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.

Exercise associated factors affecting cardiovascular health and function: from myocyte signaling to genetic variations

The exact mechanisms of the exercise induced adaptations is not lucid, but recent studies have delineated two means of signaling by which the adaptations occur (1) substrate availability signaling (metabolic stress) (2) hormone-receptor signaling. We have decided to specifically investigate two metabolic signaling enzymes [AMP-activated kinase (AMPK) and Sirtuin 1(SIRT1)] and two hormones [Adiponectin and Adrenergic stimulation].Tis based on four papers with the following conclusions: (1)Increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, mediated by AMPK. (2)The lack of optimal nutritional conditions (energetic substrates) due to a prolonged activation of AMPK can contrast the establishment of hypertrophy, possibly also by means of the negative modulation of ODC activity. (3) Our findings offer a possibile hypothesis as to the fact the the G allele on site 45 could lead to the increasd risk of Type II diabetes through a decrease in lean body mass. (4) Our results suggest that there is an ADIPOQ gene effect in relation to bone parameters. Statistical analysis show that the presence of the T allele in position 45 favors an increase in lumbar spine bone mineral content (BMC) when compared to subjects with a G allele substitution, which can be do the the increase in lean body mass in this genotype group.

Pathogenic role of IL-33-mediated eosinophil infiltration and function in experimental inflammatory bowel disease

IL-33/ST2 axis is known to promote Th2 immune responses and has been linked to several autoimmune and inflammatory disorders, including inflammatory bowel disease (IBD), and recent evidences show that it can regulate eosinophils (EOS) infiltration and function. Based also on the well documented relationship between EOS and IBD, we assessed the role of IL-33-mediated eosinophilia and ileal inflammation in SAMP1/YitFc (SAMP) murine model of Th1/Th2 chronic enteritis, and we found that IL-33 is related to inflammation progression and EOS infiltration as well as IL-5 and eotaxins increase. Administering IL-33 to SAMP and AKR mice augmented eosinophilia, eotaxins mRNA expression and Th2 molecules production, whereas blockade of ST2 and/or typical EOS molecules, such as IL-5 and CCR3, resulted in a marked decrease of inflammation, EOS infiltration, IL-5 and eotaxins mRNA expression and Th2 cytokines production. Human data supported mice’s showing an increased colocalization of IL-33 and EOS in the colon mucosa of UC patients, as well as an augmented IL-5 and eotaxins mRNA expression, when compared to non-UC. Lastly we analyzed SAMP raised in germ free (GF) condition to see the microbiota effect on IL-33 expression and Th2 responses leading to chronic intestinal inflammation. We found a remarkable decrease in ileal IL-33 and Th2 cytokines mRNA expression as well as EOS infiltration in GF versus normal SAMP with comparable inflammatory scores. Moreover, EOS depletion in normal SAMP didn’t affect IL-33 mRNA expression. These data demonstrate a pathogenic role of IL-33-mediated eosinophilia in chronic intestinal inflammation, and that blockade of IL-33 and/or downstream EOS activation may represent a novel therapeutic modality to treat patients with IBD. Also they highlight the gut microbiota role in IL-33 production, and the following EOS infiltration in the intestinal mucosa, confirming that the microbiota is essential in mounting potent Th2 response leading to chronic ileitis in SAMP.

Erythropoietin reduces pathogenic humoral immunity by inhibiting T Follicular Helper cell differentiation and function

A large fraction of organ transplant recipients develop anti-donor antibodies (DSA), with accelerated graft loss and increased mortality. We tested the hypothesis that erythropoietin (EPO) reduces DSA formation by inhibiting T follicular helper (TFH) cells. We measured DSA levels, splenic TFH, TFR cells, germinal center (GC), and class switched B cells, in murine models of allogeneic sensitization, allogeneic transplantation and in parent-to-F1 models of graft versus host disease (GVHD). We quantified the same cell subsets and specific antibodies, upon EPO or vehicle treatment, in wild type mice and animals lacking EPO receptor selectively on T or B cells, immunized with T-independent or T-dependent stimuli. In vitro, we tested the EPO effect on TFH induction. We isolated TFH and TFR cells to perform in vitro assay and clarify their role. EPO reduced DSA levels, GC, class switched B cells, and increased the TFR/TFH ratio in the heart transplanted mice and in two GVHD models. EPO did also reduce TFH and GC B cells in SRBC-immunized mice, while had no effect in TNP-AECM-FICOLL-immunized animals, indicating that EPO inhibits GC B cells by targeting TFH cells. EPO effects were absent in T cells EPOR conditional KO mice, confirming that EPO affects TFH in vivo through EPOR. In vitro, EPO affected TFH induction through an EPO-EPOR-STAT5-dependent pathway. Suppression assay demonstrated that the reduction of IgG antibodies was dependent on TFH cells, sustaining the central role of the subset in this EPO-mediated mechanism. In conclusion, EPO prevents DSA formation in mice through a direct suppression of TFH. Development of DSA is associated with high risk of graft rejection, giving our data a strong rationale for studies testing the hypothesis that EPO administration prevents their formation in organ transplant recipients. Our findings provide a foundation for testing EPO as a treatment of antibody mediated disease processes.

Role of CARM1 (PRMT4) in high grade serous ovarian cancer metabolism and function of PRMT5 in ARID1A- deficient endometrial cancer invasion

The arginine methyltransferase CARM1 (PRMT4) is amplified and overexpressed in ~20% of high-grade serous ovarian cancer (HGSOC) and correlates with a poor survival. Therapeutic approaches based on CARM1 expression remain to be an unmet need. Here we show that fatty acid metabolism represents a metabolic vulnerability for HGSOC in a CARM1 expression status dependent manner. CARM1 promotes the de novo synthesis of fatty acids and monounsaturated fatty acids (MUFAs). The disruption of MUFAs synthesis by inhibition of SCD1 results in excessive accumulation of cytotoxic saturated fatty acids and it is synthetic lethal with CARM1 expression. Collectively, our data show that the pharmacological inhibition of MUFAs synthesis via SCD1 inhibition represents a therapeutic strategy for CARM1-high HGSOC. Another arginine methyltransferase, PRMT5, has been identified by our CRISPR screening analysis as a promising candidate for invasive ARID1A-deficient endometrial cancer. Endometrial Cancer frequently harbor somatic inactivating mutation of ARID1A that can promote an invasive phenotype. Our in vitro approach validated the CRISPR screening showing that both PRTM5 knock down and its pharmaceutical inhibition specifically hamper the invasion of ARID1A inactivated cells. Mechanistically, PRMT5 directly regulates the epithelia to mesenchymal transition pathway genes interacting with the SWI/SNF complexes. Moreover, in vivo experiments showed that PRMT5 inhibition contrasted the myometrium invasion highlighting PRMT5 inhibition as promising therapeutic strategy for ARID1A- inactivated aggressive endometrial cancer.

Statistical properties of Radio Halos and the re-acceleration model

Galaxy clusters occupy a special position in the cosmic hierarchy as they are the largest bound structures in the Universe. There is now general agreement on a hierarchical picture for the formation of cosmic structures, in which galaxy clusters are supposed to form by accretion of matter and merging between smaller units. During merger events, shocks are driven by the gravity of the dark matter in the diffuse barionic component, which is heated up to the observed temperature. Radio and hard-X ray observations have discovered non-thermal components mixed with the thermal Intra Cluster Medium (ICM) and this is of great importance as it calls for a “revision” of the physics of the ICM. The bulk of present information comes from the radio observations which discovered an increasing number of Mpcsized emissions from the ICM, Radio Halos (at the cluster center) and Radio Relics (at the cluster periphery). These sources are due to synchrotron emission from ultra relativistic electrons diffusing through µG turbulent magnetic fields. Radio Halos are the most spectacular evidence of non-thermal components in the ICM and understanding the origin and evolution of these sources represents one of the most challenging goal of the theory of the ICM. Cluster mergers are the most energetic events in the Universe and a fraction of the energy dissipated during these mergers could be channelled into the amplification of the magnetic fields and into the acceleration of high energy particles via shocks and turbulence driven by these mergers. Present observations of Radio Halos (and possibly of hard X-rays) can be best interpreted in terms of the reacceleration scenario in which MHD turbulence injected during these cluster mergers re-accelerates high energy particles in the ICM. The physics involved in this scenario is very complex and model details are difficult to test, however this model clearly predicts some simple properties of Radio Halos (and resulting IC emission in the hard X-ray band) which are almost independent of the details of the adopted physics. In particular in the re-acceleration scenario MHD turbulence is injected and dissipated during cluster mergers and thus Radio Halos (and also the resulting hard X-ray IC emission) should be transient phenomena (with a typical lifetime <» 1 Gyr) associated with dynamically disturbed clusters. The physics of the re-acceleration scenario should produce an unavoidable cut-off in the spectrum of the re-accelerated electrons, which is due to the balance between turbulent acceleration and radiative losses. The energy at which this cut-off occurs, and thus the maximum frequency at which synchrotron radiation is produced, depends essentially on the efficiency of the acceleration mechanism so that observations at high frequencies are expected to catch only the most efficient phenomena while, in principle, low frequency radio surveys may found these phenomena much common in the Universe. These basic properties should leave an important imprint in the statistical properties of Radio Halos (and of non-thermal phenomena in general) which, however, have not been addressed yet by present modellings. The main focus of this PhD thesis is to calculate, for the first time, the expected statistics of Radio Halos in the context of the re-acceleration scenario. In particular, we shall address the following main questions: • Is it possible to model “self-consistently” the evolution of these sources together with that of the parent clusters? • How the occurrence of Radio Halos is expected to change with cluster mass and to evolve with redshift? How the efficiency to catch Radio Halos in galaxy clusters changes with the observing radio frequency? • How many Radio Halos are expected to form in the Universe? At which redshift is expected the bulk of these sources? • Is it possible to reproduce in the re-acceleration scenario the observed occurrence and number of Radio Halos in the Universe and the observed correlations between thermal and non-thermal properties of galaxy clusters? • Is it possible to constrain the magnetic field intensity and profile in galaxy clusters and the energetic of turbulence in the ICM from the comparison between model expectations and observations? Several astrophysical ingredients are necessary to model the evolution and statistical properties of Radio Halos in the context of re-acceleration model and to address the points given above. For these reason we deserve some space in this PhD thesis to review the important aspects of the physics of the ICM which are of interest to catch our goals. In Chapt. 1 we discuss the physics of galaxy clusters, and in particular, the clusters formation process; in Chapt. 2 we review the main observational properties of non-thermal components in the ICM; and in Chapt. 3 we focus on the physics of magnetic field and of particle acceleration in galaxy clusters. As a relevant application, the theory of Alfv´enic particle acceleration is applied in Chapt. 4 where we report the most important results from calculations we have done in the framework of the re-acceleration scenario. In this Chapter we show that a fraction of the energy of fluid turbulence driven in the ICM by the cluster mergers can be channelled into the injection of Alfv´en waves at small scales and that these waves can efficiently re-accelerate particles and trigger Radio Halos and hard X-ray emission. The main part of this PhD work, the calculation of the statistical properties of Radio Halos and non-thermal phenomena as expected in the context of the re-acceleration model and their comparison with observations, is presented in Chapts.5, 6, 7 and 8. In Chapt.5 we present a first approach to semi-analytical calculations of statistical properties of giant Radio Halos. The main goal of this Chapter is to model cluster formation, the injection of turbulence in the ICM and the resulting particle acceleration process. We adopt the semi–analytic extended Press & Schechter (PS) theory to follow the formation of a large synthetic population of galaxy clusters and assume that during a merger a fraction of the PdV work done by the infalling subclusters in passing through the most massive one is injected in the form of magnetosonic waves. Then the processes of stochastic acceleration of the relativistic electrons by these waves and the properties of the ensuing synchrotron (Radio Halos) and inverse Compton (IC, hard X-ray) emission of merging clusters are computed under the assumption of a constant rms average magnetic field strength in emitting volume. The main finding of these calculations is that giant Radio Halos are naturally expected only in the more massive clusters, and that the expected fraction of clusters with Radio Halos is consistent with the observed one. In Chapt. 6 we extend the previous calculations by including a scaling of the magnetic field strength with cluster mass. The inclusion of this scaling allows us to derive the expected correlations between the synchrotron radio power of Radio Halos and the X-ray properties (T, LX) and mass of the hosting clusters. For the first time, we show that these correlations, calculated in the context of the re-acceleration model, are consistent with the observed ones for typical µG strengths of the average B intensity in massive clusters. The calculations presented in this Chapter allow us to derive the evolution of the probability to form Radio Halos as a function of the cluster mass and redshift. The most relevant finding presented in this Chapter is that the luminosity functions of giant Radio Halos at 1.4 GHz are expected to peak around a radio power » 1024 W/Hz and to flatten (or cut-off) at lower radio powers because of the decrease of the electron re-acceleration efficiency in smaller galaxy clusters. In Chapt. 6 we also derive the expected number counts of Radio Halos and compare them with available observations: we claim that » 100 Radio Halos in the Universe can be observed at 1.4 GHz with deep surveys, while more than 1000 Radio Halos are expected to be discovered in the next future by LOFAR at 150 MHz. This is the first (and so far unique) model expectation for the number counts of Radio Halos at lower frequency and allows to design future radio surveys. Based on the results of Chapt. 6, in Chapt.7 we present a work in progress on a “revision” of the occurrence of Radio Halos. We combine past results from the NVSS radio survey (z » 0.05 − 0.2) with our ongoing GMRT Radio Halos Pointed Observations of 50 X-ray luminous galaxy clusters (at z » 0.2−0.4) and discuss the possibility to test our model expectations with the number counts of Radio Halos at z » 0.05 − 0.4. The most relevant limitation in the calculations presented in Chapt. 5 and 6 is the assumption of an “averaged” size of Radio Halos independently of their radio luminosity and of the mass of the parent clusters. This assumption cannot be released in the context of the PS formalism used to describe the formation process of clusters, while a more detailed analysis of the physics of cluster mergers and of the injection process of turbulence in the ICM would require an approach based on numerical (possible MHD) simulations of a very large volume of the Universe which is however well beyond the aim of this PhD thesis. On the other hand, in Chapt.8 we report our discovery of novel correlations between the size (RH) of Radio Halos and their radio power and between RH and the cluster mass within the Radio Halo region, MH. In particular this last “geometrical” MH − RH correlation allows us to “observationally” overcome the limitation of the “average” size of Radio Halos. Thus in this Chapter, by making use of this “geometrical” correlation and of a simplified form of the re-acceleration model based on the results of Chapt. 5 and 6 we are able to discuss expected correlations between the synchrotron power and the thermal cluster quantities relative to the radio emitting region. This is a new powerful tool of investigation and we show that all the observed correlations (PR − RH, PR − MH, PR − T, PR − LX, . . . ) now become well understood in the context of the re-acceleration model. In addition, we find that observationally the size of Radio Halos scales non-linearly with the virial radius of the parent cluster, and this immediately means that the fraction of the cluster volume which is radio emitting increases with cluster mass and thus that the non-thermal component in clusters is not self-similar.

“Nanoglial interfaces: nanostructured materials, interfaces and devices to unveil the role of astrocytes in brain function and dysfunction”

The role of non-neuronal brain cells, called astrocytes, is emerging as crucial in brain function and dysfunction, encompassing the neurocentric concept that was envisioning glia as passive components. Ion and water channels and calcium signalling, expressed in functional micro and nano domains, underpin astrocytes’ homeostatic function, synaptic transmission, neurovascular coupling acting either locally and globally. In this respect, a major issue arises on the mechanism through which astrocytes can control processes across scales. Finally, astrocytes can sense and react to extracellular stimuli such as chemical, physical, mechanical, electrical, photonic ones at the nanoscale. Given their emerging importance and their sensing properties, my PhD research program had the general goal to validate nanomaterials, interfaces and devices approaches that were developed ad-hoc to study astrocytes. The results achieved are reported in the form of collection of papers. Specifically, we demonstrated that i) electrospun nanofibers made of polycaprolactone and polyaniline conductive composites can shape primary astrocytes’ morphology, without affecting their function ii) gold coated silicon nanowires devices enable extracellular recording of unprecedented slow wave in primary differentiated astrocytes iii) colloidal hydrotalcites films allow to get insight in cell volume regulation process in differentiated astrocytes and to describe novel cytoskeletal actin dynamics iv) gold nanoclusters represent nanoprobe to trigger astrocytes structure and function v) nanopillars of photoexcitable organic polymer are potential tool to achieve nanoscale photostimulation of astrocytes. The results were achieved by a multidisciplinary team working with national and international collaborators that are listed and acknowledged in the text. Collectively, the results showed that astrocytes represent a novel opportunity and target for Nanoscience, and that Nanoglial interface might help to unveil clues on brain function or represent novel therapeutic approach to treat brain dysfunctions.