Amenagement du territoire autour des sites Seveso: comparaison entre les approches en Italie et en region Wallonne

Il continuo verificarsi di gravi incidenti nei grandi impianti industriali ha spinto gli Stati membri dell’Unione Europea a dotarsi di una politica comune in materia di prevenzione dei grandi rischi industriali. Anche a seguito della pressione esercitata dall’opinione pubblica sono state implementate, nel corso degli ultimi quarant’anni, misure legislative sempre più efficaci per la prevenzione e la mitigazione dei rischi legati ad attività industriali particolarmente pericolose. A partire dagli ultimi anni dello scorso secolo, l’Unione Europea ha emanato una serie di direttive che obbligano gli Stati membri ad essere garanti della sicurezza per l’uomo e per l’ambiente nelle zone circostanti a stabilimenti a rischio di incidente rilevante. In quest’ottica è stata pubblicata nel 1982 la Direttiva Seveso I [82/501/EEC], che è stata ampliata nel 1996 dalla Direttiva Seveso II [96/82/CE] ed infine emendata nel dicembre 2003 dalla Direttiva Seveso III [2003/105/CE]. Le Direttive Seveso prevedono la realizzazione negli Stati membri di una valutazione dei rischi per gli stabilimenti industriali che sono suscettibili a incendi, esplosioni o rilasci di gas tossici (quali, ad esempio, le industrie chimiche, le raffinerie, i depositi di sostanze pericolose). La Direttiva Seveso II è stata trasposta in legge belga attraverso “l’Accord de Coopération” del 21 giugno 1999. Una legge federale nel giugno del 2001 [M.B. 16/06/2001] mette in vigore “l’Accord de Coopération”, che è stato in seguito emendato e pubblicato il 26 aprile del 2007 [M.B. 26/04/2007]. A livello della Regione Vallona (in Belgio), la tematica del rischio di incidente rilevante è stata inclusa nelle disposizioni decretali del Codice Vallone della Pianificazione Territoriale, dell’Urbanismo e del Patrimonio [CWATUP]. In questo quadro la Regione Vallona ha elaborato in collaborazione con la FPMs (Faculté Polytechnique de Mons) una dettagliata metodologia di analisi del rischio per gli stabilimenti a rischio di incidente rilevante. In Italia la Direttiva Seveso II è stata recepita dal Decreto Legislativo n°334 emanato nell’agosto del 1999 [D. Lgs. 334/99], che ha introdotto per la prima volta nel quadro normativo italiano i concetti fondamentali di “controllo dell’urbanizzazione” e “requisiti minimi di sicurezza per la pianificazione territoriale”. Il Decreto Legislativo 334/99 è attualmente in vigore, modificato ed integrato dal Decreto Legislativo n°238 del 21 settembre 2005 [D. Lgs. 238/05], recepimento italiano della Direttiva Seveso III. Tra i decreti attuativi del Decreto Legislativo 334/99 occorre citare il Decreto Ministeriale n°151 del 2001 [D. M. 151/01] relativo alla pianificazione territoriale nell’intorno degli stabilimenti a rischio di incidente rilevante. L’obiettivo di questo lavoro di tesi, che è stato sviluppato presso la Faculté Polytechnique di Mons, è quello di analizzare la metodologia di quantificazione del rischio adottata nella Regione Vallona, con riferimento alla pianificazione territoriale intorno agli stabilimenti a rischio di incidente rilevante, e di confrontarla con quella applicata in Italia. La metodologia applicata in Vallonia è di tipo “probabilistico” ovvero basata sul rischio quale funzione delle frequenze di accadimento e delle conseguenze degli scenari incidentali. Il metodo utilizzato in Italia è “ibrido”, ovvero considera sia le frequenze che le conseguenze degli scenari incidentali, ma non la loro ricomposizione all’interno di un indice di rischio. In seguito al confronto teorico delle due metodologie, se ne è effettuato anche una comparazione pratica tramite la loro applicazione ad un deposito di GPL. Il confronto ha messo in luce come manchino, nella legislazione italiana relativa agli stabilimenti a rischio di incidente rilevante, indicazioni di dettaglio per la quantificazione del rischio, a differenza di quanto accade nella legislazione belga. Ciò lascia all’analista di rischio italiano una notevole arbitrarietà nell’effettuare ipotesi ed assunzioni che rendono poi difficile la comparazione del rischio di stabilimenti differenti. L’auspicio è che tale lacuna possa essere rapidamente superata.

Surface insertion in a tetrahedral mesh using the meccano method

[EN]This work presents an innovative method to insert an open surface in a tetrahedral mesh. The insertion of a surface in a mesh can be done with 2 different approaches: introduce the surface to the geometry before generating the mesh, or insert the surface once the mesh is generated. This work uses the second approach. Essentially, the surface is first approximated by a set of faces of the existing mesh. This set is refined to obtain a more accurate approximation. Finally, the set is processed to satisfy some topological properties and projected to the actual surface. The strategy is based on a mesh generated by the Meccano Method…

Study of the building and decorative materials and techniques used in Hungarian Roman sites

The aims of this research were: - To identify the characteristics, properties and provenance of the building and decorative material found in three Hungarian Roman sites: Nagyharsány, Nemesvámos-Balácapuszta and Aquincum - To provide a database of information on the different sites - To have an overview of main conservation strategies applied in Hungary. Geological studies, macroscopical and microscopical observations, XRD investigations, physical and chemical analyses allowed us to define the characteristics and properties of the different kinds of collected materials. Building stones sampled from Nagyharsány site showed two different kinds of massive limestone belonging to the areas surrounding the villa. Also Building stones sampled from Nemesvámos-Balácapuszta Roman villa proved to be compatible with limestone belonging to local sources. Mural painting fragments show that all samples are units composed of multilayered structures. Mosaic tesserae can be classified as following: -Pale yellow , blackish and pink tesserae are comparable with local limestone; -White tessera, composed of marble, was probably imported from distant regions of the Empire, as the usual practice of Romans. Mortars present different characteristics according to the age, the site and the functions: -Building mortars are generally lime based, white or pale yellow in colour, present a high percentage of aggregates represented by fine sand; -Supporting mortars from both mosaics and mural paintings are reddish or pinkish in colour, due to the presence of high percentage of brick dust and tiles fragments, and present a higher content of MgO. Although the condition of the sites, there is an insignificant content of soluble salts. Database The whole study has allowed us to provide work sheets for each samples, including all characteristics and properties. Furthermore, all sites included in the frame of the research have been described and illustrated on the base of their floor plans, material and construction methodologies. It can be concluded that: 1. In Nagyharsány Archaeological site, it is possible to define a sequence of different construction phases on the base of the study of building material and mortars. The results are comparable with the chronology of the site provided by the archaeologists 2. The material used for construction was of local origin while the more precious ones, used for decorative elements, were probably imported from long distance 3. Construction techniques in Hungary mainly refer to the usual Roman knowledge and practice (Vitruvius); few differences have been found 4. The database will represent an archive for Archaeologists, Historians and Conservators dealing with Roman period in Hungary.

Coastal flood vulnerability assessment with geomatic methods: Test sites of western Thailand, Sydney (Australia) and aeolian islands (south tyrrhenian sea, Italy)

The work undertaken in this PhD thesis is aimed at the development and testing of an innovative methodology for the assessment of the vulnerability of coastal areas to marine catastrophic inundation (tsunami). Different approaches are used at different spatial scales and are applied to three different study areas: 1. The entire western coast of Thailand 2. Two selected coastal suburbs of Sydney – Australia 3. The Aeolian Islands, in the South Tyrrhenian Sea – Italy I have discussed each of these cases study in at least one scientific paper: one paper about the Thailand case study (Dall’Osso et al., in review-b), three papers about the Sydney applications (Dall’Osso et al., 2009a; Dall’Osso et al., 2009b; Dall’Osso and Dominey-Howes, in review) and one last paper about the work at the Aeolian Islands (Dall’Osso et al., in review-a). These publications represent the core of the present PhD thesis. The main topics dealt with are outlined and discussed in a general introduction while the overall conclusions are outlined in the last section.

Molecular interactions: metal ions and protein chaperones in the urease system from Helicobacter pylori

Although nickel is a toxic metal for living organisms in its soluble form, its importance in many biological processes recently emerged. In this view, the investigation of the nickel-dependent enzymes urease and [NiFe]-hydrogenase, especially the mechanism of nickel insertion into their active sites, represent two intriguing case studies to understand other analogous systems and therefore to lead to a comprehension of the nickel trafficking inside the cell. Moreover, these two enzymes have been demonstrated to ensure survival and colonization of the human pathogen H. pylori, the only known microorganism able to proliferate in the gastric niche. The right nickel delivering into the urease active site requires the presence of at least four accessory proteins, UreD, UreE, UreF and UreG. Similarly, analogous process is principally mediated by HypA and HypB proteins in the [NiFe]-hydrogenase system. Indeed, HpHypA and HpHypB also have been proposed to act in the activation of the urease enzyme from H. pylori, probably mobilizing nickel ions from HpHypA to the HpUreE-HpUreG complex. A complete comprehension of the interaction mechanism between the accessory proteins and the crosstalk between urease and hydrogenase accessory systems requires the determination of the role of each protein chaperone that strictly depends on their structural and biochemical properties. The availability of HpUreE, HpUreG and HpHypA proteins in a pure form is a pre-requisite to perform all the subsequent protein characterizations, thus their purification was the first aim of this work. Subsequently, the structural and biochemical properties of HpUreE were investigated using multi-angle and quasi-elastic light scattering, as well as NMR and circular dichroism spectroscopy. The thermodynamic parameters of Ni2+ and Zn2+ binding to HpUreE were principally established using isothermal titration calorimetry and the importance of key histidine residues in the process of binding metal ions was studied using site-directed mutagenesis. The molecular details of the HpUreE-HpUreG and HpUreE-HpHypA protein-protein assemblies were also elucidated. The interaction between HpUreE and HpUreG was investigated using ITC and NMR spectroscopy, and the influence of Ni2+ and Zn2+ metal ions on the stabilization of this association was established using native gel electrophoresis, light scattering and thermal denaturation scanning followed by CD spectroscopy. Preliminary HpUreE-HpHypA interaction studies were conducted using ITC. Finally, the possible structural architectures of the two protein-protein assemblies were rationalized using homology modeling and docking computational approaches. All the obtained data were interpreted in order to achieve a more exhaustive picture of the urease activation process, and the correlation with the accessory system of the hydrogenase enzyme, considering the specific role and activity of the involved protein players. A possible function for Zn2+ in the chaperone network involved in Ni2+ trafficking and urease activation is also envisaged.

Advanced applications of X-ray Absorption Spectroscopy to the study of protein metal sites.

We present a study of the metal sites of different proteins through X-ray Absorption Fine Structure (XAFS) spectroscopy. First of all, the capabilities of XAFS analysis have been improved by ab initio simulation of the near-edge region of the spectra, and an original analysis method has been proposed. The method subsequently served ad a tool to treat diverse biophysical problems, like the inhibition of proton-translocating proteins by metal ions and the matrix effect exerted on photosynthetic proteins (the bacterial Reaction Center, RC) by strongly dehydrate sugar matrices. A time-resolved study of Fe site of RC with μs resolution has been as well attempted. Finally, a further step aimed to improve the reliability of XAFS analysis has been performed by calculating the dynamical parameters of the metal binding cluster by means of DFT methods, and the theoretical result obtained for MbCO has been successfully compared with experimental data.

Strukturelle und molekulare Charakterisierung des Asialoglykoprotein-Rezeptors aus humaner Leber

Der Asialoglykoprotein-Rezeptor (ASGPR) vermittelt als integraler Bestandteil der Leberzellmembran die Endozytose von zirkulierenden Asialoglykoproteinen. Ziele dieser Arbeit waren proteinchemische Untersuchungen von funktionellem ASGPR aus humaner Leber aufgrund einer verbesserten Präparationsmethode und die rekombinante Darstellung der beiden Untereinheiten H1 und H2. In der denaturierenden SDS-PAGE erschienen H1 und H2 überwiegend als Monomere bei 46 und 50kD; nach Deglykosylierung ergaben sich Banden bei 34 und 32kD, wonach der Glykosidanteil etwa 28% beträgt. In der nicht-denaturierenden Größenausschluß-Chromatographie wurden im nativen ASGPR ausschließlich Trimere und Dimere gefunden. In Gegenwart von 2-Mercaptoethanol konnten funktionell eine aktive von einer nicht-aktiven Fraktion getrennt werden, wobei H2 in der nicht-aktiven Fraktion angereichert war, während sich H1 zu etwa gleichen Teilen in beiden Fraktionen befand. Durch zweidimensionale Auftrennung des deglykosylierten Rezeptors wurden auf Proteinebene vier Isoformen von H1 und zwei von H2 mit unterschiedlichen pI-Werten identifiziert. Der Vergleich von funktionellem ASGPR aus normaler Leber und den hepatischen Tumorzellinien HepG2 und Huh7 in der SDS-PAGE brachte Größenunterschiede von etwa sechs und vier Kilodalton hervor. Bei H1 konnte dies auf einen höheren Glykosylierungsgrad zurückgeführt werden, während H2 auch nach Behandlung mit N-GlykosidaseF ein größeres Molekulargewicht aufwies. Ein Antikörper gegen das Insertionspeptid im cytoplasmatischen Bereich einer Splice-Variante von H2 zeigte eine deutlich erhöhte Expression von H2 mit Insertion in Huh7-Zellen gegenüber natürlichem ASGPR. Da bisherige Kenntnisse über den humanen ASGPR vorwiegend aus kultivierten Hepatomzelllinien stammen, scheinen sie nicht ohne weiteres auf die Situation in normaler Leber übertragbar. Die Präparation von funktionellem H1 aus transfizierten cos7- und 293-Zellen führte zum gleichen Bandenmuster wie beim natürlichen ASGPR. Mit einem Enzymimmunoassay wurde die Eignung von rekombinantem H1 zur Detektion von Antikörpern gegen ASGPR in 177 von 178 Patientenseren gezeigt. Da durch Präinkubation mit rekombinantem Antigen die Reaktivität mit natürlichem Rezeptor inhibiert werden konnte, trägt H1 hauptsächlich die antigenen Stellen des ASGPR.

Evaluation of the effects of insertion of viscous dampers in Moment Resisting Frames

This research work faces the problem of insertion of viscous dampers into Moment Resisiting Frames (MRF) for maximum efficiency in mitigation of the seismic effects. The work would lead to a precise design indication. The fundamental result of the thesis consists in showing that, even for moment-resisting structures, you can design a system of added viscous dampers able to achieve target levels of performances. Ie given the reduction factor in the seismic response, discover the characteristics of the viscous dampers which allow to achieve it.

Strukturelle und funktionelle Untersuchungen zur Bedeutung der endogenen retroviralen Insertion HERV-K(C4) im C4-Gen des Menschen

Der Längenpolymorphismus des C4-Gens beruht auf der An- oder Abwesenheit einer 6.4 kb langen Insertion im Intron 9. Es handelt sich dabei um einen eigenständigen bisher noch nicht beschriebenen Virus-Typ, der alle Sequenzmerkmale der Familie der humanen endogenen Retroviren (HERV) trägt und zu den HERV-K Viren gehört. Der Provirus wurde als HERV-K(C4) bezeichnet. Die Orientierung dieses retroviralen Elements ist entgegengesetzt zu der Transkriptionsrichtung des C4-Gens. Mittels RT-PCR, RNase Protection Assays und Northern-Blot Analysen konnte der Nachweis von HERV-K(C4)-Antisense mRNA-Transkripten in verschiedenen humanen Zellinien und Geweben erbracht werden. Die retroviralen Transkripte schlossen am 5'- und 3'-Ende Sequenzen des C4-Exon 9 und Exon 10 ein, so daß diese wahrscheinlich "readthrough" Transkripte darstellen, die durch einen 5' des LTR2 gelegenen Promotor initiiert oder im Zusammenhang mit der C4-Expression transkribiert und reguliert werden. Weiterhin konnten insgesamt 4 HERV-K(C4)-mRNA Spezies, einschließlich einer Vollängen-RNA detektiert werden. Die drei subgenomischen mRNAs werden vermutlich durch einfaches und mehrfaches Spleißen generiert. Die quantitative Analyse in verschiedenen humanen Zellinien ergab, daß HERV-K(C4) durchschnittlich mit einer Kopienanzahl zwischen ca.1 bis 100 Transkripten in einer Zelle vorkommt, so daß es sich um low abundance mRNAs handelt. Mittels eines Reportergen-System konnte eine Aktivität des LTR2-Promotors in der Sense-Orientierung des Retrovirus nachgewiesen werden, die nach Stimulation mit IFN- signifikant abnahm. Ein humanes Modell-Systems wurde etabliert, um die Theorie einer Antisense-Abwehr gegen exogene Retroviren in HepG2-Zellen zu überprüfen. Die Theorie basiert auf dem Nachweis von HERV-K(C4)-Antisense-Transkripten, die über eine Heteroduplexbildung mit der Sense-mRNA von verwandten, infektiösen Retroviren eine mögliche Blockierung deren Translation erwirken könnten. Es konnte eine signifikante Abnahme der retroviralen Expression von bis zu 45% nach steigenden Dosen an IFN- in HepG2-Zellen nachgewiesen werden. Der funktionell aktive 3'-LTR-Sense Promotor sowie der Nachweis von HERV-K(C4)-Antisense Transkripten sprechen für die bedeutende Rolle von HERV-K(C4) bei der Genregulation und Schutz gegen exogene Retroviren, wodurch eine Selektion stattgefunden hat.

Algorithms for UAS insertion in civil air space

One of the most interesting challenge of the next years will be the Air Space Systems automation. This process will involve different aspects as the Air Traffic Management, the Aircrafts and Airport Operations and the Guidance and Navigation Systems. The use of UAS (Uninhabited Aerial System) for civil mission will be one of the most important steps in this automation process. In civil air space, Air Traffic Controllers (ATC) manage the air traffic ensuring that a minimum separation between the controlled aircrafts is always provided. For this purpose ATCs use several operative avoidance techniques like holding patterns or rerouting. The use of UAS in these context will require the definition of strategies for a common management of piloted and piloted air traffic that allow the UAS to self separate. As a first employment in civil air space we consider a UAS surveillance mission that consists in departing from a ground base, taking pictures over a set of mission targets and coming back to the same ground base. During all mission a set of piloted aircrafts fly in the same airspace and thus the UAS has to self separate using the ATC avoidance as anticipated. We consider two objective, the first consists in the minimization of the air traffic impact over the mission, the second consists in the minimization of the impact of the mission over the air traffic. A particular version of the well known Travelling Salesman Problem (TSP) called Time-Dependant-TSP has been studied to deal with traffic problems in big urban areas. Its basic idea consists in a cost of the route between two clients depending on the period of the day in which it is crossed. Our thesis supports that such idea can be applied to the air traffic too using a convenient time horizon compatible with aircrafts operations. The cost of a UAS sub-route will depend on the air traffic that it will meet starting such route in a specific moment and consequently on the avoidance maneuver that it will use to avoid that conflict. The conflict avoidance is a topic that has been hardly developed in past years using different approaches. In this thesis we purpose a new approach based on the use of ATC operative techniques that makes it possible both to model the UAS problem using a TDTSP framework both to use an Air Traffic Management perspective. Starting from this kind of mission, the problem of the UAS insertion in civil air space is formalized as the UAS Routing Problem (URP). For this reason we introduce a new structure called Conflict Graph that makes it possible to model the avoidance maneuvers and to define the arc cost function of the departing time. Two Integer Linear Programming formulations of the problem are proposed. The first is based on a TDTSP formulation that, unfortunately, is weaker then the TSP formulation. Thus a new formulation based on a TSP variation that uses specific penalty to model the holdings is proposed. Different algorithms are presented: exact algorithms, simple heuristics used as Upper Bounds on the number of time steps used, and metaheuristic algorithms as Genetic Algorithm and Simulated Annealing. Finally an air traffic scenario has been simulated using real air traffic data in order to test our algorithms. Graphic Tools have been used to represent the Milano Linate air space and its air traffic during different days. Such data have been provided by ENAV S.p.A (Italian Agency for Air Navigation Services).

Identification of the N-Linked Glycosylation Sites of the Transcription Factor Rest and Effect of Glycosylation on DNA Binding and Transcriptional Activity

REST is a zinc-finger transcription factor implicated in several processes such as maintenance of embryonic stem cell pluripotency and regulation of mitotic fidelity in non-neuronal cells [Chong et al., 1995]. The gene encodes for a 116-kDa protein that acts as a molecular platform for co-repressors recruitment and promotes modifications of DNA and histones [Ballas, 2005]. REST showed different apparent molecular weights, consistent with the possible presence of post-translational modifications [Lee et al., 2000]. Among these the most common is glycosylation, the covalent attachment of carbohydrates during or after protein synthesis [Apweiler et al., 1999] My thesis has ascertained, for the first time, the presence of glycan chians in the transcription factor REST. Through enzymatic deglycosylation and MS, oligosaccharide composition of glycan chains was evaluated: a complex mixture of glycans, composed of N-acetylgalactosamine, galactose and mannose, was observed thus confirming the presence of O- and N-linked glycan chains. Glycosylation site mapping was done using a 18O-labeling method and MS/MS and twelve potential N-glycosylation sites were identified. The most probable glycosylation target residues were mutated through site-directed mutagenesis and REST mutants were expressed in different cell lines. Variations in the protein molecular weight and mutant REST ability to bind the RE-1 sequence were analyzed. Gene reporter assays showed that, altogether, removal of N-linked glycan chains causes loss of transcriptional repressor function, except for mutant N59 which showed a slight residual repressor activity in presence of IGF-I. Taken togheter these results demonstrate the presence of complex glycan chians in the transcription factor REST: I have depicted their composition, started defining their position on the protein backbone and identified their possible role in the transcription factor functioning. Considering the crucial role of glycosylation and transcription factors activity in the aetiology of many diseases, any further knowledge could find important and interesting pharmacological application.

Synthesis of Modified Amino Acids and Insertion in Peptides and Mimetics. Structural Aspects and Impact on Biological Activity.

I studied the effects exerted by the modifications on structures and biological activities of the compounds so obtained. I prepared peptide analogues containing unusual amino acids such as halogenated, alkylated (S)- or (R)-tryptophans, useful for the synthesis of mimetics of the endogenous opioid peptide endomorphin-1, or 2-oxo-1,3-oxazolidine-4-carboxylic acids, utilized as pseudo-prolines having a clear all-trans configuration of the preceding peptide bond. The latter gave access to a series of constrained peptidomimetics with potential interest in medicinal chemistry and in the field of the foldamers. In particular, I have dedicated much efforts to the preparation of cyclopentapeptides containing D-configured, alfa-, or beta-aminoacids, and also of cyclotetrapeptides including the retro-inverso modification. The conformational analyses confirmed that these cyclic compounds can be utilized as rigid scaffolds mimicking gamma- or beta-turns, allowing to generate new molecular and 3D diversity. Much work has been dedicated to the structural analysis in solution and in the receptor-bound state, fundamental for giving a rationale to the experimentally determined bioactivity, as well as for predicting the activity of virtual compounds (in silico pre-screen). The conformational analyses in solution has been done mostly by NMR (2D gCosy, Roesy, VT, molecular dynamics, etc.). A special section is dedicated to the prediction of plausible poses of the ligands when bound to the receptors by Molecular Docking. This computational method proved to be a powerful tool for the investigation of ligand-receptor interactions, and for the design of selective agonists and antagonists. Another practical use of cyclic peptidomimetics was the synthesis and biological evaluation of cyclic analogues of endomorphin-1 lacking in a protonable amino group. The studies revealed that a inverse type II beta-turn on D-Trp-Phe constituted the bioactive conformation.

Genomische Analyse von natürlichen Killerzell-Rezeptorgenen in nicht-humanen Primaten

Natürliche Killerzell-Rezeptoren, die MHC-Klasse-I-Moleküle binden, sind im Leukozyten Rezeptor Komplex (LRC) und im Natürlichen Killer Komplex (NKC) kodiert. Die Bindung klassischer MHC-Klasse-I-Moleküle erfolgt im Menschen durch die im LRC kodierten polymorphen Killerzell-Immunglobulin-ähnlichen Rezeptoren (KIR) und in Nagetieren durch die im NKC kodierten polymorphen C-Typ Lektin-ähnlichen Ly49-Rezeptoren. Die ebenfalls im NKC kodierten C-Typ Lektin-ähnlichen CD94/NKG2-Rezeptoren sowie der NKG2D-Rezeptor sind sowohl im Menschen als auch in Nagetieren konserviert und wenig polymorph. Im Rahmen dieser Arbeit wurde das CD94-Ly49L-Intervall der NKC-Region in einem Neuweltaffen, dem Weißbüschelaffen (Callithrix jacchus), sowie einem Feuchtnasenaffen, dem Grauen Mausmaki (Microcebus murinus), über Screening von BAC-Banken und Sequenzanalyse von BAC-Contigs untersucht. Das CD94-Ly49L-Intervall im Weißbüschelaffen hat eine Länge von 171 kb und weist orthologe Gene zu den humanen NKC-Genen auf. Eine Ausnahme bildet das Gen NKG2CE, welches äquidistant zu den humanen Genen NKG2C und NKG2E ist. NKG2F und Ly49L sind Pseudogene. Expressionsanalysen der NKC-Gene in neun Weißbüschelaffen-Individuen lieferten einen mäßigen Grad an allelischen Polymorphismen. Alternative Spleißprodukte wurden für CD94, NKG2D und NKG2A identifiziert. Für NKG2A wurden verschiedene Transkripte mit potentiell unterschiedlichen Translationsstartpunkten gefunden. Im Grauen Mausmaki beträgt die Länge des CD94-Ly49L-Intervalls 489 kb. CD94 und die NKG2-Gene sind vervielfacht und wesentlich polymorpher als im Menschen und im Weißbüschelaffen. Expressionsanalysen der NKC-Gene wurden im Grauen Mausmaki und einem weiteren madagassischen Lemuren, dem Schwarzweißen Vari (Varecia variegata), durchgeführt und zeigten, dass CD94 und die NKG2-Gene im Vari ebenfalls vervielfacht sind. Die NKG2-Moleküle der Lemuren weisen unterschiedliche Kombinationen an aktivierenden und inhibierenden Signalmotiven auf und üben somit möglicherweise diverse Funktionen aus. Ly49L stellt in den Lemuren einen potentiell funktionellen inhibierenden Rezeptor dar und NKG2D besitzt im Vergleich zum humanen NKG2D-Protein eine verkürzte Zytoplasmaregion. Alternative Spleißprodukte der NKC-Gene existieren auch in den Lemuren. Darüber hinaus wurden mehrere CD94-Gene in einem weiteren Feuchtnasenaffen, dem Potto (Perodicticus potto) und einem Trockennasenaffen, dem Philippinen-Koboldmaki (Tarsius syrichta), nachgewiesen. Ein Alu-Element, welches ausschließlich in Intron 4 der CD94-Sequenzen des Philippinen-Koboldmakis auftritt, deutet darauf hin, dass sich CD94 in der Linie der Koboldmakis und in der Linie der Feuchtnasenaffen unabhängig voneinander vervielfacht hat. Die vervielfachten, polymorphen CD94/NKG2-Rezeptoren der niederen Primaten stellen möglicherweise das funktionelle Äquivalent zu den polymorphen KIR der höheren Primaten und den polymorphen Ly49-Rezeptoren der Nagetiere dar.

Analysing the possible influence of transposon TCl4.7 insertion on the function of the genome of Cydia pomonella granulovirus

CpGV-MCp5 is a natural mutant of the Cydia pomonella Granulovirus (Mexican isolate) (CpGV-M) that harbors an insect host transposon termed TCl4.7 in its genome. TCl4.7 is located between the open reading frames Cp15 and Cp16 and separates two homologous regions hr3 and hr4, which have been recently shown to be origins of replication of CpGV-M. The MCp5 has a significant replication disadvantage in the presence of the wild-type CpGV-M. In this study, the possible effects of TCl4.7 transposon insertion on the genome function of its insertion site has been analysed. The role of Cp15 and Cp16 in the context of the virus infection cycle was examined by generating a CpGV-Bacmid (CpBAC) and Cp15 knock-out (CpBACCp15KO) and Cp16 knock-out (CpBACCp16KO) mutants. The mutant CpBACCp15KO was not able to replicate in CM larvae suggesting that Cp15 was essential for virus replication. In contrast, the mutant CpBACCp16KO infected CM larvae and produced viable occlusion bodies (OBs) demonstrating that Cp16 is a non-essential gene for virus in vivo infection of C. pomonella. The temporal transcription of Cp15 and Cp16, as well as of Cp31 (F protein) as a control, was analysed using RT-PCR and quantitative real-time PCR. It suggested a general delay or reduction of gene transcription of MCp5 compared to the parental CpGV-M. Western blot analyses using anti-Cp15 and anti-Cp16 polyclonal antibodies, however, did not show any immuno-reactive response. Thus, a direct influence of TCl4.7 on the expression of Cp15 and Cp16 could not be substantiated. To investigate whether the interruption of hr3 and hr4 palindromes affects the virus replication, two mutant bacmids with a deletion of hr3 and hr4 (CpBAChr3/hr4-KO) and another with an insertion of a Kanamycin resistance cassette between hr3 and hr4 (CpBAChr3-kan-hr4) were generated. Both mutant bacmids replicated and produced infectious virus OBs, which did not significantly differ in their median lethal concentration (LC50) and median survival time (ST50) compared to the parental CpBAC. Interestingly, the mutant CpBAChr3-kan-hr4 was very effectively out-competed by parental CpBAC, when CM larvae were co-infected with known ratios of OBs of CpBAC and the mutant CpBAChr3-kan-hr4. These observations suggested a functional co-operation between hr3 and hr4 which was interrupted by the KanR insertion in CpBAChr3-kan-hr4 and possibly by TCl4.7 transposon insertion in the mutant MCp5. This hypothesis may explain the observed replication disadvantage of the mutants MCp5 and CpBAChr3-kan-hr4 in the presence of the parental viruses CpGV-M and CpBAC, respectively.