Discharge properties of single neurons in the dorsal nucleus of the lateral lemniscus of the rat.

The aim of the present study was to characterize the discharge properties of single neurons in the dorsal nucleus of the lateral lemniscus (DNLL) of the rat. In the absence of acoustic stimulation, two types of spontaneous discharge patterns were observed: units tended to fire in a bursting or in a nonbursting mode. The distribution of units in the DNLL based on spontaneous firing rate followed a rostrocaudal gradient: units with high spontaneous rates were most commonly located in the rostral part of the DNLL, whereas in the caudal part units had lower spontaneous discharge rates. The most common response pattern of DNLL units to 200 ms binaural noise bursts contained a prominent onset response followed by a lower but steady-state response and an inhibitory response in the early-off period. Thresholds of response to noise bursts were on average higher for DNLL units than for units recorded in the inferior colliculus under the same experimental conditions. The DNLL units were arranged according to a mediolateral sensitivity gradient with the lowest threshold units in the most lateral part of the nucleus. In the rat, as in other mammals, the most common DNLL binaural input type was an excitatory response to contralateral ear stimulation and inhibitory response to ipsilateral ear stimulation (EI type). Pure tone bursts were in general a more effective stimulus compared to noise bursts. Best frequency (BF) was established for 97 DNLL units and plotted according to their spatial location. The DNLL exhibits a loose tonotopic organization, where there is a concentric pattern with high BF units located in the most dorsal and ventral parts of the DNLL and lower BF units in the middle part of the nucleus.

Analysis of GPCR properties of Frizzled receptors and establishment of the "in vitro" platform for screening of Frizzled agonists/antagonists as potential therapeutic agents

Morphogens of the Wnt protein family are the secreted lipoglycoprotein ligands which initiate several pathways heavily involved in the coordination of various developmental stages of organisms in the majority of animal species. Deregulation of these pathways in the adult leads to formation and sustaining of multiple types of cancer. The latter notion is reinforced by the fact that the very discovery of the first Wnt ligand was due to its role as the causative factor of carcinogenic transformation (Nusse and Varmus, 1982). Nowadays our knowledge on Wnt signaling has "moved with the times" and these pathways were identified to be often crucial for tumor formation, its interactions with the microenvironment, and promotion of the metastases (Huang and Du, 2008; Zerlin et al., 2008; Jessen, 2009). Thus the relevance of the pathway as the target for drug development has further increased in the light of modern paradigms of the complex cancer treatments which target also spreading and growth- promoting factors of tumors by specific and highly efficient substances (Pavet et al., 2010). Presently the field of the Wnt-targeting drug research is almost solely dominated by assays based on transcriptional activation induced by the signaling. This approach resulted in development of a number of promising substances (Lee et al., 2011). Despite its effectiveness, the method nevertheless suffers from several drawbacks. Among the major ones is the fact that this approach is prone to identify compounds targeting rather downstream effectors of the pathway, which are indiscriminately used by all the subtypes of the Wnt signaling. Additionally, proteins which are involved in several signaling cascades and not just the Wnt pathway turn out as targets of the new compounds. These issues increase risks of side effects due to off-target interactions and blockade of the pathway in healthy cells. In the present work we put forward a novel biochemical approach for drug development on the Wnt pathway. It targets Frizzleds (Fzs) - a family of 7-transmbembrane proteins which serve as receptors for Wnt ligands. They offer unique properties for the development of highly specific and effective drugs as they control all branches of the Wnt signaling. Recent advances in the understanding of the roles of heterotrimeric G proteins downstream from Fzs (Katanaev et al., 2005; Liu et al., 2005; Jernigan et al., 2010) suggest application of enzymatic properties of these effectors to monitor the receptor-mediated events. We have applied this knowledge in practice and established a specific and efficient method based on utilization of a novel high-throughput format of the GTP-binding assay to follow the activation of Fzs. This type of assay is a robust and well-established technology for the research and screenings on the GPCRs (Harrison and Traynor, 2003). The conventional method of detection involves the radioactively labeled non-hydrolysable GTP analog [35S]GTPyS. Its application in the large-scale screenings is however problematic which promoted development of the novel non-radioactive GTP analog GTP-Eu. The new molecule employs phenomenon of the time-resolved fluorescence to provide sensitivity comparable to the conventional radioactive substance. Initially GTP-Eu was tested only in one of many possible types of GTP-binding assays (Frang et al., 2003). In the present work we expand these limits by demonstrating the general comparability of the novel label with the radioactive method in various types of assays. We provide a biochemical characterization of GTP-Eu interactions with heterotrimeric and small GTPases and a comparative analysis of the behavior of the new label in the assays involving heterotrimeric G protein effectors. These developments in the GTP-binding assay were then applied to monitor G protein activation by the Fz receptors. The data obtained in mammalian cultured cell lines provides for the first time an unambiguous biochemical proof for direct coupling of Fzs with G proteins. The specificity of this interaction has been confirmed by the experiments with the antagonists of Fz and by the pertussis toxin-mediated deactivation. Additionally we have identified the specificity of Wnt3a towards several members of the Fz family and analyzed the properties of human Fz-1 which was found to be the receptor coupled to the Gi/o family of G proteins. Another process playing significant role in the functioning of every GPCR is endocytosis. This phenomenon can also be employed for drug screenings on GPCRs (Bickle, 2010). In the present work we have demonstrated that Drosophila Fz receptors are involved in an unusual for many GPCRs manifestation of the receptor-mediated internalization. Through combination of biochemical approaches and studies on Drosophila as the model organism we have shown that direct interactions of the Fzs and the α-subunit of the heterotrimeric G protein Go with the small GTPase Rab5 regulate internalization of the receptor in early endosomes. We provide data uncovering the decisive role of this self-promoted endocytosis in formation of a proper signaling output in the canonical as well as planar cell polarity (PCP) pathways regulated by Fz. The results of this work thus establish a platform for the high-throughput screening to identify substances active in the cancer-related Wnt pathways. This methodology has been adjusted and applied to provide the important insights in Fz functioning and will be instrumental for further investigations on the Wnt-mediated pathways.

Bayesian Markov chain Monte Carlo inversion of geophysical data for the purpose of estimating the hydraulic properties of the unsaturated zone

Ground-penetrating radar (GPR) has the potential to provide valuable information on hydrological properties of the vadose zone because of their strong sensitivity to soil water content. In particular, recent evidence has suggested that the stochastic inversion of crosshole GPR data within a coupled geophysical-hydrological framework may allow for effective estimation of subsurface van-Genuchten-Mualem (VGM) parameters and their corresponding uncertainties. An important and still unresolved issue, however, is how to best integrate GPR data into a stochastic inversion in order to estimate the VGM parameters and their uncertainties, thus improving hydrological predictions. Recognizing the importance of this issue, the aim of the research presented in this thesis was to first introduce a fully Bayesian inversion called Markov-chain-Monte-carlo (MCMC) strategy to perform the stochastic inversion of steady-state GPR data to estimate the VGM parameters and their uncertainties. Within this study, the choice of the prior parameter probability distributions from which potential model configurations are drawn and tested against observed data was also investigated. Analysis of both synthetic and field data collected at the Eggborough (UK) site indicates that the geophysical data alone contain valuable information regarding the VGM parameters. However, significantly better results are obtained when these data are combined with a realistic, informative prior. A subsequent study explore in detail the dynamic infiltration case, specifically to what extent time-lapse ZOP GPR data, collected during a forced infiltration experiment at the Arrenaes field site (Denmark), can help to quantify VGM parameters and their uncertainties using the MCMC inversion strategy. The findings indicate that the stochastic inversion of time-lapse GPR data does indeed allow for a substantial refinement in the inferred posterior VGM parameter distributions. In turn, this significantly improves knowledge of the hydraulic properties, which are required to predict hydraulic behaviour. Finally, another aspect that needed to be addressed involved the comparison of time-lapse GPR data collected under different infiltration conditions (i.e., natural loading and forced infiltration conditions) to estimate the VGM parameters using the MCMC inversion strategy. The results show that for the synthetic example, considering data collected during a forced infiltration test helps to better refine soil hydraulic properties compared to data collected under natural infiltration conditions. When investigating data collected at the Arrenaes field site, further complications arised due to model error and showed the importance of also including a rigorous analysis of the propagation of model error with time and depth when considering time-lapse data. Although the efforts in this thesis were focused on GPR data, the corresponding findings are likely to have general applicability to other types of geophysical data and field environments. Moreover, the obtained results allow to have confidence for future developments in integration of geophysical data with stochastic inversions to improve the characterization of the unsaturated zone but also reveal important issues linked with stochastic inversions, namely model errors, that should definitely be addressed in future research.

Permeability properties of ENaC selectivity filter mutants.

The epithelial Na(+) channel (ENaC), located in the apical membrane of tight epithelia, allows vectorial Na(+) absorption. The amiloride-sensitive ENaC is highly selective for Na(+) and Li(+) ions. There is growing evidence that the short stretch of amino acid residues (preM2) preceding the putative second transmembrane domain M2 forms the outer channel pore with the amiloride binding site and the narrow ion-selective region of the pore. We have shown previously that mutations of the alphaS589 residue in the preM2 segment change the ion selectivity, making the channel permeant to K(+) ions. To understand the molecular basis of this important change in ionic selectivity, we have substituted alphaS589 with amino acids of different sizes and physicochemical properties. Here, we show that the molecular cutoff of the channel pore for inorganic and organic cations increases with the size of the amino acid residue at position alpha589, indicating that alphaS589 mutations enlarge the pore at the selectivity filter. Mutants with an increased permeability to large cations show a decrease in the ENaC unitary conductance of small cations such as Na(+) and Li(+). These findings demonstrate the critical role of the pore size at the alphaS589 residue for the selectivity properties of ENaC. Our data are consistent with the main chain carbonyl oxygens of the alphaS589 residues lining the channel pore at the selectivity filter with their side chain pointing away from the pore lumen. We propose that the alphaS589 side chain is oriented toward the subunit-subunit interface and that substitution of alphaS589 by larger residues increases the pore diameter by adding extra volume at the subunit-subunit interface.

A further examination of the distinction between dependency-oriented and achievement-oriented parental psychological control: Psychometric properties of the DAPCS with French-speaking late adolescents

Psychological control refers to parental behaviors that intrude on the psychological and emotional development of the child. In 2010, Soenens and colleagues proposed a distinction between two domain-specific expressions of psychological control, that is, Dependency-oriented Psychological Control (DPC) and Achievement-oriented Psychological Control (APC). The aim of this study was to evaluate the factor structure, reliability, and convergent validity of the French form of the Dependency-oriented and Achievement-oriented Psychological Control Scale (DAPCS; Soenens, Vansteenkiste, and Luyten, 2010) in a sample of late adolescents (N = 291, mean age = 21.65). Confirmatory factor analyses confirmed the hypothesized two-factor solution of the DAPCS for paternal as well as for maternal ratings. Moreover, high indices of internal consistency indicated that both subscales produced reliable scores. Further, convergent validity was confirmed by theoretically consistent associations between the DAPCS' subscales and well-established assessments of general parenting style dimensions. Finally, results evidenced gender specific patterns supporting the relevance of domain differentiation in the assessment of psychological control. Overall, the results of this study indicated that the French form of the DAPCS might be a useful instrument to assess two domainspecific types of parental psychological control among French-speaking adolescents.

CNDOL: A fast and reliable method for the calculation of electronic properties of very large systems. Applications to retinal binding pocket in rhodopsin and gas phase porphine.

Very large molecular systems can be calculated with the so called CNDOL approximate Hamiltonians that have been developed by avoiding oversimplifications and only using a priori parameters and formulas from the simpler NDO methods. A new diagonal monoelectronic term named CNDOL/21 shows great consistency and easier SCF convergence when used together with an appropriate function for charge repulsion energies that is derived from traditional formulas. It is possible to obtain a priori molecular orbitals and electron excitation properties after the configuration interaction of single excited determinants with reliability, maintaining interpretative possibilities even being a simplified Hamiltonian. Tests with some unequivocal gas phase maxima of simple molecules (benzene, furfural, acetaldehyde, hexyl alcohol, methyl amine, 2,5 dimethyl 2,4 hexadiene, and ethyl sulfide) ratify the general quality of this approach in comparison with other methods. The calculation of large systems as porphine in gas phase and a model of the complete retinal binding pocket in rhodopsin with 622 basis functions on 280 atoms at the quantum mechanical level show reliability leading to a resulting first allowed transition in 483 nm, very similar to the known experimental value of 500 nm of "dark state." In this very important case, our model gives a central role in this excitation to a charge transfer from the neighboring Glu(-) counterion to the retinaldehyde polyene chain. Tests with gas phase maxima of some important molecules corroborate the reliability of CNDOL/2 Hamiltonians.

Properties of functional brain networks correlate with frequency of psychogenic non-epileptic seizures.

Abnormalities in the topology of brain networks may be an important feature and etiological factor for psychogenic non-epileptic seizures (PNES). To explore this possibility, we applied a graph theoretical approach to functional networks based on resting state EEGs from 13 PNES patients and 13 age- and gender-matched controls. The networks were extracted from Laplacian-transformed time-series by a cross-correlation method. PNES patients showed close to normal local and global connectivity and small-world structure, estimated with clustering coefficient, modularity, global efficiency, and small-worldness (SW) metrics, respectively. Yet the number of PNES attacks per month correlated with a weakness of local connectedness and a skewed balance between local and global connectedness quantified with SW, all in EEG alpha band. In beta band, patients demonstrated above-normal resiliency, measured with assortativity coefficient, which also correlated with the frequency of PNES attacks. This interictal EEG phenotype may help improve differentiation between PNES and epilepsy. The results also suggest that local connectivity could be a target for therapeutic interventions in PNES. Selective modulation (strengthening) of local connectivity might improve the skewed balance between local and global connectivity and so prevent PNES events.

Effect of rufinamide on gating properties of voltage-gated sodium channel Na(v)1.7

Background: Voltage-gated sodium channels (Nav1.x) are important players in chronic pain. A particular interest has grown in Nav1.7, expressed in nociceptors, since mutations in its gene are associated to two inherited pain syndromes or insensitivity to pain. Rufinamide, a drug used to treat refractory epilepsy such as the Lennox-Gastaut syndrome, has been shown to reduce the number of action potentials in cortical neurons without completely blocking Na channels. Aim: The goal of this study was to investigate the effect of rufinamide on Nav1.7 current. Methods and results: Whole-cell patch clamp experiments were performed using HEK293 cells stably expressing Nav1.7. Rufinamide significantly decreased peak sodium current by 28.3, 21.2 and 12.5% at concentrations of 500, 100 and 50μM respectively (precise EC50 could not be calculated since higher rufinamide concentrations could not be achieved in physiological buffer solution). No significant difference on the V1/2 of voltage-dependence of activation was seen; however a shift in the steady-state inactivation curve was observed (-82.6 mV to -88.8 mV and -81.8 to -87.6 mV for 50 and 100 μM rufinamide respectively, p <0.005). Frequency-dependent inhibition of Nav1.7 was also influenced by the drug. One hundred μM rufinamide reduced the peak sodium current (in % of the peak current taken at the first sweep of a train of 50) from 90.8 to 80.8% (5Hz), 88.7 to 71.8% (10 Hz), 69.1 to 49.2% (25 Hz) and 22.3 to 9.8% (50 Hz) (all p <0.05). Onset of fast inactivation was not influenced by the drug since no difference in the time constant of current decay was observed. Conclusion: In the concentration range of plasma level in human treated for epilepsy, 15 μM, rufinamide only minimally blocks Nav1.7. However, it stabilizes the inactivated state and exerts frequencydependent inhibition of Nav1.7. These pharmacological properties may be of use in reducing ectopic discharges as a causal and symptom related contributor of neuropathic pain syndrome.

A cohort-based analysis of the evolution of lifetime prevalence of recourse to prostitution by men in the general population of Switzerland, 1987-2000

Background: Despite their relevance to the prevention of sexually transmitted infections, there are few data on the frequency of recourse to prostitution in the male population in Switzerland. Using data gathered for the evaluation of the Swiss AIDS prevention strategy, we analysed net aggregate change and cohort-based change in lifetime prevalence of recourse to prostitution. Methods: Seven repeated cross-sectional telephone surveys of the general population aged 17-45 years (17-30 years only for the 1987 and 1988 surveys) were undertaken from 1987 to 2000 providing information on sexual behaviour including men's recourse to prostitution (total n¼9318). Age categories were: 17-20, 21-25, 26-30, 31-35, 36-40 and 41-45 years. Prevalence at 17-30 years was available in all surveys and prevalence at 41-45 was available for 1989-2000, though not for the same cohorts. Intra-cohort increase in prevalence over 10 years was analysed using truncated information for cohorts aged 21-25 and 26-30 years in 1987 and 1990. Population estimates were computed with 95% confidence intervals (CI). Results: No net change occurred in the 17-45 years male population prevalence between 1989 (17.6%, CI ¼ 15.4; 20.0) and 2000 (17.7%, CI ¼ 15.6; 20.0). The median starting prevalence of recourse to prostitution at age 17-20 was 4.8% (in 1989, CI ¼ 2.0; 9.7) and the range was from 1.8 (in 1994) to 10.4% (in 1990). The median ending prevalence at age 41-45 was 21.9% (in 1994, CI 16.7; 27.9) and the range was from 17.9 (in 2000) to 26.1% (in 1992). No clear trend was observed in either starting or ending prevalence. Intra-cohort evolution of the 1997 and 1990 cohorts was very similar. Conclusions: Based on available data, there was no net (aggregate) change in the prevalence of recourse to prostitution by males in Switzerland between 1989 and 2000. Within the time frame available, intra-cohort evolution was also very similar.

Nucleic acid-binding properties of the RRM-containing protein RDM1.

RDM1 (RAD52 Motif 1) is a vertebrate protein involved in the cellular response to the anti-cancer drug cisplatin. In addition to an RNA recognition motif, RDM1 contains a small amino acid motif, named RD motif, which it shares with the recombination and repair protein, RAD52. RDM1 binds to single- and double-stranded DNA, and recognizes DNA distortions induced by cisplatin adducts in vitro. Here, we have performed an in-depth analysis of the nucleic acid-binding properties of RDM1 using gel-shift assays and electron microscopy. We show that RDM1 possesses acidic pH-dependent DNA-binding activity and that it binds RNA as well as DNA, and we present evidence from competition gel-shift experiments that RDM1 may be capable of discrimination between the two nucleic acids. Based on reported studies of RAD52, we have generated an RDM1 variant mutated in its RD motif. We find that the L119GF --> AAA mutation affects the mode of RDM1 binding to single-stranded DNA.

In vivo measurement of tissue oxygenation by time-resolved luminescence spectroscopy: advantageous properties of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate.

Measuring tissue oxygenation in vivo is of interest in fundamental biological as well as medical applications. One minimally invasive approach to assess the oxygen partial pressure in tissue (pO2) is to measure the oxygen-dependent luminescence lifetime of molecular probes. The relation between tissue pO2 and the probes' luminescence lifetime is governed by the Stern-Volmer equation. Unfortunately, virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity. For that reason, we studied the oxygen sensitivity and phototoxicity of dichlorotris(1, 10-phenanthroline)-ruthenium(II) hydrate [Ru(Phen)] using a dedicated optical fiber-based, time-resolved spectrometer in the chicken embryo chorioallantoic membrane. We demonstrated that, after intravenous injection, Ru(Phen)'s luminescence lifetime presents an easily detectable pO2 dependence at a low drug dose (1 mg∕kg) and low fluence (120 mJ∕cm2 at 470 nm). The phototoxic threshold was found to be at 10 J∕cm2 with the same wavelength and drug dose, i.e., about two orders of magnitude larger than the fluence necessary to perform a pO2 measurement. Finally, an illustrative application of this pO2 measurement approach in a hypoxic tumor environment is presented.

Psychometric properties of the French version of the Zuckerman-Kuhlman Personality Questionnaire

This instrumental study was designed to investigate the psychometric properties of the French version and the cross-language replicability of the Zuckerman-Kuhlman Personality Questionnaire (ZKPQ) at the factor- and at the facet-level. The ZKPQ is an instrument aimed at assessing the five basic factors of Zuckerman's Alternative Five-Factor Model (AFFM). Subjects were 843 French-speaking Swiss, mainly students. At the factor-level, the reliability ranged from .73 to .87 and at the facet level, the reliability ranged from .57 to .77. Differences between genders are congruent with those found in the American sample. Women scored higher on N-Anx, and lower on ImpSS, and Act. A series of exploratory factor analyses supported the overall five-factor structure and the structure at the facet-level. The correlations among the scales support that the five basic factors of the AFFM are orthogonal. Targeted factor analyses and congruence coefficients show high cross-language replicability at the factor- and at the facet-level. The adequacy of the model at the factor- and facet-level was tested using confirmatory factor analyses. The results show that the French version of the ZKPQ is a reliable and valid instrument and has a high cross-language replicability.

Determination of chemical composition and its relationship with optical properties of Ti-N and Ti-V-N sputtered thin films

In the investigation of thin films of transition metal nitrides, an essential role is played by the accurate determination of their chemical composition. Actually the chemical composition depends on the deposition parameters and influences the optical properties. These relations are illustrated in thin films of TiNx and (Ti1-yVy)N-x deposited by reactive magnetron sputtering from composite targets of the elements. By variation of the nitrogen partial pressure and the target composition, different samples have been obtained. The chemical composition has been measured by electron probe microanalysis at low irradiation voltages. The optical properties are evaluated by ex-situ ellipsometry. Using the screened Drude model, they are correlated with the differences in composition. Adding vanadium or nitrogen in Ti-N is shown to have the same effect on the optical properties.