Thesis advisor: Steven D. Bruner / The enediyne antitumor antibiotics are produced by complex biosynthetic machinery in acetomycetes. This dissertation will focus on the study of three enzymes involved in key steps in the biosynthesis of two enediynes, neocarzinostatin and C-1027. Neocarzinostatin is biosynthesized by a number of enzymes that synthesize and decorate the enediyne core and the peripheral moieties. NcsB1 is one enzyme involved in functionalizing the naphthoic acid portion of neocarzinostatin, a key group involved in binding to target DNA duplexes. The enzyme has been shown to be a promiscuous (<italic>S</italic>)-adenosylmethionine-dependent <italic>O</italic>-methyltransferase responsible for methylating a variety of hydroxynaphthoic acids. Multiple crystal structures of NcsB1 cocomplexed to substrate and/or cofactor have been solved. These structures revealed a displacement of the C-terminal domain when not bound to substrate, a movement that likely opens up the active site for naphthoate binding. Additionally, the ternary complex structure of 1,4-dihydroxynaphthoic acid, (<italic>S</italic>)-adenosylhomocysteine, and NcsB1 was solved and showed a rotation of this alternate substrate in the binding pocket, allowing for methylation. These results led us to probe NcsB1 activity using active site mutants, demonstrating altered substrate specificity and revealing key residues in substrate binding. The final step of neocarzinostatin biosynthesis involves multiple enzymes that convergently assemble the multiple biosynthetic intermediates to form the chromophore. NcsB2, originally proposed to catalyze the attachment of the naphthoic acid moiety to the enediyne core, has been characterized <italic>in vitro</italic>. Studies into its substrate specificity as an adenylation domain led to a revised biosynthetic pathway of 2-hydroxy-7-methoxy-5-methyl naphthoic acid. Instead of catalyzing the attachment of an enzyme bound naphthoic acid to the enediyne core, NcsB2 was found to act as a CoA-ligase, activating a variety of naphthoic acids and forming a naphthoyl-CoA intermediate. The results of these studies present an outstanding opportunity to produce novel analogs of neocarzinostatin by manipulating its biosynthesis. C-1027 is an architecturally similar enediyne that is also biosynthesized in a convergent route. C-1027 is a member of a class of enediynes that contains a functionalized &beta;-tyrosine derived from L-tyrosine. The first catalytic step towards this beta-tyrosine moiety is achieved by <italic>Sg</italic>TAM, a tyrosine aminomutase that catalyzes a 2,3-amino shift on L-tyrosine to form (<italic>S</italic>)-&beta;-tyrosine. The first X-ray crystal structure of <italic>Sg</italic>TAM was recently solved by our group, revealing structural homology to ammonia lyases. Through site-directed mutagenesis, X-ray crystallography, and biochemical analysis, residues that influence the mechanism by which <italic>Sg</italic>TAM catalyzes this difficult transformation were explored. From these studies, the enzymatic base and other pertinent residues involved in catalysis have been identified. In addition, residues that close the tunnel leading to the active site, thought to play a key role in mutase activity, were probed. Further study of rational mutants of <italic>Sg</italic>TAM will allow us to engineer its activity to alter its substrate specificity and the type of product it produces. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Steven D. Bruner / Thesis advisor: Mary Roberts / The dioxygenase DpgC performs a key step in the biosynthesis of 3,5-dihydroxyphenylglycine (DPG), a nonproteogenic amino acid found in the vancomycin family of antibiotics. Remarkably, DpgC performs a 4-electron oxidation without the use of metals or cofactors. The tools of synthetic organic chemistry, enzymology and structural biology were used to study this enzyme. We have solved the first structure of an enzyme of this oxygenase class, in complex with a bound substrate mimic. The structure confirms the absence of cofactors, and electron density consistent with molecular oxygen is located adjacent to the site of oxidation on the substrate. The use of a designed, synthetic substrate analog allowed us to gain unique insights into the chemistry of oxygen activation. We systematically probed the importance of active site residues by engineering conservative changes using site-directed mutagenesis. The kinetic parameters of these constructs imply that the phenolic hydroxyls of the substrate are of particular importance. These conclusions were verified by kinetic evaluation of synthetic substrate analogs. We have synthesized cyclopropyl substrate derivatives to probe the electron transfer step. The single electron oxidation should produce a radical species capable of opening the cycloproyl ring, thus providing a handle of detection. Our results resolve the unique and complex chemistry of DpgC, a key enzyme in the biosynthetic pathway of an important class of antibiotics. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

A dissertation submitted to the Faculty of Science of the University of Witwatersrand, Johannesburg, in full fulfilment of the requirements for the degree of Master of Science. May 2017 / Nanomaterials (NMs) have been shown to exhibit unique physical and chemical properties that are highly size and shape-dependent. The ability to control synthesis of nanoparticles (NPs) with particular shapes and sizes can lead to exciting new applications or enhancements of current systems in the fields of optics, electronics, catalytics, biomedicine and biotechnology. Due to increased chemical pollution as well as health concerns, biological synthesis of NMs has quickly emerged as potentially being an eco-friendly, scalable, and clean alternative to chemical and physical synthesis. In this study, the inference that the heavy metal-resistant bacteria, Paenibacillus castaneae, has the propensity to synthesize metal NPs was validated. NP formation was achieved after the exposure of bacterial cell biomass or cell-free extracts (CFE) to excess metal ion precursors in solution. These include lead nitrate and calcium sulphate dehydrate, gold (III) chloride trihydrate and silver nitrate, respectively. All reactions were incubated at 37 °C for 72 h at 200 rpm and observed for a colour change. UV–visible (UV-Vis) spectral scans (200 nm – 900 nm) were measured on a Jasco V-630 UV-Vis spectrophotometer. For scanning electron microscopy (SEM), samples were fixed, dehydrated and loaded onto carbon-coated aluminium stubs. The stubs were then sputter-coated with either Au/Pd or Cr and analysed on the FEI Nova Nanolab 600 FEG-SEM/FIB. Size distribution analysis was done using transmission electron microscopy (TEM) using the FEI Tecnai T12 TEM and Image J software. Powder X-ray diffraction measurements were carried out on a Rigaku Miniflex-II X-ray diffractrometer. Colour changes indicative of the synthesis of PbS, Au and Ag NPs were observed as a white precipitate (PbS), purple (Au) and yellow-brown (Ag) colour, respectively. This was confirmed by absorbance peaks at 325 nm and 550 nm (PbS), 595 nm (Au) and 440 nm (Ag) from UV-Vis analyses. Exposure of P. castaneae biomass and CFE to PbS ions in solution resulted in the production of nanospheres, irregularly-shaped NPs, nanorods, nanowires as well as large nanoflowers. Exposure of P. castaneae biomass to Au3+ ions in solution produced Au nanospheres, nanotriangles, nanohexagons, nanopentagons and nanopolyhedrons. Ag/AgCl NP production occurred using both the P. castaneae biomass and CFE, and resulted in the synthesis of nanospheres only. This is the first report of the biosynthesis of such a diverse set of anisotropic NPs by P. castaneae. It is also the first instance in which anisotropic PbS nanorods and nanowires, 3-D Au nanoprisms as well as “rough” Ag/AgCl nanospheres were bacterially produced. This study serves as an eco-friendly approach for the synthesis of NPs that is a simple yet amenable method for the large-scale commercial production of nanoparticles with technical relevance. This in turn expands the limited knowledge surrounding the biological synthesis of heavy metal NMs. / MT 2017

Orientador: Sandro Roberto Valentini / Coorientador: Cleslei Fernando Zanelli / Banca: Mário Henrique Bengtson / Banca: Carla Columbano de Oliveira / Resumo: O fator de tradução de eucariotos 5A (eIF5A) é altamente conservado em arqueas e eucariotos e sofre uma modificação pós-traducional única e essencial chamada hipusinação. Embora tenha sido sugerida inicialmente uma função para eIF5A no início da tradução, foi na etapa de elongação que sua função foi melhor demonstrada. Estudos prévios mostraram que eIF5A tem algum papel na translocação de proteínas pela via co-traducional e que mutantes desse fator possuem níveis aumentados de proteínas envolvidas com o estresse de retículo endoplasmático (RE). Desta forma, nesse trabalho, foi estudada a correlação entre eIF5A e a via de resposta ao estresse de RE (Unfolded Protein Response - UPR). Considerando que o fator de transcrição Hac1 é um elemento essencial da via UPR em Saccharomyces cerevisiae, foi inicialmente verificado se eIF5A afetava o splicing citoplasmático do mRNA de HAC1, o qual é dependente da ativação de Ire1, um sensor de proteínas desenoveladas no interior do RE. Para isto, foi realizada a quantificação dos níveis de mRNA maduro e imaturo de HAC1 por meio de ensaios de qPCR e RT-PCR semiquantitativa, análises estas que não revelaram qualquer diferença de comportamento do mutante hyp2-3 de eIF5A em relação ao selvagem. Por outro lado, diferentes mutantes de eIF5A mostraram sensibilidade a DTT e resistência a tunicamicina, um comportamento ainda não descrito na literatura e que difere de mutantes da via de SRP (via co-traducional de translocação de proteínas para o RE). ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The eukaryotic translation factor 5A (eIF5A) is highly conserved in archaea and eukaryotes and undergoes a unique and essential post-translational modification called hypusination. Although a function for eIF5A was initially suggested at the initiation of translation, it was in the elongation step that its function was best demonstrated. Previous studies have shown that eIF5A has a role in protein translocation by the co-translational pathway and that mutants of this factor have increased levels of proteins involved with endoplasmic reticulum (ER) stress. Therefore, in this work, the correlation of eIF5A with the stress response pathway of ER (the Unfolded Protein Response - UPR) was studied. Considering that the Hac1 transcription factor is an essential element of the UPR pathway in Saccharomyces cerevisiae, it was first verified whether eIF5A affects the cytoplasmic splicing of the HAC1 mRNA, which is dependent on the activation of Ire1, a sensor of unfolded proteins in the RE. To test it, we did the quantification of the mature and immature mRNA levels of HAC1 by means of qPCR and semi-quantitative RT-PCR assays, which did not reveal any difference in the behavior of the eIF5A mutant hyp2-3 in comparison to the wild-type. On the other hand, different mutants of eIF5A show sensitivity to DTT and resistance to tunicamycin, a behavior not yet described in the literature and that differs from mutants of the SRP pathway (co-translocation pathway for RE) and also this behavior of eIF5A mutants was not modified by overexpression of SRP. In addition, the eIF5A mutant phenotype in DTT and tunicamycin differs from that of knockout strains of ribosomal proteins, knockout of a ribosome biogenesis factor... (Complete abstract click electronic access below) / Mestre

Experimental studies indicate that culturing chondrocytes on biodegradable polymeric scaffolds may yield“engineered" cartilage for the replacement of tissue lost to injury or diseases such as osteoarthritis. A method of estimating the outcome of cell-polymer cultures would aid in the design and evaluation of engineered tissue for therapeutic use. The goals of this project were to develop, validate, and apply first-generation mathematical models that describe the kinetics of extracellular matrix (ECM) deposition and scaffold degradation in cell-polymer constructs cultured in vitro. The ECM deposition model is based on a product-inhibition mechanism and predicts an asymptotic, exponential increase in the concentration of ECM molecules found in cartilage, including collagen and glycosaminoglycans (GAG). The scaffold degradation model uses first-order kinetics to describe the hydrolysis of biodegradable polyesters in systems not limited by diffusion. Each model was fit to published data describing the accumulation of GAG and collagen, as well as the degradation of poly glycolic acid (PGA) and poly lactic acid (PLA), respectively. As experimental validation, cell-polymer constructs (n = 24) and unseeded scaffolds (n = 24) were cultured in vitro, and biochemical assays for GAG and collagen content, as well as scaffold mass measurements, were performed at 1, 2, 4, 6, 8, or 10 weeks of culture (n = 8 per time point). The mathematical models demonstrate a moderate to strong goodness of fit with the previously published data and our experimental results (R2=0.75-0.99). These models were also combined to predict the temporal evolution of total construct mass with reasonable accuracy (30% RMS deviation). In ongoing work, estimates of biochemical composition derived from these models are being proposed to predict the mechanical properties and functionality of the constructs. This modeling scheme may be useful in elucidating more specific mechanisms governing ECM accumulation. Given their potential predictive power, these models may also reduce the cost of performing long-term culture experiments.

The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H2O2), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H2O2 treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Melanin was shown to play no role in the protection of the pathogen from immune cell recognition and killing by alveolar macrophages, with similar degrees of engulfment, and spore viabilities, of mutant and wild-type strains after phagocytosis. Contrary to expectations, the albino PKS-deficient mutant was significantly more virulent than the melanised wild-type strain during pathogenicity studies in the invertebrate infection model Galleria mellonella, with levels of virulence restored to near wild-type levels in the complemented strain ΔLppks1::hph:PKS. Taken together, the results presented in this thesis show that melanin protects L. prolificans from UV radiation and from oxidative killing by H2O2, consistent with its survival in extreme environmental habitats. However, melanin was 3 not found to play a role in the resistance of the pathogen to the antifungal drug amphotericin B, to protect the fungus from immune cell recognition or killing by alveolar macrophages, or to its pathogenicity.

by Li Tin Yau. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 56-60). / Acknowledgment --- p.i / Table of Contents --- p.ii / Abstract --- p.iii / Abbreviation --- p.iv / Chapter I --- Introduction --- p.1 / Chapter I-1 --- General Background --- p.1 / Chapter I-2 --- Mechanistic Aspect of Glycosidase Inhibition --- p.4 / Chapter I-3 --- Previous Syntheses of Valienamine and its Diastereomers --- p.7 / Chapter II --- Results and Discussion --- p.16 / Chapter II-1 --- General Strategy --- p.16 / Chapter II-2 --- Synthesis of Tribenzyl ether69 --- p.18 / Chapter II-3 --- Synthesis of Diol67 --- p.20 / Chapter II-4 --- "Synthesis of N,O,O, O, O-Pentaacetylvalienamine30 and its 2-Epimer62" --- p.22 / Chapter II-5 --- Synthesis of Diacetate66 --- p.26 / Chapter II-6 --- Synthesis of the Valienamine Derivatives 63 and64 --- p.30 / Chapter III --- Conclusion --- p.34 / Chapter IV --- Experimental --- p.36 / Chapter V --- Reference --- p.56

Cui, Yong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 73-84). / Abstracts in English and Chinese. / Thesis/Assessment Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vi / Table of Contents --- p.vii / List of Figures --- p.xi / List of Supplemental Tables --- p.xiii / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- The plant secretory and endocytosis pathways --- p.2 / Chapter 1.2 --- Rab proteins --- p.4 / Chapter 1.2.1 --- Overview of the small GTPases --- p.4 / Chapter 1.2.2 --- Function of Rab proteins in Arabidopsis --- p.6 / Chapter 1.3 --- Prevacuolar compartments --- p.9 / Chapter 1.3.1 --- PVCs in mammalian and yeast cells --- p.9 / Chapter 1.3.2 --- PVCs in plant cells --- p.9 / Chapter 1.4 --- Vacuolar Sorting Receptors --- p.10 / Chapter 1.5 --- Project objectives --- p.10 / Chapter CHAPTER 2 --- Early and Late Prevacuolar Compartments in Arabidopsis thaliana Cells --- p.12 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.2 --- MATERIALS AND METHODS --- p.19 / Chapter 2.2.1 --- Plasmid Construction --- p.19 / Chapter 2.2.2 --- Plants materials and growth conditions --- p.19 / Chapter 2.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.20 / Chapter 2.2.4 --- Confocal imaging studies --- p.21 / Chapter 2.3 --- RESULTS --- p.23 / Chapter 2.3.1 --- Organelle markers serve as a tool to study biogenesis and turnover of PVCs --- p.23 / Chapter 2.3.2 --- AtRab5 and AtRab7 proteins show distinct but closely associated patterns in the PVC-to-Vacuole pathway --- p.26 / Chapter 2.3.3 --- AtRab5 and AtRab7 proteins localize on the distinct organellein Arabidopsis thaliana protoplasts --- p.32 / Chapter 2.3.4 --- AtRab5 proteins are closely associated with AtRab7 proteins --- p.35 / Chapter 2.3.5 --- ARA7-Q69L proteins recruit a SNARE complex onto the enlarged PVCs --- p.37 / Chapter 2.4 --- Discussion --- p.40 / Chapter 2.4.1 --- PVC dynamics in Arabidopsis cells --- p.40 / Chapter 2.4.2 --- AtVSR and its point mutation form defined different stages of PVCs in Arabidopsis thaliana protoplasts --- p.41 / Chapter 2.4.3 --- AtRab7 proteins localized on the tonoplast and newly defined late PVCs --- p.41 / Chapter CHAPTER 3 --- AtRab7 proteins play a critical role in mediating vacuolar trafficking in Arabidopsis thaliana Cells --- p.43 / Chapter 3.1 --- Introduction --- p.44 / Chapter 3.2 --- MATERIALS AND METHODS --- p.45 / Chapter 3.2.1 --- Plasmid Construction --- p.45 / Chapter 3.2.2 --- Plants materials and growth conditions --- p.45 / Chapter 3.2.3 --- Transient Expression of Arabidopsis suspension cultured cells --- p.45 / Chapter 3.2.4 --- Confocal imaging studies --- p.45 / Chapter 3.2.5 --- Drug treatment --- p.46 / Chapter 3.3 --- RESULTS --- p.48 / Chapter 3.3.1 --- Mutations at GTP-binding motifs and the effector domain affect the subcellular localization of AtRabG3e --- p.48 / Chapter 3.3.2 --- "AtRabG3e-T22N induced vacuolation of YFP-ARA7 marked PVCs, which remains separated from ER, Golgi and TGN but colocalizes with early PVC markers" --- p.51 / Chapter 3.3.3 --- AtRab7-T22N inhibits vacuolar trafficking of cargo proteins --- p.54 / Chapter 3.3.4 --- Wortmannin-induced vacuolation of late PVCs in transgenic plants --- p.57 / Chapter 3.4 --- Discussion --- p.59 / Chapter 3.4.1 --- The proper targeting of AtRab7 proteins --- p.59 / Chapter 3.4.2 --- AtRab5 and AtRab7 proteins are essential for vacuolar protein trafficking --- p.59 / Chapter CHAPTER 4 --- Summary and Future Perspectives --- p.61 / Chapter 4.1 --- Summary --- p.62 / Chapter 4.1.1 --- Localization of AtRab5 and AtRab7 proteins on different populations of PVCs --- p.62 / Chapter 4.1.2 --- Functions of AtRab7 proteins in Arabidopsis cells --- p.63 / Chapter 4.1.3 --- The Rab conversion maturation model --- p.63 / Chapter 4.2 --- Future perspectives --- p.64 / References --- p.73

This work identifies transcription factors (TF) controlling triacylglycerol (TAG) synthesis and accumulation in plant tissues. TAG plays vital role in plants and are used by humans. Most plants accumulate oil in the seed, but some species accumulate oil in other tissues. The Wrinkled1 (WRI1) TF has been shown to regulate oil accumulation in multiple species and tissues. Here, four WRI homologues in avocado were identified, their phylogeny was examined and three of them were cloned into expression vectors for further characterization. However, WRI1 likely does not act alone in regulation of TAG accumulation in plants. Additional candidate TFs were identified by using transcriptome data from a variety of species, and cloned into expression vectors. Future studies will be able to use this information to better understand regulation of TAG accumulation, which will allow increased oil accumulation in plants for various human uses.

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