion is occurring. Cypress in a basin swamp exhibited signs of salinity stress with low MQY and elevated NPQ values compared to Cypress at other sites. Cypress leaves at the basin swamp also had the highest Na+, lipid peroxidation, and proline content compared to plants at other sites. Detached Cypress leaf experiments were conducted to explore the mechanisms of salt tolerance. Detached cypress leaves were first exposed to elevated NaCl concentrations for 24, 48, or 72 hours. Elevated salinity caused a decrease in leaf transpiration for all times tested. Total peroxidase activity exhibited an increase in response to salt stress after 48 hours. Enhanced peroxidase activity was found to be associated with the induction of a ~37 kDa peroxidase isoform. Treatment of leaves with clofibrate caused an increase in activity of the ~37 kDa peroxidase. Pre-treatment of leaves with brefeldin A (BFA) blocked the induction of the ~37 kDa peroxidase associated with salt stress. Pre-treatment of Cypress leaves with diphenyliodonium (DPI) blocked the decrease in transpiration associated with salt stress, suggesting that H2O2 is enzymatically produced within the stomata in response to salt stress
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Changsawang, Narin. "The effects of growth conditions on the elemental and biochemical composition of the diatom Thalassiosira weissflogii and the haptophyte Emiliania huxleyi." Thesis, University of Essex, 2015. http://repository.essex.ac.uk/15697/.
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A change in environmental conditions often leads to changes of physiology and biochemical composition of microalgae. Temperature and light intensity are important environmental factors regulating the growth of microalgae. In this study, the elemental and biochemical composition were measured in 2 marine microalgae under different temperatures and light intensities in nutrient replete and deplete conditions. The effect of temperature was observed in the marine haptophyte Emiliania huxleyi (CCMP 1516) at nutrient replete semi-continuous cultures. Triplicate cultures were incubated different temperature from 14 to 22oC and under photon flux densities (PFD) 600 μmol photons m−2 s−1. The growth rate (GR) of E. huxleyi increased with temperature. Cell volume varied with temperature, being about 40% smaller at higher temperature (22oC). Cellular chlorophyll a (chl a), nitrogen, phosphorus, and carbon contents were also lower at 22oC than other temperatures. Protein, total amino acids from free and combined amino acid, and total pigments [mol accessory pigment (mol chl a)-1] were decreased with increasing temperature; however, the opposite response was observed in fatty acids. In addition to the effect of combined temperature and light intensity was investigated in the marine diatom Thalassiosira weissflogii (CCMP 1056) under nutrient-limited semi-continuous cultures. The cultures were incubated at 16 and 26°C and PFD of 50 ± 10 (low light; LL) and 500 ± 10 (high light; HL) μmol photons m−2 s−1. HL incubated-cultures were diluted at 50% day-1 and LL incubated-cultures were diluted at 25% day-1. The GR were largely set by dilution rate (nitrogen limitation), but not by temperature and irradiance. The GR were around 0.72 d-1 in HL placed-cultures and 0.32 d-1 in the LL placed-cultures. Temperature did not affect mean cell size, whereas mean cell size decreased with increased irradiance by 20 to 29 %. Both temperature and irradiance influenced cellular chl a, carbon and chl a specific light absorption. Cellular nitrogen and phosphorus varied with temperature and irradiance. Protein, total amino acid (free and combined amino acid) and total fatty acid increased with increased temperature and irradiance; however, the opposite response was found in carbohydrate. Overall, temperature and light affected elemental and biochemical composition in 2 marine microalgae. Both relationship of the chlorophyll (chl):carbon (C) and RNA:protein ratio and growth rate in E. huxleyi under variable temperature positively supported a bio-optical and growth rate hypothesis respectively. However, the opposite response was found in T. weissflogii. Instead the C:chl and RNA:protein ratio and growth rate in T. weissflogii under variable irradiance positively supported a bio-optical and growth rate hypothesis.
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Al, Mohamad Zakriya Ali E. "Quantitative assessment of the biochemical composition of equine cartilage using 7T ultra-high field magnetic resonance imaging (MRI) techniques." Thesis, University of Glasgow, 2016. http://theses.gla.ac.uk/8227/.
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Equine fetlock region disease is responsible for significant morbidity and mortality. Diagnosis of sesamoidean ligament, cartilage and subchondral bone injury has been obtained by clinical MRI. Low-field MRI provides images helpful in the investigation of MCPJ/MTPJ region pathology in horses in the clinical setting but the greater resolution of high and ultra-field MR images has the potential to aid interpretation through a better understanding of MRI anatomy. Quantitative MRI could provide a non-invasive technique to determine tissue biochemical properties associated with the early onset of articular cartilage degenerative conditions such as osteoarthritis. So far, ultra-high field MRI has not been used in equine research and practice. However, recently 3T MRI has been introduced in equine hospitals in Europe and the US. The general objectives of this project, which utilised cadaver limbs, was to improve understanding of the MRI anatomy of the equine MCPJ/MTPJ region and to evaluate the use of MRI for the non-invasive, quantitative assessment of articular cartilage from the same region. The first specific objective was to describe the appearance of the normal anatomy of the equine MCPJ/MTPJ region, especially the SDFT & DDFT and DSLs, using high field (1.5T) and ultra high field (7T) MRI and to compare the images obtained with the two systems. The second objective was to determine the accuracy and precision of articular cartilage thickness measurements using 1.5T and 7T MRI and comparing the measurements with those made from histological sections of the MCPJ/MTPJ. The third objective was to measure T1 & T2 MRI sequence relaxation times for normal horse articular cartilage pre and post gadolinium contrast (dGEMRIC) administration and to determine their correlation with GAG concentration, including a description of topographical variation. The fourth objective was to compare sodium concentration in normal equine MCPJ/MTPJ articular cartilage measured using 7T MR imaging with a dual tuned quadrature 23Na/1H coil with the biochemical properties (sodium concentration determined by flame photometry and GAG concentration). The final objective was to evaluate MR sodium imaging for the assessment of enzymatically degraded equine cartilage. The findings demonstrated that 7T MRI produces high resolution images, which enable better evaluation of the hard and soft tissues of the equine MCPJ/MTPJ region than images from lower field MR systems and which permit accurate and precise articular cartilage thickness measurements to be made. Moreover, it was found that the dGEMRIC technique appears to provide a feasible quantitative tool for evaluating the articular cartilage properties. However, the quantitative parameters determined by the dGEMRIC method cannot fully characterise the biochemical properties of the cartilage. Moreover, delayed gadolinium-enhanced (dGEMRIC) techniques are time consuming, requiring relatively long incubation and scanning times. The measurement of T2 time is a very complex method. The work described in the last chapters demonstrated that sodium MRI was significantly correlated with the biochemical properties of the equine articular cartilage. Therefore the sodium MRI technique showed promise in imaging articular cartilage and providing useful information on the biochemical properties of the cartilage.
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Matchett, Ashley A. "Genetic and biochemical analysis of materials from a medieval population from Ynys Mon North Wales." Thesis, University of Central Lancashire, 2011. http://clok.uclan.ac.uk/3880/.
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The archaeological excavation of the early medieval site at Towyn-Y-Capel on the island of Anglesey (Ynys Môn) in North Wales, UK, provided the opportunity to study a large population (122 skeletons) at a site that was in use over a period of up to 550 years (650 -1200 AD). Samples of skeletal materials for this study were taken directly from the site itself .The osteological condition of skeletal material was variable across the site. In general, the upper burials in particular were in the poorest condition, and were mainly fragmented and dispersed due to the ongoing site erosion and diagenetic processes. Conversely, lower “cist” burials were in far better condition. The assessment of skeletal sample condition was used to select materials chosen for genetic analysis, and 44% (54) of the skeletal population were selected for analysis of appropriate samples of tooth and bone. The gross morphology of samples was assessed and 87% of bones and teeth were considered to be in good or fair condition, according to the gross preservation index (GPI) used, while only 2% of bones and no teeth were considered to be in excellent condition. In addition to GPI, a novel technique called Qualitative Light Fluoresence (QLF), based on autofluoresence, was used to ascertain the surface condition of the teeth. Compared to the fluorescence of modern enamel, there was a net loss of 21.8% fluorescence, although the degree of fluorescence from one sample to another varied (with a standard deviation from the mean of 24.973). Histological sections taken from non-human bone finds from the site generally varied less than that indicated by the gross morphology, showing good to excellent histological preservation. Further to gross and histological morphology, ten skeletal samples were selected for detailed investigations, and were analysed for amino acid racemisation and amino acid composition. All samples tested had D/L enatomer Aspartic acid ratio less than 0.1, although 50% of the samples had D/L enatiomer Aspartic acid ratio over 0.08, which indicated that the recovery of aDNA from these skeletal samples was feasible, although the biological condition of the teeth was fairly degraded. The inorganic element profile of the same ten samples showed no discernable anomalies, either due to diet or diagenesis. To consolidate genographic research, strontium isotope analysis was performed and, from the small population subset, three anomalous ratios were found. Two of these were high (Skeletons 33 and 60), indicating that these individuals had spent their childhoods in areas with high strontium ratios, representative of precambrian rock types, possibly older than those of the Holyhead Rock group, such as in Northern Scotland or Norway. The skeletal samples yielding the lowest strontium ratio (Skeleton 52) are of compelling interest, since the ratio is indicative of upbringing in only one place in the North Atlantic, namely Iceland. In this study, DNA recovery was performed on teeth and bones from the site, after extensive decalcification of samples, and also extraction and optimisation trials. Amplification of DNA extracted from teeth samples was generally more successful than for bone samples. A random amplification based polymorphic (RAPD) DNA technique was utilised to “fingerprint” human and animal samples with limited success. Contamination and template variation are likely causes for the lack of success. Amplification using several primers specific for human HV1 & 2 mtDNA targets was also met with limited success. The results show that 14.8% of the skeletal teeth samples were amplified, and these were not commonly reproducible. DNA spiking trials demonstrated that some of the samples were affected by inhibition. Independent confirmation of 9 of 10 successful samples was attained by sequencing, and although sequences were highly degraded, an attempt was made at determining the haplogroups from the sequenced HV1 haplotypes based on likelihood. Generally, the site showed a high predominance of Haplotype K (5) followed by H (2) and U (2) haplogroup profiles.
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Chou, Lan-Szu. "Isolation and Biochemical Characterization of Acid- and Bile- Tolerant Strains of Lactobacillus Acidophilus and Bifidobacterium Bifidum." DigitalCommons@USU, 1997. https://digitalcommons.usu.edu/etd/5427.
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Lactic acid bacteria have been reported to be used as a health adjunct in food for u many years. However, these health benefits have not been proven. and how these bacteria pass through the digestion process and remain viable in the human intestinal tract is still not clear. The aim of this work was to isolate mutants from Lactobacillus acidophilus or Bifidobacterium bifidum that could tolerate the conditions of the digestion process (low pH and bile conduction) and to characterize these isolated mutants.
Acid- and bile-tolerant mutants of L. acidophilus were isolated from parental strains successfully using natural selection techniques. These mutants survived and grew at conditions of pH 3.5 with 0.2% mixed bile salts added. After the selection, phenotypic characterization was identified to further clarify desirable traits for use as probiotic adjuncts in foods. These phenotypic characteristics included protease, aminopeptidase, ß-galactosidase, and bile salt hydrolase activity. Based on different protease, aminopeptidase, and ß-galactosidase activity, selected acid- and bile-tolerant mutants contained different growth characteristics compared with their parents. All the isolates tested showed different bile salt hydrolase activity, and this activity was not strain and medium dependent.
Plasmid profiles and fatty acid analysis were conducted to provide more information of these acid/bile tolerant isolates and whether or not they were mutants from their parent strains rather than only adapted variants. Results showed the acid-/bile-tolerant isolates contained different plasmid profiles and cell wall fatty acids compared with their parents, which indicated these isolates were mutants. Protein expression by two-dimensional gel electrophoresis showed different protein expression patterns between acid- and bile-tolerant mutants and their parents. fm1her suggesting these isolates were mutants. We observed the protein production in parent strains decreased as the pH decreased. and protein expression in mutants remained the same as pH decreased.
Two of the proposed health benefits of probiotic bacteria are anticholesterol activity and antimicrobial activity. These were evaluated using selected acid- and bile-tolerant mutants. Results showed no decrease of cholesterol in the test medium during bacterial growth. The observed antimicrobial activity was due to the presence of active cells. and this may relate to the acid production during cell growth and not to the production of antimicrobial substances.
We concluded that the acid-/bile-tolerant isolates were mutants, and they survived and grew better in harsh environments compared with their parent strains. These mutants may be useful as a food adjunct in the future, but further study is needed to establish their use and possible probiotic benefits.
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Halling, Linder Cecilia. "Biochemical and functional properties of mammalian bone alkaline phosphatase isoforms during osteogenesis." Doctoral thesis, Linköpings universitet, Avdelningen för mikrobiologi och molekylär medicin, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-127100.
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The human skeleton is a living and dynamic tissue that constantly is being renewed in a process called bone remodeling. Old bone is resorbed by osteoclasts and new bone is formed by osteoblasts. Bone is a composite material made up by mineral crystals in the form of hydroxyapatite (calcium and phosphate) that provides the hardness of bone, and collagen fibrils that provides elasticity and flexibility. Alkaline phosphatase (ALP) is a family of enzymes that is present in most species and catalyzes the hydrolysis of various phosphomonoesters at alkaline pH. Despite the generalized use of ALP as a biochemical marker of bone formation, the precise function of bone ALP (BALP) is only now becoming clear. Three circulating human BALP isoforms (B1, B2, and B/I) can be distinguished in healthy individuals and a fourth isoform (B1x) has been discovered in patients with chronic kidney disease and in bone tissue. Paper I. Three endogenous phosphocompounds, (i.e., inorganic pyrophosphate (PPi), pyridoxal 5′-phosphate (PLP) and phosphoethanolamine (PEA)), have been suggested to serve as physiological substrates for BALP. The BALP isoforms display different catalytic properties towards PPi and PLP, which is attributed to their distinct N-linked glycosylation patterns. The catalytic activity, using PEA as substrate, was barely detectable for all BALP isoforms indicating that PEA is not a physiological substrate for BALP. Paper II. Mouse serum ALP is frequently measured and interpreted in mammalian bone research. However, little is known about the circulating ALPs in mice and their relation to human ALP. We characterized the circulating and tissue-derived mouse ALP isozymes and isoforms from mixed strains of wild-type and knockout mice. All four BALP isoforms (B/I, B1x, B1, and B2) were identified in mouse serum and bone tissues, in good correspondence with those found in human bones. All mouse tissues, except liver, contained significant ALP activities. This is a notable difference as human liver contains vast amounts of ALP. Paper III. The objective of this study was to investigate the binding properties of human collagen type I to human BALP, including the two BALP isoforms B1 and B2, together with ALP from human liver, human placenta and E. coli. A surface plasmon resonance-based analysis showed that BALP binds stronger to collagen type I in comparison with ALPs expressed in non-mineralizing tissues. The B2 isoform binds significantly stronger to collagen type I in comparison with the B1 isoform, indicating that glycosylation differences in human ALPs are of crucial importance for protein–protein interactions with collagen type I. Paper IV. Tartrate-resistant acid phosphatase (TRAP) is highly expressed in osteoclasts and frequently used as a marker of bone resorption. Intriguingly, recent studies show that TRAP is also expressed in osteoblasts and osteocytes. TRAP displays enzymatic activity towards the endogenous substrates for BALP, i.e., PPi and PLP. Both TRAP and BALP can alleviate the inhibitory effect of osteopontin on mineralization by dephosphorylation, which suggests a novel role for TRAP in skeletal mineralization.
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Bolton, Shawna N. "Forensic taphonomy : investigating the post mortem biochemical properties of cartilage and fungal succession as potential forensic tools." Thesis, University of Wolverhampton, 2015. http://hdl.handle.net/2436/579577.
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Post mortem interval (PMI – the time elapsed since death and discovery) is important to medicolegal investigations. It helps to construct crucial time lines and assists with the identification of unknown persons by inclusion or exclusion of a suspect’s known movements. Accurate methodologies for establishing PMI are limited to about 48-hours. Such methods involve use of increasing levels of potassium in vitreous humour, and algor mortis. This study is two-fold. Firstly, it explores the biomolecular changes in degrading porcine cartilage buried in soil environments and its potential to determine PMI in the crucial two days to two months period. Trotters were interred in a number of graves at two distinct locations exhibiting dissimilar soil environments. Weekly disinterments (for 6 weeks) resulted in dissection for cartilage samples which were processed for protein immunoblot analyses and cell vitality assays. Results demonstrate that aggrecan, a major structural proteoglycan, produces high (230kDa) and low (38kDa) molecular weight cross-reactive polypeptides (CRPs) within cartilage extracellular matrix. The 230kDa CRP degrades in a reproducible manner irrespective of the different soil environments utilised. As PMI increases, aggrecan diminishes and degrades forming heterogeneous subpopulations with time. Immunodetection of aggrecan ceases when joint exposure to the soil environment occurs. At this time, aggrecan is metabolised by soil microbes. The molecular breakdown of cartilage proteoglycans has potential for use as a reliable indicator of PMI, irrespective of differing soil environments, beyond the 48-hours period. Likewise, vitality assays also demonstrated viable chondrocytes for as long as 35 PM days. The second component of this study examined the fungal activity associated with trotters buried below ground. Results indicate that fungal growth was considerably influenced by soil chemistry and changes in the environment. Fungal colonisation did not demonstrate temporal patterns of succession. The results of this study indicate that cartilage has the potential to prolong PMI determination well beyond the current 48- and 100-hour limitations posed by various other soft tissue methods. Moreover, the long-term post mortem viability of chondrocytes presents an opportunity to explore DNA extraction from these cells for the purpose of establishing a positive identification for unidentified remains. On the contrary, the growth and colonisation patterns of post putrefactive fungi in relation to decomposing porcine trotters proved to be futile for estimating PMI. Therefore, fungi may not be a suitable candidate for evaluating PMI during the early phase fungal activity.
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Bergendahl, Christina. "Development of competence in biochemical experimental work : Assessment of complex learning at university level." Doctoral thesis, Umeå : Dept. of Chemistry, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-313.
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Martinenko, Evgeny. "Prediction of survival of early stages lung cancer patients based on ER beta cellular expressions and epidemiological data." Master's thesis, University of Central Florida, 2011. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4796.
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We attempted a mathematical model for expected prognosis of lung cancer patients based on a multivariate analysis of the values of ER-interacting proteins (ERbeta) and a membrane bound, glycosylated phosphoprotein MUC1), and patients clinical data recorded at the time of initial surgery. We demonstrate that, even with the limited sample size available to use, combination of clinical and biochemical data (in particular, associated with ERbeta and MUC1) allows to predict survival of lung cancer patients with about 80% accuracy while prediction on the basis of clinical data only gives about 70% accuracy. The present work can be viewed as a pilot study on the subject: since results confirm that ER-interacting proteins indeed inuence lung cancer patients' survival, more data is currently being collected.ID: 030646185; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2011.; Includes bibliographical references (p. 32-33).
M.S.
Masters
Mathematics
Sciences
Mathematical Science
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Ross, Trinette Noel. "Evaluation of bone biochemical markers and inflammatory markers in yearlings fed varying ratios of omega-6 and omega-3 polyunsaturated fatty acids." [College Station, Tex. : Texas A&M University, 2006. http://hdl.handle.net/1969.1/ETD-TAMU-1036.
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Schlipf, Daniel M. "BIOMOLECULE LOCALIZATION AND SURFACE ENGINEERING WITHIN SIZE TUNABLE NANOPOROUS SILICA PARTICLES." UKnowledge, 2015. http://uknowledge.uky.edu/cme_etds/44.
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Mesoporous silica materials are versatile platforms for biological catalysis, isolation of small molecules for detection and separation applications. The design of mesoporous silica supports for tailored protein and biomolecule interactions has been limited by the techniques to demonstrate biomolecule location and functionality as a function of pore size. This work examines the interaction of proteins and lipid bilayers with engineered porous silica surfaces using spherical silica particles with tunable pore diameters (3 – 12 nm) in the range relevant to biomolecule uptake in the pores, and large particle sizes (5 - 15 µm) amenable to microscopy imaging
The differentiation of protein location between the external surface and within the pore, important to applications requiring protein protection or catalytic activity in pores, is demonstrated. A protease / fluorescent protein system is used to investigate protein location and protection as a function of pore size, indicating a narrow pore size range capable of protein protection, slightly larger than the protein of interest and approaching the protease dimensions. Selective functionalization, in this case exterior-only surface functionalization of mesoporous particles with amines, is extended to larger pore silica materials. A reaction time dependent functionalization approach is demonstrated as the first visually confirmed, selective amine functionalization method in protein accessible supports.
Mesoporous silica nanoparticles are effective supports for lipid bilayer membranes and membrane associated proteins for separations and therapeutic delivery, although the role of support porosity on membrane fluidity is unknown. Transport properties of bilayers in lipid filled nanoparticles as a function of pore diameter and location in the particle are measured for the first time. Bilayer diffusivity increases with increasing pore size and is independent of bilayer location within the core, mid or cap of the particle, suggesting uniform long range bilayer mobility in lipid filled pores. Application of lipid bilayers on mesoporous silica was examined for membrane associated proteins A unique method to adhere functional proteins in lipid bilayers on mesoporous silica particles is established using vesicles derived from cell plasma membranes and their associated proteins. This method of membrane protein investigation retains proteins within native lipid membranes, stabilizing proteins for investigation on supports.
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Gupta, Prachi. "SYNTHESIS AND CHARACTERIZATION OF ANTIOXIDANT CONJUGATED POLY(ΒETA-AMINO ESTER) MICRO/NANOGELS FOR THE SUPPRESSION OF OXIDATIVE STRESS." UKnowledge, 2016. http://uknowledge.uky.edu/cme_etds/58.
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Oxidative stress is a pathophysiological condition defined by an increased production of reactive oxygen species (ROS), which can result in the growth arrest of cells followed by cell disintegration or necrosis. A number of small molecule antioxidants (e.g. curcumin, quercetin and resveratrol) are capable of directly scavenging ROS, thereby short-circuiting the self-propagating oxidative stress state. However, poor solubility and rapid 1st pass metabolism results in overall low bioavailability and acts as a barrier for its use as a drug to suppress oxidative stress efficiently.
To overcome this limitation, these small molecule antioxidants were covalently conjugated into poly(β-amino ester) (PβAE) cross-linked networks to formulate prodrug gel microparticles and nanoparticles (nanogels). Being hydrolytically degradable in nature, these PβAE crosslinked systems released antioxidants in their original structural form in a sustained controlled fashion.
Both quercetin and curcumin-PβAE nanogels showed prolonged suppression of cellular oxidative stress induced by H2O2. Curcumin PβAE nanogels also demonstrated protection against mitochondrial oxidative stress induced by H2O2 and polychlorinated biphenyls.
Curcumin-PβAE gel microparticles were also developed as a platform to treat oral mucositis through a local antioxidant delivery route. The same synthesis chemistry was transferred to formulate resveratrol PβAE gel microparticles for topical applications, to treat UV radiation induced oxidative stress. Both formulations showed suppression of induced oxidative stress. An in vivo trial with curcumin-PβAE microparticles further showed relatively reduced the severity of induced oral mucositis (OM) in hamster check pouch as compared to placebo.
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Larsson, Birgitta. "Physiological Importance of 1,25(OH)2D3 Membrane-Initiated and Nuclear Actions in Chickens as a Function of Growth, and Maturation in Male and Female Chickens." DigitalCommons@USU, 2002. https://digitalcommons.usu.edu/etd/5500.
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It is well established that l,25(OH)2D3 is a major regulator of calcium homeostasis. The steroid exerts its effects on its target organs by two mechanisms, a slow mechanism mediated by nuclear vitamin D receptors (nVDR), and a rapid mechanism mediated by the l,25(OH)2D3 membrane-associated rapid response steroid binding protein (l,25D3-MARRS bp). In this dissertation, the physiological relevance of membrane initiated steroid signaling was investigated by studying the correlation of age in male and female chickens with the magnitude of responses to l,25(OH)2D3 in duodena from 7-, 14-, 28-, and 58-week-old birds. Measurements of l,25(OH)2D3 (130 pM) responsiveness as a function of age showed an age-related decrease in intestinal Ca2+ transport for both male and female birds. Western analyses on isolated basal lateral membranes (BLM) indicated a decreased expression of 1,25D3-MARRS bp with increasing age in male chickens, while its expression increased with age in female birds. Saturation analyses of [3H]l,25(OH)2D3 binding to BLM revealed an allosteric interaction identified as cooperative binding for both male and female birds. The degree of positive cooperativity increased with increasing age. Both genders showed a significant increase in dissociation constant (Kd) with increasing age, indicating decreasing affinity. No age-related changes in the number of binding sites, Bmax, were observed in male chickens, while female birds showed an increase in Bmax with increasing age. Data obtained in parallel binding analyses of [3H]l,25(OH)2D3 to nuclear fraction nVDR in contrast, indicated an absence of cooperative binding and an absence of significant changes in Kd or Bmax with age for both male and female birds. Membrane-initiated signal transduction by l,25(OH)2D3 was assessed by determination of protein kinase C (PKC) and A (PKA) activities. Both male and female birds showed an age-related decrease in stimulation of PKC activity in response to l,25(OH)2D3, while PKA activity increased. In conclusion, this dissertation demonstrates that there is a decrease in l,25(OH)2D3-induced intestinal calcium uptake as a function of age in duodena of both male and female chickens, which can be correlated to a decreased affinity for l,25(OH)2D3, and a decreased PKC activity.
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Jia, Yang. "FORMS OF SUPPLEMENTAL SELENIUM IN VITAMIN-MINERAL MIXES DIFFERENTIALLY AFFECT SEROLOGICAL AND HEPATIC PARAMETERS OF GROWING BEEF STEERS GRAZING ENDOPHYTE-INFECTED TALL FESCUE." UKnowledge, 2019. https://uknowledge.uky.edu/animalsci_etds/97.
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Consumption of endophyte-infected tall fescue results in a syndrome of negatively altered physiological systems, collectively known as fescue toxicosis. Another challenge to endophyte-infected tall fescue -based beef cattle operations is that the soils often are selenium (Se) poor, necessitating the need to provide supplemental Se. To test the general hypothesis that different forms of supplemental Se would ameliorate the negative effects of fescue toxicosis, predominately-Angus steers (BW = 183 ± 34 kg) were randomly selected from herds of fall-calving cows grazing an endophyte-infected tall fescue pasture and consuming vitamin-mineral mixes that contained 35 ppm Se as sodium selenite (ISe), SELPLEX (OSe), or an 1:1 blend of ISe and OSe (MIX). Steers were commonly weaned and depleted of Se for 98 d. Steers were assigned (n = 8 per treatment) to the same Se-form treatments upon which they were raised and subjected to summer-long common grazing of an endophyte-infected tall fescue pasture (0.51 ppm ergot alkaloids: ergovaline plus ergovalinine; 10.1 ha). Selenium treatments were administered by daily top-dressing 85 g of vitamin-mineral mix onto 0.23 kg soyhulls, using in-pasture Calan gates. The first project objective was to determine the effect of forms of supplemental Se on whole blood Se, serum prolactin, liver glutamine synthetase (GS) activity, carcass parameters, and growth performance (Experiment 1). In Experiment 1, whole blood Se increased for all treatments from day 0 to 22 and then did not change. Across periods, MIX and OSe steers had greater whole blood Se than ISe steer. Compared to ISe steers, MIX and OSe steers had more serum prolactin. Liver GS mRNA, protein content, and activity were greater in MIX and OSe steers than ISe steers. However, the ADG and carcass parameters were not affected by Se treatments. The second project objective was to determine the effect of forms of supplemental Se on serum clinical parameters of Experiment 1 steers (Experiment 2). In Experiment 2, across periods, MIX steers had more serum albumin than OSe, and ISe steers, respectively. Serum alkaline phosphatase (ALP) activity was greater in MIX and OSe steers. In addition, blood urea nitrogen (BUN), serum sodium, phosphorus, and magnesium concentration were affected by Se treatments. Partial correlation analysis revealed that serum albumin, BUN, and ALP activity were correlated with whole blood Se concentration. The third project objective was to evaluate the hepatic transcriptome profiles of Experiment 1 steers using microarray and targeted RT-PCR analyses (Experiment 3). In Experiment 3, bioinformatic analysis of microarray data indicated that hepatic glutamate/glutamine, proline, arginine, and citrulline metabolism was affected by different forms of supplemental Se. The mRNA expression of critical proteins involved in glutamate/glutamine (GLS2, GLUD1, GLUL), proline (PYCR1, ALDH18A1), and urea (ARG1, ARG2, OAT, NAGS, OTC, ORNT1) metabolism were differentially expressed by Se treatments. Collectively, we conclude that consumption of 3 mg Se/d as OSe or MIX forms of Se in vitamin-mineral mixes 1) increased whole blood Se content, an indicator of greater whole-body Se assimilation; 2) increased serum prolactin, albumin, and ALP, the reduction of which are hallmarks of fescue toxicosis; and 3) altered hepatic nitrogen metabolism, as indicated by changes in key enzymes of glutamate/glutamine, proline, and urea metabolism. However, 4) these positive effects on metabolic parameters were not accompanied by increased growth performance.
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Ahn-Jarvis, Jennfier H. "Development of a standardized functional soy product for cancer prevention trials:Phase II evaluation of isoflavone bioavailability in men with asymptomatic prostate cancer." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1357255127.
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Liu, Yunhao. "Structural and biochemical analysis of HutD from Pseudomonas fluorescens SBW25 : a thesis submitted in fulfilment of the requirements for the degree of Master of Science in Molecular Biosciences at Massey University, Auckland, New Zealand." Massey University, 2009. http://hdl.handle.net/10179/1074.
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Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of growing on histidine as the sole source of carbon and nitrogen. Expression of histidine utilization (hut) genes is controlled by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the direct inducer. Recent genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut locus, which encodes a highly conserved hypothetical protein. Previous genetic analysis showed that hutD is involved in hut regulation, in such a way that it prevents overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth phenotype in minimal medium with histidine as the sole carbon and nitrogen source. While the genetic evidence supporting a role of hutD in hut regulation is strong, nothing is known of the mechanism of HutD action. Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified HutD was subjected to chemical and structural analysis. Analytic size-exclusion chromatography indicated that HutD forms a dimer in the elution buffer. The crystal structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of one subdomain and two ß-barrel domains. Comparative structural analysis revealed a conserved binding pocket. The interaction of formate with a highly conserved residue Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with carboxylic group(s) such as histidine, urocanate or formyl-glutamate. The hypothesis that HutD functions via binding to urocanate, the hut inducer, was tested. Experiments using a thermal shift assay and isothermal titration calorimetry (ITC) analysis suggested that HutD binds to urocanate but not to histidine. However, the signal of HutD-urocanate binding was very weak and detected only at high urocanate concentration (53.23 mM), which is not physiologically relevant. The current data thus does not support the hypothesis of HutD-urocanate binding in vivo. Although the HutD-urocanate binding was not confirmed, this work has laid a solid foundation for further testing of the many alternative hypotheses regarding HutD function.
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Tao, Jing. "Treatment of sanitary sewer overflow using fixed media bioreactors." Columbus, Ohio : Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1228071779.
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Authimoolam, Sundar Prasanth. "BIOMIMETIC ORAL MUCIN FROM POLYMER MICELLE NETWORKS." UKnowledge, 2015. http://uknowledge.uky.edu/cme_etds/51.
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Mucin networks are formed by the complexation of bottlebrush-like mucin glycoprotein with other small molecule glycoproteins. These glycoproteins create nanoscale strands that then arrange into a nanoporous mesh. These networks play an important role in ensuring surface hydration, lubricity and barrier protection. In order to understand the functional behavior in mucin networks, it is important to decouple their chemical and physical effects responsible for generating the fundamental property-function relationship. To achieve this goal, we propose to develop a synthetic biomimetic mucin using a layer-by-layer (LBL) deposition approach. In this work, a hierarchical 3-dimensional structures resembling natural mucin networks was generated using affinity-based interactions on synthetic and biological surfaces. Unlike conventional polyelectrolyte-based LBL methods, pre-assembled biotin-functionalized filamentous (worm-like) micelles was utilized as the network building block, which from complementary additions of streptavidin generated synthetic networks of desired thickness. The biomimetic nature in those synthetic networks are studied by evaluating its structural and bio-functional properties. Structurally, synthetic networks formed a nanoporous mesh. The networks demonstrated excellent surface hydration property and were able capable of microbial capture. Those functional properties are akin to that of natural mucin networks. Further, the role of synthetic mucin as a drug delivery vehicle, capable of providing localized and tunable release was demonstrated. By incorporating antibacterial curcumin drug loading within synthetic networks, bacterial growth inhibition was also demonstrated. Thus, such bioactive interfaces can serve as a model for independently characterizing mucin network properties and through its role as a drug carrier vehicle it presents exciting future opportunities for localized drug delivery, in regenerative applications and as bio-functional implant coats.
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Sims, Lynn. "Biochemical Studies of ABCE1." Doctoral diss., University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5501.
Full textAbstract:
The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases >30 fold from ~30 [micro]M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.Ph.D.
Doctorate
Biology
Sciences
Biomedical Sciences
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Djerdjouri, Nour-Eddine. "Hydrogen peroxide delignification in a biomimetic system based on manganese peroxidase." Diss., Available online, Georgia Institute of Technology, 2005, 2003. http://etd.gatech.edu/theses/available/ipstetd-1017/.
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Thakkar, Jay. "Biochemical Evaluation of Lignin-like Molecules." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/199.
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Current anticoagulants carry a serious risk of bleeding complications. In addition, narrow therapeutic index, drug interactions, immunological reactions, toxicity and high cost to benefit ratio limits the effective use of these drugs in patients with thrombotic conditions.Heparin is the most widely used anticoagulant. We hypothesized that one of the major drawback of heparins, its non-specific interaction with the plasma proteins arises as a result of negative charges. To reduce these non-specific interactions, our laboratory designed sulfated low molecular weight lignin (LMWL) like biomacromolecules, which were found to be direct inhibitors of thrombin and factor Xa, acting through a unique exosite-2 mediated process. To elucidate the structural basis of this mechanism, we studied unsulfated and size fractionated LMWLs. Detailed enzyme inhibition studies with sulfated and unsulfated LMWLs of ferulic and caffeic acid oligomers revealed that sulfation was not absolutely critical for dual inhibition property and smaller oligomers can yield a potent anticoagulant. Mechanistically, unsulfated LMWLs retained exosite-2 mediated inhibition mechanism. A major advantage expected of the unsulfated LMWLs is the possibility that orally bioavailable anticoagulants may become possible.To identify target specific structures within the heterogeneous population of sulfated LMWLs, we prepared sulfated β-O-4-linked oligomer using chemical synthesis. Enzyme inhibition studies revealed that the sulfated β-O-4 LMWL were highly selective direct inhibitors of thrombin. These results show for the first time that specific structural features on LMWL scaffold dictate inhibition specificity. Studies in plasma and blood display highly promising anticoagulant profile for further studies in animals. To further study the LMWL scaffold as macromolecular mimetic of heparin; we investigated their effect in preventing cellular infection by herpes simplex virus-1 (HSV-1). Based on previous findings on sulfated lignins a size-dependent study on unsulfated LMWLs was done. The unsulfated lignins were found to not only inhibit HSV-1 entry into mammalian cells, but were more potent than sulfated lignins. Interestingly, shorter chains were found to be as active as the longer ones, suggesting that structural features, in addition to carboxylate groups, may be important. It can be expected that unsulfated lignins also antagonize the entry of other enveloped viruses, like HIV-1 and HCV that utilize heparan sulfate to gain entry into cells. The results further present major opportunities for developing lignin-based antiviral formulations for topical use.
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Zhou, Jin Chuan. "Biochemical characterisation of KDM2A." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:030faa1d-5d3c-4066-9e9f-a44cd13cf85c.
Full textAbstract:
Mammalian genomes are characterised by unique regions of non-methylated DNA known as CpG islands (CGIs). These genomic elements are characterised by a high density of CpGs and an elevated GC content compared to the surrounding, bulk of the genome. CGIs are prevalently associated with the 5’ end of genes and represent key nucleation sites where specific transcription factors and chromatin modifiers are recruited to impact on gene function. This thesis is focused at understanding the biochemical properties of the recently discovered H3K36-specific histone demethylase, KDM2A. This enzyme is specifically recruited to CGIs but how it interfaces with local chromatin in vivo remains unknown. Using defined chromatin templates in vitro, this study demonstrates that KDM2A binding to DNA relies on a zinc finger CXXC domain that preferentially recognizes non-methylated CpGs. In particular, nucleosomes represent a major barrier to KDM2A binding and chromatin substrates are interpreted by the CXXC domain through specific interaction with CpGs within linker DNAs. Moreover, the adjacent PHD domain does not contribute to KDM2A binding to chromatin. Together these observations suggest that sequence, methylation status and accessibility of DNA define how CGI chromatin is interpreted by CXXC domain proteins. In particular, the precise targeting of KDM2A to CGIs contributes to the creation of a unique chromatin architecture that highlights gene regulatory regions within large and complex mammalian genomes.
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Soda, Takahiro. "Converging biochemical pathways in psychiatric disorders." Thesis, Massachusetts Institute of Technology, 2012. http://hdl.handle.net/1721.1/73775.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2012.Cataloged from PDF version of thesis.
Includes bibliographical references.
According to the World Health Organization, neuropsychiatric diseases account for approximately one third of years lost to disability. Yet, despite this huge disease burden, there is a lack of new treatments under development: approved treatments all essentially target the same target(s), if the target itself is known. There is now considerable evidence for a common set of heritable risk for psychiatric disorders including schizophrenia, bipolar disorder, as well as autism. Many of these risk alleles affect genes implicated in neuronal development with known roles at an early stage; these genes would have an effect on the individual before the onset of overt symptoms or diagnosis. Furthermore, many of the genes identified are known to participate in established pathways that are relevant for neuronal development and function. It is important then to address the causality between these signaling pathways that are important for neurodevelopment, and the risk of developing neuropsychiatric disorder. The work presented in this thesis represents two projects that aim to work toward this goal. The first project pertains to the mechanisms of transcriptional repression by DISC1 on ATF4-mediated gene transcription. The second project presents some initial steps towards uncovering the role of BCL9 in neuronal development.
by Takahiro Soda.
Ph.D.
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Pooler, Amy Melissa. "Regulation of biochemical pathways involved in neurodegeneration." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/31177.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Brain and Cognitive Sciences, 2005.Includes bibliographical references.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss. Although much is known about how AD affects the brain, the cause of this disease remains elusive. Current AD treatments target symptoms of the disease but do not prevent or slow the underlying neurodegeneration. Therefore, research into the biochemical mechanisms of AD is necessary in order to develop a better understanding of how to treat it. Misprocessing of the amyloid precursor protein (APP) in the brains of AD patients leads to accumulation of the amyloidogenic peptide AD. A soluble APP fragment (APPS) is formed when APP is cleaved within the AP region, thereby preventing AP formation. Activation of 5-HT2A or 5-HT2c receptors has been shown to increase APPS secretion in vitro; therefore, we determined whether activation of these receptors might have a similar effect in vivo. We found that a 5-HT2A/2c agonist affected brain APP metabolism in guinea pigs by increasing CSF levels of APPS and, following chronic treatment, by decreasing levels of AP. Our data indicate that activation of brain 5-HT2c receptors may be useful for treating AD by reducing AP production. Traumatic brain injury is a risk factor for AD, although the reason is unknown. To explore this relationship, we examined the effect of the inflammatory mediator PGE2 on production of APP in cultured microglia. We found that PGE2 treatment stimulated APP overexpression and that this effect was likely mediated by the prostaglandin EP2 receptor and the cAMP signaling cascade. Therefore, EP2 receptor antagonists may constitute an additional target for prevention of AD following brain injury.
(cont.) The neuropathology associated with AD includes neuritic dystrophy and degeneration. Therefore, restoration of neuritic growth and repair of phospolipid membranes may be important for treating AD. We found that treatment of NGF- differentiated PC 12 cells with the phospholipid precursor uridine enhanced neurite outgrowth by both enhancing phosphatide biosynthesis and by stimulating a G-protein receptor-coupled signaling pathway. Subsequently, we found that the HMG-CoA reductase inhibitor pravastatin enhanced neurite outgrowth in rat hippocampal neurons, not by affecting cholesterol synthesis, but by inhibition of isoprenoid formation. Stimulation of neurite growth by either uridine or statins may reduce AD risk by averting neuritic dystrophy and degeneration. However, further studies must be conducted to determine whether they are able to affect neuritic processes in vivo.
by Amy Melissa Pooler.
Ph.D.