Proteins, which regulate most of the biological activities, perform their functions through their unique three-dimensional structures. The folding process of this three dimensional structure from one dimensional sequence is not well understood. The available facts infer that the protein structures are mostly conserved while sequences are more tolerant to mutations i.e. a number of sequences can adopt the same fold. These arch of optimal sequences for a chosen conformation is known as inverse protein folding and this thesis takes this approach to solve the enigmatic problem. This thesis presents a protein sequence design method based on the native state topology of protein structure. The structural importance of the amino acid positions has been converted into the topological parameter of the protein conformation. This scheme of extraction of topology of structures has been successfully applied on three dimensional lattice structures and in turn sequences with minimum energy for a given structure are obtained. This technique along with the reduced amino cid alphabet(A reduced amino acid alphabet is any clustering of twenty amino acids based on some measure of the irrelative similarity) has been applied on the protein structures and hence designed optimal amino acid sequences for a given structure. These designed sequences are energetically much better than the native amino acid sequence. The utility of this method is further confirmed by showing the similarity between naturally occurring and the designed sequences. In summary, a computationally efficient method of designing optimal sequences for a given structure is given. The physical interaction energy between the amino acids is an important part of study of protein-protein interaction, structure prediction, modeling and docking etc. The local environment of amino acids makes a difference between the same amino acid pairs in the protein structure and so the pair-wise interaction energy of amino acid residues should depend on the irrespective environment. A local environment depended knowledge based potential energy function is developed in this thesis. Two different environments, one of these is the local degree (number of contacts) and the other is the secondary structural element of amino acids, have been considered. The investigations have shown that the environment-based interaction preferences for amino acids is able to provide good potential energy functions which perform exceedingly well in discriminating the native structure from the structures with random interactions. Further, the membrane proteins are located in a completely different physico-chemical environment with different amino acid composition than the water soluble proteins. This work provides reliable potential energy functions which take care of different environment for the investigation(model/predict) of the structure of helical membrane proteins. Three different environments, parallel and perpendicular to the lipid bilayer and number of amino acid contacts, are explored to analyze the environmental effects on the potential functions. These environment dependent scoring functions perform exceedingly well indiscriminating the native sequence from a set of random sequences. Hydrophobicity of amino acids is a measure of buriedness or exposure to the aqueous environment. The lack of uniformity within the protein environment gives rise to the different values of hydrophobicity for the same amino acids, which completely depends on its location inside the protein.The contact based environment dependent hydrophobicity values of all amino acids, separately for globular and membrane proteins, have also been evaluated in this thesis. Apart from developing scoring functions, the packing of helices in membrane proteins is investigated by an approach based on the local backbone geometry and side chain atom-atom contacts of amino acids. A parameter defined in this study is able to capture the essential features of inter-helical packing, which may prove to be useful in modeling of helical membrane proteins. In conclusion, this thesis has described a novel technique to design the energetically minimized amino acid sequences which can fold in to a given conformation. Also the environment dependent interaction preference of amino acids in globular proteins is captured an efficient manner. Specially, the environment dependent scoring function for helical membrane proteins is a first successful attempt in this direction.

Chemical biology encompasses the study and manipulation of biological system using chemistry, often by virtue of small molecules or unnatural amino acids. Much insight has been gained into the mechanisms of biological processes with regards to protein structure and function, metabolic processes and changes between healthy and diseased states. As an ever expanding field, developing new tools to interact with and impact biological systems is an extremely valuable goal. Herein, work is described towards the synthesis of a small library of heterocyclic-containing small molecules and the mechanistic details regarding the interesting and unexpected chemical compounds that arose; an alternative set of non-toxic copper catalyzed azide-alkyne click conditions for in vivo metabolic labelling; and the synthesis of an unnatural amino acid for further chemical modification via [3+2] cycloadditions with nitrones upon incorporation into a peptide of interest. Altogether, these projects strive to supplement pre-existing methodology for the synthesis of small molecule libraries and tools for metabolic labelling, and thus provide further small molecules for understanding biological systems.

Dans notre groupe, nous nous intéressons au développement de peptides contenant des acides γ-aminés. Comme d’autres peptides contenant des acides aminés non naturels, ils ont montré leur capacité à posséder des conformations stables et/ou des propriétés biologiques intéressantes. De plus, ces peptides sont généralement résistant à la protéolyse. Dans l’objectif de synthétiser des acides -diaminés sous la forme d’un seul stéréoisomère, nous avons développé une voie de synthèse reposant sur une réaction de Blaise suivie d’une réduction diastéréosélective. En appliquant cette méthode, nous avons synthétisé des acides β,γ-diaminés dérivés de la D-phénylalanine et de l’acide L-glutamique. Le premier a été utilisé pour concevoir des analogues d’un peptide antimicrobien, la gramicidine S. Comparé à la molécule parent, les analogues ont montré une cytotoxicité beaucoup moins importante pour les cellules hôtes tout en conservant une activité antibactérienne intéressante. Cette étude nous a donné de meilleures connaissances pour développer d’autres analogues de la gramicidine S ainsi que d’autres peptides antimicrobiens. Nous avons également effectué de nombreuses optimisations pour synthétiser de façon efficace des acides β,γ-diaminés cycliques à partir de l’acide L-glutamique. Les oligomères incorporant ces acides β,γ-diaminés et des acides α-aminés ont montré un fort potentiel pour l’adoption de conformations stables. Ces études vont être poursuivies. / In our group, we are interested in developing peptides containing β,γ-diamino acids . Along with many other peptides containing unnatural amino acids, they have shown the ability to possess stable conformations and/or interesting biological activities. Moreover, those peptides are usually more resistant to proteolysis. In order to synthesize stereopure γ-amino acids, we have developed a synthetic route using Blaise reaction and subsequent diastereoselective reduction as key reactions. Through applying this method, we have synthesized β,γ-diamino acids derived from D-phenylalanine and L-glutamic acid. The former β,γ-diamino acid was used for designing antimicrobial peptide gramicidin S analogues. Compared with mother molecule, the analogues exerted much less host cell cytotoxicity while remaining interesting antibacterial activity. Meanwhile, it gave us more knowledge for further developing analogues of gramicidin S as well as other antimicrobial peptides. We also paid lots of effort to efficiently synthesize cyclic β,γ-diamino acids starting from L-glutamic acid. Interestingly, when oligomers incorporating this β,γ-diamino acids and α-amino acids, they have shown the potential to adopt stable conformations. The following studies will be continuously investigated.

The protein folding problem is examined. Specifically, the problem of predicting protein secondary structure from the amino acid sequence is investigated. A literature study is presented into the protein folding process and the different techniques that currently exist to predict protein secondary structures. These techniques include the use of expert rules, statistics, information theory and various computational intelligence techniques, such as neural networks, nearest neighbour methods, Hidden Markov Models and Support Vector Machines. A pattern recognition technique based on statistical analysis is developed to predict protein secondary structure from the amino acid sequence. The technique can be applied to any problem where an input pattern is associated with an output pattern and each element in both the input and output patterns can take its value from a set with finite cardinality. The technique is applied to discover the role that small sequences of amino acids play in the formation of protein secondary structures. By applying the technique, a performance score of Q8 = 59:2% is achieved, with a corresponding Q3 score of 69.7%. This compares well with state of the art techniques, such as OSS-HMM and PSIPRED, which achieve Q3 scores of 67.9% and 66.8% respectively, when predictions on single sequences are made. / Dissertation (MEng)--University of Pretoria, 2009. / Electrical, Electronic and Computer Engineering / unrestricted

On the chromosomal DNA of the microorganism Achromobacter sp. CCM 4824, which was gained in the Laboratory of enzyme technology MBU AVCR v.v.i., there was identified a gene coding an enzyme capable of hydrolyzation of semi-synthetic β-lactam antibiotics ampicillin and cephalosporin with a D-phenylglycine as a side chain. This enzyme belongs to a group of α-amino acid esterases, which are interesting because of a potential use in kinetically controled synthesis of β-lactam antibiotics. In several aspects α-amino acid esterases might be better than actually used penicillin acylases and that is why these enzymes are subjects of a big interest. The gene for α-amino acid esterase coded by chromosomal DNA was cloned, characterized and heterologously expressed in constructed highly-producing bacterial system Escherichia coli BL21(DE3)JM5. Produced enzyme was purified and its properties important for possible use in the production of semi-synthetic β-lactam antibiotics were determined. Key words: alpha-amino acid ester hydrolase, Achromobacter sp., recombinant expression system, β-lactam antibiotics

This diploma thesis deals with the evaluation of clinical manifestations in infants with allergic colitis and their overall nutritional status, anthropometry, blood count and antibodies against cow's milk proteins. Allergic colitis is one of the most common manifestations of infant allergy to cow`s milk protein with gastrointestinal involvement. The theoretical part summarizes the anthropometric evaluation of growth and development, infant nutrition, the most important laboratory indicators of nutritional status. The part is generally devoted to food allergies, mainly allergy to cow's milk protein. The practical part deals with the evaluation of a sample of examined patients in gastroenterology outpatient clinics and nutritional outpatient clinic of the Department of Pediatrics and Adolescent Medicine of the 1st Faculty of Medicine, Charles University and General Hospital in Prague. The observed group of infants with allergic colitis includes infants exclusively breastfed, breastfed by a mother following a non-dairy diet, infants on an extensive hydrolyzate or on an amino acid formula. The main goal of the diploma thesis was to trace the most common clinical manifestations and whether there are any changes in their nutritional status during the disease and its treatment. A total of 45 randomly...

Summary: Many primary human tumors and tumor cell lines highly express human L-type amino acid transporter 1 (hLAT1); cancerous cells in vivo are strongly linked to LAT1 expression. Synthetic chemistry and in vitro screening efforts have afforded a variety of novel and highly hLAT1 selective compounds, such as JPH203 1. In a recent report, we demonstrated that 1 has potent in vitro and in vivo activity. JPH203 was intravenously administered to produce significant growth inhibition against HT-29 tumors transplanted in nude mice. The current work develops a robust LC/MS-MS method to monitor 1 and its major Phase II metabolite N-acetyl-JPH203 2 from biological samples. We have conducted in vitro and in vivo experiments and the major scientific findings are: i) the major route of biotransformation of 1 is Phase II metabolism to produce 2; ii) metabolite 2 is formed in various organs/tissues (i.e. blood, liver, kidney); and iii) as dogs, which are deficient in NAT genes, do not produce 2, the dog will not be an appropriate toxicological model to evaluate 1.

Atmospheric nucleation is a process of phase transformation like liquid water transforming into solid or gas phase water, which serves as a significant impact on many atmospheric and technological processes. During the process of the atmospheric nucleation, certain 3D molecular models for atmospheric nucleation will be generated, which are main mixtures of water molecules and hexanol molecules. Analyzing these 3D molecular models can promote the understanding for the nucleation and growth of the particles and phases in a multi-component mixture, as well as for the changes in climate and weather. Therefore, the research for atmospheric nucleation can be transformed into the research for the 3D molecular visualizations and comparisons, which are the similarity calculations. Unfortunately, the research on understanding atmospheric nucleation processes is restricted due to the lack of efficient visual data exploration tools. In this paper, the issue of lacking efficient data visualization tools is tackled by implementing our own application to visualize the atmospheric nucleation. The similarity calculation for these 3D molecules is implemented in order to analyze and compare the atmospheric nucleation processes and molecular models. Admittedly, there are various 3D molecular similarity calculation algorithms, such as clique-detection algorithms and point matching, etc; however, these algorithms are specifically utilized in the fields of protein amino-acids and pharmacophore. Due to the large scale of the atmospheric nucleation data, GPU (Graphical Processing Units) is employed in order to significantly reduce the computation times. This is achieved by utilizing CUDA (Compute Uniform Device Architecture) technology which allows us to execute our algorithm in a parallel method. Furthermore, in this research, the knowledge of hypertree visualization is intended to be utilized to enhance the previously developed web-based visualization and analysis tool that allows remote users to effectively mine the wealth of particle-based nucleation simulation data. The research goal is to speed up knowledge discovery and improve users' productivity through effective data visualization technique and more friendly user interface design. Meanwhile, a feasible parallel computing solution is developed to overcome the slow response due to expensive large data pre-processing. The core research of my thesis is to calculate the similarity between the distinct 3D molecules.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20303号 / 薬科博第72号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 川端 猛夫, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

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