In this study, effects of different carbon sources and their feeding strategies on recombinant human growth hormone (rhGH) production by Pichia pastoris were investigated by means of cell growth, recombinant protein production and expression levels of hGH and alcohol oxidase (AOX) genes. In this content, firstly, the strain to be used for high level rhGH production was selected between the two phenotypes, i.e., P. pastoris hGH-Mut+ and P. pastoris hGH-MutS. In this selection both phenotypes were compared in two different media containing glycerol/methanol or sorbitol/methanol and P. pastoris-hGH-Mut+ strain grown on medium containing 30 g/L sorbitol with 1% (v/v) methanol was found to have the highest hGH expression level and rhGH production level, 9.84x109 copies/mg CDW and 120 mg/L, respectively. Thereafter, effects of sorbitol, mannitol, fructose, lactose, sucrose, citric acid, lactic acid and acetic acid were investigated by using P. pastoris hGH-Mut+ strain in laboratory scale bioreactors. Among them sorbitol and sucrose were selected to be compared for production in pilot scale bioreactors by adding them batch-wise at the beginning of induction phase with fed batch methanol feeding scheme at &amp / #956 / =0.03h-1. It was shown that sucrose does not support cell growth as sorbitol although it does not repress recombinant protein production. Then three different feeding strategies were applied to develop sorbitol/methanol mixed feeding i) single sorbitol addition at t=0, ii) besides at t=0, adding second batch-wise sorbitol at t=9 h, iii) giving pulse methanol at t=24 h to trigger AOX promoter. These three strategies were compared with a production without addition of co-substrate sorbitol. Substrate consumption, cell growth, recombinant protein production and expression levels of hGH and AOX were investigated for these different feeding strategies. The highest cell concentration was achieved in third strategy as 55 g/L where the highest extracellular rhGH production (301 mg/L) was achieved in the second strategy, with addition of two times of sorbitol. For this highest recombinant protein production case, overall cell and product yield on total substrate were found as 0.17 g/g and 1.71 mg/g, respectively. Moreover, the highest hGH and AOX expression levels were obtained in this strategy.

Le paludisme constitue un problème majeur de santé publique en zone tropicale et subtropicale. La morbidité ainsi que la mortalité sont principalement dues au parasite Plasmodium falciparum transmis à l'homme par la piqûre de moustiques femelles du genre Anopheles. Dans le but d'orienter au mieux les stratégies d'élimination du paludisme et d'une meilleure évaluation de l'efficacité des méthodes de lutte, les indicateurs mesurant le risque de transmission doivent être plus sensibles. Il a été montré que la réponse anticorps humaine contre des protéines/peptides salivaires d'Anopheles représente un bio-marqueur d'exposition aux piqûres de moustiques et pouvait être un indicateur de la transmission du paludisme. Toutefois cet outil doit être optimisé. Ce travail a ainsi un double objectif : i) valider la protéine salivaire CE5 comme bio-marqueur d'exposition aux piqûres d'Anopheles et comme indicateur évaluant l'efficacité de stratégie de lutte anti-vectorielle, et 2) identifier de nouvelles protéines salivaires comme candidat bio-marqueur spécifique à l'exposition de l'homme aux seules piqûres infectantes d'Anopheles. Tout d'abord, nous avons démontré que la réponse anticorps IgG contre la protéine CE5 pourrait être un indicateur du contact homme-vecteur, complémentaire et très sensible, en mesurant l'exposition de l'homme aux piqûres d'Anopheles et un outil évaluant l'efficacité, à court terme, des moustiquaires imprégnées d'insecticide. Par la suite, les méthodes de protéomique 2D-DIGE et de spectrométrie de masse ont permis d'identifier cinq protéines salivaires (gSG6 , gSG1b , TRIO , SG5 et la forme longue D7) qui sont surexprimées dans les glandes salivaires d'An. gambiae infectées par P. falciparum. Des peptides de chaque protéine, définis in silico, apparaissent antigéniques chez des individus exposés aux piqûres d'Anopheles, après évaluation par la technique d'épitope mapping. L'ensemble de ces travaux est non seulement une première étape pour optimiser cet outil immuno-épidémiologique évaluant le contact homme-vecteur mais démontre également la possibilité de définir un nouveau bio-marqueur qui serait spécifique des piqûres infectantes d'Anopheles. / Malaria is a major public health problem in tropical and subtropical areas. Morbidity and mortality are mainly due to Plasmodium falciparum transmitted to human individuals by the bite of female Anopheles mosquitoes. In order to orientate appropriate strategies for malaria elimination and for a better evaluation of the efficacy of control methods, the indicators measuring the risk of transmission should be more sensitive. It has been shown that the human antibody response against Anopheles salivary proteins/peptides represents a biomarker of exposure to mosquito bites and could be an indicator of malaria transmission. However, this tool must be optimized. This work has thus two objectives: i) to validate the salivary protein cE5 as biomarker of exposure to Anopheles bites and as an indicator for evaluating the efficacy of vector control strategy, and 2) to identify new salivary proteins as a candidate biomarker only specific to human exposure to infective bites of Anopheles.First, we demonstrated that the IgG antibody response to cE5 protein could be an indicator of human-vector contact, complementary and very sensitive, measuring the human exposure to Anopheles bites and a tool evaluating the short-term efficacy of insecticide treated nets. Subsequently, the proteomic methods, 2D - DIGE and mass spectrometry, allowed to identify five salivary proteins (gSG6, gSG1b, TRIO, SG5 and the long form D7) which are overexpressed in the salivary glands of An . gambiae infected by wild P. falciparum. Peptides for each protein, identified in silico, appear antigenic in individuals exposed to Anopheles bites, after the evaluation by the epitope mapping technique.Altogether, this work is not only the first step to optimize this immuno-epidemiological tool assessing the human-vector contact, but also demonstrates the possibility to define a new biomarker specific to the infective bites of Anopheles.

Soil carbon (C) and nitrogen (N) are predominantly in organic form. Proteins/ peptides, as an important organic form of N, constitute a substantial part of soil organic matter. On one hand, proteins/peptides are an important N source for plants and microorganisms, particularly in soils where inorganic N is limited. On the other hand, their stabilization in soils by forming organo-mineral associates or macromolecule complex reduces the C loss as CO2 into the atmosphere. Therefore, studies on the turnover, abundance, composition, and stability of proteins/peptides are of crucial importance to agricultural productivity and environmental sustainability. In the first part of this study, the bioavailability and distribution of amino acids, (building block of proteins/peptides), were investigated, in soils across the North-South and West-East transects of continental United States. The second part of this study aimed to understand the variations of organic C speciation in soils of continental United States. Previous investigations of the interactions between soil minerals and proteins/peptides were mostly limited to batch sorption experiments in labs, seldom of which gave the details at the molecular scales. Therefore, in the third part of this study, the molecular orientation of self-assembled oligopeptides on mineral surfaces was investigated by employing synchrotron based polarization-dependent Near Edge X-ray Adsorption Fine Structure Spectroscopy (NEXAFS) techniques. Specific aims of this study were: 1) to assess potentially bioavailable pool of proteinaceous compounds and the immediately bioavailable pool of free amino acids in surface and subsurface soils of various ecosystems; 2) to evaluate the relationship between environmental factors and levels/composition of the two pools; 3) to investigate the organic C speciation in soils of various land use; and 4) to understand molecular level surface organization of small peptides on mineral surfaces. The levels of free amino acids and hydrolysable amino acids which represent the potentially bioavailable pool of proteinaceous compounds in A-horizon soils were significantly high than in C-horizon soils due to the accumulation of organic matter in surface. On average, free amino acids accounted for less than 4 % of hydrolysable amino acids which represent the total proteinaceous compounds in soils. The composition of free amino acids was significantly different between surface soil and subsurface soil and was significantly influenced by mean annual temperature and precipitation. A relatively uniform composition of hydrolysable amino acids was observed irrespective of a wide range of land use. Significant variations were observed for the levels of free and hydrolysable amino acids along mean annual temperature and precipitation gradients, as well as among vegetation types of continental USA, suggesting levels of free and hydrolysable amino acids were associated with the above-ground biomass and root distribution. Organic C speciation investigation revealed the presence of carboxylic-C (38%), aliphatic-C (~ 22%), aromatic-C (~ 18%), O/N-alkyl-C (~ 16%), and phenolic-C (< 6%). Factors such as temperature and vegetation cover were revealed in this study to account for the fluctuations of the proportions of aromatic-C and phenolic-C, in particular. Phenolic-C may serve as a good indicator for the effect of temperature or vegetation on the composition of SOC. The average composition of soil organic C, over the continental scale, was relatively uniform over various soil ecosystems and between two soil horizons irrespective of surface organic C content. Polarization dependent NEXAFS analysis showed the oligopeptides tend to orient on mineral surface with an average tilt angle of 40 ° between the molecular chain and the mineral surface. / Ph. D.

Functional characterization of biochemically-isolated proteins is a central task in the biochemical and genetic description of the biology of cells and tissues. Protein identification by mass spectrometry consists of associating an isolated protein with a specific gene or protein sequence in silico, thus inferring its specific biochemical function based upon previous characterizations of that protein or a similar protein having that sequence identity. By performing this analysis on a large scale in conjunction with biochemical experiments, novel biological knowledge can be developed. The study presented here focuses on mass spectrometry-based proteomics of organisms with unsequenced genomes and corresponding developments in biological sequence database searching with mass spectrometry data. Conventional methods to identify proteins by mass spectrometry analysis have employed proteolytic digestion, fragmentation of resultant peptides, and the correlation of acquired tandem mass spectra with database sequences, relying upon exact matching algorithms; i.e. the analyzed peptide had to previously exist in a database in silico to be identified. One existing sequence-similarity protein identification method was applied (MS BLAST, Shevchenko 2001) and one alternative novel method was developed (MultiTag), for searching protein and EST databases, to enable the recognition of proteins that are generally unrecognizable by conventional softwares but share significant sequence similarity with database entries (~60-90%). These techniques and available database sequences enabled the characterization of the Xenopus laevis microtubule-associated proteome and the Dunaliella salina soluble salt-induced proteome, both organisms with unsequenced genomes and minimal database sequence resources. These sequence-similarity methods extended protein identification capabilities by more than two-fold compared to conventional methods, making existing methods virtually superfluous. The proteomics of Dunaliella salina demonstrated the utility of MS BLAST as an indispensable method for characterization of proteins in organisms with unsequenced genomes, and produced insight into Dunaliella?s inherent resilience to high salinity. The Xenopus study was the first proteomics project to simultaneously use all three central methods of representation for peptide tandem mass spectra for protein identification: sequence tags, amino acids sequences, and mass lists; and it is the largest proteomics study in Xenopus laevis yet completed, which indicated a potential relationship between the mitotic spindle of dividing cells and the protein synthesis machinery. At the beginning of these experiments, the identification of proteins was conceptualized as using ?conventional? versus ?sequence-similarity? techniques, but through the course of experiments, a conceptual shift in understanding occurred along with the techniques developed and employed to encompass variations in mass spectrometry instrumentation, alternative mass spectrum representation forms, and the complexities of database resources, producing a more systematic description and utilization of available resources for the characterization of proteomes by mass spectrometry and advanced informatic approaches. The experiments demonstrated that proteomics technologies are only as powerful in the field of biology as the biochemical experiments are precise and meaningful.

The Protein Data Bank (PDB) is the definitive electronic repository for experimentally-derived protein structures, composed mainly of those determined by X-ray crystallography. Approximately 200 new structures are added weekly to the PDB, and at the time of writing, it contains approximately 97,000 structures. This represents an expanding wealth of high-quality information but there seem to be few bioinformatics tools that consider and analyse these data as an ensemble. This thesis explores the development of three efficient, fast algorithms and software implementations to study protein structure using the entire PDB. The first project is a crystal-form matching tool that takes a unit cell and quickly (< 1 second) retrieves the most related matches from the PDB. The unit cell matches are combined with sequence alignments using a novel Family Clustering Algorithm to display the results in a user-friendly way. The software tool, Nearest-cell, has been incorporated into the X-ray data collection pipeline at the Diamond Light Source, and is also available as a public web service. The bulk of the thesis is devoted to the study and prediction of protein disorder. Initially, trying to update and extend an existing predictor, RONN, the limitations of the method were exposed and a novel predictor (called MoreRONN) was developed that incorporates a novel sequence-based clustering approach to disorder data inferred from the PDB and DisProt. MoreRONN is now clearly the best-in-class disorder predictor and will soon be offered as a public web service. The third project explores the development of a clustering algorithm for protein structural fragments that can work on the scale of the whole PDB. While protein structures have long been clustered into loose families, there has to date been no comprehensive analytical clustering of short (~6 residue) fragments. A novel fragment clustering tool was built that is now leading to a public database of fragment families and representative structural fragments that should prove extremely helpful for both basic understanding and experimentation. Together, these three projects exemplify how cutting-edge computational approaches applied to extensive protein structure libraries can provide user-friendly tools that address critical everyday issues for structural biologists.