Desde que su genoma fue completamente secuenciado en el año 1995, Mycoplasma genitalium es un patógeno humano que por sus características se considera el modelo ideal para poder estudiar el conjunto mínimo de genes necesarios para mantener todas las rutas biológicas indispensables en una célula crecida en laboratorio. A pesar de su simplicidad, confirmada por el tamaño reducido de su genoma (580 kb y tan solo 482 genes) y la falta de una pared celular, M.genitalium presenta una protrusión de la membrana que se extiende de uno de los polos de la célula, llamada Organela Terminal (TO), presente también en otras especies de Mycoplasmas. Diversos experimentos han puesto en evidencia como la TO esta involucrada en la adherencia, en la división celular, en la patogenicidad y motilidad de ‘gliding’ características de algunas especies de Mycoplasmas. El presente trabajo está centrado en la identificación y caracterización de las interacciones proteína-proteína en el ‘wheel complex’, una ultra-estructura con forma de cuenco que ha sido localizada en la en el extremo más proximal de la TO, cerca del cuerpo celular. También se resolvió a resolución atómica la estructura de MG491, una de las cuatro proteínas que se han localizado en el ‘wheel complex’. Como primer paso para llegar a entender el funcionamiento de la TO, se realizó un experimento de ‘screening’ mediante resonancia plasmónica de superficie (SPR) para detectar las posibles interacciones entre las proteínas MG200, MG219, MG386 y MG491. Además se emplearon resonancia magnética nuclear (RMN) y cromatografía de exclusión molecular (GCF) para corroborar y caracterizar las interacciones identificadas. El ensayo reveló que las cuatro proteínas participan en interacciones muy específicas a través de una extensa red de interacciones fuertes y débiles. En particular, la caja EAGR de MG200, un dominio característico que se encuentra sólo en algunas de las proteínas de TO, interactúa con 25 aminoácidos localizados en la región C-terminal de MG491, y que resultan ser necesarios para el correcto funcionamiento de MG491, como demostrado por experimentos realizados in vivo. El dominio C-terminal de MG200 se une a la región N-terminal de MG219. MG219 también interactúa a través de su dominio C-terminal con MG386 C-terminal, una región que contiene tres cajas EAGR no idénticas.Después de la introducción de cuatro metioninas en la secuencia de MG491, la estructura cristalina se resolvió por difracción anómala simple (SAD) de un constructo que incluye los primeros 308 residuos (MG491-Nt_63-205). La estructura reveló que MG491 forma un dímero de heterodímeros estructurales en los que los monómeros interactúan con los dos monomeros vecinos a través de dos interfaces diferentes. Los dos heterodímero, que tienen la mayor superficie de contacto, están relacionados por una transformación de ~ 72 ° de rotación y una pequeña traslación. En particular, dos conformaciones alternativas de un loop de 19 residuos localizado en la interfaz entre monómeros parecen ser cruciales para la estabilidad de los heterodímeros.Las interacciones identificadas entre las cuatro proteínas que forman el ‘wheel complex’, junto con la estructura de MG491-Nt_63-205 y los resultados obtenidos in vivo, proporcionan nuevos datos sobre la organización y el funcionamiento del ‘wheel complex’ y de toda la Organella Terminal. Esta información también se puede ampliar para el análisis de la ultraestructura de M. genitalium a medida que se determina por microscopía electrónica y por tomografía. / Since its genome was completely sequenced in 1995, human pathogen Mycoplasma genitalium has drawn the attention of the scientific community because its characteristics make it a perfect candidate to study the minimal set of genes necessary and sufficient to sustain all indispensable biological pathways in a viable cell. Despite its genomic simplicity (with a genome of 580 Kb and 482 protein coding genes, 387 considered essentials), M.genitalium presents a membrane extension protruding from one pole of the cell body, called Terminal Organelle (TO), that is present also in several other species of Mycoplasmas. Terminal Organelles have been demonstrated to be involved in adherence, cell division, pathogenicity and gliding motility. The present work is focused on the identification and characterization of protein-protein interactions in the wheel complex, a bowl-shaped ultra-structure within the Terminal Organelle that is located at the most proximal end, close to the cell body. The structure of MG491, one of the four proteins known to be part of the wheel complex, was also determined at close to atomic resolutions. As a first step to understand the functioning and assembling of the Terminal Organelle, mid-throughput Surface Plasmon Resonance (SPR) screening assays were used to detect positive binary interactions among wheel complex proteins MG200, MG219, MG386 and MG491. In addition, nuclear magnetic resonance (NMR) and gel filtration chromatography (GCF) were employed to corroborate and characterize the identified interactions. The assay revealed that the four proteins participate in very specific interactions via an extensive network of transient and stable interactions. In particular, MG200 EAGR box, a characteristic domain only found in proteins from the Terminal Organelle, interacts with a 25 amino acid residues from the C-terminal region of MG491, which has been demonstrated by in vivo experiments to be crucial for MG491 function. In addition, MG200 C-terminal region binds to the N terminal domain of MG219. The latter also interacts through its C-terminal domain with MG386 C-terminal, a region containing three non-identical EAGR boxes. After the introduction of four methionines in MG491 sequence, the crystal structure was solved by single-wavelength anomalous dispersion (SAD) from a construct spanning residues 1 to 308 (MG491-Nt-63-205). The structure revealed that MG491 forms a two-fold dimer of structural hetero-dimers in which monomers interact with its two neighbour subunits through two different interfaces. Subunits from the heterodimer presenting the largest surface contact are related by a transformation of ~72° rotation and a small screw translation. In particular, two alternative conformations of a 19 residues loop, which locates at the interface between monomers, appear instrumental for the stability of heterodimers. Interactions identified relating the four proteins forming the wheel complex, together with the unique structural peculiarities determined from the MG491 structure and the in vivo results, shed light into the organization and functioning of the wheel complex and of the whole Terminal Organelle. This information can also be extended to the analysis of the ultrastructure from M.genitalium as it is being determined by electron-microscopy and electron-tomography.

A continuous column crystalliser has been designed and built using a Schildknecht-type Archimedean transportation screw within a stainless steel jacket. Problems concerned with the surface finish on the inner walls of the jacket which affect crystal transportation have been investigated, leading to effective operation of the apparatus. The process of column crystallisation may be considered as a multistage separation and purification technique; it is applicable to both aqueous and organic systems. In the present work the examination has been undertaken by firstly continuously crystallising a flow of feed solution in the column. Thesecrystals are transported along the screw, melted and some of the melt liquid allowed to flow countercurrently to the crystals thus washing them by reflux. The remainder of the melt liquid is removed as pure product, the impure product provided by the now impure reflux liquid being removed from the opposite extremity of the column. Separations in aqueous solutions which have been investigated include the desalination of brine, concentration of metal salts, the extraction of deuteriai.moxide from a heavy water/water mixture and the concentration of ethanol in water. The purification of organic solvents included the removal of cyclohexane from benzene and the extraction of para-xylene from 0-, m-, p-xylene and ethylbenzene. The unit operations involved in column crystallisation fall into the categories of heat transfer, mass transfer and mixing. In order to examine how dependent the efficient operation of the column is on these factors, the system has been optimised for organic, aqueous and mixed organic/aqueous cases. The variables studied including:- (a) the speed of rotation of the transportation screw (b) the length of the column (c) the attitude of the column (ie crystals transported upwards or downwards). (d) the position of the feed into the column (e) the ratio of the products removal rates (f) the rate of production of crystals Since the scale-up of equipment is an ultimate aim of the present investigation it is necessary to study the results mathematically. Where possible, this examination has been undertaken thus establishing the major factors involved in the separation and purification scheme. It should also be possible to determine whether absolute optimisation of operation is attainable - i.e. does separation increase as the crystal rate increases to infinity or is an optimum separation eventually attained?

The Cambridge structural database (CSD) is a vast resource for crystallographic information. As of 1st January 2009 there are more than 469,611 crystal structures available in the CSD. This work is centred on a program dSNAP which has been developed at the University of Glasgow. dSNAP is a program that uses statistical methods to group fragments of molecules into groups that have a similar conformation. This work is aimed at applying methods to reduce the number of variables required to describe the geometry of the fragments mined from the CSD. To this end, the geometric definition employed by dSNAP was investigated. The default definition is total geometries which are made up of all angles and all distances, including all non-bonded distances and angles. This geometric definition was investigated in a comparative manner with four other definitions. There were all angles, all distances, bonded angles and distances and bonded angles, distances and torsion angles. These comparisons show that non-bonded information is critical to the formation of groups of fragments with similar conformations. The remainder of this work was focused in reducing the number of variables required to group fragments having similar conformations into distinct groups. Initially a method was developed to calculate the area of triangles between three atoms making up the fragment. This was employed systematically as a means of reducing the total number of variables required to describe the geometry of the fragments. Multivariate statistical methods were also applied with the aim of reducing the number of variables required to describe the geometry of the fragment in a systematic manner. The methods employed were factor analysis and sparse principal components analysis. Both of these methods were used to extract important variables from the original default geometric definition, total geometries. The extracted variables were then used as input for dSNAP and were compared with the original output. Biplots were used to visualise the variables describing the fragments. Biplots are multivariate analogues to scatter plots and are used to visualise how the fragments are related to the variables describing them. Owing to the large number of variables that make up the definition factor analysis was applied to extract the important variables before the biplot was calculated. The biplots give an overview of the correlation matrix and using these plots it is possible to select variables that are influencing the formation of clusters in dSNAP .

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