Computational methods for the analysis of protein structure and function

The vast majority of known proteins have not yet been experimentally characterized and little is known about their function. The design and implementation of computational tools can provide insight into the function of proteins based on their sequence, their structure, their evolutionary history and their association with other proteins. Knowledge of the three-dimensional (3D) structure of a protein can lead to a deep understanding of its mode of action and interaction, but currently the structures of <1% of sequences have been experimentally solved. For this reason, it became urgent to develop new methods that are able to computationally extract relevant information from protein sequence and structure. The starting point of my work has been the study of the properties of contacts between protein residues, since they constrain protein folding and characterize different protein structures. Prediction of residue contacts in proteins is an interesting problem whose solution may be useful in protein folding recognition and de novo design. The prediction of these contacts requires the study of the protein inter-residue distances related to the specific type of amino acid pair that are encoded in the so-called contact map. An interesting new way of analyzing those structures came out when network studies were introduced, with pivotal papers demonstrating that protein contact networks also exhibit small-world behavior. In order to highlight constraints for the prediction of protein contact maps and for applications in the field of protein structure prediction and/or reconstruction from experimentally determined contact maps, I studied to which extent the characteristic path length and clustering coefficient of the protein contacts network are values that reveal characteristic features of protein contact maps. Provided that residue contacts are known for a protein sequence, the major features of its 3D structure could be deduced by combining this knowledge with correctly predicted motifs of secondary structure. In the second part of my work I focused on a particular protein structural motif, the coiled-coil, known to mediate a variety of fundamental biological interactions. Coiled-coils are found in a variety of structural forms and in a wide range of proteins including, for example, small units such as leucine zippers that drive the dimerization of many transcription factors or more complex structures such as the family of viral proteins responsible for virus-host membrane fusion. The coiled-coil structural motif is estimated to account for 5-10% of the protein sequences in the various genomes. Given their biological importance, in my work I introduced a Hidden Markov Model (HMM) that exploits the evolutionary information derived from multiple sequence alignments, to predict coiled-coil regions and to discriminate coiled-coil sequences. The results indicate that the new HMM outperforms all the existing programs and can be adopted for the coiled-coil prediction and for large-scale genome annotation. Genome annotation is a key issue in modern computational biology, being the starting point towards the understanding of the complex processes involved in biological networks. The rapid growth in the number of protein sequences and structures available poses new fundamental problems that still deserve an interpretation. Nevertheless, these data are at the basis of the design of new strategies for tackling problems such as the prediction of protein structure and function. Experimental determination of the functions of all these proteins would be a hugely time-consuming and costly task and, in most instances, has not been carried out. As an example, currently, approximately only 20% of annotated proteins in the Homo sapiens genome have been experimentally characterized. A commonly adopted procedure for annotating protein sequences relies on the "inheritance through homology" based on the notion that similar sequences share similar functions and structures. This procedure consists in the assignment of sequences to a specific group of functionally related sequences which had been grouped through clustering techniques. The clustering procedure is based on suitable similarity rules, since predicting protein structure and function from sequence largely depends on the value of sequence identity. However, additional levels of complexity are due to multi-domain proteins, to proteins that share common domains but that do not necessarily share the same function, to the finding that different combinations of shared domains can lead to different biological roles. In the last part of this study I developed and validate a system that contributes to sequence annotation by taking advantage of a validated transfer through inheritance procedure of the molecular functions and of the structural templates. After a cross-genome comparison with the BLAST program, clusters were built on the basis of two stringent constraints on sequence identity and coverage of the alignment. The adopted measure explicity answers to the problem of multi-domain proteins annotation and allows a fine grain division of the whole set of proteomes used, that ensures cluster homogeneity in terms of sequence length. A high level of coverage of structure templates on the length of protein sequences within clusters ensures that multi-domain proteins when present can be templates for sequences of similar length. This annotation procedure includes the possibility of reliably transferring statistically validated functions and structures to sequences considering information available in the present data bases of molecular functions and structures.

Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ε-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 µM for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ΔpH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 µM, and for ADP, increasing from 0 up to a few µM. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few µM for ADP and at 400 µM for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ΔpH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ε-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ε mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ε-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ε-overexpressing E.coli strains, which carried either the WT ε subunit or the ε88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ε appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Numerical models of trench migration for lateral heterogeneous subducting plates

The aim of this Thesis is to investigate the effect of heterogeneities within the subducting plate on the dynamics of subduction. In particular, I study the motion of the trench for oceanic and continental subduction, first, separately, and, then, together in the same system to understand how they interact. The understanding of these features is fundamental to reconstruct the evolution of complex subduction zones, such as the Central Mediterranean. For this purpose, I developed 2D and 3D numerical models of oceanic and continental subduction where the rheological, geometrical and compositional properties of the plates are varied. In these models, the trench and the overriding plate move self-consistently as a function of the dynamics of the system. The effect of continental subduction on trench migration is largely investigated. Results from a parametric study showed that despite different rheological properties of the plates, all models with a uniform continental crust share the same kinematic behaviour: the trench starts to advance once the continent arrives at the subduction zone. Hence, the advancing mode in continental collision scenarios is at least partly driven by an intrinsic feature of the system. Moreover, the presence of a weak lower crust within the continental plate can lead to the occurrence of delamination. Indeed, by changing the viscosity of the lower crust, both delamination and slab detachment can occur. Delamination is favoured by a low viscosity value of the lower crust, because this makes the mechanical decoupling easier between crust and lithospheric mantle. These features are observed both in 2D and 3D models, but the numerical results of the 3D models also showed that the rheology of the continental crust has a very strong effect on the dynamics of the whole system, since it influences not only the continental part of plate but also the oceanic sides.

Residual function, spontaneous reorganisation and treatment plasticity in homonymous visual field defects

This thesis will focus on the residual function and visual and attentional deficits in human patients, which accompany damage to the visual cortex or its thalamic afferents, and plastic changes, which follow it. In particular, I will focus on homonymous visual field defects, which comprise a broad set of central disorders of vision. I will present experimental evidence that when the primary visual pathway is completely damaged, the only signal that can be implicitly processed via subcortical visual networks is fear. I will also present data showing that in a patient with relative deafferentation of visual cortex, changes in the spatial tuning and response gain of the contralesional and ipsilesional cortex are observed, which are accompanied by changes in functional connectivity with regions belonging to the dorsal attentional network and the default mode network. I will also discuss how cortical plasticity might be harnessed to improve recovery through novel treatments. Moreover, I will show how treatment interventions aimed at recruiting spared subcortical pathway supporting multisensory orienting can drive network level change.

Methods for objective and interpretative evaluation of function and motor control: gait in perturbed condition and in the aquatic therapy

Gait analysis allows to characterize motor function, highlighting deviations from normal motor behavior related to an underlying pathology. The widespread use of wearable inertial sensors has opened the way to the evaluation of ecological gait, and a variety of methodological approaches and algorithms have been proposed for the characterization of gait from inertial measures (e.g. for temporal parameters, motor stability and variability, specific pathological alterations). However, no comparative analysis of their performance (i.e. accuracy, repeatability) was available yet, in particular, analysing how this performance is affected by extrinsic (i.e. sensor location, computational approach, analysed variable, testing environmental constraints) and intrinsic (i.e. functional alterations resulting from pathology) factors. The aim of the present project was to comparatively analyze the influence of intrinsic and extrinsic factors on the performance of the numerous algorithms proposed in the literature for the quantification of specific characteristics (i.e. timing, variability/stability) and alterations (i.e. freezing) of gait. Considering extrinsic factors, the influence of sensor location, analyzed variable, and computational approach on the performance of a selection of gait segmentation algorithms from a literature review was analysed in different environmental conditions (e.g. solid ground, sand, in water). Moreover, the influence of altered environmental conditions (i.e. in water) was analyzed as referred to the minimum number of stride necessary to obtain reliable estimates of gait variability and stability metrics, integrating what already available in the literature for over ground gait in healthy subjects. Considering intrinsic factors, the influence of specific pathological conditions (i.e. Parkinson’s Disease) was analyzed as affecting the performance of segmentation algorithms, with and without freezing. Finally, the analysis of the performance of algorithms for the detection of gait freezing showed how results depend on the domain of implementation and IMU position.

Low codimensional intrinsic regular submanifolds in the Heisenberg group H^n

The thesis mainly concerns the study of intrinsically regular submanifolds of low codimension in the Heisenberg group H^n, called H-regular surfaces of low codimension, from the point of view of geometric measure theory. We consider an H-regular surface of H^n of codimension k, with k between 1 and n, parametrized by a uniformly intrinsically differentiable map acting between two homogeneous complementary subgroups of H^n, with target subgroup horizontal of dimension k. In particular the considered submanifold is the intrinsic graph of the parametrization. We extend various results of Ambrosio, Serra Cassano and Vittone, available for the case when k = 1. We prove that the uniform intrinsic differentiability of the parametrizing map is equivalent to the existence and continuity of its intrinsic differential, to the local existence of a suitable approximating family of Euclidean regular maps, and, when the domain and the codomain of the map are orthogonal, to the existence and continuity of suitably defined intrinsic partial derivatives of the function. Successively, we present a series of area formulas, proved in collaboration with V. Magnani. They allow to compute the (2n+2−k)-dimensional spherical Hausdorff measure and the (2n+2−k)-dimensional centered Hausdorff measure of the parametrized H-regular surface, with respect to any homogeneous distance fixed on H^n. Furthermore, we focus on (G,M)-regular sets of G, where G and M are two arbitrary Carnot groups. Suitable implicit function theorems ensure the local existence of an intrinsic parametrization of such a set, at any of its points. We prove that it is uniformly intrinsically differentiable. Finally, we prove a coarea-type inequality for a continuously Pansu differentiable function acting between two Carnot groups endowed with homogeneous distances. We assume that the level sets of the function are uniformly lower Ahlfors regular and that the Pansu differential is everywhere surjective.