Thesis (Ph. D.)--Columbia University, 1939. / Vita. Includes bibliographical references.

Nucleotidylyltransferase inhibitors are designed to target enzymes responsible for one step of cell wall biosynthesis in Gram-positive, Gram-negative and mycobacteria. Glucose 1-phosphate thymidylyltransferase Cps2L/RmlA (EC 2.7.7.24) is an enzyme essential for the growth and proliferation of many bacteria, including Mycobacterium tuberculosis. Cps2L/RmlA serves to couple glucose 1-phosphate and deoxythymidine triphosphate to form deoxythymidine diphosphoglucose. dTDP-?-L-rhamnose acts as an inhibitor of RmlA. Phosphonates are synthetic analogues of natural phosphates that have shown widespread ability to probe biological systems. Several approaches were investigated toward the synthesis of dTDP-?-L-rhamnose analogues, which incorporated phosphonate functionality into their scaffolds. A series of L-rhamnose phosphonate and ketosephosphonate analogues, with varying degrees of fluorination about the 1C position, were synthesized. These compounds were evaluated as potential inhibitors of thymidylyltransferase Cps2L, the first enzyme in the L-rhamnose biosynthetic pathway, and a novel antibiotic target. Enzyme-inhibitor and enzyme-substrate binding experiments were performed using WaterLOGSY NMR spectroscopy for the phosphonate-based compounds and known enzyme sugar nucleotide substrates. IC50 values were measured and Ki values were calculated for the compounds determined to be inhibitors. New insights were gained into the binding promiscuity of various enzymes within the L-rhamnose biosynthetic pathway (Cps2L, RmlB-D) and the mechanism of inhibition for the most potent inhibitor, L-rhamnose-1C-phosphonate. Thiophosphates are analogues of natural phosphates in which the P—O bond has been replaced with a P—S bond. Methods were investigated for the preparation of O and S-glucosyl thiophosphates. A series new protected glucosyl thiophosphate compounds were synthesized and characterized as precursors to glucose-1-thiophopshate, a probable glucose 1-phosphate substrate analogue for Cps2L.

Thesis (Ph. D.)--University of Michigan, 1933. / Caption title: Pentose metabolism III. The rate of absorption of l-rhamnose and the formation of glycogen in the organism of the white rat after oral administration of l-rhamnose, by Alfred K. Silberman and Howard B. Lewis. "Reprinted from the Journal of Biological Chemistry, vol. 101, no. 3 ... August, 1933." Bibliography: p. 750-751.

Thesis (PH. D.)--University of Michigan, 1933. / Caption title: Pentose metabolism III. The rate of absorption of l-rhamnose and the formation of glycogen in the organism of the white rat after oral administration of l-rhamnose, by Alfred K. Silberman and Howard B. Lewis. "Reprinted from the Journal of Biological Chemistry, vol. 101, no. 3 ... August, 1933." Bibliography: p. 750-751.

Even though the structure of the plant cell wall is by and large quite well characterized, its synthesis and regulation remains largely obscure. However, it is accepted that the building blocks of the polysaccharidic part of the plant cell wall are nucleotide sugars. Thus to gain more insight into the cell wall biosynthesis, in the first part of this thesis, plant genes possibly involved in the nucleotide sugar interconversion pathway were identified using a bioinformatics approach and characterized in plants, mainly in Arabidopsis. For the computational identification profile hidden markov models were extracted from the Pfam and TIGR databases. Mainly with these, plant genes were identified facilitating the “hmmer” program. Several gene families were identified and three were further characterized, the UDP-rhamnose synthase (RHM), UDP-glucuronic acid epimerase (GAE) and the myo-inositol oxygenase (MIOX) families. For the three-membered RHM family relative ubiquitous expression was shown using variuos methods. For one of these genes, RHM2, T-DNA lines could be obtained. Moreover, the transcription of the whole family was downregulated facilitating an RNAi approach. In both cases a alteration of cell wall typic polysaccharides and developmental changes could be shown. In the case of the rhm2 mutant these were restricted to the seed or the seed mucilage, whereas the RNAi plants showed profound changes in the whole plant. In the case of the six-membered GAE family, the gene expressed to the highest level (GAE6) was cloned, expressed heterologously and its function was characterized. Thus, it could be shown that GAE6 encodes for an enzyme responsible for the conversion of UDP-glucuronic acid to UDP-galacturonic acid. However, a change in transcript level of variuos GAE family members achieved by T-DNA insertions (gae2, gae5, gae6), overexpression (GAE6) or an RNAi approach, targeting the whole family, did not reveal any robust changes in the cell wall. Contrary to the other two families the MIOX gene family had to be identified using a BLAST based approach due to the lack of enough suitable candidate genes for building a hidden markov model. An initial bioinformatic characterization was performed which will lead to further insights into this pathway. In total it was possible to identify the two gene families which are involved in the synthesis of the two pectin backbone sugars galacturonic acid and rhamnose. Moreover with the identification of the MIOX genes a genefamily, important for the supply of nucleotide sugar precursors was identified. In a second part of this thesis publicly available microarray datasets were analyzed with respect to co-responsive behavior of transcripts on a global basis using nearly 10,000 genes. The data has been made available to the community in form of a database providing additional statistical and visualization tools (http://csbdb.mpimp-golm.mpg.de). Using the framework of the database to identify nucleotide sugar converting genes indicated that co-response might be used for identification of novel genes involved in cell wall synthesis based on already known genes. / Obwohl der Aufbau der pflanzlichen Zellwand im Großen und Ganzen relativ gut charakterisiert ist, ist relativ wenig über ihre Synthese bekannt. Allgemein akzeptiert ist jedoch, dass die Nukleotidzucker die Vorstufe für den polysaccharidären Teil der Zellwand stellen. Im Rahmen der vorliegenden Arbeit wurden neue Kandidatengene für die Zellwandbiosynthese mittels bioinformatorischer Analysen ermittelt und deren Rolle in Pflanzen, hauptsächlich Arabidopsis thaliana untersucht. Zur Identifizierung von Arabidopsis thaliana Kandidatengenen des Nukleotidzucker-Stoffwechselweges wurden „hidden Markov Modelle“ für Gene desselben aus den Datenbanken Pfam und TIGR extrahiert. Unter anderem wurden diese dann unter Zuhilfenahme des Programms hmmer zur Identifikation von pflanzlichen Genen benutzt. Es wurden einige Genfamilien identifiziert und drei von diesen wurden weiter charakterisiert. Hierbei handelte sich um eine UDP-Rhamnose Synthase Familie (RHM), eine UDP-Glucuronsäurepimerase Familie (GAE) und eine myo-Inositol Oxygenase Familie (MIOX). Für die RHM Kandidatengenfamilie, mit drei Mitgliedern, wurde die relativ ubiquitäre Expression aller Gene mittels verschiedener Methoden gezeigt und für eines der Gene, RHM2, konnten T-DNA Linien bezogen werden. Außerdem wurde die Transkription der gesamten Familie mittels eines RNAi Konstruktes herunter geregelt. In beiden Fällen konnte eine Veränderung von zellwandtypischen Polysacchariden sowie schwere Entwicklungsstörungen gezeigt werden. Diese waren bei der rhm2 Funktionsverlustpflanze auf den Samenschleim bzw. den Samen reduziert, bei den RNAi Pflanzen hingegen war die gesamte Pflanze betroffen. Im Falle der zweiten Kandidatengenfamilie, GAE, wurde das höchst-exprimierte Gen (GAE6) kloniert, heterolog exprimiert und die Funktion charakterisiert. So konnte gezeigt werden, dass GAE6 für ein Enzym kodiert, welches UDP-Glukuronsäure in UDP-Galakturonsäure wandelt. Allerdings zeigten Pflanzen mit veränderter Transkriptmenge, erreicht durch T-DNA Insertionen (gae2, gae5, gae6), Überexpression (GAE6) oder RNAi / keine robuste Veränderung der Zellwand. Die letzte betrachtete Kandiatengenfamilie myo-Inositol Oxygenase wurde im Gegensatz zu den beiden anderen Familien, durch eine BLAST Suche gefunden, da zur Zeit der Durchführung noch zu wenig myo-inositol Oxygenasen bekannt waren, um daraus „hidden Markov Modelle“ abzuleiten. Dennoch konnten erste bioinformatorische Analysen zu dieser Genfamilie gemacht werden. Insgesamt gesehen wurden in diesem Teil der Arbeit die beiden Genfamilien identifiziert und charkterisiert, die bei der Synthese der beiden Pektinrückgradzucker Rhamnose und Galakturonsäure die tragende Rolle spielen. Weiterhin wurde mit der Identifizierung der MIOX Genfamilie, eine Genfamilie identifiziert, die wichtige Vorstufen in der Synthese der Nukleotidzucker liefert. In einem zweiten Teil der Arbeit wurden öffentlich zugängliche Mikroarray-Daten durch ihr Gleich -oder Ungleichverhalten charakterisiert. Dieses erfolgte auf globaler Ebene für zunächst fast 10.000 Gene. Die Daten wurden in Form einer allgemein zugänglichen Datenbank der Allgemeinheit zur Verfügung gestellt (http://csbdb.mpimp-golm.mpg.de). Eine Anwendung der Methode auf Gene des Nukleotidzuckerstoffwechsels, deutet darauf hin, dass so neue Kandiatengene, die bei der Zellwandsynthese eine Rolle spielen, von bereits bekannten Genen abgeleitet werden können.

Bacterial microcompartments (BMCs) are proteinaceous metabolic compartments found in a wide range of bacteria, whose function it is to encapsulate pathways for the breakdown of various carbon sources, whilst retaining toxic and volatile intermediates formed from substrate breakdown. Examples of these metabolic processes are the 1,2- propanediol-breakdown pathway in Salmonella enterica (Pdu microcompartment), as well as the ethanolamine breakdown pathway in Clostridium difficile (Eut microcompartment). Some of the major challenges to exploiting BMCs as a tool in biotechnology are understanding how enzymes are targeted to microcompartments, as well as being able to engineer the protein shell of BMCs to make synthetic microcompartments that allow specific enzyme pathways to be targeted to their interior. Finally, the metabolic burden imposed by the production of large protein complexes requires a detailed knowledge of how the expression of these systems are controlled. This project explores the structure and biochemistry of an essential BMC pathway enzyme, the acylating propionaldehyde dehydrogenase. With crystal structures of the enzyme with the cofactors in the cofactor binding site and biochemical data presented to confirm the enzyme's substrate. The project also focuses on the creation of synthetic biology tools to enable BMC engineering with a modular library of BMC shell protein parts; forward engineered ribosome binding sites (RBS) fused to BMC aldehyde dehydrogenase localisation sequences. The parts for this library were taken from the BMC loci found in Clostridium phytofermentans and Salmonella enterica. Using a synthetic biology toolkit will allow the rapid prototyping of BMC constructs for use in metabolic engineering. The shell protein parts were used to generate a number of transcriptional units, to assess the effect of overexpression of individual BMC shell components on the morphology of BMCs and the effect these had on their host chassis. Different strength forward engineered RBS and localisation constructs have been designed to assess the possibility of controlling the levels of heterologous proteins targeted to the interior of microcompartment shell to allow metabolic engineering of encapsulated pathways. Along with looking at overexpression of a single shell protein, to assess viability of BMCs as scaffold-like structures, recombinant BMCs can be explored for their utility in bioengineering and their potential role in generating biofuels.

No description available.

The objective of the field of Synthetic Biology is to implement novel functionalities in a biological context or redesign existing biological systems. To achieve this, it employs tried and tested engineering principles, such as standardisation and the design-build-test cycle. A crucial part of this process is the convergence of modelling and experiment. The aim of this thesis is to improve the design principles employed by Synthetic Biology in the context of Gene Regulatory Networks (GRNs). Small Ribonucleic Acids (sRNAs), in particular, are focussed on as a mechanism for post-transcriptional expression regulation, as they present great potential as a tool to be harnessed in GRNs. Modelling sRNA regulation and its interaction with its associated chaperone Host-Factor of Bacteriophage Qβ (Hfq) is investigated. Inclusion of Hfq is found to be necessary in stochastic models, but not in deterministic models. Secondly, feedback is core to the thesis, as it presents a means to scale-up designed systems. A linear design framework for GRNs is then presented, focussing on Transcription Factor (TF) interactions. Such frameworks are powerful as they facilitate the design of feedback. The framework supplies a block diagram methodology for visualisation and analysis of the designed circuit. In this context, phase lead and lag controllers, well-known in the context of Control Engineering, are presented as genetic motifs. A design example, employing the genetic phase lag controller, is then presented, demonstrating how the developed framework can be used to design a genetic circuit. The framework is then extended to include sRNA regulation. Four GRNs, demonstrating the simplest forms of genetic feedback, are then modelled and implemented. The feedback occurs at three different levels: autoregulation, through an sRNA and through another TF. The models of these GRNs are inspired by the implemented biological topologies, focussing on steady state behaviour and various setups. Both deterministic and stochastic models are studied. Dynamic responses of the circuits are also briefly compared. Data is presented, showing good qualitative agreement between models and experiment. Both culture level data and cell population data is presented. The latter of these is particularly useful as the moments of the distributions can be calculated and compared to results from stochastic simulation. The fit of a deterministic model to data is attempted, which results in a suggested extension of the model. The conclusion summarises the thesis, stating that modelling and experiment are in good qualitative agreement. The required next step is to be able to predict behaviour quantitatively.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Mestre em Engenharia Química / Os surfactantes produzidos por micro-organismos apresentam as mesmas características dos surfactantes sintéticos, sendo capazes de reduzir a tensão superficial em soluções aquosas e em misturas de hidrocarbonetos Neste trabalho estudou-se a produção de biossurfactante por fermentação de gorduras provenientes de flotadores de estação de tratamento de efluentes de abatedouros de aves e suínos, contendo 19,48 g/L de gordura e 0,2 g/L de nitrogênio. Avaliou-se a produção de raminolipídeo empregando duas linhagens de Pseudomonas aeruginosa (ATCC 9027 e 10145) em relação às variáveis de processo: concentração de efluente gorduroso, concentração de nitrogênio, pela adição de nitrato de amônio, e concentração de levedura cervejeira autolisada residual, aplicando um planejamento fatorial (PF) e um planejamento composto central (PCC). Os experimentos ocorreram em um reator batelada, modelo B. Braun International com volume útil de 1,5 L para fermentações em 48 horas com nível de aeração de 0,5 vvm, velocidade de agitação de 550 rpm e concentração inicial do micro-organismo de 1,0 ± 0,2 g/L de células. Os resultados do PF mostraram que a linhagem Pseudomonas aeruginosa ATCC 10145 apresentou-se como a mais eficiente para produzir biossurfactante em relação à ATCC 9027, pois nas mesmas condições das variáveis de processo, a linhagem ATCC 10145 conseguiu produzir 0,94 g/L a mais de raminose e reduzir em 0,8 dina/cm a tensão superficial do meio em relação ao resultado obtido pela ATCC 9027. Além disso, a linhagem ATCC 10145 apresentou índice de emulsificação de 100% e crescimento celular 1,17 g/L a mais que ATCC 9027, apresentando um produção do raminolipídeo parcialmente associada ao crescimento celular, com o melhor tempo de fermentação de 48 horas. Com a linhagem escolhida pelo PF, fez-se uma otimização dos resultados em um PCC. As variáveis utilizadas no PCC foram: concentração de efluente, concentração de nitrato de amônio e concentração de levedura cervejeira autolisada residual. Na otimização dos resultados, a melhor concentração de efluente para a produção do biossurfactante com Pseudomonas aeruginosa ATCC 10145 foi de 60 g/L, com concentração de levedura cervejeira autolisada residual de 15 g/L, sem a adição de nitrato de amônio como fonte de nitrogênio, alcançando 3,84g/L de raminose e 100% de índice de emulsificação.

No description available.