Amino acid availability plays an important role in modulating the activity of T-cells. One of the pathways employed by T-cells to sense nutrient levels is the “mammalian target of rapamycin” (mTOR) pathway that is inhibited in response to nutrient depletion. Indoleamine 2,3-dioxygenase (IDO) is the first and rate-limiting enzyme along the tryptophan catabolising kynurenine pathway. T-cells are very sensitive to lack of this essential amino acid in their microenvironment and this confers strong immunomodulatory properties to cells expressing active IDO. It therefore has a significant physiological role as a homeostatic mechanism used in mammalian organisms to dampen excessive activation of the immune system but is also used as an immune evasion mechanism by many cancers. In this study, we investigated the IDO inhibitory properties and mechanism of action of the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) that potentially forms a negative feedback loop in the kynurenine pathway. We studied the molecule in enzymatic assays, in live cells and discovered that it inhibits IDO in an indirect way via the formation of hydrogen peroxide. Secondly, we looked at the effects of tryptophan and its metabolites on T-cell proliferation and mTOR activity, and discovered a metabolite that inhibits T-cell proliferation. Lastly we examined mechanisms of T-cell suppression employed by myeloid derived suppressor cells (MDSCs), focusing on their ability to deplete amino acids from their microenvironment. We were able to exclude tryptophan consumption as a suppressive mechanism and established that by manipulating extracellular concentrations of several amino acids other than arginine and cysteine – that are known to be utilised by MDSCs - we were able to reduce their inhibitory properties. In summary, we have described in detail how 3-HAA inhibits IDO in in vitro assays, outlined how some tryptophan metabolites can inhibit T-cell proliferation, and clarified aspects of suppressive mechanism employed by MDSCs.

Conjugation reactions on proteins have been used to access various post-translational modifications, for targeted delivery of drugs, for microscopy, and in studying receptor-ligand interactions. However, the ability to modify native proteins is constrained by the reactive functionalities of naturally occurring amino acids. This has driven research into the incorporation of unnatural amino acids (UAAs) into proteins. Research in this area has been motivated both by the possibility of increasing the breadth of chemical techniques for protein modification by introducing novel 'bio-orthogonal' reactive groups via UAA incorporation, as well as generating well-defined conjugates by the site-selective incorporation of these UAAs into proteins. The objective of this thesis is to both expand the diversity of UAAs for access to new metal-mediated reactions on proteins, as well as to utilise these reactions to reveal functional information about a range of biological systems. A brief introduction into current protein conjugation and UAA incorporation methods will be made in Chapter 1. In Chapter 2, the genetic incorporation of alkene-bearing UAAs into recombinant proteins expressed in both bacterial and mammalian systems is discussed. This technique is demonstrated to enable Ru-catalysed olefin cross-metathesis (CM) reactions on the resultant proteins. This work builds upon previously established methods to chemically incorporate CM handles onto proteins. The rational design of UAAs, as well as assays and modelling studies to screen them for recognition by the cellular incorporation machinery are discussed in detail. The expression of a range of alkene-tagged recombinant proteins, their complete characterisation, as well as the development of a more general protocol for on-protein CM is elucidated. In Chapter 3, the utility of UAA incorporation to probe mammalian cell translation systems is examined. Incorporation of an azide-bearing UAA, in addition to heavy stable-isotope labelled amino acids is used to uncover a previously unreported system of protein synthesis in mammalian cell nuclei, along with rapid metabolic degradation of the synthesised peptides. Various orthogonal methods for the detection of this system as well as possible reasons for its conservation are discussed. In Chapter 4, UAA incorporation and metal-mediated bioconjugation reactions are utilised in the development of a novel and generally applicable proteomics technique. This technique is used to determine quantitative changes in cell proteomes in response to external stimuli, and may be applied to systems to which traditional proteomics techniques cannot, such as ex vivo primary cells. Finally, in Chapter 5, further applications of UAA incorporation are discussed. Preliminary results are reported in efforts to use UAAs in the vibrational Raman microscopic imaging of biological systems, in generating HIV vaccines, and inducing T-cell stimulation.

In this thesis, the response of DNA and RNA to linear and torsional mechanical stress is studied using coarse-grained models. Inspired by single-molecule assays developed over the last two decades, the end-to-end extension, buckling and torque response behaviour of the stressed molecules is probed under conditions similar to experimentally used setups. Direct comparison with experimental data yields excellent agreement for many conditions. Results from coarse-grained simulations are also compared to the predictions of continuum models of linear polymer elasticity. A state diagram for supercoiled DNA as a function of twist and tension is determined. A novel confomational state of mechanically stressed DNA is proposed, consisting of a plectonemic structure with a denaturation bubble localized in its end-loop. The interconversion between this novel state and other, known structural motifs of supercoiled DNA is studied in detail. In particular, the influence of sequence properties on the novel state is investigated. Several possible implications for supercoiled DNA structures in vivo are discussed. Furthermore, the dynamical consequences of coupled denaturation and writhing are studied, and used to explain observations from recent single molecule experiments of DNA strand dynamics. Finally, the denaturation behaviour, topology and dynamics of short DNA minicircles is studies using coarse-grained simulations. Long-range interactions in the denaturation behaviour of the system are observed. These are induced by the topology of the system, and are consistent with results from recent molecular imaging studies. The results from coarse-grained simulations are related to modelling of the same system in all-atom simulations and a local denaturation model of DNA, yielding insight into the applicability of these different modelling approaches to study different processes in nucleic acids.

Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.

This thesis is concerned with the development of methodology for the asymmetric synthesis of a range of amino polyol containing compounds. Chapter 1 highlights the abundance of the amino polyol motif in nature, the wide range of biological activities displayed by amino polyol containing compounds, and their occurrence in drug molecules. A variety of different methods for the synthesis of stereodefined amino polyols is then discussed. Chapter 2 details a full investigation into the doubly diastereoselective conjugate addition reactions of the antipodes of lithium N-benzyl-N-(alpha-methylbenzyl)amide to enantiopurealpha,beta-unsaturated esters which contain a dioxolane unit. The “matched” conjugate addition reactions were further coupled with a highly diastereoselective in situ enolate oxidation using camphorsulfonyloxaziridine for the synthesis of keyalpha-hydroxy-beta-amino ester intermediates. Subsequent cyclisation and further elaboration allowed access to a range of amino polyol containing compounds including imino sugars, amino sugars, and amino acids. Chapter 3 extends the investigation into the doubly diastereoselective lithium amide conjugate addition reaction to enantiopure alpha,beta-unsaturated esters which contain two dioxolane units. A full assessment into the conjugate addition of the antipodes of lithium N-benzyl-N-(alpha-methylbenzyl)amide to a series of D-pentose derived alpha,beta-unsaturated esters is reported. Subsequent elaboration of thebeta-amino ester products of these conjugate addition reactions resulted in the synthesis of (2'S,3'S,4'R)-dihydroxyhomoproline and (2'S,3'R,4'S)-dihydroxyhomoproline. Chapter 4 describes the asymmetric syntheses of protected forms of APTO and AETD, the 2,4,5-trihydroxy substitutedbeta-amino acid residues found within the hexapeptide marine natural products microsclerodermins C, D and E. The optimised synthetic routes to APTO and AETD involved three key steps: a diastereoselective aminohydroxylation [via conjugate addition of lithium (R)-N-benzyl-N-(alpha-methylbenzyl)amide to an achiralalpha,beta-unsaturated ester followed by in situ enolate oxidation with camphorsulfonyloxaziridine], a diastereoselective dihydroxylation, and an olefination. Chapter 5 contains full experimental procedures and characterisation data for all compounds synthesised in chapters 2, 3 and 4.

Title: Effect of protein (amino acid) ingestion on muscle protein synthesis following resistance exercise. Purpose: The main objective of this thesis is to verify the three basic factors of the amount, type and timing of protein intake based on scientific studies and literature, to provide the most objective and accurate information and procedure on the methodology of nutrition and supplementation associated with the intake of protein / amino acids after strength training and how it all affects muscle synthesis. Summery: The theoretical part of the thesis, discusses the factors affecting muscle protein synthesis, which stimulate growth and tissue regeneration, based on optimal stress response. Logically, it starts from general, i.e. the explanation of terms such as muscle tissue, the stimulation of muscle tissue and its manifestations and changes, nutrition factors and muscle stimulation, the mechanism of dietary factors (proteins/amino acids), specificity of protein/ amino acids in their application to answer the three key issues, which are summarized in the section named scientific studies, which focuses on the effect of intake of protein/ amino acids, in relationship to the efficiency of protein synthesis after strength training. The section summarizes, in detail, the questions of timing,...

Nutrient availability regulates eukaryotic cell growth. This study focuses on two signaling pathways, involved in sensing amino acids and carbon sources, which allow cells to respond appropriately to their presence. The first part of this study shows that Ssy1, a plasma membrane localized sensor in the Ssy1-Ptr3-Ssy5 (SPS) amino acid sensing pathway, can detect 19 common L-amino acids with different potencies and affinities based on the physiochemical structure of amino acids. Substituents around alpha carbon are critical for amino acid sensing by Ssy1. Furthermore, a high concentration of cysteine is toxic to cells. Inactivation of SPS signaling confers resistance to cysteine. The second part focuses on the regulation of Hap4, the regulatory subunit of the Hap2/3/4/5 transcriptional factor complex. Many components of the 25-subunit Mediator complex negatively regulate HAP4 expression. Srb8 undergoes post-translational modification in response to changes of the carbon source. Gal11 and Med3 positively regulate HAP4 expression.

Chiral molecules play a central role in our daily life and in nature, for instance the different enantiomers or diastereomers of a chiral molecule may show completely different biological activity. For this reason, it is a vital goal for synthetic chemists to design selective and efficient methodologies that allow the synthesis of the desired enantiomer. In this context, it is highly important that the concept of green chemistry is considered while designing new approaches that eventually will provide more environmental and sustainable chemical synthesis.The aim of this thesis is to develop the concept of combining transition metal catalysis and aminocatalysis in one process (dual catalysis). This strategy would give access to powerful tools to promote reactions that were not successful with either transition metal catalyst or the organocatalyst alone. The protocols presented in this thesis based on organocatalytic transformations via enamine or iminium intermediates or both, in combination with transition metal catalysis, describes new enantioselective organocatalytic procedures that afford valuable compounds with high chemo- and enantioselectivity from inexpensive commercial available starting materials. In paper I, we present a successful example of dual catalysis: the combination of transition metal activation of an electrophile and aminocatalyst activation of a nucleophile via enamine intermediate. In paper II, the opposite scenario is presented, here the transition metal activates the nucleophile and the aminocatalyst activates the electrophile via an iminium intermediate. In paper III,we present a domino Michael/carbocyclisation reaction that is catalysed by a chiral amine (via iminium/enamine activation) in combination with a transition metal catalysts activation of an electrophile. In paper IV, the concept of dual catalysis was further extended and applied for the highly enantioselective synthesis of valuable structural scaffolds, namely poly-substituted spirocyclic oxindoles. Finally, in paper V the concept of dual catalysis was expanded, by investigating more challenging and environmentally benign processes, such as the successful combination of a heterogeneous palladium and amine catalysts for the highly enantioselective synthesis of functionalised cyclopentenes, containing an all carbonquaternary stereocenter, dihydrofurans and dihydropyrrolidines.

This diploma thesis deals with the study of adsorption of low-molecular components of organic substances produced by phytoplankton - AOM (Algal Organic Matter) on AC (Activated Carbon) during drinking water treatment. For the experimental purpose in this thesis, there were used selected amino acids as low molecular substances of AOM that are difficult to remove by conventional water treatment process by coagulation. As adsorbent, there was used a detailed characterized of granulated activated carbon (GAC) - Filtrasorb TL 830 (FTL830) which is intended directly for the purpose of water treatment. There were realizing the equilibrium batch adsorption experiments with three different model amino acids - arginine (Arg), phenylalanine (Phe) and aspartic acid (Asp). There was investigated the efficiency of removing amino acid depending on the solution temperature and pH. Results of the adsorption experiments have shown that the temperature affects the adsorption efficiency. Adsorption is essentially described as an exothermic process but the adsorption of Arg and Phe from an aqueous solution to GAC occurs more efficiently at higher temperatures. It means that the adsorption is the endothermic process. In the case of Arg adsorption, the temperature was found to influence adsorption efficiency less than...

O tratamento atual indicado para casos de envenenamentos por peçonhas é a administração intravenosa de antivenenos, produzidos através da hiperimunização de animais. Entretanto, os antivenenos disponíveis podem, algumas vezes, não proteger os pacientes e causar reações de hipersensibilidade. Fosfolipases A2 (PLA2), L-aminoácido oxidases (LAAO), metalo e serinoproteases são os principais componentes de peçonhas ofídicas e contribuem para a neurotoxicidade, hemorragia, hemólise, miotoxicidade, cardiotoxicidade e formação de edemas. Foram empregados ensaios para avaliar as atividades das enzimas presentes na peçonha de serpentes da espécie Lachesis muta rhombeata e aquele para atividade de protease foi otimizado. A tecnologia de Phage display foi empregada para a seleção de fagos-anticorpos capazes de reconhecer a peçonha bruta. Os fagos foram amplificados em Escherichia coli TG1 e usados para infectar E. coli HB2151, a qual produz fragmentos de anticorpos humanos solúveis. Estes foram purificados e utilizados em testes de inibição de alguns dos componentes tóxicos da peçonha. Os testes de atividade para PLA2, protease e Laminoácido oxidase foram padronizados com sucesso e as 3 proteínas mostraram elevada atividade enzimática. Após otimização, a quantidade de peçonha necessária para o ensaio de protease foi reduzida em 25 vezes. A massa molecular de PLA2 foi estimada em 17 kDa e as massas moleculares de proteases foram estimadas em 40, 35 e 24 kDa, através de zimogramas. O método de bio panning foi eficiente para a seleção de fagos-anticorpos contra a peçonha bruta. Diversos fragmentos de anticorpos foram purificados e incubados com a peçonha bruta para testar suas capacidades de neutralização sobre cada enzima. Cinco clones demonstraram-se hábeis em inibir a PLA2 através da inibição da hemólise. O clone 4E inibiu 100% da hemólise durante as duas horas de ensaio quando pré-incubado na proporção 2:1 (scFv:peçonha). Os clones 2C e 4E inibiram 100% durante uma hora quando pré-incubados na proporção 1:1 e os clones 2F e 9F inibiram a hemólise parcialmente. Outros testes serão conduzidos para a seleção de clones capazes de neutralizar as demais enzimas, os quais, juntamente com os clones já selecionados, serão analisados através de ensaios in vivo. Espera-se que eles possam contribuir para a construção de um novo antiveneno capaz de superar algumas das dificuldades associadas às técnicas de imunoterapia convencionais / The current treatment for animal envenoming is the intravenous administration of antivenoms, produced by animal hyperimmunization. Unfortunately, available antivenoms sometimes do not protect patients and may cause hypersensitivity reactions. Phospholipases A2 (PLA2), L-amino acid oxidases, metallo and serine proteases are considered the most important snake venom components and contribute to neurotoxicity, hemorrhage, hemolysis, myotoxicity, edematogeny and cardiotoxicity. Assays for evaluating the enzymes present in Lachesis muta rhombeata venom were developed and the protease one was optimized. Phage display technology was used to select phage antibodies able to recognize the crude venom. Phages were amplified in Escherichia coli TG1 and used to infect E. coli HB2151, which produces human antibody fragments. Inhibition tests aiming the neutralization of some toxic components of the venom were performed using purified antibody fragments. Activity assays for evaluating PLA2, protease and L-aminoacid oxidase were successfully performed and all enzymes showed high activity levels. The molecular mass of PLA2 was estimated in 17 kDa and the molecular mass of proteases were estimated in 40, 35 and 24 kDa, by zymography. After optimizing the conditions for proteolytic assay, it was possible to use 25 times less venom than it was necessary at first. The bio panning method was efficient for selecting specific phage antibodies against the crude venom. Several clones were selected to infect HB2151 and to produce soluble antibody fragments, which were purified and incubated with the venom to test their inhibition capacity over each enzyme. Five clones demonstrated ability to neutralize PLA2 by inhibiting hemolysis. The clone 4E could inhibit 100% of hemolysis for over 2 hours when preincubated at the ratio 2:1 (scFv:venom). Clones 2C and 4E could inhibit 100% for 1 hour when preincubated at the ratio 1:1 and clones 2F e 9F could inhibit partially. Other tests will be performed to select clones able to neutralize other enzymes and, together with the clones already selected, will be evaluated by in vivo experiments. It is expected that they may contribute to the construction of a potential new antivenom able to overcome some of the problems associated with conventional immunotherapy