Influence of Ser and Thr residues in the geometry of transmembrane helices : implications on the structure and function of G protein-coupled receptors

Consultable des del TDXTítol obtingut de la portada digitalitzadaEn aquesta tesi s'apliquen eines bioinformàtiques a l'estudi de determinats sistemes biològics. En particular, l'estudi teòric de la influència de determinats aminoàcids sobre l'estructura i la dinàmica dels elements d'estructura secundària de les proteïnes s'aplica a la modelització per homologia dels receptors acoblats a proteïna G (GPCRs) i a l'estudi dels seus mecanismes d'activació. Se sap que determinats residus, com prolina, serina o treonina, provoquen distorsions locals en l'estructura de les hèlices a. L'anàlisi de bases de dades de seqüències de segments transmembrana mostra com certes combinacions d'aquests residus són més comunes que d'altres, i que algunes d'elles estan sobre-representades de manera significativa, mentre que d'altres estan clarament sots-representades. La restricció d'aquesta anàlisi de seqüències a la regió transmembrana dels GPCRs de la Classe A mostra com aquestes combinacions es troben en posicions específiques i, a més, es troben conservades en certes subfamílies de receptors. L'estructura i la dinàmica de les hèlices transmembrana que contenen aquestes combinacions de prolina i serina o treonina s'han estudiat mitjançant simulacions de dinàmica molecular en un entorn hidrofòbic explícit. Els resultats mostren com algunes d'aquestes combinacions indueixen distorsions importants en l'estructura de l'hèlix a, degut al seu efecte desestabilitzador de la xarxa de ponts d'hidrogen que dóna estabilitat a l'hèlix. Aquests resultats s'han aplicat a la construcció d'un model tridimensional del receptor de quimiocines CCR5 , utilitzant tècniques de modelització molecular per homologia. En aquest model es proposa que les hèlices transmembrana (TMH) 2 i 3 del receptor CCR5 són estructuralment diferents del patró de rodopsina. TMH2 està més doblegada degut a la presència d'un motiu Thr-X-Pro, que, a més, fa que aquesta hèlix es doblegui cap a TMH3. Així doncs, es proposa que, en aquest receptor, aquestes dues hèlices interaccionen. Aquesta interacció estaria mediada per la presència de residus hidrofòbics conservats i específics en les dues hèlices. Aquestes hipòtesis han estat posades a prova mitjançant experiments de mutagènesi dirigida, gràcies a la col·laboració amb l'Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. Els resultats experimentals permeten establir la hipòtesi que la interfície TMH2-TMH3 participa en l'activació induïda per quimiocines del receptor CCR5. Com a conclusió, aquesta tesi pretén mostrar com, mitjançant la utilització d'eines bioinformàtiques, és possible traduir les seqüències primàries de proteïnes i les interaccions a nivell atòmic en estructures tridimensionals de proteïnes. A més, aquesta tesi mostra que, encara que l'estructura tridimensional de la rodopsina bovina és un patró útil per la modelització per homologia de GPCRs, s'han de tenir en compte de manera explícita les especificitats de seqüència de cada receptor per tal de construir models de receptors particulars. Aquestes especificitats de seqüència consisteixen en patrons de seqüència conservats en determinades famílies, que es tradueixen en divergències estructurals. Entre aquests patrons de seqüència, es proposa que els residus de serina i treonina, sols o combinats amb residus de prolina propers, poden modular la geometria de les TMHs, degut a la seva capacitat d'interferir amb la xarxa de ponts d'hidrogen que dóna estabilitat a les hèlices a. Finalment, es proposa que la influència dels motius de serina, treonina i prolina en l'estructura de les TMHs pot estar relacionada amb els processos d'activació dels GPCRs de la Classe A i, possiblement, d'altres proteïnes de membrana. En els GPCRs, aquests motius poden haver evolucionat per tal d'adaptar uns mecanismes d'activació conservats als lligands característics de cada família de receptors.This thesis is framed in the study of particular biological systems through the use of bioinformatics. In particular, the theoretical study of the influence of certain amino acids on the structure and dynamics of the secondary structure elements of proteins has been applied to homology modelling of G protein-coupled receptors (GPCRs) and to the study of their mechanisms of activation. Certain residues, as proline, serine or threonine, are known to induce local distortions in the a-helical structure. Analysis of sequence databases of transmembrane segments evidence that certain combinations of these residues are more common than others, and that some of them are significantly over-represented, while others are clearly under-represented. The focusing this sequence analysis on the transmembrane region of Class A GPCRs illustrates that these combinations are located in some specific locations and conserved within certain subfamilies of receptors. The structure and dynamics of transmembrane a-helices containing these combinations of proline and serine or threonine have been studied using molecular dynamics simulations in an explicit hydrophobic environment. The results show how some of these combinations induce significant distortions in the a-helical structure, due to their effect on the hydrogen bond network that stabilizes the helix. These results have been applied to the building of a three-dimensional model of the chemokine CCR5 receptor, using homology modelling techniques. In this model, transmembrane helices (TMH) 2 and 3 of CCR5 are proposed to be different from the bovine rhodopsin template. TMH2 is more bent due to the presence of a Thr-X-Pro motif, which, in turn, induces this helix to lean towards TMH3. As a consequence, an interaction between these two helices is proposed for this particular receptor. This interaction would be mediated through the presence of specific and conserved hydrophobic and aromatic residues in both helices. These hypothesis have been tested through site-directed mutagenesis experiments, thanks to a collaboration with the Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire (IRIBHN), Université Libre de Bruxelles. The experimental results let us to hypothesize that the TMH2-TMH3 interface is involved in the chemokine-induced activation of the CCR5 receptor. As a conclusion, this thesis aims to show how through the use of bioinformatics tools, primary sequences of proteins and interactions at an atomic level can be translated to three-dimensional protein structures. In addition, this thesis illustrates that, even though the three-dimensional structure of bovine rhodopsin is a very useful template for homology modelling of GPCRs, the sequence specificities of each receptor have to be explicitly taken into account in order to build models. These sequence specificities consist in sequence patterns conserved within certain families, which are translated into structural divergences. Among these sequence patterns, we hypothesize that serine and threonine, alone or combined with nearby proline residues, can modulate the geometry of TMHs, due to its capability to interfere with the hydrogen bond network that stabilize a-helices. Finally, we propose that the influence of serine, threonine and proline motifs in the structure of TMHs may be related to processes of activation in the Class A of GPCRs, and, possibly, other membrane proteins as well. In GPCRs, these motifs may have evolved in order to adapt a conserved mechanism of activation of the G protein to the cognate ligands of each receptor family

Somatic cell count in dairy herds

Over the last two decades the dairy industry has encountered some major changes and as a result the farmer’s attention has shifted from a cow- towards a herd-level approach by means of close monitoring of herd level parameters. The overall aim of this thesis was to critically review how useful current somatic cell count (SCC) parameters are for monitoring udder health on herd level by producers and processors, and if necessary to suggest improvements and recommendations for the use of these parameters. For a large number of farms worldwide the bulk SCC (BMSCC) is the SCC parameter available to monitor trends in udder health. In this thesis it was assessed if bulk SCC (BMSCC) reflects the average SCC of all individual lactating cows, the correlation with the prevalence of cows with a subclinical mastitis, and if the sampling interval and variation of the BMSCC data affected the accuracy to describe measured detailed series of BMSCC data. Subsequently it was examined which cow factors, management practices, and seasonal fluctuations contributed to the average herd somatic cell count (HSCC) and if these factors found in studies in Europe and the United States also applied on farms in a different environment such as on Australian dairy farms. Finally, to support decision regarding udder health a model to predict of the average herd somatic cell count of the next month was developed. Briefly stated, the main conclusions of the studies described in this thesis are: - BMSCC is a less useful tool to monitor udder health when individual herd recording data are available; - The difference between BMSCC and the average SCC of all lactating cows is with up to 40%; - When BMSCC data are the only data available to monitor udder health at herd level, it should be anticipated that it is likely that it will underestimate the prevalence of subclinicalmastitis; - When BMSCC is used to detect a trend in average HSCC, the error in describing a series of BMSCC data increases significantly when sampling interval exceeds 4 d; - When BMSCC data is used as the outcome variable in management studies, a proportion of farms may be inaccurately classified as too low and as a consequence, association between BMSCC and certain management practices could be incorrect; - The contribution of management, herd characteristics and seasonal variables are different per level of the average HSCC and therefore when advice on alterations in management is given, the level of average HSCC should be taken into account; - Management style might be equally important for the impact of a management practice as the implementation itself; - Management practices applied in the EU and United States will have a similar effect on udder health under Australian conditions; - An accurate prediction of the average HSCC for the subsequent month is possible when information on management, herd characteristics and seasonal variation is taken into account

Strategic test-day recording regimes to estimate lactation yield in tropical dairy animals

Background In developing dairy sectors, genetic improvement programs have limited resources and recording of herds is minimal. This study evaluated different methods to estimate lactation yield and sampling schedules with fewer test-day records per lactation to determine recording regimes that (1) estimate lactation yield with a minimal impact on the accuracy of selection and (2) optimise the available resources. Methods Using Sahiwal cattle as a tropical dairy breed example, weekly milk records from 464 cows were used in a simulation study to generate different shaped lactation curves. The daily milk yields from these simulated lactation curves were subset to equally spaced (weekly, monthly and quarterly) and unequally spaced (with four, five or six records per lactation) test-day intervals. Lactation yield estimates were calculated from these subsets using two methods: the test-interval method and Wood’s (Nature 216:164-165, 1967) lactation curve model. Using the resulting lactation yields, breeding values were predicted and comparisons were made between the sampling regimes and estimation methods. Results The results show that, based on the mean square error of prediction, use of Wood’s lactation curve model to estimate total yield was more accurate than use of the test-interval method. However, the differences in the ranking of animals were small, i.e. a 1 to 5% difference in accuracy. Comparisons between the different test-day sampling regimes showed that, with the same number of records per lactation (for example, quarterly and four test-days), strategically timed test-days can result in more accurate estimates of lactation yield than test-days at equal intervals. Conclusions An important outcome of these results is that combining Wood’s model for lactation yield estimation and as few as four, five or six strategically placed test-day records can produce estimates of lactation yield that are comparable with estimates based on monthly test-day records using the test-interval method. Furthermore, calculations show that although using fewer test-days results in a decrease in the accuracy of selection, it does provide an opportunity to progeny-test more sires. Thus, using strategically timed test-days and Wood’s model to estimate lactation yield, can lead to a more efficient use of the allocated resources

Selecting an appropriate genetic evaluation model for selection in a developing dairy sector

This study aimed to identify genetic evaluation models (GEM) to accurately select cattle for milk production when only limited data are available. It is based on a data set from the Pakistani Sahiwal progeny testing programme which includes records from five government herds, each consisting of 100 to 350 animals, with lactation records dating back to 1968. Different types of GEM were compared, namely: (1) multivariate v. repeatability model when using the first three lactations, (2) an animal v. a sire model, (3) different fixed effects models to account for effects such as herd, year and season; and (4) fitting a model with genetic parameters fixed v. estimating the genetic parameters as part of the model fitting process. Two methods were used for the comparison of models. The first method used simulated data based on the Pakistani progeny testing system and compared estimated breeding values with true breeding values. The second method used cross-validation to determine the best model in subsets of actual Australian herd-recorded data. Subsets were chosen to reflect the Pakistani data in terms of herd size and number of herds. Based on the simulation and the cross-validation method, the multivariate animal model using fixed genetic parameters was generally the superior GEM, but problems arise in determining suitable values for fixing the parameters. Using mean square error of prediction, the best fixed effects structure could not be conclusively determined. The simulation method indicated the simplest fixed effects structure to be superior whereas in contrast, the cross-validation method on actual data concluded that the most complex one was the best. In conclusion it is difficult to propose a universally best GEM that can be used in any data set of this size. However, some general recommendations are that it is more appropriate to estimate the genetic parameters when evaluating for selection purposes, the animal model was superior to the sire model and that in the Pakistani situation the repeatability model is more suitable than a multivariate

Veterinary advice for entrepreneurial Dutch dairy farmers : From curative practice to coach-consultant: what needs to be changed?

Dairy farms are tending to become larger, with a milk quota of more than 8 tons a year, and are managed by entrepreneurial dairy farmers with their own specific characteristics and farming style. Some Dutch veterinary practices appear unable to respond to this different style and often do not serve such farms or lose them as client. Moreover, the veterinary curriculum often focuses on traditional, family-run, smaller dairy operations and not on larger farms, which raises the question whether newly qualified veterinary practitioners are adequately trained to provide these entrepreneurial farmers with the services they require. This article addresses the characteristics of entrepreneurial dairy farmers and those of cattle practitioners, to determine whether cattle practitioners need to acquire other skills to better prepare them for their coaching-consultant tasks on larger dairy farms