Parallel modeling of the electric field distribution in the brain

The term "Brain Imaging" identi�es a set of techniques to analyze the structure and/or functional behavior of the brain in normal and/or pathological situations. These techniques are largely used in the study of brain activity. In addition to clinical usage, analysis of brain activity is gaining popularity in others recent �fields, i.e. Brain Computer Interfaces (BCI) and the study of cognitive processes. In this context, usage of classical solutions (e.g. f MRI, PET-CT) could be unfeasible, due to their low temporal resolution, high cost and limited portability. For these reasons alternative low cost techniques are object of research, typically based on simple recording hardware and on intensive data elaboration process. Typical examples are ElectroEncephaloGraphy (EEG) and Electrical Impedance Tomography (EIT), where electric potential at the patient's scalp is recorded by high impedance electrodes. In EEG potentials are directly generated from neuronal activity, while in EIT by the injection of small currents at the scalp. To retrieve meaningful insights on brain activity from measurements, EIT and EEG relies on detailed knowledge of the underlying electrical properties of the body. This is obtained from numerical models of the electric �field distribution therein. The inhomogeneous and anisotropic electric properties of human tissues make accurate modeling and simulation very challenging, leading to a tradeo�ff between physical accuracy and technical feasibility, which currently severely limits the capabilities of these techniques. Moreover elaboration of data recorded requires usage of regularization techniques computationally intensive, which influences the application with heavy temporal constraints (such as BCI). This work focuses on the parallel implementation of a work-flow for EEG and EIT data processing. The resulting software is accelerated using multi-core GPUs, in order to provide solution in reasonable times and address requirements of real-time BCI systems, without over-simplifying the complexity and accuracy of the head models.

Supramolecular order and dynamics of functional materials studied by solid state NMR

The goal of this thesis was the investigation of the structure, conformation, supramolecular order and molecular dynamics of different classes of functional materials (phthalocyanine, perylene and hexa-peri-hexabenzocoronene derivatives and mixtures of those), all having planar aromatic cores modified with various types of alkyl chains. The planar aromatic systems are known to stack in the solid and the liquid-crystalline state due to p-p interactions forming columnar superstructures with high one-dimensional charge carrier mobility and potential application in photovoltaic devices. The different functionalities attached to the aromatic cores significantly influence the behavior of these systems allowing the experimentalists to modify the structures to fine-tune the desired thermotropic properties or charge carrier mobility. The aim of the presented studies was to understand the interplay between the driving forces causing self-assembly by relating the structural and dynamic information about the investigated systems. The supramolecular organization is investigated by applying 1H solid state NMR recoupling techniques. The results are related with DSC and X-ray scattering data. Detailed information about the site-specific molecular dynamics is gained by recording spinning sideband patterns using 1H-1H and 13C-1H solid state NMR recoupling techniques. The determined dipole-dipole coupling constants are then related with the coupling constants of the respective rigid pairs, thus providing local dynamic order parameters for the respective moieties. The investigations presented reveal that in the crystalline state the preferred arrangement in the columnar stack of discotic molecules modified with alkyl chains is tilted. This leads to characteristic differences in the 1H chemical shifts of otherwise chemically equivalent protons. Introducing branches and increasing the length of the alkyl chains results in lower mesophase transitions and disordered columnar stacks. In the liquid-crystalline state some of the discs lose the tilted orientation, others do not, but all start a rapid rotation about the columnar axis.

Trascriptome analysis and functional genomics of Neisseria meningitidis in human blood

Neisseria meningitidis (Nm) is the major cause of septicemia and meningococcal meningitis. During the course of infection, it must adapt to different host environments as a crucial factor for survival. Despite the severity of meningococcal sepsis, little is known about how Nm adapts to permit survival and growth in human blood. A previous time-course transcriptome analysis, using an ex vivo model of human whole blood infection, showed that Nm alters the expression of nearly 30% of ORFs of the genome: major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. Starting from these data, mutagenesis studies of a subset of up-regulated genes were performed and the mutants were tested for the ability to survive in human whole blood; Nm mutant strains lacking the genes encoding NMB1483, NalP, Mip, NspA, Fur, TbpB, and LctP were sensitive to killing by human blood. Then, the analysis was extended to the whole Nm transcriptome in human blood, using a customized 60-mer oligonucleotide tiling microarray. The application of specifically developed software combined with this new tiling array allowed the identification of different types of regulated transcripts: small intergenic RNAs, antisense RNAs, 5’ and 3’ untranslated regions and operons. The expression of these RNA molecules was confirmed by 5’-3’RACE protocol and specific RT-PCR. Here we describe the complete transcriptome of Nm during incubation in human blood; we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. In addition the tiling array analysis demonstrated that Nm expresses a set of new transcripts, not previously identified, and suggests the presence of a circuit of regulatory RNA elements used by Nm to adapt to proliferate in human blood.

Lesion detection and classification in breast cancer: evaluation of approaches based on morphological features, tracer kinetic modelling and semi-quantitative parameters in MR functional imaging (DCE-MRI)

The diagnosis, grading and classification of tumours has benefited considerably from the development of DCE-MRI which is now essential to the adequate clinical management of many tumour types due to its capability in detecting active angiogenesis. Several strategies have been proposed for DCE-MRI evaluation. Visual inspection of contrast agent concentration curves vs time is a very simple yet operator dependent procedure, therefore more objective approaches have been developed in order to facilitate comparison between studies. In so called model free approaches, descriptive or heuristic information extracted from time series raw data have been used for tissue classification. The main issue concerning these schemes is that they have not a direct interpretation in terms of physiological properties of the tissues. On the other hand, model based investigations typically involve compartmental tracer kinetic modelling and pixel-by-pixel estimation of kinetic parameters via non-linear regression applied on region of interests opportunely selected by the physician. This approach has the advantage to provide parameters directly related to the pathophysiological properties of the tissue such as vessel permeability, local regional blood flow, extraction fraction, concentration gradient between plasma and extravascular-extracellular space. Anyway, nonlinear modelling is computational demanding and the accuracy of the estimates can be affected by the signal-to-noise ratio and by the initial solutions. The principal aim of this thesis is investigate the use of semi-quantitative and quantitative parameters for segmentation and classification of breast lesion. The objectives can be subdivided as follow: describe the principal techniques to evaluate time intensity curve in DCE-MRI with focus on kinetic model proposed in literature; to evaluate the influence in parametrization choice for a classic bi-compartmental kinetic models; to evaluate the performance of a method for simultaneous tracer kinetic modelling and pixel classification; to evaluate performance of machine learning techniques training for segmentation and classification of breast lesion.

Functional polymer coatings — My-Patterning and My-Analysis

In dieser Arbeit werden Strukturen beschrieben, die mit Polymeren auf Oberflächen erzeugt wurden. Die Anwendungen reichen von PMMA und PNIPAM Polymerbürsten, über die Restrukturierung von Polystyrol durch Lösemittel bis zu 3D-Strukturen, die aus PAH/ PSS Polyelektrolytmultischichten bestehen. Im ersten Teil werden Polymethylmethacrylat (PMMA) Bürsten in der ionischen Flüssigkeit 1-Butyl-3-Methylimidazolium Hexafluorophospat ([Bmim][PF6]) durch kontrollierte radikalische Polymerisation (ATRP) hergestellt. Kinetische Untersuchungen zeigten ein lineares und dichtes Bürstenwachstum mit einer Wachstumsrate von 4600 g/mol pro nm. Die durchschnittliche Pfropfdichte betrug 0.36 µmol/m2. Als Anwendung wurden Mikrotropfen bestehend aus der ionischen Flüssigkeit, Dimethylformamid und dem ATRP-Katalysator benutzt, um in einer definierten Geometrie Polymerbürsten auf Silizium aufzubringen. Auf diese Weise lässt sich eine bis zu 13 nm dicke Beschichtung erzeugen. Dieses Konzept ist durch die Verdampfung des Monomers Methylmethacrylat (MMA) limitiert. Aus einem 1 µl großen Tropfen aus ionischer Flüssigkeit und MMA (1:1) verdampft MMA innerhalb von 100 s. Daher wurde das Monomer sequentiell zugegeben. Der zweite Teil konzentriert sich auf die Strukturierung von Oberflächen mit Hilfe einer neuen Methode: Tintendruck. Ein piezoelektrisch betriebenes „Drop-on-Demand“ Drucksystem wurde verwendet, um Polystyrol mit 0,4 nl Tropfen aus Toluol zu strukturieren. Die auf diese Art und Weise gebildeten Mikrokrater können Anwendung als Mikrolinsen finden. Die Brennweite der Mikrolinsen kann über die Anzahl an Tropfen, die für die Strukturierung verwendet werden, eingestellt werden. Theoretisch und experimentell wurde die Brennweite im Bereich von 4,5 mm bis 0,21 mm ermittelt. Der zweite Strukturierungsprozess nutzt die Polyelektrolyte Polyvinylamin-Hydrochlorid (PAH) und Polystyrolsulfonat (PSS), um 3D-Strukturen wie z.B. Linien, Schachbretter, Ringe, Stapel mit einer Schicht für Schicht Methode herzustellen. Die Schichtdicke für eine Doppelschicht (DS) liegt im Bereich von 0.6 bis 1.1 nm, wenn NaCl als Elektrolyt mit einer Konzentration von 0,5 mol/l eingesetzt wird. Die Breite der Strukturen beträgt im Mittel 230 µm. Der Prozess wurde erweitert, um Nanomechanische Cantilever Sensoren (NCS) zu beschichten. Auf einem Array bestehend aus acht Cantilevern wurden je zwei Cantilever mit fünf Doppelschichten PAH/ PSS und je zwei Cantilever mit zehn Doppelschichten PAH/ PSS schnell und reproduzierbar beschichtet. Die Massenänderung für die individuellen Cantilever war 0,55 ng für fünf Doppelschichten und 1,08 ng für zehn Doppelschichten. Der daraus resultierende Sensor wurde einer Umgebung mit definierter Luftfeuchtigkeit ausgesetzt. Die Cantilever verbiegen sich durch die Ausdehnung der Beschichtung, da Wasser in das Polymer diffundiert. Eine maximale Verbiegung von 442 nm bei 80% Luftfeuchtigkeit wurde für die mit zehn Doppelschichten beschichteten Cantilever gefunden. Dies entspricht einer Wasseraufnahme von 35%. Zusätzlich konnte aus den Verbiegungsdaten geschlossen werden, dass die Elastizität der Polyelektrolytmultischichten zunimmt, wenn das Polymer gequollen ist. Das thermische Verhalten in Wasser wurde im nächsten Teil an nanomechanischen Cantilever Sensoren, die mit Poly(N-isopropylacrylamid)bürsten (PNIPAM) und plasmapolymerisiertem N,N-Diethylacrylamid beschichtet waren, untersucht. Die Verbiegung des Cantilevers zeigte zwei Bereiche: Bei Temperaturen kleiner der niedrigsten kritischen Temperatur (LCST) ist die Verbiegung durch die Dehydration der Polymerschicht dominiert und bei Temperaturen größer der niedrigsten kritischen Temperatur (LCST) reagiert der Cantilever Sensor überwiegend auf Relaxationsprozesse innerhalb der kollabierten Polymerschicht. Es wurde gefunden, dass das Minimum in der differentiellen Verbiegung mit der niedrigsten kritischen Temperatur von 32°C und 44°C der ausgewählten Polymeren übereinstimmt. Im letzten Teil der Arbeit wurden µ-Reflektivitäts- und µ-GISAXS Experimente eingeführt als neue Methoden, um mikrostrukturierte Proben wie NCS oder PEM Linien mit Röntgenstreuung zu untersuchen. Die Dicke von jedem individuell mit PMMA Bürsten beschichtetem NCS ist im Bereich von 32,9 bis 35,2 nm, was mit Hilfe von µ-Reflektivitätsmessungen bestimmt wurde. Dieses Ergebnis kann mit abbildender Ellipsometrie als komplementäre Methode mit einer maximalen Abweichung von 7% bestätigt werden. Als zweites Beispiel wurde eine gedruckte Polyelektrolytmultischicht aus PAH/PSS untersucht. Die Herstellungsprozedur wurde so modifiziert, dass Goldnanopartikel in die Schichtstruktur eingebracht wurden. Durch Auswertung eines µ-GISAXS Experiments konnte der Einbau der Partikel identifiziert werden. Durch eine Anpassung mit einem Unified Fit Modell wurde herausgefunden, dass die Partikel nicht agglomeriert sind und von einer Polymermatrix umgeben sind.

A clustering method for robust and reliable large scale functional and structural protein sequence annotation

Bioinformatics, in the last few decades, has played a fundamental role to give sense to the huge amount of data produced. Obtained the complete sequence of a genome, the major problem of knowing as much as possible of its coding regions, is crucial. Protein sequence annotation is challenging and, due to the size of the problem, only computational approaches can provide a feasible solution. As it has been recently pointed out by the Critical Assessment of Function Annotations (CAFA), most accurate methods are those based on the transfer-by-homology approach and the most incisive contribution is given by cross-genome comparisons. In the present thesis it is described a non-hierarchical sequence clustering method for protein automatic large-scale annotation, called “The Bologna Annotation Resource Plus” (BAR+). The method is based on an all-against-all alignment of more than 13 millions protein sequences characterized by a very stringent metric. BAR+ can safely transfer functional features (Gene Ontology and Pfam terms) inside clusters by means of a statistical validation, even in the case of multi-domain proteins. Within BAR+ clusters it is also possible to transfer the three dimensional structure (when a template is available). This is possible by the way of cluster-specific HMM profiles that can be used to calculate reliable template-to-target alignments even in the case of distantly related proteins (sequence identity < 30%). Other BAR+ based applications have been developed during my doctorate including the prediction of Magnesium binding sites in human proteins, the ABC transporters superfamily classification and the functional prediction (GO terms) of the CAFA targets. Remarkably, in the CAFA assessment, BAR+ placed among the ten most accurate methods. At present, as a web server for the functional and structural protein sequence annotation, BAR+ is freely available at http://bar.biocomp.unibo.it/bar2.0.

Functional and Genetic characterization of new genomic islands from an E. coli strain associated with neonatal meningitis.

Enterobacteriaceae genomes evolve through mutations, rearrangements and horizontal gene transfer (HGT). The latter evolutionary pathway works through the acquisition DNA (GEI) modules of foreign origin that enhances fitness of the host to a given environment. The genome of E. coli IHE3034, a strain isolated from a case of neonatal meningitis, has recently been sequenced and its subsequent sequence analysis has predicted 18 possible GEIs, of which: 8 have not been previously described, 5 fully meet the pathogenic island definition and at least 10 that seem to be of prophagic origin. In order to study the GEI distribution of our reference strain, we screened for the presence 18 GEIs a panel of 132 strains, representative of E. coli diversity. Also, using an inverse nested PCR approach we identified 9 GEI that can form an extrachromosomal circular intermediate (CI) and their respective attachment sites (att). Further, we set up a qPCR approach that allowed us to determine the excision rates of 5 genomic islands in different growth conditions. Four islands, specific for strains appertaining to the sequence type complex 95 (STC95), have been deleted in order to assess their function in a Dictyostelium discoideum grazing assays. Overall, the distribution data presented here indicate that 16 IHE3034 GEIs are more associated to the STC95 strains. Also the functional and genetic characterization has uncovered that GEI 13, 17 and 19 are involved in the resistance to phagocitation by Dictyostelium d thus suggesting a possible role in the adaptation of the pathogen during certain stages of infection.

Identification and functional characterization of human cytomegalovirus Fc binding proteins

ABSTRACT Human cytomegalovirus (HCMV) employs many different mechanisms to escape and subvert the host immune system surveillance. Among these different mechanisms the role of human IgG Fc receptors (FcγR) in HCMV pathogenesis is still unclear. In mammalians, FcγRs are expressed on the surface of all haematopoietic cells and have a multifaceted role in regulating the activity of antibodies to generate a well-balanced immune response. Viral proteins with Fcγ binding ability are highly diffuse among herpesviruses. They interfere with the host receptors functions in order to counteract immune system recognition. So far, two human HCMV Fcγ binding proteins have been described: UL119 and RL11. This work was aimed to the identification and characterization of HCMV Fcγ binding proteins. The study is divided in two parts: first the characterization of UL119 and RL11; second the identification and characterization of novel HCMV Fcγ binding proteins. Regarding the first part, we demonstrated that both UL119 and RL11 internalize Fcγ fragments from transfected cells surface through a clathrin dependent pathway. In infected cells both proteins were found in the viral assembly complex and on virions surface as envelope associated glycoproteins. Moreover, internalized Fcγ in infected cells do not undergo lysosomal degradation but rather traffic in early endosomes up to the viral assembly complex. Regarding the second part, we were able to identify two novels Fcγ binding protein coded by CMV: RL12 and RL13. The latter was also further characterized as recombinant protein in terms of cellular localization, Fc binding site and IgG internalization ability. Finally binding specificity of both RL12 and RL13 seems to be confined to human IgG1 and IgG2. Taken together, these data show that HCMV codes for up to 4 FcγR and that they could have a double role both on virus and on infected cells.

Functional hydrogels

Hydrogels are used in a variety of applications in daily life, such as super absorbers, contact lenses and in drug delivery. Functional hydrogels that allow the incorporation of additional functionalities have enormous potential for future development. The properties of such hydrogels can be diversified by introducing responsiveness to external stimuli. These crosslinked polymers are known to respond to changes in temperature, pH and pressure, as well as chemical and electrical stimuli, magnetic fields and irradiation. From this responsive behavior possible applications arise in many fields like drug delivery, tissue engineering, purification and implementation as actuators, biosensors or for medical coatings. However, their interaction with biomaterial and way of functioning are yet not fully understood. Therefore, thorough investigations regarding their optical, mechanical and chemical nature have to be conducted. A UV-crosslinkable polymer, consisting of N-isopropylacrylamide, methacrylic acid and the UV-crosslinker 4-benzoylphenyl methacrylate was synthesized. Its composition, determined by a comprehensive NMR study, is equivalent to the composition of the monomer mixture. The chemical characteristics were preserved during the subsequently formation of hydrogel films by photo-crosslinking as proved by XPS. For the optical characterization, e.g. the degree of swelling of very thin films, the spectroscopy of coupled long range surface plasmons is introduced. Thicker films, able to guide light waves were analyzed with combined surface plasmon and optical waveguide mode spectroscopy (SPR/OWS). The evaluation of the data was facilitated by the reverse Wentzel-Kramers-Brillouin (WKB) approximation. The meshsize and proper motion of the surface anchored hydrogels were investigated by fluorescence correlation spectroscopy (FCS), micro photon correlation spectroscopy (µPCS) and SPR/OWS. The studied gels exhibit a meshsize that allowed for the diffusion of small biomolecules inside their network. For future enhancement of probing diffusants, a dye that enables FRET in FCS was immobilized in the gel and the diffusion of gold-nanoparticles embedded in the polymer solution was studied by PCS. These properties can be conveniently tuned by the crosslinking density, which depends on the irradiation dose. Additionally, protocols and components for polymer analogous reactions based on active ester chemistry of the hydrogel were developed. Based on these syntheses and investigations, the hydrogel films are applied in the fields of medical coatings as well as in biosensing as matrix and biomimetic cushion. Their non-adhesive properties were proved in cell experiments, SPR/OWS and ToF-SIMS studies. The functionality and non-fouling property of the prepared hydrogels allowed for adaption to the needs of the respective application.

Opportunistic Data Gathering and Dissemination in Urban Scenarios

In the era of the Internet of Everything, a user with a handheld or wearable device equipped with sensing capability has become a producer as well as a consumer of information and services. The more powerful these devices get, the more likely it is that they will generate and share content locally, leading to the presence of distributed information sources and the diminishing role of centralized servers. As of current practice, we rely on infrastructure acting as an intermediary, providing access to the data. However, infrastructure-based connectivity might not always be available or the best alternative. Moreover, it is often the case where the data and the processes acting upon them are of local scopus. Answers to a query about a nearby object, an information source, a process, an experience, an ability, etc. could be answered locally without reliance on infrastructure-based platforms. The data might have temporal validity limited to or bounded to a geographical area and/or the social context where the user is immersed in. In this envisioned scenario users could interact locally without the need for a central authority, hence, the claim of an infrastructure-less, provider-less platform. The data is owned by the users and consulted locally as opposed to the current approach of making them available globally and stay on forever. From a technical viewpoint, this network resembles a Delay/Disruption Tolerant Network where consumers and producers might be spatially and temporally decoupled exchanging information with each other in an adhoc fashion. To this end, we propose some novel data gathering and dissemination strategies for use in urban-wide environments which do not rely on strict infrastructure mediation. While preserving the general aspects of our study and without loss of generality, we focus our attention toward practical applicative scenarios which help us capture the characteristics of opportunistic communication networks.

Life cycle assessment in construction field: A life cycle cost analysis of reinforcement concrete bridge

The present work is included in the context of the assessment of sustainability in the construction field and is aimed at estimating and analyzing life cycle cost of the existing reinforced concrete bridge “Viadotto delle Capre” during its entire life. This was accomplished by a comprehensive data collection and results evaluation. In detail, the economic analysis of the project is performed. The work has investigated possible design alternatives for maintenance/rehabilitation and end-of-life operations, when structural, functional, economic and also environmental requirements have to be fulfilled. In detail, the economic impact of different design options for the given reinforced concrete bridge have been assessed, whereupon the most economically, structurally and environmentally efficient scenario was chosen. The Integrated Life-Cycle Analysis procedure and Environmental Impact Assessment were also discussed in this work. The scope of this thesis is to illustrate that Life Cycle Cost analysis as part of Life Cycle Assessment approach could be effectively used to drive the design and management strategy of new and existing structures. The final objective of this contribution is to show how an economic analysis can influence decision-making in the definition of the most sustainable design alternatives. The designers can monitor the economic impact of different design strategies in order to identify the most appropriate option.

Advanced data deduplication techniques and their application

Data deduplication describes a class of approaches that reduce the storage capacity needed to store data or the amount of data that has to be transferred over a network. These approaches detect coarse-grained redundancies within a data set, e.g. a file system, and remove them.rnrnOne of the most important applications of data deduplication are backup storage systems where these approaches are able to reduce the storage requirements to a small fraction of the logical backup data size.rnThis thesis introduces multiple new extensions of so-called fingerprinting-based data deduplication. It starts with the presentation of a novel system design, which allows using a cluster of servers to perform exact data deduplication with small chunks in a scalable way.rnrnAfterwards, a combination of compression approaches for an important, but often over- looked, data structure in data deduplication systems, so called block and file recipes, is introduced. Using these compression approaches that exploit unique properties of data deduplication systems, the size of these recipes can be reduced by more than 92% in all investigated data sets. As file recipes can occupy a significant fraction of the overall storage capacity of data deduplication systems, the compression enables significant savings.rnrnA technique to increase the write throughput of data deduplication systems, based on the aforementioned block and file recipes, is introduced next. The novel Block Locality Caching (BLC) uses properties of block and file recipes to overcome the chunk lookup disk bottleneck of data deduplication systems. This chunk lookup disk bottleneck either limits the scalability or the throughput of data deduplication systems. The presented BLC overcomes the disk bottleneck more efficiently than existing approaches. Furthermore, it is shown that it is less prone to aging effects.rnrnFinally, it is investigated if large HPC storage systems inhibit redundancies that can be found by fingerprinting-based data deduplication. Over 3 PB of HPC storage data from different data sets have been analyzed. In most data sets, between 20 and 30% of the data can be classified as redundant. According to these results, future work in HPC storage systems should further investigate how data deduplication can be integrated into future HPC storage systems.rnrnThis thesis presents important novel work in different area of data deduplication re- search.

Functional characterization of the placenta specific protein PLAC1 and its use for cancer immunotherapy

Summary Antibody-based cancer therapies have been successfully introduced into the clinic and have emerged as the most promising therapeutics in oncology. The limiting factor regarding the development of therapeutical antibody vaccines is the identification of tumor-associated antigens. PLAC1, the placenta-specific protein 1, was categorized for the first time by the group of Prof. Sahin as such a tumor-specific antigen. Within this work PLAC1 was characterized using a variety of biochemical methods. The protein expression profile, the cellular localization, the conformational state and especially the interacting partners of PLAC1 and its functionality in cancer were analyzed. Analysis of the protein expression profile of PLAC1 in normal human tissue confirms the published RT-PCR data. Except for placenta no PLAC1 expression was detectable in any other normal human tissue. Beyond, an increased PLAC1 expression was detected in several cancer cell lines derived of trophoblastic, breast and pancreatic lineage emphasizing its properties as tumor-specific antigen. rnThe cellular localization of PLAC1 revealed that PLAC1 contains a functional signal peptide which conducts the propeptide to the endoplasmic reticulum (ER) and results in the secretion of PLAC1 by the secretory pathway. Although PLAC1 did not exhibit a distinct transmembrane domain, no unbound protein was detectable in the cell culture supernatant of overexpressing cells. But by selective isolation of different cellular compartments PLAC1 was clearly enriched within the membrane fraction. Using size exclusion chromatography PLAC1 was characterized as a highly aggregating protein that forms a network of high molecular multimers, consisting of a mixture of non-covalent as well as covalent interactions. Those interactions were formed by PLAC1 with itself and probably other cellular components and proteins. Consequently, PLAC1 localize outside the cell, where it is associated to the membrane forming a stable extracellular coat-like structure.rnThe first mechanistic hint how PLAC1 promote cancer cell proliferation was achieved identifying the fibroblast growth factor FGF7 as a specific interacting partner of PLAC1. Moreover, it was clearly shown that PLAC1 as well as FGF7 bind to heparin, a glycosaminoglycan of the ECM that is also involved in FGF-signaling. The participation of PLAC1 within this pathway was approved after co-localizing PLAC1, FGF7 and the FGF7 specific receptor (FGFR2IIIb) and identifying the formation of a trimeric complex (PLAC1, FGF7 and the specific receptor FGFR2IIIb). Especially this trimeric complex revealed the role of PLAC1. Binding of PLAC1 together with FGF7 leads to the activation of the intracellular tyrosine kinase of the FGFR2IIIb-receptor and mediate the direct phosphorylation of the AKT-kinase. In the absence of PLAC1, no FGF7 mediated phosphorylation of AKT was observed. Consequently the function of PLAC1 was clarified: PLAC1 acts as a co-factor by stimulating proliferation by of the FGF7-FGFR2 signaling pathway.rnAll together, these novel biochemical findings underline that the placenta specific protein PLAC1 could be a new target for cancer immunotherapy, especially considering its potential applicability for antibody therapy in tumor patients.

Graph-based analysis of brain resting-state fMRI data in nocturnal frontal lobe epileptic patients

Il lavoro che ho sviluppato presso l'unità di RM funzionale del Policlinico S.Orsola-Malpighi, DIBINEM, è incentrato sull'analisi dati di resting state - functional Magnetic Resonance Imaging (rs-fMRI) mediante l'utilizzo della graph theory, con lo scopo di valutare eventuali differenze in termini di connettività cerebrale funzionale tra un campione di pazienti affetti da Nocturnal Frontal Lobe Epilepsy (NFLE) ed uno di controlli sani. L'epilessia frontale notturna è una peculiare forma di epilessia caratterizzata da crisi che si verificano quasi esclusivamente durante il sonno notturno. Queste sono contraddistinte da comportamenti motori, prevalentemente distonici, spesso complessi, e talora a semiologia bizzarra. L'fMRI è una metodica di neuroimaging avanzata che permette di misurare indirettamente l'attività neuronale. Tutti i soggetti sono stati studiati in condizioni di resting-state, ossia di veglia rilassata. In particolare mi sono occupato di analizzare i dati fMRI con un approccio innovativo in campo clinico-neurologico, rappresentato dalla graph theory. I grafi sono definiti come strutture matematiche costituite da nodi e links, che trovano applicazione in molti campi di studio per la modellizzazione di strutture di diverso tipo. La costruzione di un grafo cerebrale per ogni partecipante allo studio ha rappresentato la parte centrale di questo lavoro. L'obiettivo è stato quello di definire le connessioni funzionali tra le diverse aree del cervello mediante l'utilizzo di un network. Il processo di modellizzazione ha permesso di valutare i grafi neurali mediante il calcolo di parametri topologici che ne caratterizzano struttura ed organizzazione. Le misure calcolate in questa analisi preliminare non hanno evidenziato differenze nelle proprietà globali tra i grafi dei pazienti e quelli dei controlli. Alterazioni locali sono state invece riscontrate nei pazienti, rispetto ai controlli, in aree della sostanza grigia profonda, del sistema limbico e delle regioni frontali, le quali rientrano tra quelle ipotizzate essere coinvolte nella fisiopatologia di questa peculiare forma di epilessia.

Expression, localization, and functional model of cholesterol transporters in lactating and nonlactating mammary tissues of murine, bovine, and human origin

Members of the ATP-binding cassette (ABC) transporters play a pivotal role in cellular lipid efflux. To identify candidate cholesterol transporters implicated in lipid homeostasis and mammary gland (MG) physiology, we compared expression and localization of ABCA1, ABCG1, and ABCA7 and their regulatory genes in mammary tissues of different species during the pregnancy-lactation cycle. Murine and bovine mammary glands (MGs) were investigated during different functional stages. The abundance of mRNAs was determined by quantitative RT-PCR. Furthermore, transporter proteins were localized in murine, bovine, and human MGs by immunohistochemistry. In the murine MG, ABCA1 mRNA abundance was elevated during nonlactating compared with lactating stages, whereas ABCA7 and ABCA1 mRNA profiles were not altered. In the bovine MG, ABCA1, ABCG1, and ABCA7 mRNAs abundances were increased during nonlactating stages compared with lactation. Furthermore, associations between mRNA levels of transporters and their regulatory genes LXRalpha, PPARgamma, and SREBPs were found. ABCA1, ABCG1, and ABCA7 proteins were localized in glandular MG epithelial cells (MEC) during lactation, whereas during nonlactating stages, depending on species, the proteins showed distinct localization patterns in MEC and adipocytes. Our results demonstrate that ABCA1, ABCG1, and ABCA7 are differentially expressed between lactation and nonlactating stages and in association with regulatory genes. Combined expression and localization data suggest that the selected cholesterol transporters are universal MG transporters involved in transport and storage of cholesterol and in lipid homeostasis of MEC. Because of the species-specific expression patterns of transporters in mammary tissue, mechanisms of cholesterol homeostasis seem to be differentially regulated between species.