Dissertations / Theses on the topic 'High protein'

In the past decade, automatisation has led to an immense increase of data in biology. Next generation sequencing techniques will produce a vast amount of sequences across all species in the coming years. In many cases, identifying the function and biological role of a protein from its sequence can be a complicated and time-intensive task. The identification of a protein's interaction partners is a tremendous help for understanding the biological context in which it is involved. In order to fully characterise a protein-protein interaction (PPIs), it is necessary to know the three-dimensional structure of the interacting partners. Despite optimisation efforts from projects such as the Protein Structure Initivative, determining the structure of a protein through crystallography remains a time- and cost-intensive procedure. The primary aim of the research described in this dissertation was to produce a World Wide Web resource that facilitates visual exploration and validation (or questioning) of data derived from functional genomics experiments, by building upon existing structural information about direct physical PPIs. Secondary aims were (i) to demonstrate the utility of the new resource, and (ii) its application in biological research. We created a database that emphasises specifically the intersection between the PPIs-results emerging from the structural biology and functional genomics communities. The BISC database holds BInary SubComplexes and Modellable Interactions in current functional genomics databases (BICS-MI). It is publicly available at hyyp://bisc.cse.ucsc.edu. BISC is divided in three sections that deliver three types of information of interest to users seeking to investigate or browse PPIs. The template section (BISCHom and BISCHet) is devoted to those PPIs that are characterised in structural detail, i.e. binary SCs extracted from experimentally determined three-dimensional structures. BISCHom and BISCHet contain the homodimeric (13,583 records) and heterodimeric (5612 records) portions of these, respectively. Besides interactive, embedded Jmol displays emphasising the interface, standard information and links are provided, e.g. sequence information and SPOP classification for both partners, interface size and energy scores (PISA). An automated launch of the MolSurfer program enables the user to investigate electrostatic and hydrophobic correlation between the partners, at the inter-molecular interface. The modellable interactions section (BISC0MI) identifies potentially modellable interactions in three major functional genomics interaction databases (BioGRID), IntAct, HPRD). To create BISC-MI all PPIs that are amenable to automated homology modelling based on conservative similarity cut-offs and whose partner protein sequences have recrods in the UniProt database, have been extracted. The modellable interaction services (BISC-MI Services) section offers, upon user request, modelled SC-structures for any PPIs in BISC-MI. This is enabled through an untomated template-based (homology) modelling protocol using the popular MODELLER program. First, a multiple sequence alignment (MSA) is generated using MUSCLE, between the target and homologous proteins collected from UniProt (only reviewed proteins from organisms whose genome has been completely sequenced are included to find putative orthologs). Then a sequence-to-profile alignment is generated to integrate the template structure in the MSA. All models are produced upon user request to ensure that the most recent sequence data for the MSAs are used. Models generated through this protocol are expected to be more accurate generally than models offered by other automated resources that rely on pairwise alignments, e.g. ModBase. Two small studies were carried out to demonstrate the usability and utility of BISC in biological research. (1) Interaction data in functional genomics databases often suffers from insufficient experimental and reporting standards. For example, multiple protein complexes are typically recorded as an inferred set of binary interactions. Using the 20S core particle of the yeast proteasome as an example, we demonstrate how the BISC Web resource can be used as a starting point for further investigation of such inferred interactions. (2) Malaria, a mosquito-borne disease, affects 3500-500 million people worldwide. Still very little is known about the malarial parasites' genes and their protein functions. For Plasmodium falciparum, the most lethal among the malaria parasites, only one experimentally derived medium scale PPIs set is available. The validity of this set has been doubted in the the malarial biologist community. We modelled and investigated eleven binary interactions from this set using the BISC modelling pipeline. Alongside we compared the BISC models of the individual partners to those obtained from ModBase.

Fundamental to all biological processes is the interplay between biomolecules such as proteins and nucleic acids. Studies of interactions should therefore be more informative than mere detection of expressed proteins. Preferably, such studies should be performed in material that is as biologically and clinically relevant as possible, i.e. in primary cells and tissues. In addition, to be able to take into account the heterogeneity of such samples, the analyses should be performed in situ to retain information on the sub-cellular localization where the interactions occur, enabling determination of the activity status of individual cells and allowing discrimination between e.g. tumor cells and surrounding stroma. This requires assays with an utmost level of sensitivity and selectivity. Taking these issues into consideration, the in situ proximity-ligation assay (in situ PLA) was developed, providing localized detection of proteins, protein-protein interactions and post-translational modifications in fixed cells and tissues. The high sensitivity and selectivity afforded by the assay's requirement for dual target recognition in combination with powerful signal amplification enables visualization of single protein molecules in intact single cells and tissue sections. To further increase the usefulness and application of in situ PLA, the assay was adapted to high content analysis techniques such as flow cytometry and high content screening. The use of in situ PLA in flow cytometry offers the possibility for high-throughput analysis of cells in solution with the unique characteristics offered by the assay. For high content screening, it was demonstrated that in situ PLA can enable cell-based drug screening of compounds affecting post-translational modifications and protein-protein interactions in primary cells, offering superior abilities over current assays. The methods presented in this thesis provide powerful new tools to study proteins in genetically unmodified cells and tissues, and should offer exciting new possibilities for molecular biology, diagnostics and drug discovery. 

Proteins and protein-based materials are used for a wide range of therapeutic, diagnostic, and biotechnological applications. Still, the inherent instability of proteins in non-native environments greatly limits the applications in which they are effective. In order to increase their utility, proteins are often modified, either biologically or chemically, to manipulate their bioactivity and stability profiles. In this work, covalent attachment of polymers to the enzyme chymotrypsin was used to predictably tailor protein bioactivity and stability. Specifically, atom transfer radical polymerization (ATRP) based polymer-based protein engineering (PBPE) was used to grow polymers directly from the surface of chymotrypsin. First, the temperature responsive polymers poly(N-isopropyl acrylamide) (pNIPAM), which has a lower critical solution temperature (LCST) and poly(dimethylamino propane sulfonate) (pDMAPS), which has an upper critical solution temperature (UCST), were separately grown from chymotrypsin. The temperature responsive properties of the polymers were conserved in the protein-polymer conjugates, and chymotrypsin bioactivity, productivity, and substrate specificity were predictably tailored at different temperatures depending on the structural organization of the polymers. Next, a dual block polymer-chymotrypsin conjugate was synthesized by growing poly(sulfobetaine methacrylamide) (pSBAm)-block-pNIPAm conjugates from the surface of chymotrypsin. The CT-pSBAm-b-pNIPAm conjugates showed temperature dependent kinetics, due to UCST or LCST driven polymer collapse at high and low temperature. Most interestingly, the dual block conjugates were dramatically more stable than native chymotrypsin to low pH. In order to further investigate the effect of polymer conjugation on chymotrypsin stability at low pH, four distinct and uniquely charged polymers were grown from the surface of chymotrypsin. With these new conjugates, we confirmed that chymotrypsin low pH stability was dependent on the chemical structure of polymers covalently attached to chymotrypsin. Indeed, positively charged polymers stabilized chymotrypsin to low pH, but negatively charged and amphiphilic polymers destabilized the enzyme. Lastly, after developing strategies for low pH stabilization, new protein-polymer conjugates with the chemical permeation enhancer 1-phenylpiperazine were designed to enable protein transport across the intestinal epithelium. Bovine serum albumin-poly(oligoethylene methacrylate)-block-poly(phenylpiperazine acrylamide) BSA-pOEGMA-b-pPPZ conjugates induced dose dependent increases in Caco-2 monolayer permeability and transported across an in vitro intestinal monolayer model with low cell toxicity.

Protein-Protein Interaction (PPI) Research currently generates an extraordinary amount of publications and interest in fellow computer scientists and biologists alike because of the underlying potential of the source material that researchers can work with. PPI networks are the networks of protein complexes formed by biochemical events or electrostatic forces serving a biological function [1]. Since the analysis of the protein networks is now growing, we have more information regarding protein, genomes and their influence on life. Today, PPI networks are used to study diseases, improve drugs and understand other processes in medicine and health that will eventually help mankind. Though PPI network research is considered extremely important in the field, there is an issue – we do not have enough people who have enough interdisciplinary knowledge in both the fields of biology and computer science; this limits our rate of progress in the field. Most biologists that are not expert coders need a way of calculating graph values and information that will help them analyze the graphs better without having to manipulate the data themselves. In this research, I test a few ways of achieving results through the use of available frameworks and algorithms, present the results and compare each method’s efficacy. My analysis takes place on very large datasets where I calculate several centralities and other data from the graph using different metrics, and I also visualize them in order to gain further insight. I also managed to note the significance of MPI and multithreading on the results thus obtained that suggest building scalable tools will help improve the analysis immensely.

Thesis (M.S.)--University of Alabama at Birmingham, 2008.
Title from PDF title page (viewed Sept. 21, 2009). Additional advisors: Martha W. Bidez, W. Michael Carson, Richard A. Gray, W. William Wilson. Includes bibliographical references.

Computing has pushed a paradigm shift in many disciplines, including structural biology and chemistry. This change has been mainly driven by the increase in performance of computers, the capacity of dealing with huge amounts of experimental and analysis data and the development of new algorithms. Thanks to these advances, our understanding on the chemistry that supports life has increased and it is even more sophisticated that we had never imagined before. Proteins play a major role in nature and are often described as the factories of the cell as they are involved in virtually all important function in living organisms. Unfortunately, our understanding of the function of many proteins is still very poor due to the actual limitations in experimental techniques which, at the moment, they can not provide crystal structure for many protein complexes. The development of computational tools as protein-protein docking methods could help to fill this gap. In this thesis, we have presented a new protein-protein docking method, LightDock, which supports the use of different custom scoring functions and it includes anisotropic normal analysis to model backbone flexibility upon binding process. Second, several interesting web-based tools for the scientific community have been developed, including a web server for protein-protein docking, a web tool for the characterization of protein-protein interfaces and a web server for including SAXS experimental data for a better prediction of protein complexes. Moreover, the optimizations made in the pyDock protocol and the increase in th performance helped our group to score in the 5th position among more than 60 participants in the past two CAPRI editions. Finally, we have designed and compiled the Protein-Protein (version 5.0) and Protein-RNA (version 1.0) docking benchmarks, which are important resources for the community to test and to develop new methods against a reference set of curated cases.
Gràcies als recents avenços en computació, el nostre coneixement de la química que suporta la vida ha incrementat enormement i ens ha conduït a comprendre que la química de la vida és més sofisticada del que mai haguéssim pensat. Les proteïnes juguen un paper fonamental en aquesta química i són descrites habitualment com a les fàbriques de les cèl·lules. A més a més, les proteïnes estan involucrades en gairebé tots els processos fonamentals en els éssers vius. Malauradament, el nostre coneixement de la funció de moltes proteïnes és encara escaig degut a les limitacions actuals de molts mètodes experimentals, que encara no són capaços de proporcionar-nos estructures de cristall per a molts complexes proteïna-proteïna. El desenvolupament de tècniques i eines informàtiques d’acoblament proteïna-proteïna pot ésser crucial per a ajudar-nos a reduir aquest forat. En aquesta tesis, hem presentat un nou mètode computacional de predicció d’acoblament proteïna-proteïna, LightDock, que és capaç de fer servir diverses funcions energètiques definides per l’usuari i incloure un model de flexibilitat de la cadena principal mitjançant la anàlisis de modes normals. Segon, diverses eines d’interès per a la comunitat científica i basades en tecnologia web han sigut desenvolupades: un servidor web de predicció d’acoblament proteïna-proteïna, una eina online per a caracteritzar les interfícies d’acoblament proteïna-proteïna i una eina web per a incloure dades experimentals de tipus SAXS. A més a més, les optimitzacions fetes al protocol pyDock i la conseqüent millora en rendiment han propiciat que el nostre grup de recerca obtingués la cinquena posició entre més de 60 grups en les dues darreres avaluacions de l’experiment internacional CAPRI. Finalment, hem dissenyat i compilat els banc de proves d’acoblament proteïna-proteïna (versió 5) i proteïna-ARN (versió 1), molt importants per a la comunitat ja que permeten provar i desenvolupar nous mètodes i analitzar-ne el rendiment en aquest marc de referència comú.

Cytochrome P450 monooxygenases, one of the most important classes of heme-thiolate proteins, have attracted considerable interest in the biochemical community because of its catalytic versatility, substrate diversity and great number in the superfamily. Although P450s are capable of catalyzing numerous difficult oxidation reactions, the relatively low stability, low turnover rates and the need of electron-donating cofactors have limited their practical biotechnological and pharmaceutical applications as isolated enzymes. The goal of this study is to tailor such heme-thiolate proteins into efficient biocatalysts with high specificity and selectivity by protein engineering and to better understand the structure-function relationship in cytochromes P450. In the effort to engineer P450cam, the prototype member of the P450 superfamily, into an efficient peroxygenase that utilizes hydrogen peroxide via the “peroxide-shunt” pathway, site-directed mutagenesis has been used to elucidate the critical roles of hydrophobic residues in the active site. Various biophysical, biochemical and spectroscopic techniques have been utilized to investigate the wild-type and mutant proteins. Three important P450cam variants were obtained showing distinct structural and functional features. In P450camV247H mutant, which exhibited almost identical spectral properties with the wild-type, it is demonstrated that a single amino acid switch turned the monooxygenase into an efficient preoxidase by increasing the peroxidase activity nearly one thousand folds. In order to tune the distal pocket of P450cam with polar residues, Leu 246 was replaced with a basic residue, lysine, resulting in a mutant with spectral features identical to P420, the inactive species of P450. But this inactive-species-like mutant showed catalytic activities without the facilitation of any cofactors. By substituting Gly 248 with a histidine, a novel Cys-Fe-His ligation set was obtained in P450cam which represented the very rare case of His ligation in heme-thiolate proteins. In addition to serving as a convenient model for hemoprotein structural studies, the G248H mutant also provided evidence about the nature of the axial ligand in cytochrome P420 and other engineered hemoproteins with thiolate ligations. Furthermore, attempts have been made to replace the proximal ligand in sperm whale myoglobin to construct a heme-thiolate protein model by mimicking the protein environment of cytochrome P450cam and chloroperoxidase.

In the field of protein research in general and the pharmaceutical industry in particular, it is now necessary to perform measurements of the secondary structure of proteins on many samples simultaneously, for instance to screen for molecules that stabilize proteins or to evaluate the action of multiple environmental conditions. In this context, we have proposed a new approach to evaluate the secondary structure of proteins on a very large scale (approximately 2000 to 4000 samples / cm2), by combining infrared imaging and 2D printing of protein microarrays. In view of the large amount of data, in a first step, methods for automating the extraction of spectra of interest from microarray infrared images and for automating the processing of the spectra were developed. Since the estimation of the secondary structure from infrared spectra is based on the construction of prediction models by chemometric methods, a relevant set of proteins for calibration was mandatory. A protein bank consisting of 92 commercially available proteins, the structure of which was well characterized by X-ray crystallography, was established for this purpose. After the development of predictive models for secondary structure determination and the validation of the protein microarray approach, we tried to optimize the models to improve the secondary structure prediction by different approaches as secondary structure definition, partial deuteration or subtraction of side chain contribution to the spectra. On the other hand, dealing with non-native structures not present in the reference protein library was a challenge. We took the opportunity to analyze the structural modifications of a subset of our protein library subjected to moderate denaturation conditions. Multivariate curve resolution-alternating least squares (MCR-ALS) was used to model a new spectral component appearing in the protein set subjected to denaturing conditions, which could represent a potential spectroscopic marker of aggregation and could allow a semi-quantitative evaluation of the aggregation. While the assessment of secondary structure was well established in the first part of this work, tertiary structure and stability are also critical. Hydrogen / deuterium exchange (HDX) is a potential approach for studying the structure and dynamics of proteins. In the last part of this work, we built a device which allowed to follow the HDX exchange kinetics simultaneously on the entire microarray. In conclusion, protein microarray FTIR imaging opens the door to high throughput analysis of protein secondary structure without any labelling and would allow better understanding of three-dimensional structure and dynamics of proteins through recording HDX curves.
Dans le domaine de la recherche sur les protéines et de l'industrie pharmaceutique, il s’avère désormais nécessaire d'effectuer des mesures de la structure secondaire des protéines sur de nombreux échantillons simultanément, de cribler des molécules qui stabilisent les protéines, ou d'évaluer l'action de multiples conditions environnementales. Dans ce contexte, nous avons proposé une nouvelle approche pour évaluer la structure secondaire des protéines à très grande échelle (environ 2 000 à 4 000 échantillons / cm2), en associant l'imagerie infrarouge et l'impression 2D de damiers de protéines. Dans un premier temps, des méthodes d'automatisation de l'extraction des spectres d'intérêt à partir des images infrarouges des damiers et d'automatisation des spectres ont été développées. L'estimation de la structure secondaire à partir des spectres infrarouges étant basée sur la construction de modèles de prédiction à partir de méthodes chimiométriques, un ensemble pertinent de protéines pour l'étape de calibration était obligatoire. Une banque de protéines constituée de 92 protéines disponibles dans le commerce, dont la structure était bien caractérisée par cristallographie aux rayons X, a été constituée dans ce but. Après élaboration des modèles prédictifs de la structure secondaire et la validation de l'approche des damiers de protéines, nous avons tenté d'optimiser les modèles pour améliorer les prédictions de structure secondaire par différentes approches. D'autre part, traiter des protéines présentant une structure jamais rencontrée dans les structures natives de notre bibliothèque de protéines de référence constituait un défi. Nous avons saisi l'opportunité d'analyser les modifications structurales d'un sous-ensemble de notre bibliothèque de protéines, caractérisé par un contenu structurel secondaire très différent en le soumettant à des conditions de dénaturation modérées La méthode de résolution de courbes multivariées des moindres carrés alternés (MCR-ALS) a été utilisée pour modéliser une nouvelle composante spectrale apparaissant dans l'ensemble protéique soumis à des conditions dénaturantes, et a permis de révéler un marqueur spectroscopique potentiel d'agrégation protéique permettant une évaluation semi-quantitative de son contenu. Alors que l’évaluation de la structure secondaire a été bien établie dans la première partie de ce travail, la structure tertiaire et la stabilité sont également critiques. L'échange hydrogène / deutérium (HDX) est une approche potentielle pour l’étude de la structure et de la dynamique des protéines. Dans la dernière partie de ce travail, nous avons construit un dispositif qui a permis de suivre la cinétique d’échange HDX simultanément sur l'ensemble d’un damier. En conclusion, l'imagerie FTIR de damiers de protéines ouvre la porte à une analyse à haut débit de la structure secondaire des protéines et permettrait de mieux comprendre la structure et la dynamique tridimensionnelles grâce à l'enregistrement des courbes HDX.
Doctorat en Sciences
info:eu-repo/semantics/nonPublished

This thesis describes a series of four experiments designed to evaluate the role of the supply of protein in livestock grazing high quality pasture during mating and during pregnancy. The first two studies investigated the effects of high crude protein content of spring or autumn re-growth pasture on the reproductive performance of dairy cows and of ewes at mating. The last two studies investigated how the dietary supply of protein, body condition and their interactions contribute to the breakdown of immunity during the peri-parturient period in ewes and investigated underlying endocrine mechanisms. In the first study (Chapter 3) cows were blood sampled via the tail vein during the breeding period in spring. Plasma was then analysed for urea concentration. Cows with high plasma urea (HPU) or low plasma urea (LPU) were defined as those with plasma urea concentrations of ≥ or < 44.9 mg/dl respectively. Lactating cows (n = 200) were also categorized into high milk producers (HMP) or low milk producers (LPM) relative to an average daily yield of 26.6 l/d. Pasture clipping showed an average pasture CP (crude protein) content of 223 g/kg DM. Concentrations of plasma urea ranged from 26.6 to 64.4 mg/dl. No correlation was observed between plasma urea concentration and either reproductive indicators or milk parameters. Mean blood urea concentration of HPU cows was 50.8 compared to 38.5 mg/dl in LPU cows. There was a trend for more animals (P = 0.09) in the HPU group than in the LPU group not to return to oestrus. Cumulative pregnancy rate in HPU and LPU was similar except at week 6 after the start of mating when more (P < 0.01) HPU than LPU cows were pregnant. Calving to conception interval, calving interval and interval between conception and first service were similar (P > 0.05) between HPU and LPU cows. Gestation length, calving rate, milk yield and milk components were also similar (P > 0.05) between LPU and HPU cows. There was no difference (P > 0.05) in plasma urea concentrations between HMP and LMP milk producers. However, calving to conception interval, interval between calving and first service and calving interval were longer (P < 0.001), submission rate higher (P < 0.001) and NRR (Non-return rate) higher (P < 0.05) in LMP than HMP. The number of services, the interval between first and second service, gestation length and CR (calving rate) were similar (P > 0.05) between HMP and LMP cows. HMP had lower (P < 0.001) milk protein and fat concentrations than LMP cows. This information indicates that, despite the fact that plasma urea was consistently higher than levels in the literature which have been associated with reduced fertility in dairy cows; no impairment of reproductive performance was observed. In the second experiment (Chapter 4) mature and dry Coopworth ewes were blocked by weight, body condition and previous prolificacy (high, HP vs low twinning frequency, LP) into two groups and thereafter randomly allocated to diet which were designed to provided either 1) high protein (163 g/kg DM, ryegrass/red clover pasture, HPP) or low protein (119 g/kg DM, hay and barley grain, HB) supply at joining. These were designed to provide high and low plasma urea concentration. Over a period of 17 days, ewes recorded as mated were examined by laparoscopy, at which time there was no difference in blood urea concentration (58.6 vs 56.1 mg/dl) between HPP and HB groups. Fifty days after the start of joining the number of foetuses present was counted using ultrasonography. As a consequence of lack of difference in the plasma urea concentration, irrespective of treatment group, individual animals were categorized into high (HU) and low plasma urea (LU) status based on whether plasma urea concentration was higher or lower than the sample mean of 51.5 mg urea/dl. Lambs which weighed greater than the mean plus one standard deviation for their litter size were classified as oversize. Ovulation rate and conception rate were similar (P > 0.05) between HPP and BH and between HU and LU ewes. Ewes with previous high reproductive performance (HP) as would be expected had higher ovulation rate (P < 0.001) and conception rate (P < 0.01) than LP ewes. Embryo losses was not (P = 0.06) different between HB and HPP ewes. Urea category (HU vs LU) did not (P > 0.05) influence embryo mortality. Foetal loss, neonatal loss, total reproductive loss and mean lamb birth weight was were not affected by diet, nor urea category (P > 0.05). Single ovulations had tended (P = 0.08) to contribute to higher embryo loss compared to multiple ovulations, and, single foetuses suffered higher (P<0.001) losses compared to multiples. While the study did not achieve large differences in plasma urea concentrations between diets, the levels of plasma urea operating were high yet reproductive wastage rates were similar to those recorded in the literature. Together with similar apparent lack of effect on a high plasma urea environment, the data suggest either that previous findings from controlled studies have a more complex aetiology or that pastoral animals can adapt to high tissue ammonia/urea status. The third trial (Chapter 5) was designed to provide information on the supply of amino acids to the abomasum from protein supplementation which have previously been found to overcome dietary scarcity associated with limitation of peri-parturient increase in FEC. Twin-suckling ewes were fitted with rumen and abomasal cannulae and grazed a ryegrass/clover sward (C) or the same sward but with a 500 g/h/d protein supplement (S). The trial was designed as a cross-over with two 14 day adaptation periods followed by two five-day digesta-sampling periods. All ewes were treated with anthelmintic 14 days after lambing. Weekly analysis of blood glucose was carried on whole blood and analysis of amino acids in plasma. The flows of amino acids (AA) and dry matter (DM) at the abomasum were measured during both sampling periods using intra-ruminally infused markers. Live weight and faecal egg count (FEC) were recorded weekly. Diurnal variation in AA flow at the abomasum peaked between 12:00 and 15:00 h and was greatest in S ewes. Flows of AA, including DAPA, were increased by supplementation by 16%, while sulphur amino acids (SAA) were the most enhanced (by 21%) and flows of leucine, lysine, glutamine and aspartate were increased by about 20%. There were significant time effects in rumen and abomasal pH (P < 0.01; in both cases in both periods) reflecting increase in pH after 09.00 h. During Period II, rumen pH in digesta of C ewes was significantly higher (P < 0.001) than that of S ewes (6.7 ± 0.05 vs 6.4 ± 0.05 for C and S ewes, respectively). Plasma AA concentrations (P < 0.01) were lower in S ewes 21 days after parturition, but similar (P > 0.05) to those of C ewes at other times. Forty-three days after lambing (after cross over), the order was reversed as plasma methionine and cysteine concentrations of C ewes became low (P < 0.05). These changes in plasma AA were accompanied by changes in body condition score between day 23 and 70 post-partum whereby C ewes lost more body condition than S ewes. There was evidence for a lower FEC in S ewes, being 46 vs. 670 epg, respectively for S and C groups (P = 0.08) 21 days after anthelmintic treatment. There were higher (P < 0.05) blood glucose levels in C compared to S ewes at day +35 relative to lambing which was reversed and significantly higher (P < 0.01) for S ewes by day +56 from lambing (after treatments were reversed). There was no significant effect of treatment on live weight and lamb performance. There are limited data in amino acid supply on lactating ewes on pasture and the present study contributes additional information on the supply of amino acids at the abomasum. The prediction that flow rates that sulphur amino acids may have been enhanced to the greatest degree could be significant since sulphur amino acids are needed for the synthesis of glutathione for immune response. It can be calculated that supplementation to supply the quantities of S-amino acid at pasture would be needed, since it would not be possible for sheep to increase pasture intake to achieve similar S-amino acid flow. Increase in bypass amino acids in S ewes at certain times in the day probably suggests influence by protein supplementation at certain times of the grazing cycle. Reduced plasma free amino acids at day +21 relative to lambing, may indicate sparing of body protein breakdown by protein supplementation. However, the difference in blood glucose on day 35 and day 56 may indicate re-adjustment of hormonal settings, responsible for nutrient partitioning. The last study (Chapter 6) used ewes during the peri-parturient period on pasture. Eighty pregnant ewes were allocated into four groups balanced for anticipated number of lambs. Group 1 had a high body condition score (BCS) of 4.0 which was maintained throughout pregnancy by pasture allowance (HM; n = 20). Group 2 (n= 40) had medium body condition (BCS 3.0) and were split into two subgroups; one was offered pasture to allow gain of condition (MH; n = 20) and the second allowed to lose condition by offering a low grazing allowance (ML; n = 20). Group 3 were thin ewes (BCS 2.4) and pasture allowance was designed to maintain this condition (LM; n = 20). These feeding regimes were maintained for 3 weeks from week -8 of pregnancy. During week -5 to -4 all ewes were acclimatized to a protein supplement (60 g/d). A glucose tolerance test (GTT) was conducted during week -4 after which half of the ewes in each group were offered a protein supplement at the rate of 500 g/d, creating –S and –NS groups. During wk -2, a second GTT was carried out. Animals were treated with an anthelmintic 3 wks before lambing, and were then challenged with a dose of 10 000 Teladorsagia circumcincta larvae on weeks -2 and -1 relative to lambing. Weekly recording of FEC, live weight and body condition was carried out. Lambs were weighed within 24 h of birth and again at 44 and 65 d of age. Computed tomography body scanning was carried out on ewes at weeks -8, -3 and +8 relative to lambing. There were no differences (P > 0.05) in lamb performance due to body condition or protein supplementation. FEC of all groups was low (≈ 9 peg) and there was no (P > 0.05) significant difference between ewes of different body condition or due to effects of protein supplementation. Ewes bearing/bearing multiple lambs had the highest FEC at day -32 and +12 relative to lambing, which was significant (P < 0.05) on the latter date. There were no significant effects of supplementation on parasite status. There were differences in basal plasma glucose concentration between groups (P < 0.001), being highest in HM/S and least in ML/NS ewes and was generally higher (P <0.001) during GTT 2 than GTT 1. Ewes carrying a single foetus had higher (P <0.001) basal glucose than those carrying multiple lambs (2.2 vs. 1.7 mmol/L, respectively). Other plasma glucose response indexes were similar (P <0.05) between groups. There were differences in insulin responsiveness between groups (P < 0.001), being highest in MH/S and least in ML/S ewes. Insulin responsiveness tended (P = 0.06) to be lower during GTT 1 than GTT 2, but was higher (P < 0.01) in ewes carrying singles than multiples. There was tendency for higher though non-significant, basal insulin concentrations in HM ewes. Insulin trends over time after glucose infusion suggest greater insulin response at GTT 1. Basal insulin was not correlated with CT muscle weight. Despite differences in body muscle mass at the start of the trial and differences induced by nutrition during late pregnancy, positive gains in muscle mass occurred during early pregnancy and muscle mass was similar in all groups by day 56 of lactation. Animals with greatest fat content at parturition (HM) mobilised the greatest amount and those with least fat (LM) deposited fat during lactation. Further experimentation may consider the use of the hyperinsulinemic-euglycemic clamp approach to more precisely estimate whether hormonal re-setting through insulin resistance may be involved in relaxation of immunity during the peri-parturient period.

This thesis presents several approaches to the study of the folding and dynamics of proteins, with an emphasis on examples form immunoglobulin-like fold. The first part describes the application of atomic force microscopy to studying the mechanical unfolding of proteins. Most studies to date have used methods such as chemical or thermal denaturation, thus force represents a new type of “denaturant” which adds to the description of the folding energy landscape. Furthermore it is a single-molecule method with the potential to resolve parallel folding pathways, for example. The technique has been used to study the mechanical unfolding of the RNase barnase which has already been extensively characterised by more conventional methods. Barnase is shown to be folded and stable in the protein construct used, yet it unfolds at very low forces compared to proteins with mechanical function (such as titin 127). Phi-value analysis by protein engineering has allowed the detailed characterization of folding pathways by bulk methods. A method is presented and justified which circumvents the large uncertainties in unfolding rates obtained from kinetic fits to AFM data. This has been applied to the domain titin 127, a protein from muscle, showing that the protein unfolds through an intermediate and with a very native-like transition state under force, contrasting with previous bulk studies. The second part of the work concerns the comparison of TNfn3 and FNfn10, belonging to the fibronectin type III superfamily and, like 127, members of the immunoglobulin-like fold. These two domains have very similar structure, yet present a very different response to mutation, which has led to the suggestion that FNfn10 has greater structural “plasticity”. The dynamics of these domains are addressed by means of equilibrium hydrogen exchange measurements, which support the notion that the periphery of FNfn10 is more flexible. The core dynamics have been investigated using recently developed NMR side-chain dynamics experiments, and the results compared to molecular dynamics simulations.

Proteins play important roles in most cellular functions, such as, replication, transcription regulation, signal transduction, for catalyzing chemical reaction, etc. Technologies developed to identify proteins rely either on observing their own properties such as charge, size, mass to charge ratio or sequence composition; or on using affinity reagents that recognize specific protein targets. Immunoassays utilizing functionalized affinity reagents are powerful for targeted proteomics. Among them, DNA-assisted immunoassays in which affinity reagents are labeled with DNA molecules, offer some unique advantages. In this thesis, I will present works to improve current DNA-assisted immunoassays such as proximity ligation assays (PLA), as well as to take advantage of DNA reactions to adress other problems. In paper I, a new solid support (MBC-Ts) was functionalized with antibodies and used in the solid-phase PLA for detection of VEGF. The assay using MBC-Ts was compared among the commercially available solid supports in different matrices and it was shown to exhibit enhanced limit of detection in complex matrices. In paper II, a two-step protocol was described to prepare high-quality probes used in homogeneous and in situ PLA by purifying DNA-labeled affinity reagents from unconjugated affinity reagents and excess oligonucleotides. In paper III, PLA was applied on a capillary western blotting instrument so that both the sensitivity and specificity of the original assay were improved. In paper IV, a new method was introduced to profile protein components in individual protein complexes by DNA-barcoded antibodies. This method has been used to profile protein complexes such as surface proteins on individual secreted vesicles.

La fonction des protéines est intimement liée à leur structure et à leur dynamique. La Résonance Magnétique Nucléaire est une technique de choix permettant d'étudier ces deux aspects à une résolution atomique. La relaxation du spin des noyaux d'azote-15 permet de quantifier ces mouvements aux échelles de temps pico- nanosecondes grâce à la determination de la fonction de densité spectrale, décrivant les mouvements du vecteur NH amide. Il est essentiel de mesurer les vitesses de relaxation à des champs magnétiques faibles pour mieux décrire les mouvements nanoseconde. De telles mesures sont possibles grâce à la relaxométrie haute-résolution et ont été réalisées sur l'ubiquitine. Celles-ci ont permis la caractérisation de mouvements nanoseconde dans les parties flexibles de l'ubiquitine. L'interprétation des données de relaxation pour des protéines désordonnées requiert le développement de modèles spécifiques à ces protéines. Nous avons développé une approche, appelée IMPACT, permettant une reconstruction mathématique de la fonction de densité spectrale. Appliquée au facteur de transcription Engrailed 2, cette approche a permis d'accéder à la distribution d'échelles de temps ps-ns à partir de données de relaxation à haut champ. Cette approche, combinée à des mesures de relaxométrie sur la région C-terminale de la protéine Artemis, devrait permettre d'obtenir une représentation fidèle et précise de la dynamique d'une protéine désordonnée. De plus, nous avons étudié la cinétique et la thermodynamique de l'interaction entre Artemis et la Ligase IV. Nos travaux ont permis de développer de nouvelles approches pour l'analyse de larges ensembles de données de relaxation
The intimate relation between the structure, dynamics and function of biomolecules is widely recognized. NMR is a unique technique to extract information on both structure and dynamics at atomic resolutions. Measurements of nitrogen-15 nuclear spin relaxation allow a quantitative description of motions on pico-nanosecond timescales through the characterization of the spectral density function (SDF), which describes the motions of amide bonds in proteins. The SDF has to be sampled at low magnetic fields, inappropriate for protein NMR, in order to obtain a better description of motions. Such measurements are possible by the use of high-resolution relaxometry. Such measurements on Ubiquitin highlight the sub- and low-nanosecond motions in flexible regions. The classical models for the interpretation of relaxation data in proteins are not well suited for intrinsically disordered proteins (IDPs) and require the development of new approaches. We developed a new approach, called IMPACT, based on a mathematical reconstruction of the distribution of correlation times from the experimental SDF. We have applied IMPACT to the transcription factor Engrailed 2. Our method allowed an unprecedented description of the distribution of pico- to nanosecond motions in IDPs. The IMPACT approach will be combined with high-resolution relaxometry measurements on the C-terminal region of the protein Artemis to provide information on an IDP. In addition, we have described the kinetics and thermodynamics of the interaction of Artemis with the DNA Binding Domain of Ligase IV.Overall, this work contributes to the development of new concepts for the interpretation of extensive nuclear spin relaxation data in proteins

Over a decade after the completion of the human genome, researchers around the world are still wondering what information is hidden in the genome. Although the sequences of all human genes are known, it is still almost impossible to determine much more than the primary protein structure from the coding sequence of a gene. As a result of that, the need for recombinantly produced proteins to study protein structure and function is greater than ever. The main objective of this thesis has been to improve protein production, particularly using Escherichia coli. To improve protein production in Escherichia coli there are a number of different parameters to consider. Two very important parameters in the process of protein production are transcription and translation. To study the influence of differences in transcription rate, target proteins with different characteristics were produced under control of three promoters of different strength (lacUV5, trc and T7). Analyzing the total amount of target protein as well as the amount of soluble protein demonstrated the benefits of using a strong promoter such as T7. However, protein production is also highly dependent on translational efficiency, and a drawback associated with the use of Escherichia coli as host strain is that codons rarely used in this host can have a negative effect on the translation. The influence of using a strain supplied with genes for rare codon tRNAs, such as Rosetta(DE3), instead of the standard host strain BL21(DE3), was therefore evaluated. By using Rosetta(DE3) an improved protein yield for many of the poorly produced proteins was achieved, but more importantly the protein purity was significantly increased for a majority of the proteins. For further understanding of the underlying causes of the positive effects of Rosetta(DE3), the improved purity was thoroughly studied. The cause of this improvement was explained by the fact that Rosetta(DE3) has a significantly better read through of the full sequence during translation and thereby less truncated versions of the full-length protein is formed.  Moreover, the effect of supplementation of rare tRNAs was shown to be highly dependent on the target gene sequence. Surprisingly, it was not the total number of rare codons that determined the benefit of using Rosetta(DE3), instead it was shown that rare arginine codons and to some extent also rare codon clusters had a much bigger impact on the final outcome. As a result of the increased interest in large-scale studies in the field of proteomics, the need for high-throughput protein production pipelines is greater than ever. For that purpose, a protein production pipeline that allows handling of nearly 300 different proteins per week was set up within the Swedish Human Protein Atlas project. This was achieved by major and minor changes to the original protocol including protein production, purification and analysis. By using this standard setup almost 300 different proteins can be produced weekly, with an overall success rate of 81%. To further improve the success rate it has been shown that by adding an initial screening step, prior high-throughput protein production, unnecessary protein production can be avoided. A plate based micro-scale screening protocol for parallel production and verification of 96 proteins was developed. In that, protein production was performed using the EnBase® cultivation technology followed by purification based on immobilized metal ion affinity chromatography. The protein products were finally verified using matrix-assisted laser desorption ionization time-of-flight MS. By using this method, proteins that will be poorly produced can be sorted out prior high-throughput protein production.

QC 20131120

Much like Ultra High Temperature (UHT) processed milk, UHT processed high protein beverages suffer from storage defects including gelation and sedimentation. Although these beverages are made with milk proteins, the food system is very different and much more complex from that of milk. Thus, one cannot assume that the mechanism of development of storage defects is the same as in milk. As such, the goal of this project was to investigate the factors affecting storage stability of high protein beverages. As a first step, a method was developed to facilitate the study of milk protein solutions by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) Spectroscopy. Scanning the proteins in solution followed by subtraction of the contribution to the absorbance by water provided the most reliable and repeatable results. Next, the suitability of the data for various forms of spectral enhancement was assessed. Finally the method was applied in a series of experiments in order to determine if the data could provide numerical information. Thermodynamic data for heating of β-lactoglobulin was obtained using three methods: transmission-FTIR spectroscopy in D2O, ATR-FTIR spectroscopy in D2O and in H2O. All techniques provided equivalent results, indicating that analyzing proteins in water by ATR-FTIR spectroscopy can be used to provide quantitative information. A number of different protein products (α-lactalbumin, β-lactoglobulin, calcium caseinate, WPC, WPI, MPC, MPI) and combinations of these proteins were UHT processed and their storage stability was observed. Unfortunately, the changes were too subtle to be detected by FTIR spectroscopy and we had to rely more heavily on visual observations. From these observations it was determined that altering the protein combinations did not prevent sedimentation of caseinates. However, the presence of β-lactoglobulin changed the consistency of the sediment. Additionally, it was
Tout comme dans le cas du lait ultra haute température (UHT), les protéines contenues des les breuvages hyper protéinés sont affectées par le traitement à haute température. Ce type de traitement thermique mène à la formation de gel et de sédiments dans ces boissons. Bien que ces breuvages soient à base de protéines laitières, la composition chimique de ces boissons demeure très différente et beaucoup plus complexe que celle du lait. Ainsi, on peut supposer que le mécanisme de formation menant à des défauts de conservation est différent de celui du lait ultra haute température. Le but de ce projet était de se familiariser avec les facteurs affectant la stabilité de conservation des breuvages hyper protéinés.La première étape du projet consistait à développer une méthode pour étudier les solutions hyper protéinées à base de lait en utilisant la spectroscopie infrarouge à transformée de Fourier en réflectance totale atténuée (RTA-IRTF). Il a été déterminé que le balayage des protéines en solution suivi d'une soustraction du spectre de l'eau était la méthode la plus fiable et la plus reproductible. Une fois l'acquisition des données brutes complétée, celles-ci étaient transformées par amélioration spectrale et évaluées pour déterminer si elles pouvaient être utilisées de manière quantitative. Les données thermodynamiques sur le chauffage de la β-lactoglobuline ont été obtenus en utilisant trois méthodes: IRTF à transmission dans D2O, RTA-IRTF dans D2O et dans H2O. Ces trois méthodes ont produit des résultats équivalents indiquant que de l'information quantitative pouvait être obtenue lors de l'analyse des protéines en solution aqueuse en utilisant la spectroscopie RTA-IRTF. Un traitement UHT a été appliqué sur différentes permutations de protéines et nous avons essayé de suivre le vieillissement des protéines avec la méthode développée. Malheureusement

To understand the cellular processes, knowledge of the localization and function of proteins are essential. There are several high throughput ventures examining the human proteome. However, there are some bottlenecks in these ventures. For example the production and expression of soluble proteins for analysis. Another obstacle for affinity proteomics is the generation of high quality antibodies, invaluable tools in biotechnological applications. The objective in this thesis was to facilitate protein purification and sample preparation before analysis and downstream applications. We also aimed to attain more information on what constitutes an ideal immunogen, and on how different immune systems respond to a common amino acid sequence.   In one of the projects an automated purification set-up was developed to ensure high recovery of up to milligram amounts of protein with high purity. The system allowed up to 60 recombinant proteins to be purified under both native and denaturing conditions. In another project, the same developed set-up was additionally shown to work with an alternative chromatography resin with small adjustments. Instead of immobilized metal ion affinity chromatography, used in the first project, ion exchange chromatography was applied under denaturing conditions, with good results. To further automate the production line in high throughput projects, an automated sample preparation was set up for mass spectrometry and e.g. gel electrophoresis analysis. We showed that a crude cell lysate could be used as input in the magnetic bead based system, and totally absent from manual handling, the output was purified and buffer exchanged samples ready for mass spectrometry analysis, as well as a fraction of sample that could be used for complementary analyses, for example gel electrophoresis to determine the protein concentration and purity.   The other objective was – as noted – to gain better comprehension of antibody generation to foreign proteins, and to shed more light over how to design a good antigen. First was a solubility assay developed that determined the remaining fraction of soluble protein after reduction of the concentration of denaturing agent. The assay was performed in a 96 deep well plate, and only instrumentation available in a standard laboratory was necessary. The fact that the assay could be automated on a pipetting robot, increased the throughput and reduced the necessary manual handling. Obtained information on antigen solubility was correlated to the cognate antibody titers. At average the antibody yield was higher when a soluble antigen was used for immunization. Also, the probability of failing in eliciting an immune response was increased if an insoluble antigen was used. However, the antibody titers in each solubility class were highly diverse, and thus also some insoluble antigens were found that provoked the immune system. To further examine the differences between different B cell repertoires, a massive epitope mapping was performed with more than 400 different antisera reacting to the same amino acid sequence. Antigenic hot spot regions were discovered, as well as regions depleted in antibody recognition. However, in one third of the antisera the most abundant antigenic region did not elicit any binding of antibodies. This further validates the conclusion that good antigen design is essential, however is it not certain the outcome of immunizations can ever be determined a priori due to the variability between hosts. An alternative to immunization is selection of affinity reagents by phage display. In the last project an initial parallelized set-up selected antibody fragments that showed high specificity and were compatible with several biotechnological applications, making the set-up a promising alternative to conventional immunization in proteome-wide endeavors.
QC 20101102

In the field of proteomics, proteins are analyzed and quantified in high numbers. Protein analysis is of great importance and can for example generate information regarding protein function and involvement in disease. Different strategies for protein analysis and quan- tification have emerged, suitable for different applications. The focus of this thesis lies on protein identification and quantification using different setups and method development has a central role in all included papers. The presented research can be divided into three parts. Part one describes the develop- ment of two different screening methods for His6-tagged recombinant protein fragments. In the first investigation, proteins were purified using immobilized metal ion affinity chro- matography in a 96-well plate format and in the second investigation this was downscaled to nanoliter-scale using the miniaturized sample preparation platform, integrated selective enrichment target (ISET). The aim of these investigations was to develop methods that could work as an initial screening step in high-throughput protein production projects, such as the Human Protein Atlas (HPA) project, for more efficient protein production and purification. In the second part of the thesis, focus lies on quantitative proteomics. Protein fragments were produced with incorporated heavy isotope-labeled amino acids and used as internal standards in absolute protein quantification mass spectrometry experiments. The aim of this investigation was to compare the protein levels obtained using quanti- tative mass spectrometry to mRNA levels obtained by RNA sequencing. Expression of 32 different proteins was studied in six different cell lines and a clear correlation between protein and mRNA levels was observed when analyzing genes on an individual level. The third part of the thesis involves the antibodies generated within the HPA project. In the first investigation a method for validation of antibodies using protein immunoenrichment coupled to mass spectrometry was described. In a second study, a method was developed where antibodies were used to capture tryptic peptides from a digested cell lysate with spiked in heavy isotope-labeled protein fragments, enabling quantification of 20 proteins in a multiplex format. Taken together, the presented research has expanded the pro- teomics toolbox in terms of available methods for protein analysis and quantification in a high-throughput format.

QC 20141022

Affinity proteomics has experienced rapid development over the last two decades and one of the most promising platforms to emerge are the protein microarrays. The combination of affinity reagents and miniaturisation enables assays for simultaneous high throughput and sensitive protein analysis. Due to the combination of these desrable properties, a multitude of protein array platforms for rapid and efficient study of proteomes and protein interactions are in use today. Although the protein microarray field has more than two decades of history to look back on the development of new protein microarray platforms continues to this day and beyond. In the paper I in this thesis, a microarray of eluates from dried blood spot samples collected from neonates were designed and utilised for detection of complement factor 3 (C3) deficiency. The data acquired from the microarrays platform were compared to C3 levels obtained through enzyme-linked immunosorbant assay (ELISA), and the microarray assay were found to separate the C3 deficient samples from the controls. The conclusion of this investigation was that the microarray platform would be suitable for high-throughput screening of C3 deficiency in neonates. Paper II outlines the work in developing a multiplex platform for validation of affinity reagents. A set of 398 affinity binders, originating from five research groups, were profiled against 432 antigens and representing both polyclonal rabbit antibodies, monoclonal mouse antibodies, and recombinant single-chain variable fragments. Approximately 50% of the binders were found to preferably recognise their intended target while 10% of the binders did not generate any, or low, signals with their respective targets. For paper III, a reverse phase array (RPPA) platform using fluorescence-based detection of IgA deficiency in over 2.000 samples where validated on a label-free detection system and ELISA. The data from the label-free platform and the RPPA were found agree well with each other while data from ELISA did with neither of them. It was found that the label-free platform proved to be well-suited for detection of IgA in serum. Paper IV describes one of the world’s largest protein microarrays containing 21.120 recombinant protein fragments. We describe some of the possible applications of these large-scale arrays, such as binding profiles for the validation of antibodies with 11.520 and 21.120 recombinant proteins, as well as screening for autoimmunity in human serum samples.

QC 20150528

Introduction and Background Aventure AB has created a "between-meal beverage" with high protein and energy content aimed at older and/or physically active individuals. Aventure AB wished to transform this high energy drink to a plant-based product, because of the growing trend of plant-based diets. Aim The aim of this project was to replace the protein source in the beverage ”Skaka & smaka - strawberry taste”, while maintaining good flavour and texture. The original beverage contains whey protein, which was to be replaced with a vegetable source. Materials and methods A new plant-based beverage was developed from the original recipe. Four different alternative protein sources were added and evaluated with regard to taste, visual look, pH, dry matter, viscosity, brix, protein and energy content. To beverages based on different protein sources, four different juice concentrates were added in varying combinations and concentrations. Aromas were also added and a change of the fruit-purée recipe was made. The salt concentration was modified. The final product was evaluated through a consumer sensory analysis at a sports centre in Lund. The participants were asked to judge it by first impression, colour, odour, flavour, texture and overall impression on a 9-point hedonic scale. 76 individuals participated and compared the original beverage with the new plant-based one. Results and Discussion A beverage containing pea protein with a combination of a new fruit purée with a reduced amount of strawberries but an increased amount of bananas was selected as the most promising candidate. Further improvement of this beverage included addition of a juice concentrate mixture (6 mL/100 g), containing 50 % apple concentrate + 50 % lime concentrate. The salt content in the beverage was 0.03 g salt/100 g beverage. The sensory analysis revealed a significant difference regarding the first impression and flavour in favour of the original beverage, while a significant difference in colour appeared in favour of the new one containing pea protein. 19.6 % of the participating women, and 22.5 % of the men claimed that they would buy the new plant-based beverage. Conclusion A plant-based version of Skaka & smaka has been developed, and the sensory analysis indicates that the new product has potential on today's market. However, the beverage requires further development to satisfy all the needs within the target group.

The high mobility group protein HMGA2 is an architectural transcription factor, which is expressed during embryogenesis. Aberrant expression causes benign and malignant tumor formation. The protein possesses three "AT hook" domains and an acidic Cterminal. HMGA2 is natively unstructured, however it forms a homodimer. In this study site-directed mutagenesis was used to create single methionine mutants, HMGA2Q37M, HMGA2I71M and HMGA2Q85M. These mutants were cross-linked using EDC and then cleaved using CNBr to determine which domains are involved in homodimer formation. Our results indicate that the second "AT hook" domain may interact with the C-terminal. We then labeled a peptide containing the C-terminal (CTP) with tetramethylrhodamine-5- maleimide (TRM). We found that the CTP-TMR binds to HMGA2Α95-108, which lacks the C-terminal. These results suggest that the C-terminal is required for homodimer formation. The techniques used within this study can be applied to forensics and with further research HMGA2 may have a forensic application.

Tuberculosis (TB) is an endemic health-crisis, particularly in sub-Saharan Africa. The rise of multiand extensively-drug resistant Mycobacterium tuberculosis (Mtb), the causative agent of TB, has led to further developments in understanding the physiology of Mtb during infection, as well as searching for novel drug targets, in order to combat the disease. Our understanding of cells, both eukaryotic and prokaryotic, has changed substantially in the last 50 years, incorporating the role of stable and transient protein-protein interactions which govern cell function and behaviour. Although there are many in vivo and in vitro methods for studying protein-protein interactions, they suffer from the lack of ability to distinguish physiological interactions from interactions that occur which are not physiologically relevant to the cell. Structure-based methods for determining protein interactions have the benefit of screening out false positives whilst simultaneously assessing the possible biological function of the protein complex in question. This study sought to assess different high-throughput methods for capturing stable, water soluble protein complexes from M. smegmatis (Msm), a close relative of Mtb, for structural characterisation by low-resolution transmission electron microscopy (EM). The use of partial biochemical fractionation was assessed, which produced low-resolution structures of glutamine synthetase I, bacterioferritin, and Encapsulin. These structures were unambiguously identified through a combination of fitting of homologous crystal structures into the low-resolution maps, and information obtained by liquid chromatography mass spectrometry (LC-MS/MS) of bands isolated from native- and SDS-PAGE gels. Since Encapsulin is likely to participate in the Msm oxidative stress response and functions to enclose the target proteins DyP-type peroxidase (DyP) and ferritin-family protein (BrfB), optimal conditions for cryo-EM were tested for further efforts to obtain a high-resolution structure. Furthermore, hypotheses were generated for the function of Mtb and Msm Encapsulin based on the Msm Encapsulin structure obtained with the aid of a crystal structure homologue; these related to the mode of cargo binding and pore selectivity. A single-step purification method was also assessed through grid blotting on blue native (BN) PAGE using GroEL as a test protein. The hydrophobicity and charge of the EM copper grid was tested to find the optimal grid property for particle transfer. This established that particles of GroEL could be transferred from BN-PAGE onto an EM copper grid and a successful negative stain reconstruction was obtained. In summary, the pipeline from purifying protein complexes to generating hypotheses based on structure was successfully investigated in Msm, which will aid in the production of novel drug targets for Mtb as well as in the application to other organisms.

Doutoramento em Biomedicina
Protein synthesis is central to life and is being intensively studied at various levels. The exception is mRNA translational fidelity whose study has been hampered by technical difficulties in detecting amino acid misincorporations in proteins. Few genes have so far been associated to the control of protein synthesis fidelity and it is unclear how many genes control this biological process. We investigated the role of RNA modification by RNA modifying enzymes (RNAmods) in protein synthesis efficiency and accuracy. Our hypothesis was that RNAmods that modify tRNA nucleosides (tRNAmods) have a significant impact on protein synthesis through modulation of codonanticodon interactions. To address this issue, we focused our work on tRNAmods involved in the modification of tRNA anticodons. The biology of these enzymes is still poorly understood, but they are involved in RNA processing, stability and function and their deregulation is associated with cancer, neurodegenerative, metabolic and other diseases. We have set up a yeast genetic screen and used mass-spectrometry methods to determine the role of tRNAmods on proteome homeostasis. Our work identified a subgroup of yeast tRNAmods that play essential roles in protein synthesis fidelity and folding. The genes that encode insoluble proteins isolated from yeast cells lacking U34 modification were enriched in codon sites that are decoded by the hypomodified tRNAs. These aggregated proteins also participate in specific biological processes, suggesting that tRNAmods are linked to specific physiological pathways. Interestingly, we detected amino acid misincorporations at the codon sites decoded by the anticodons of the hypomodified tRNAs, demonstrating that tRNA U34 modifications control translational error rate.
A síntese proteica é central para a vida e tem sido extensivamente estudada a vários níveis. Contudo, o estudo da fidelidade da tradução do mRNA tem progredido lentamente devido a dificuldades técnicas na deteção de incorporações incorretas de aminoácidos nas proteínas. Poucos genes têm sido associados com o controlo da fidelidade da síntese proteica e não é evidente quais os genes que controlam este processo biológico. Nesta tese investigámos o papel da modificação dos nucleósidos do RNA na eficiência e precisão da síntese proteica. A nossa hipótese é que as enzimas que modificam nucleósidos do tRNA (tRNAmods) têm um impacto significativo na síntese proteica através da modulação das interações codão-anticodão. A biologia das tRNAmods e das modificações do tRNA são ainda pouco conhecidas, mas estão envolvidas na estabilidade e função do RNA e mutações nos seus genes causam doenças neurodegenerativas, metabólicas, cancro, entre outras. Neste projeto realizámos um rastreio genético em levedura com o objetivo de identificar tRNAmods que asseguram a homeostase do proteoma (proteostase) e usámos espectrometria de massa para clarificar o papel das tRNAmods na fidelidade da síntese proteica. Os resultados do estudo genético mostram que um sub-grupo de tRNAmods envolvidas na modificação de nucleósidos do anticodão do tRNA são essenciais para manter a estabilidade do proteoma. Outras tRNAmods estudadas não produziram impactos visíveis na proteostase. Os genes de proteínas agregadas que isolámos a partir de células de levedura com tRNAs hipomodificados são enriquecidos em codões descodificados por estes tRNAs. Os nossos dados mostram também que tais proteínas participam em processos biológicos específicos e têm níveis de aminoácidos errados mais elevados que as células wild-type. Estes dados mostram que certas modificações do tRNA são essenciais para a fisiologia celular, estabilidade do proteoma e fidelidade da síntese proteica.

Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection.
Grand Challenges Canada
Revisión por pares

Orientador: Yoon Kil Chang
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Os análogos de carne obtidos por extrusão termoplástica de proteínas vegetais são caracterizados pelo seu elevado teor proteico e estrutura semelhante às fibras da carne, envolvendo diversos tipos de ligações e/ou interações químicas entre as proteínas. O objetivo deste trabalho foi avaliar as características tecnológicas e físico-químicas de análogos de carne, à base de isolado proteico de soja, obtidos por processo de extrusão termoplástica a alta umidade (AU) e baixa umidade (BU). Para cada condição de umidade foi utilizado um Delineamento Composto Central Rotacional de três variáveis independentes (glúten vital, umidade de condicionamento e temperatura de extrusão). As variáveis dependentes avaliadas foram a textura instrumental, cor instrumental, capacidade de absorção de água, índice de solubilidade em água, capacidade de absorção de óleo, índice de dispersibilidade de proteína, energia mecânica específica e o tipo de interações proteicas. Estas interações foram avaliadas através de sete tipos de solventes específicos: (i) tampão fosfato para as proteínas no estado nativo; (ii) dodecil sulfato de sódio para as interações hidrofóbicas e iônicas; (iii) Triton 100X para as interações hidrofóbicas; (iv) ureia para as interações hidrofóbicas e pontes de hidrogênio; (v) ß-mercaptoetanol para as ligações dissulfeto; e (vi) ß-mercaptoetanol e ureia e (vii) dodecil sulfato de sódio e ureia, para avaliar o efeito sinérgico entre os sistemas. O ponto otimizado (caracterizado principalmente por promover maiores valores de L* e de capacidade de absorção de água, menores valores de índice de solubilidade em água, de capacidade de absorção de óleo, de desnaturação proteica e valores intermediários de textura instrumental e de energia mecânica específica) foi processado juntamente com uma amostra controle para ambos os processos com o intuito de validar os modelos matemáticos e avaliar as possíveis alterações na morfologia dos análogos de carne, na massa molecular das proteínas, na composição de aminoácidos totais e na desnaturação proteica. As melhores condições de processamento foram obtidos para os análogos de carne contendo de 12 e 5 % de glúten vital, 58 e 18 % de umidade de condicionamento e 135 e 100 °C para a temperatura de extrusão, para o processo AU e BU, respectivamente. As principais interações proteína-proteína encontradas nos análogos de carne foram as ligações dissulfeto e ligações de hidrogênio para o processo AU e as ligações dissulfeto e interações iônicas para o processo BU. A adição de glúten vital promoveu uma aparência mais lisa e melhor orientação na estrutura das fibras. Verificou-se que ocorreu aumento nas proteínas de baixa massa molecular e diminuição nas proteínas de alta massa molecular. No perfil de aminoácidos totais houve maior variação negativa para os aminoácidos essenciais (triptofano e treonina), semi essenciais (cisteína) e não essenciais (serina), indicando que houve redução no valor nutricional. As estruturas secundárias (a-hélice, ß-folha, ß-volta e a estrutura desordenada) mostraram alteração na sua conformação devido à desnaturação proteica e formação de novos agregados
Abstract: Meat analogue obtained by termoplastic extrusion of vegetable proteins are characterized by its high protein levels and structure similar to meat fibers, which comprises many types of chemical bonds and/or interactions between proteins. The aim of this work was to evaluate the technological and physico-chemical characteristics of meat analogue based on isolated soy protein obtained by thermoplastic extrusion process at high moisture (HM) and low moisture (LM) content. For each moisture condition was used a Central Rotational Composite Design with three independent variables (vital gluten, moisture content and extrusion temperature). The dependent variables evaluated were instrumental texture, instrumental color, water absorption capacity, water solubility index, oil absorption capacity, protein dispersibility index, specific mechanical energy, and the type of protein interactions. These interactions were evaluated using seven specific solvents types: (i) phosphate buffer for proteins in native state; (ii) sodium dodecil sulphate for hydrophobic and ionic interactions; (iii) Triton 100X for hydrophobic interactions; (iv) urea for hydrophobic interactions and hydrogen bonds; (v) ß-mercaptoethanol for dissulfide bonds; and (vi) ß-mercaptoethanol and urea and (vii) sodium dodecil sulphate and urea, for the synergistic effect between the systems. The optimized point (characterized mainly by promoting higher values for L* and water absorption capacity, lower values for water solubility index, oil absoption capacity and protein denaturation and intermediate values for instrumental texture and specific mechanical energy) was processed, together with a control sample for each processes, in order to validate the mathematical models and to evaluate possibles changes in the meat analogues morphology, in the protein molecular weight, in the total amino acid composition, and in the protein denaturation. The best processing conditions were obtained for the meat analogue containing 12 and 5 % of vital gluten, 58 and 18 % of moisture content and 135 and 100 °C of extrusion temperature, for the HM and LM processes, respectively. The main protein-protein interactions found in meat analogues were the dissulfide bonds and hydrogen bonds for the LM process and the dissulfide bonds and ionic interactions for the HM process. The addition of vital gluten promoted a smoother appearance and better orientation in the fiber structure. It was found that occured an increase in the protein with low molecular weight and a reduction in the protein with high molecular weight. There were a greater negative variation for the essential (tryptophan and threonine), semi-essential (cysteine) and nonessential (serine) amino acids in the total amino acid profile, indicating a reduction of the nutritional value. The secondary structure (a-helix, ß-sheet, ß-turn and disordered structure) showed alteration in its conformation due to the protein denaturation and formation of new aggregates
Doutorado
Tecnologia de Alimentos
Doutor em Tecnologia de Alimentos

High quality reagents are preconditions for high performance protein analyses. But despite progress in some techniques, e.g. mass spectrometry, there is still a lack of affinity-based detection techniques with enhanced precision, specificity, and sensitivity. Building on the concept of multiple affinity recognition reactions and signal amplification, a proximity ligation assay (PLA) was developed as a molecular tool for analyzing proteins and their post-translational modification and interactions. PLA enhanced the analysis of protein expression levels and post-translational modifications in western blotting (Paper I), which had elevated sensitivity and specificity, and an ability to investigate protein phosphorylation. A general and straightforward method was established for the functionalization of affinity reagents through adding DNA strands to protein domains for protein analysis in medicine (Paper II). A method for protein domain-mediated conjugation was developed to simplify the use of recombinant affinity reagents, such as designed ankyrin repeat protein (DARPin), in DNA-mediated protein analyses. Alzheimer’s disease (AD) is characterized by progressive cognitive decline and memory impairment, and amyloid-beta plaques and neurofibrillary tangles (NFT) in the brain are clinical hallmarks of the disease. In order to understand the mechanisms underlying the formation of NFT, in situ PLA was used to explore the role of microtubule affinity related kinase 2 (MARK2) in phosphorylating tau protein during the pathological progress of AD (Paper III). The analyses of roles of MARK proteins 1-4 in phosphorylating tau protein in cells and in post-mortem human brains were performed in Paper IV. The focus of this thesis was the study of post-translational modifications and interactions of proteins in medicine. Procedures for high performance protein analysis in western blotting via proximity ligation were developed, and a functionalization method for recombinant affinity reagents in DNA-mediated protein analysis was established. These and other techniques were used to investigate the roles of tau-phosphorylating MARK family proteins in AD.

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Errata attached to inside front cover. Bibliography: leaves 112-166. This thesis is concerned with the interaction of the plasma factor, phospholipid transfer protein (PLTP) with HDL. The studies described in this thesis define the mechanism of the remodelling of reconstitute HDL (rHDL) by PLTP and the reasons why triglyceride enrichment of rHDL enhances the remodelling process. Other studies describe why triglyceride enrichment of rHDL enhances PLTP-mediated phospholipid transfers. In conclusion, this thesis describes the mechanism of the PLTP-mediated remodelling of rHDL and presents reasons why the remodelling is enhanced in particles that are enriched with triglyceride.

Thesis: M. Eng., Massachusetts Institute of Technology, Department of Electrical Engineering and Computer Science, 2018.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis.
Includes bibliographical references (pages 45-47).
We present cell gels, a platform for high-throughput multiplexed measurements of transcriptome and proteome in single cells. This method takes advantage of droplet barcoding and hydrogel chemistry to capture mRNAs and proteins in thousands of cells. We report the challenges of acquiring sequencing data from cell gels due to complex mechanisms underlying bead based library prep in polyacrylamide gels. We show the applications of this method in detecting intracellular proteins, sorting based on intracellular markers after cell lysis, and expanding thousands of cells in single droplets.
by Shirin Shivaei.
M. Eng.