Functional characterization of Streptococcus pyogenes pili

Group A Streptococcus is a Gram-positive human pathogen able to colonize both upper respiratory tract and skin. GAS is responsible for several acute diseases and autoimmune sequelae that account for half a million deaths worldwide every year (Cunningham et al., 2000). As other bacteria, GAS infections requires the capacity of the pathogen to adhere to host tissues and to form cell aggregates. The ability to persist in distinct host niches like the throat and the skin and to trigger infections is associated with the expression of different GAS virulence factors. GAS pili has been described as important virulence factors encoded by different FCT-operon regions. Based on this information, we decided to study the possible effect of environmental conditions that could regulate the pili expression. In this study we reported the influence of pH environment variations in biofilm formation for strains pertaining to a panel of different GAS FCT-types. The biofilm formation was promoted, excepted in the FCT-1 strains, by a changing in pH from physiological to acidic condition of growth in in vitro biofilm assay. By analyzing the possible association between biofilm formation and pH dependence, we have found that in FCT-2 and FCT-3 strains, the biofilm is promoted by pH reduction leading to an increase of pili expression. These data confirmed a direct link between pH dependent pilus expression and biofilm formation in GAS. As pili are a multi component structure we decided to investigate the functional role of one of its subunits, the AP-1 protein. AP-1 is highly conserved through the different FCT-types and suggests a possible essential role for the pili function. We focused our attention on the AP-1 protein encoded by the FCT-1 strains (M6). In particular this AP-1 protein contains the von Willebrand Factor A (VWFA) domain, which share an homology with the human VWFA domain that has been reported to be involved in adhesion process. We have demonstrated that the AP-1 protein binds to human epithelial cells by its VWFA domain, whereas the biofilm formation is mediated by the N-terminal region of AP-1 protein. Moreover, analyzing the importance of AP-1 in in vivo experiments we found a major capacity of tissue dissemination for the wild-type strain compared to the isogenic AP-1 deletion mutant. Pili have been also reported as potential vaccine candidates against Gram positive bacteria. For these reason we decided to investigate the relationship between cross reaction of sera raised against different GAS and GBS pilin subunits and the presence of a conserved Cna_B domain, in different pilin components. Our idea was to investigate if, using pilus conserved domains, a broad coverage vaccine against streptococcal infection could be possible.

Epidemiological and functional characterization of Streptococcus pneumoniae pili

Streptococcus pneumoniae is an important life threatening human pathogen causing agent of invasive diseases such as otitis media, pneumonia, sepsis and meningitis, but is also a common inhabitant of the respiratory tract of children and healthy adults. Likewise most streptococci, S. pneumoniae decorates its surface with adhesive pili, composed of covalently linked subunits and involved in the attachment to epithelial cells and virulence. The pneumococcal pili are encoded by two genomic regions, pilus islet 1 (PI-1), and pilus islet-2 (PI-2), which are present in about 30% and 16% of the pneumococcal strains, respectively. PI-1 exists in three clonally related variants, whereas PI-2 is highly conserved. The presence of the islets does not correlate with the serotype of the strains, but with the genotype (as determined by Multi Locus Sequence Typing). The prevalence of PI-1 and PI-2 positive strains is similar in isolates from invasive disease and carriage. To better dissect a possible association between PIs presence and disease we evaluated the distribution of the two PIs in a panel of 113 acute otitis media (AOM) clinical isolates from Israel. PI-1 was present in 30.1% (N=34) of the isolates tested, and PI-2 in 7% (N=8). We found that 50% of the PI-1 positive isolates belonged to the international clones Spain9V-3 (ST156) and Taiwan19F-14 (ST236), and that PI-2 was not present in the absence of Pl-1. In conclusion, there was no correlation between PIs presence and AOM, and, in general, the observed differences in PIs prevalence are strictly dependent upon regional differences in the distribution of the clones. Finally, in the AOM collection the prevalence of PI-1 was higher among antibiotic resistant isolates, confirming previous indications obtained by the in silico analysis of the MLST database collection. Since the pilus-1 subunits were shown to confer protection in mouse models of infection both in active and passive immunization studies, and were regarded as potential candidates for a new generation of protein-based vaccines, the functional characterization was mainly focused on S. pneumoniae pilus -1 components. The pneumococcal pilus-1 is composed of three subunits, RrgA, RrgB and RrgC, each stabilized by intra-molecular isopeptide bonds and covalently polymerized by means of inter-molecular isopeptide bonds to form an extended fibre. The pilus shaft is a multimeric structure mainly composed by the RrgB backbone subunit. The minor ancillary proteins are located at the tip and at the base of the pilus, where they have been proposed to act as the major adhesin (RrgA) and as the pilus anchor (RrgC), respectively. RrgA is protective in in vivo mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

Synthesis of hexa-peri-hexabenzocoronenes - from building blocks to functional materials

Discotic hexa-peri-hexabenzocoronene (HBC) derivatives have attracted intensive scientific interest due to their unique optoelectronic properties, which depends, to a large extend, upon the attached functional groups. The presented work covers the synthesis of novel HBC building blocks and new HBC derivatives as functional materials. The traditional preparation of HBC derivatives requires elaborate synthetic techniques and tremendous effort. Especially, more than 10 synthetic steps are usually necessary to approach HBCs with lower symmetries. In order to simplify the synthetic work and reduce the high costs, a novel synthetic strategy involving only four steps was developed based on 2,3,5,6-tetraphenyl-1,4-diiodobenzene intermediates and palladium catalyzed Suzuki cross coupling reactions. In order to introduce various functionalities and expand the diversity of multi-functionalizations, a novel C2v-symmetric dihalo HBC building block 2-47, which contains one iodine and one bromine in para positions, was prepared following the traditional intermolecular [4+2] Diels-Alder reaction route. The outstanding chemical selectivity between iodo and bromo groups in this compound consequently leads to lots of HBC derivatives bearing different functionalities. Directly attached heteroatoms will improve the material properties. According to the application of intramolecular Scholl reaction to a para-dimethoxy HPB, which leads to a meta-dimethoxy HBC, a phenomenon of phenyl group migration was discovered. Thereby, several interesting mechanistic details involving arenium cation intermediates were discussed. With a series of dipole functionalized HBCs, the molecular dynamics of this kind of materials was studied in different phases by DSC, 2D WAXD, solid state NMR and dielectric spectroscopies. High charge carrier mobility is an important parameter for a semiconductive material and depends on the degree of intramolecular order of the discotic molecules in thin films for HBC derivatives. Dipole – dipole interaction and hydrogen bonds were respectively introduced in order to achieve highly ordered supramolecular structure. The self-assembly behavior of these materials were investigated both in solution and solid state. Depending upon the different functionalities, these novel materials show either gelating or non-linear optical properties, which consequently broaden their applications as functional materials. In the field of conceivable electronic devices at a molecular level, HBCs hold high promise. Differently functionalized HBCs have been used as active component in the studies of single-molecular CFET and metal-SAMs-metal junctions. The outstanding properties shown in these materials promise their exciting potential applications in molecular devices.

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.

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Analysis of the immunological and functional features of the Neisserial Heparin Binding Antigen (NHBA)

Neisserial Heparin Binding Antigen (NHBA) is a surface-exposed lipoprotein ubiquitously expressed by genetically diverse Neisseria meningitidis strains and is an antigen of the multicomponent protein-based 4CMenB vaccine, able to induce bactericidal antibodies in humans and to bind heparin-like molecules. The aim of this study is to characterize the immunological and functional properties of NHBA. To evaluate immunogenicity and the contribution of aminoacid sequence variability to vaccine coverage, we constructed recombinant isogenic strains that are susceptible to bactericidal killing only by anti-NHBA antibodies and engineered them to express equal levels of selected NHBA peptides. In these recombinant strains, we observed different titres associated with the different peptide variants. These recombinant strains were then further engineered to express NHBA chimeric proteins to investigate the regions important for immunogenicity. In natural strains, anti-NHBA antibodies were found to be cross-protective against strains expressing different peptides. To investigate the functional properties of this antigen, the recombinant purified NHBA protein was tested in in vitro binding studies and was found to be able to bind epithelial cells. The binding was abolished when cells were treated specifically with heparinase III, suggesting that the interaction with the cells is mediated by heparan sulfate proteoglycans (HSPG). Mutation of the Arg-rich tract of NHBA abrogated the binding, confirming the importance of this region in mediating the binding to heparin-like molecules. In a panel of N. meningitidis strains, the deletion of nhba resulted in a reduction of adhesion with respect to each isogenic wild type strain. Furthermore, the adhesion of the wild-type strain was prevented by using anti-NHBA polyclonal sera, demonstrating the specificity of the interaction. These results suggest that NHBA could be a novel meningococcal adhesin contributing to host-cell interaction. Moreover, we analysed NHBA NalP-mediated cleavage in different NHBA peptides and showed that not all NHBA peptides are cleaved.

Template-assisted patterning of functional polymers

In this thesis, anodic aluminum oxide (AAO) membranes, which provide well-aligned uniform mesoscopic pores with adjustable pore parameters, were fabricated and successfully utilized as templates for the fabrication of functional organic nanowires, nanorods and the respective well-ordered arrays. The template-assisted patterning technique was successfully applied for the realization of different objectives:rnHigh-density and well-ordered arrays of hole-conducting nanorods composed of cross-linked triphenylamine (TPA) and tetraphenylbenzidine (TPD) derivatives on conductive substrates like ITO/glass have been successfully fabricated. By applying a freeze-drying technique to remove the aqueous medium after the wet-chemical etching of the template, aggregation and collapsing of the rods was prevented and macroscopic areas of perfectly freestanding nanorods were feasible. Based on the hole-conducting nanorod arrays and their subsequent embedding into an electron-conducting polymer matrix via spin-coating, a novel routine concept for the fabrication of well-ordered all-organic bulk heterojunction for organic photovoltaic applications was successfully demonstrated. The increased donor/acceptor interface of the fabricated devices resulted in a remarkable increase of the photoluminescence quenching compared to a planar bilayer morphology. Further, the fundamental working principle of the templating approach for the solution-based all-organic photovoltaic device was demonstrated for the first time.rnFurthermore, in order to broaden the applicability of patterned surfaces, which are feasible via the template-based patterning of functional materials, AAO with hierarchically branched pores were fabricated and utilized as templates. By pursuing the common templating process hierarchically polymeric replicas, which show remarkable similarities with interesting biostructures, like the surface of the lotus leaf and the feet of a gecko, were successfully prepared.rnIn contrast to the direct infiltration of organic functional materials, a novel route for the fabrication of functional nanowires via post-modification of reactive nanowires was established. Therefore, reactive nanowires based on cross-linked pentafluorophenylesters were fabricated by utilizing AAO templates. The post-modification with fluorescent dyes was demonstrated. Furthermore, reactive wires were converted into well-dispersed poly(N-isopropylacrylamide) (PNIPAM) hydrogels, which exhibit a thermal-responsive reversible phase transition. The reversible thermal-responsible swelling of the PNIPAM nanowires exhibited a more than 50 % extended length than in the collapsed PNIPAM state. rnLast but not least, the shape-anisotropic pores of AAO were utilized to uniformly align the mesogens of a nematic liquid crystalline elastomer. Liquid crystalline nanowires with a narrow size distribution and uniform orientation of the liquid crystalline material were fabricated. It was shown that during the transition from the nematic to the isotropic phase the rod’s length shortened by roughly 40 percent. As such these liquid crystalline elastomeric nanowires may find application, as wire-shaped nanoactuators in various fields of research, like lab-on-chip systems, micro fluidics and biomimetics.rn

Musical Training Induces Functional Plasticity in Human Hippocampus

Training can change the functional and structural organization of the brain, and animal models demonstrate that the hippocampus formation is particularly susceptible to training-related neuroplasticity. In humans, however, direct evidence for functional plasticity of the adult hippocampus induced by training is still missing. Here, we used musicians' brains as a model to test for plastic capabilities of the adult human hippocampus. By using functional magnetic resonance imaging optimized for the investigation of auditory processing, we examined brain responses induced by temporal novelty in otherwise isochronous sound patterns in musicians and musical laypersons, since the hippocampus has been suggested previously to be crucially involved in various forms of novelty detection. In the first cross-sectional experiment, we identified enhanced neural responses to temporal novelty in the anterior left hippocampus of professional musicians, pointing to expertise-related differences in hippocampal processing. In the second experiment, we evaluated neural responses to acoustic temporal novelty in a longitudinal approach to disentangle training-related changes from predispositional factors. For this purpose, we examined an independent sample of music academy students before and after two semesters of intensive aural skills training. After this training period, hippocampal responses to temporal novelty in sounds were enhanced in musical students, and statistical interaction analysis of brain activity changes over time suggests training rather than predisposition effects. Thus, our results provide direct evidence for functional changes of the adult hippocampus in humans related to musical training.

Spatial and functional relationships between air conduits and blood capillaries in the pulmonary gas exchange tissue of adult and developing chickens

The documented data regarding the three-dimensional structure of the air capillaries (ACs), the ultimate sites of gas exchange in the avian lung is contradictory. Further, the mode of gas exchange, described as cross-current has not been clearly elucidated. We studied the temporal and spatial arrangement of the terminal air conduits of the chicken lung and their relationship with the blood capillaries (BCs) in embryos as well as the definitive architecture in adults. Several visualization techniques that included corrosion casting, light microscopy as well as scanning and transmission electron microscopy were used. Two to six infundibulae extend from each atrium and give rise to numerous ACs that spread centrifugally. Majority of the ACs are tubular structures that give off branches, which anastomose with their neighboring cognates. Some ACs have globular shapes and a few are blind-ending tapering tubes. During inauguration, the luminal aspects of the ACs are characterized by numerous microvillus-like microplicae, which are formed during the complex processes of cell attenuation and canalization of the ACs. The parabronchial exchange BCs, initially inaugurated as disorganized meshworks, are reoriented via pillar formation to lie predominantly orthogonal to the long axes of the ACs. The remodeling of the retiform meshworks by intussusceptive angiogenesis essentially accomplishes a cross-current system at the gas exchange interface in the adults, where BCs form ring-like patterns around the ACs, thus establishing a cross-current system. Our findings clarify the mode of gas exchange in the parabronchial mantle and illuminate the basis for the functional efficiency of the avian lung.

Social cognition as a mediator variable between neurocognition and functional outcome in schizophrenia: empirical review and new results by structural equation modeling

Cognitive impairments are currently regarded as important determinants of functional domains and are promising treatment goals in schizophrenia. Nevertheless, the exact nature of the interdependent relationship between neurocognition and social cognition as well as the relative contribution of each of these factors to adequate functioning remains unclear. The purpose of this article is to systematically review the findings and methodology of studies that have investigated social cognition as a mediator variable between neurocognitive performance and functional outcome in schizophrenia. Moreover, we carried out a study to evaluate this mediation hypothesis by the means of structural equation modeling in a large sample of 148 schizophrenia patients. The review comprised 15 studies. All but one study provided evidence for the mediating role of social cognition both in cross-sectional and in longitudinal designs. Other variables like motivation and social competence additionally mediated the relationship between social cognition and functional outcome. The mean effect size of the indirect effect was 0.20. However, social cognitive domains were differentially effective mediators. On average, 25% of the variance in functional outcome could be explained in the mediation model. The results of our own statistical analysis are in line with these conclusions: Social cognition mediated a significant indirect relationship between neurocognition and functional outcome. These results suggest that research should focus on differential mediation pathways. Future studies should also consider the interaction with other prognostic factors, additional mediators, and moderators in order to increase the predictive power and to target those factors relevant for optimizing therapy effects.

Disorders of paravertebral lumbar muscles: from pathology to cross-sectional imaging

Paravertebral lumbar muscles are important for spine stabilization and mobility. They may be abnormal in several disorders that may be associated with pain or functional impairment. Special attention should be paid to the paravertebral muscles during imaging, so that a possible muscular disease is not overlooked, especially in patients with low back pain. This article reviews such imaging abnormalities.

Sphingosine kinase 1 is pivotal for Fc epsilon RI-mediated mast cell signaling and functional responses in vitro and in vivo

Mast cell degranulation is pivotal to allergic diseases; investigating novel pathways triggering mast cell degranulation would undoubtedly have important therapeutic potential. FcepsilonRI-mediated degranulation has contradictorily been shown to require SphK1 or SphK2, depending on the reports. We investigated the in vitro and in vivo specific role(s) of SphK1 and SphK2 in FcepsilonRI-mediated responses, using specific small interfering RNA-gene silencing. The small interfering RNA-knockdown of SphK1 in mast cells inhibited several signaling mechanisms and effector functions, triggered by FcepsilonRI stimulation including: Ca(2+) signals, NFkappaB activation, degranulation, cytokine/chemokine, and eicosanoid production, whereas silencing SphK2 had no effect at all. Moreover, silencing SPHK1 in vivo, in different strains of mice, strongly inhibited mast cell-mediated anaphylaxis, including inhibition of vascular permeability, tissue mast cell degranulation, changes in temperature, and serum histamine and cytokine levels, whereas silencing SPHK2 had no effect and the mice developed anaphylaxis. Our data differ from a recent report using SPHK1(-/-) and SPHK2(-/-) mice, which showed that SphK2 was required for FcepsilonRI-mediated mast cell responses. We performed experiments in mast cells derived from SPHK1(-/-) and SPHK2(-/-) mice and show that the calcium response and degranulation, triggered by FcepsilonRI-cross-linking, is not different from that triggered in wild-type cells. Moreover, IgE-mediated anaphylaxis in the knockout mice showed similar levels in temperature changes and serum histamine to that from wild-type mice, indicating that there was no protection from anaphylaxis for either knockout mice. Thus, our data strongly suggest a previously unrecognized compensatory mechanism in the knockout mice, and establishes a role for SphK1 in IgE-mediated mast cell responses.

Cross-modal plasticity in the human thalamus: evidence from intraoperative macrostimulations

During stereotactic functional neurosurgery, stimulation procedure to control for proper target localization provides a unique opportunity to investigate pathophysiological phenomena that cannot be addressed in experimental setups. Here we report on the distribution of response modalities to 487 intraoperative thalamic stimulations performed in 24 neurogenic pain (NP), 17 parkinsonian (PD) and 10 neuropsychiatric (Npsy) patients. Threshold responses were subdivided into somatosensory, motor and affective, and compared between medial (central lateral nucleus) and lateral (ventral anterior, ventral lateral and ventral medial) thalamic nuclei and between patients groups. Major findings were as follows: in the medial thalamus, evoked responses were for a large majority (95%) somatosensory in NP patients, 47% were motor in PD patients, and 54% affective in Npsy patients. In the lateral thalamus, a much higher proportion of somatosensory (83%) than motor responses (5%) was evoked in NP patients, while the proportion was reversed in PD patients (69% motor vs. 21% somatosensory). These results provide the first evidence for functional cross-modal changes in lateral and medial thalamic nuclei in response to intraoperative stimulations in different functional disorders. This extensive functional reorganization sheds new light on wide-range plasticity in the adult human thalamocortical system.

[Cross-talk between heart and kidney--mechanisms and management of the cardiorenal syndrome from a nephrologists view]

Renal dysfunction represents a frequent comorbidity in patients with in chronic heart failure and is not only a strong predictor of mortality, but also causally linked to the development and progression of CHF. Mechanisms involved in the cross-talk between the kidney and the heart include the up-regulated sympathetic nerve system, activation of the renin-angiotensin-aldosterone system, vasopressin release and decreased activity of arterial baroreceptors and natriuretic peptides resulting in abnormal salt and water retention. The main therapeutic goals for patients with the so-called cardiorenal syndrome is the normalization of volume status while avoiding overdiuresis and renal dysfunction as well as the implementation of an evidence-based pharmacologic treatment to improve patient outcome. If these two goals are not achieved with conventional therapy, renal replacement therapy should be discussed in an interdisciplinary approach. All current renal replacement techniques have proved to be useful in controlling hypervolemia and ameliorating functional cardiac parameters and quality of life in patients with heart failure. Nevertheless, the influence of renal replacement therapy on long-term survival of affected patients has not been addressed in large controlled studies.

Parameter-free extraction of the functional network topology from intracranial EEG recordings: a time-resolved study of graph properties in focal onset seizures

Rationale: Focal onset epileptic seizures are due to abnormal interactions between distributed brain areas. By estimating the cross-correlation matrix of multi-site intra-cerebral EEG recordings (iEEG), one can quantify these interactions. To assess the topology of the underlying functional network, the binary connectivity matrix has to be derived from the cross-correlation matrix by use of a threshold. Classically, a unique threshold is used that constrains the topology [1]. Our method aims to set the threshold in a data-driven way by separating genuine from random cross-correlation. We compare our approach to the fixed threshold method and study the dynamics of the functional topology. Methods: We investigate the iEEG of patients suffering from focal onset seizures who underwent evaluation for the possibility of surgery. The equal-time cross-correlation matrices are evaluated using a sliding time window. We then compare 3 approaches assessing the corresponding binary networks. For each time window: * Our parameter-free method derives from the cross-correlation strength matrix (CCS)[2]. It aims at disentangling genuine from random correlations (due to finite length and varying frequency content of the signals). In practice, a threshold is evaluated for each pair of channels independently, in a data-driven way. * The fixed mean degree (FMD) uses a unique threshold on the whole connectivity matrix so as to ensure a user defined mean degree. * The varying mean degree (VMD) uses the mean degree of the CCS network to set a unique threshold for the entire connectivity matrix. * Finally, the connectivity (c), connectedness (given by k, the number of disconnected sub-networks), mean global and local efficiencies (Eg, El, resp.) are computed from FMD, CCS, VMD, and their corresponding random and lattice networks. Results: Compared to FMD and VMD, CCS networks present: *topologies that are different in terms of c, k, Eg and El. *from the pre-ictal to the ictal and then post-ictal period, topological features time courses that are more stable within a period, and more contrasted from one period to the next. For CCS, pre-ictal connectivity is low, increases to a high level during the seizure, then decreases at offset. k shows a ‘‘U-curve’’ underlining the synchronization of all electrodes during the seizure. Eg and El time courses fluctuate between the corresponding random and lattice networks values in a reproducible manner. Conclusions: The definition of a data-driven threshold provides new insights into the topology of the epileptic functional networks.