Structure analysis of biologically important prokaryotic glycopolymers

Of the many post-translational modifications organisms can undertake, glycosylation is the most prevalent and the most diverse. The research in this thesis focuses on the structural characterisation of glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two domains of life (bacteria and archaea). The common theme linking these subprojects is the development and application of high sensitivity analytical techniques, primarily mass spectrometry (MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with extraordinary variety in composition and structure. A further challenge is posed by additional functionalities such as lipids whose characterisation is not always straightforward. Glycosylation in prokaryotes has a variety of different biological functions, including their important roles in the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein glycosylation will provide further targets for industrial applications, as well as insight into this post- translational modification across evolution and protein processing under extreme conditions. The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and associated MS technologies were employed to characterise the N- and O-linked glycosylation and to explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that membrane-derived lipids were present in the S- layer samples despite extensive purification, calling into question the predicted presence of covalently linked lipid. The H. volcanii N-glycosylation is mediated by the products of the agl gene cluster and the functional characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant strains of the S-layer. Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B. pseudomallei is reported to produce several polysaccharides, amongst which the already characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of this research project. MS strategies coupled with various hydrolytic and chemical derivatisation methodologies were employed to define the composition and potential sequences of the O-antigen repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen structure for atypical LPS which is remarkably different from the typical O-antigen. The development of structural analysis tools in MS and NMR applicable to the illustrated types of glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation and their modifications thus providing more informative results for pathogenicity and immunological studies as well as pathway comparisons.Open Acces

Status of the CALICE DAQ system

A data acquisition (DAQ) system is described which will be used for the next generation of prototype calorimeters using particle flow algorithms for the International Linear Collider (ILC). The design is sufficiently generic and scalable such that it should have numerous applications either for ILC detectors or elsewhere within high energy physics in general. The DAQ system will be implemented using FPGAs and built using off-the-shelf components and networking hardware with programmable FPGAs. The software for the DAQ system is based on an existing framework, DOOCS, which is a server/client object-oriented system. The design philosophy, current status of the project and its aims are presented in this report.Comment: 4 pages, 1 figure, for the LCWS08 conference proceeding

The Lyapunov matrix equation. Matrix analysis from a computational perspective

Decay properties of the solution $X$ to the Lyapunov matrix equation $AX + X A^T = D$ are investigated. Their exploitation in the understanding of equation matrix properties, and in the development of new numerical solution strategies when $D$ is not low rank but possibly sparse is also briefly discussed.Comment: This work is a contribution to the Seminar series "Topics in Mathematics", of the PhD Program of the Mathematics Department, Universit\`a di Bologna, Ital

Evolution by the vortex filament equation of curves with a corner

In this proceedings article we shall survey a series of results on the stability of self-similar solutions of the vortex filament equation. This equation is a geometric flow for curves in $\mathbb R^3$ and it is used as a model for the evolution of a vortex filament in fluid mechanics. The main theorem gives, under suitable assumptions, the existence and description of solutions generated by curves with a corner, for positive and negative times. Its companion theorem describes the evolution of perturbations of self-similar solutions up to a singularity formation infinite time, and beyond this time. We shall give a sketch of the proof. These results were obtained in collaboration with Luis Vega.Comment: 17 pages, 2 pictures, proceedings of the 40th "Journ\'ees EDP" - Biarritz 201

Inside and outside legality: A pluralistic and social construction of law

What is inside and what is outside the law? A question that is not easy to answer and that forces legal theory to ask itself about which are today the forms of normative production and which are the boundaries of law. Through the analysis of the deep transformations of the global legal order and of the issues related to the boundaries of law and to the forms of hybrid legality, the essay offers a pluralist and dynamic interpretation of the social genesis of law.¿Qué es lo que está dentro y lo que está fuera del derecho? Es un dilema difícil de solucionar, que obliga a la teoría jurídica a interrogarse acerca de las actuales formas de producción normativa y de los mismos confines del derecho. A través del análisis de las profundas transformaciones del orden jurídico global y de las problemáticas que atañen a los confines del derecho y a las formas de juridicidad híbrida, este ensayo se presenta como una lectura pluralista y dinámica de la génesis social del derecho