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Building Platforms to Genetically Encode New ChemistryJohnson, Alexander M. January 2017 (has links)
Thesis advisor: Abhishek Chatterjee / Abstract Unnatural amino acid (UAA) incorporation is a powerful tool used by biochemists to discover the nature of protein structure and function. The evolution of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs enables site-specific incorporation of UAAs proteins inside of living cells. The goal of this study was to further expand the repertoire of genetically encoded unnatural amino acids in E. coli as well as eukaryotes. We first attempted to engineer an aaRS, previously evolved for p-borono-phenylalanine (pBoF), to specifically charge 3-acetyl-p-borono-phenylalanine (AcpBoF). A randomized library of the pBoF-specific synthetases was generated and it was subjected to established selection schemes in a bacterial host. This report also describes the development of a yeast-based selection system to alter the substrate specificity of bacterial leucyl-tRNA synthetase, for genetic code expansion in eukaryotes. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
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A methodology in predicting protein tertiary structure.January 1993 (has links)
by Li Leung Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 76-81). / Acknowledgements / Abstract / Chapter 1. --- Protein modeling --- p.1 / Chapter 1.1 --- Genetic Engineering --- p.1 / Chapter 1.2 --- Protein Engineering --- p.2 / Chapter 1.2.1 --- The basic concept --- p.2 / Chapter 1.2.2 --- The importance of protein modeling --- p.3 / Chapter 1.2.3 --- Applications --- p.4 / Chapter 1.2.3.1 --- Industry --- p.4 / Chapter 1.2.3.2 --- Medicine --- p.4 / Chapter 1.3 --- The structure of protein molecule --- p.5 / Chapter 2. --- About this thesis --- p.8 / Chapter 2.1 --- Methods on protein tertiary structure prediction --- p.8 / Chapter 2.1.1 --- Energy minimization method --- p.9 / Chapter 2.1.2 --- Sequence homology method --- p.9 / Chapter 2.1.3 --- Hierarchical assembly method --- p.11 / Chapter 2.2 --- Artificial Intelligence and molecular modeling --- p.11 / Chapter 2.3 --- Computer graphics and molecule display --- p.13 / Chapter 2.3.1 --- Molecular model in computer graphics --- p.13 / Chapter 2.3.2 --- Interactive graphic operations --- p.16 / Chapter 2.4 --- The objective of this thesis --- p.17 / Chapter 3. --- Algorithms for protein secondary structure prediction --- p.20 / Chapter 3.1 --- Hydrophobicity --- p.20 / Chapter 3.2 --- Algorithms for protein secondary structure prediction --- p.22 / Chapter 3.2.1 --- The Chou and Fasman method --- p.23 / Chapter 3.2.1.1 --- Method --- p.24 / Chapter 3.2.1.2 --- Results --- p.25 / Chapter 3.2.2 --- The GOR method --- p.26 / Chapter 3.2.2.1 --- Theory --- p.26 / Chapter 3.2.2.2 --- Method and results --- p.26 / Chapter 3.3 --- A proposed algorithm --- p.28 / Chapter 3.3.1 --- Procedure of our algorithm --- p.30 / Chapter 4. --- A protein tertiary structure prediction method --- p.31 / Chapter 4.1 --- The linkage between two amino acids --- p.32 / Chapter 4.2 --- Rotation angle between two peptide planes --- p.34 / Chapter 4.2.1 --- Helical structure --- p.35 / Chapter 4.2.1.1 --- Concept --- p.35 / Chapter 4.2.1.2 --- Procedure --- p.36 / Chapter 4.2.2 --- Sheet structure --- p.37 / Chapter 4.2.3 --- Turn structure --- p.38 / Chapter 4.2.4 --- Anti-parallel sheet and turn structure --- p.40 / Chapter 4.3 --- Random factor in rotation angle of peptide planes --- p.41 / Chapter 4.4 --- Atomic size --- p.41 / Chapter 4.5 --- Tertiary structure prediction algorithm --- p.42 / Chapter 5. --- Implementation --- p.45 / Chapter 5.1 --- Hardware --- p.45 / Chapter 5.2 --- User-defined data types and data structures --- p.46 / Chapter 5.3 --- Technique in molecule displaying --- p.48 / Chapter 5.4 --- Image processing --- p.50 / Chapter 5.5 --- Options in our program --- p.52 / Chapter 5.6 --- Steps in protein tertiary structure prediction --- p.54 / Chapter 6. --- Results --- p.59 / Chapter 6.1 --- The results of protein secondary structure prediction --- p.59 / Chapter 6.2 --- The results of protein tertiary structure prediction --- p.66 / Chapter 7. --- Conclusion --- p.70 / Chapter 7.1 --- Comments on protein secondary structure prediction algorithm --- p.70 / Chapter 7.1.1 --- Advantages and disadvantages --- p.70 / Chapter 7.1.2 --- Further development --- p.71 / Chapter 7.2 --- Discussion on X-ray crystallographic data --- p.72 / Chapter 7.3 --- Comments on the protein tertiary structure prediction algorithm --- p.73 / Chapter 7.3.1 --- Advantages and disadvantages --- p.73 / Chapter 7.3.2 --- Further development --- p.74 / Chapter 7.3.2.1 --- Rotation angle between two peptide planes --- p.74 / Reference --- p.76 / Glossary --- p.82 / Appendix A An algorithm to determine hydrophobic value --- p.83 / Appendix B Chou and Fasman algorithm --- p.84 / Appendix C GOR algorithm --- p.87 / Appendix D Shading algorithm --- p.88
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Tryptophan Supplementation During Lactation on Sow ProductivityVarvel, Hallie Johnson 01 August 2019 (has links)
The objective of this study was to examine the potential effects of supplementing excess crystalline tryptophan (trp) in the lactation diets of sows. Sixty-one sows of varying parity were fed either a control diet (0.26% trp) or a treatment diet with an extra two grams of tryptophan (0.30% trp). Over the 28 day lactation period utilized by the production site, sow and litter performance were recorded. Sow performance was measured by backfat loss, blood urea nitrogen, milk composition, return to estrus, wean to estrus interval, and conception rate. Litter performance was measured as average weaning weight, number weaned, and pre-wean mortality. The control and treatment groups were further subdivided by parity for statistical analysis. Sows of parity one and two were classified as primiparous, while sows of parity three or more were classified as multiparous. There were no significant differences (P≤0.05) between the control and treatment diets even with regards to parity groups. There was one trend (0.05
0.10) in which treatment multiparous sows had higher litter weaning weight (P=0.055) than the control multiparous sows. In summary, these results indicate that increasing the tryptophan level in this lactation diet by two grams did not significantly influence sow or litter performance.
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The Photoregulation of Phenylpropnaoid Metabolism and Amino Acid Accumulation in Triticum Aestivum (Var. Fremont)Guerra, Daniel J. 01 May 1983 (has links)
Wheat (Triticum aestivum L.) grown in controlled environments with a 24-hour photoperiod was analyzed for phenylpropanoid and amino acid metabolites. Discrete spectral environments, including a metal halide, high-pressure sodium and low-pressure sodium lamps, provided both photosynthetically active radiation and phenylpropanoid inducing fluences of light . A greenhouse spectral environment supplemented with fluorescent lamps was also used to culture wheat . All. four spectral envi ronments were used to culture wheat to maturity separately. The activities of phenylalanine and tyrosine ammonia-lyase were photoinduced in wheat tissue obtained from plants grown in the metal halide, high -pressure sodium and greenhouse spectral environments. These enzyme activities are the committed catalytic step in phenylpropanoid biosynthesis and were induced in wheat tissue by fluences of light in the ultraviolet and blue regions of the spectrum. The low-pressure sodium lamp , which does not provide strong irradiance in these wavelengths , produced significantly lower ammonia-lyase activities than were observed in wheat grown within the metal halide , high-pressure sodium, or greenhouse spectral environments. These effects were not caused by phytochrome, since calculation of PfrfPtotal for the low-pressure sodium lamp was higher than the ratio obtained from metal halide or high-pressure sodium lamps. Lignin was also significantly reduced in wheat grown with low-pressure sodium lamps . Several essentialamino acids were in lower molar concentration in protein from wheat grown under low-pressure sodium lamps. However, phenylalanine and tyrosine were in significantly higher concentration in wheat grain produced in this spectral environment, and amino acid concentrations of wheat cultured with the low-pressure sodium lamp are regarded as a direct result of the spectral properties of this light source.
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Amino acid platinum(II) complexes : synthesis, characterisation and coupling to porphyrinsBond, Jacquline, University of Western Sydney, Faculty of Informatics, Science and Technology January 2000 (has links)
The study of cancer plays an important role in modern medical science. Over the years, a lot has been learnt about the properties and treatment of cancer cells. Despite the remarkable progress made in understanding the genesis of cancer, the work so far has had very little impact in the clinic especially the design of new and improved drugs. Platinum-based drugs such as cis-diamminedichloroplatinum(II) and its anolgue, carboplatin, are the most effective chemotherapeutic agents used in the treatment of testicular, ovarian, bladder and lung cancers. Nevertheless, the emergence of toxic side-effects compromises its clinical effectiveness. It is generally agreed that most of the toxic effects of platinum-based drugs arise from their lack of selectivity. This thesis reports on the development of new platinum(II) complexes bound to carrier molecules with the hope of obtaining compounds which display the cytotoxic effects only in tumour tissue. In addition, some information is included about what is known about the causes of cancer, how it kills and the current methods of treatment / Doctor of Philosophy (PhD)
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Evaluation of the threonine requirement and the bioavailability of threonine in feedstuffs in pregnant sowsLevesque, Crystal 11 1900 (has links)
Current recommendations for amino acid intake during pregnancy are for a constant amino acid intake throughout. However, the demand for amino acids changes from maternal tissue growth in early gestation to fetal, conceptus and mammary tissue development in late gestation. The availability of amino acids from feed ingredients are based on growing pig data, although recent evidence suggests that mature animals have a greater capacity to digest and absorb amino acids. Therefore, this thesis investigated the threonine requirement of sows in gestation and the availability of threonine (Thr) in common feed ingredients fed directly to sows using the indicator amino acid oxidation technique.
The Thr requirement in early gestation was determined to be 5.0 to 6.0 g/d, at least 40% below current recommended Thr requirements, whereas the requirement for Thr in late gestation was determined to be 12.3 to 13.6 g/d, close to 30% above current recommendations. These results suggest that current sow feeding recommendations (i.e. constant level of AA throughout gestation) result in over- and under-feeding AA in early and late gestation, respectively. The metabolic availability of Thr in corn and barley fed to growing pigs was 82.2 and 115.3%, respectively, whereas when fed to pregnant sows, the metabolic availability of Thr in corn and barley was 88.0 and 89.3%, respectively. The > 100% availability of Thr from barley was likely due to the effect of barley on the demand for Thr for production of mucin and mucous proteins. The results indicate that the availability of amino acids from feed ingredients is greater when fed to sows than when fed to growing pigs.
In conclusion, current sow amino acid requirement recommendations do not appropriately reflect actual amino acid demand during pregnancy. The deficiency in dietary amino acids during late gestation may result in maternal lean tissue catabolism to support fetal growth. The greater availability of amino acids from feed ingredients in sows may reduce the degree of amino acid deficiency in late gestation under current feeding programs. Application of phase feeding sows during pregnancy will more closely meet the demand for amino acids and may improve sow reproductive longevity. / Nutrition and Metabolism
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Amino acid signalling in yeast: functional analysis of the Stp transcription factorsWielemans, Kevin 05 October 2010 (has links)
The whole genome duplication (WGD) event is an intriguing mechanism from an evolutionary perspective. Such an event may be the source of new genes, functions or species. Traces of WGD event have been detected in the genome of all four eukaryotic kingdoms: plants, animals, fungi and protists. In fungi, an ancestor of Saccharomyces cerevisiae underwent an event of WGD, about 100 million years ago, after diverging from the Kluyveromyces lineage. In S. cerevisiae, only ten percent of the resulting duplicated genes survived as duplicates. In particular, some of these duplicates encodes for transcription factors in several nutrient sensing pathways.
The main subject of this thesis’s work is the external amino acid sensing system in S. cerevisiae. The detection of extracellular amino acids in yeast begins with a transporter homologue devoid of any uptake activity, the Ssy1 sensor. The binding of extracellular amino acids to Ssy1 leads to the successive activation of Ptr3 and the Ssy5 endoprotease. This endoporotease catalyses the processing of two transcriptions factors: Stp1 and Stp2. The Stp factors, released from their N-terminal cytoplasmic-anchored domains, are then translocated into the nucleus, where they activate the transcription of several amino acid permease genes (e.g. AGP1 and DIP5). Starting this work, the Stp factors were considered as functionally redundant.
We first determined that the STP1 and STP2 genes derivate from the event of WGD. The conservation of these two genes in S. cerevisiae was accompanied by a functional divergence of their products at several levels: processing sensibility, transcriptional activation capacity, target genes, cellular abundance level and stability. The Stp2 factor with its high abundance in the cells and its higher Ssy5-processing sensibility is specialized towards induction of the AGP1 gene when the external amino acid signaling is weakly stimulated. Under strong stimulation conditions, the amino acids induce cleavage-triggered destabilization of Stp2 through the proteasomal pathway and the induction of AGP1 is mediated mainly by the Stp1 transcription factor. Unlike Stp2, the Stp1 factor is characterized by its high transcriptional activation capacity and weaker sensitivity towards Ssy5-processing. The Stp factors differ also by their genetic targets. Indeed, only Stp2 regulates the expression of DIP5. Finally, we determined that the processing sensibility and the transcriptional activation capacity of each Stp factors is directly linked to their N- and C-terminal domains, respectively.
The phosphorylation states and the degradation of the Stp2 factor were also examined. The event of degradation concerns only the processed forms of this factor and takes places principally in the nucleus. Some data indicate that such an event might be important to limit the activation capacity of this factor. The role of the Stp2 phosphorylation in the external amino acid signaling pathway is still unknown but this event might be important for the Stp2 degradation or its transcriptional activity.
The unique Stp factor from Kluyveromyces lactis (Kl-Stp), a pre-WGD species, was also studied. The Kl-Stp factor shares at least two characteristic with the S. cerevisiae Stp2 factor: high sensibility towards processing and high levels of degradation. This observation leads us to conclude to that the STP genes may have been conserved after WGD though a mechanism called neofunctionalization (one of the duplicate obtained after duplication retains the ancestral function while the other evolves to perform a novel function).
Finally, a new model for the external amino acid signaling pathway that brings together all the data obtained during this thesis’s work is proposed.
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Hydrogen Bond-directed Stereospecific Interactions in (A) General Synthesis of Chiral Vicinal Diamines and (B) Generation of Helical Chirality with Amino AcidsKim, Hyunwoo 15 September 2011 (has links)
Hydrogen bonding interactions have been applied to the synthesis of chiral vicinal diamines and the generation of helical chirality. A stereospecific synthesis of vicinal diamines was developed by using the diaza-Cope rearrangement reaction driven by resonance-assisted hydrogen bonds (RAHBs). This process for making a wide variety of chiral diamines requires only a single starting chiral diamine, 1,2-bis(2-hydroxyphenyl)-1,2-diaminoethane (HPEN) and aldehydes. Experimental and computational studies reveal that this process provides one of the simplest and most versatile approaches to preparing chiral vicinal diamines including not only C2 symmetric diaryl and dialkyl diamines but also unsymmetrical alkyl-aryl and aryl-aryl diamines with excellent yields and enantiopurities.
Weak forces affecting kinetics and thermodynamics of the diaza-Cope rearrangement were systematically studied by combining experimental and computational approaches. These forces include hydrogen bonding effects, electronic effects, steric effects, and oxyanion effects.
As an example of tuning diamine catalysts, a vicinal diamine-catalyzed synthesis of warfarin is described. Detailed mechanistic studies lead to a new mechanism involving diimine intermediates. Decreasing the NCCN dihedral angle by varying the diamine structure results in an increase of the enantioselectivity up to 92% ee.
Hydrogen bonds have been used to generate helical chirality in a highly stereospecific manner with a single amino acid and 2,2′-dihydroxybenzophenone. DFT computational and experimental data including circular dichroism (CD), X-ray crystallography and 1H NMR data provide insight into the origin of the stereospecificity. A signalling dizao group can be attached to the receptor for general sensing of amino acid enantiopurity.
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Structural and Functional Insights on Regulation by Phenolic CompoundsShahinas, Dea 26 February 2009 (has links)
The shikimate pathway is a primary metabolic pathway involved in the synthesis
of aromatic compounds in plants, fungi, apicomplexan parasites and microbes.
The absence of this pathway in animals makes it ideal for the synthesis of
antimicrobial compounds and herbicides. Additionally, its branching into indole
hormone synthesis and phenylpropanoid secondary metabolism makes this
pathway attractive for metabolic engineering. Here, the focus is on the first step
of the shikimate pathway catalyzed by DAHP synthase. This step consists of the
condensation of phosphoenol pyruvate and erythrose-4-phosphate to make
DAHP, which undergoes another six catalytic steps to synthesize chorismate, the
precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP
synthase isozymes, which are known to indirectly regulate downstream pathways
in response to wounding and pathogen stress. The model presented here
proposes that DAHP synthase isozymes are regulated by the end products
tyrosine, tryptophan and phenylalanine.
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Structural and Functional Insights on Regulation by Phenolic CompoundsShahinas, Dea 26 February 2009 (has links)
The shikimate pathway is a primary metabolic pathway involved in the synthesis
of aromatic compounds in plants, fungi, apicomplexan parasites and microbes.
The absence of this pathway in animals makes it ideal for the synthesis of
antimicrobial compounds and herbicides. Additionally, its branching into indole
hormone synthesis and phenylpropanoid secondary metabolism makes this
pathway attractive for metabolic engineering. Here, the focus is on the first step
of the shikimate pathway catalyzed by DAHP synthase. This step consists of the
condensation of phosphoenol pyruvate and erythrose-4-phosphate to make
DAHP, which undergoes another six catalytic steps to synthesize chorismate, the
precursor of the aromatic amino acids. Arabidopsis thaliana contains three DAHP
synthase isozymes, which are known to indirectly regulate downstream pathways
in response to wounding and pathogen stress. The model presented here
proposes that DAHP synthase isozymes are regulated by the end products
tyrosine, tryptophan and phenylalanine.
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