Each one teaches one: characterizing active forms of proteins by single molecule force spectroscopy

This Ph.D. candidate thesis collects the research work I conducted under the supervision of Prof.Bruno Samor´ı in 2005,2006 and 2007. Some parts of this work included in the Part III have been begun by myself during my undergraduate thesis in the same laboratory and then completed during the initial part of my Ph.D. thesis: the whole results have been included for the sake of understanding and completeness. During my graduate studies I worked on two very different protein systems. The theorical trait d’union between these studies, at the biological level, is the acknowledgement that protein biophysical and structural studies must, in many cases, take into account the dynamical states of protein conformational equilibria and of local physico-chemical conditions where the system studied actually performs its function. This is introducted in the introductory part in Chapter 2. Two different examples of this are presented: the structural significance deriving from the action of mechanical forces in vivo (Chapter 3) and the complexity of conformational equilibria in intrinsically unstructured proteins and amyloid formation (Chapter 4). My experimental work investigated both these examples by using in both cases the single molecule force spectroscopy technique (described in Chapter 5 and Chapter 6). The work conducted on angiostatin focused on the characterization of the relationships between the mechanochemical properties and the mechanism of action of the angiostatin protein, and most importantly their intertwining with the further layer of complexity due to disulfide redox equilibria (Part III). These studies were accompanied concurrently by the elaboration of a theorical model for a novel signalling pathway that may be relevant in the extracellular space, detailed in Chapter 7.2. The work conducted on -synuclein (Part IV) instead brought a whole new twist to the single molecule force spectroscopy methodology, applying it as a structural technique to elucidate the conformational equilibria present in intrinsically unstructured proteins. These equilibria are of utmost interest from a biophysical point of view, but most importantly because of their direct relationship with amyloid aggregation and, consequently, the aetiology of relevant pathologies like Parkinson’s disease. The work characterized, for the first time, conformational equilibria in an intrinsically unstructured protein at the single molecule level and, again for the first time, identified a monomeric folded conformation that is correlated with conditions leading to -synuclein and, ultimately, Parkinson’s disease. Also, during the research work, I found myself in the need of a generalpurpose data analysis application for single molecule force spectroscopy data analysis that could solve some common logistic and data analysis problems that are common in this technique. I developed an application that addresses some of these problems, herein presented (Part V), and that aims to be publicly released soon.

Rare and complicated forms of infantile hemangiomas: new therapeutic outlooks

Infantile hemangiomas (IHs) are the most common benign neoplastic pathology of childhood; their natural history generally involves three phases: after the onset, which usually occurs in the first weeks of life, there is the proliferation phase where the IH reaches its maximum development and it is followed by the spontaneous involution which leads to the IH regression. The duration and the extent of these phases may vary widely even though in most of the cases the involution process begins around twelve months of life and the regression, complete or partial, is completed around seventh-ninth year of life. The majority of the IHs does not require any treatment. However, 10%-20% is likely to develop serious complications, functional impairments or aesthetic alterations and entail a timely treatment. Although there is no treatment protocol currently shared, therapies usually used in cases with a complication risk consist in: systemic or intralesional steroids as a first choice; interferon α, vincristine and/or bleomicin as second or third choice and/or surgical treatment. Propranolol, a non-selective beta-blocker, has been used for cardiovascular diseases even in childhood for decades. Since 2008 it has been widely used in the IHs treatment, although it is still "off-label". In literature there are hundreds of cases and some clinical studies that show the effectiveness and safety of this drug for this indication. Thanks to a multidisciplinary team (Dermatologists, Cardiologists, Paediatricians, and Radiologists) of S. Orsola-Malpighi Hospital, a clinical study, which has been previously approved by the ethics committee, is carried out in order to evaluate the efficacy and safety of systemic propranolol in the treatment of IHs in paediatric age. At the end of 2012, 78 patients underwent this treatment: the results we have obtained so far show a good efficacy and safety profile in agreement with the data provided by the literature.

Optimization of molecular and crystalline forms of drugs, agrochemicals, pesticides in relation to activity, bioavailability, patentability and to the fabrication of polymorphs, solvates, co-crystals with green chemistry methods

This doctorate was funded by the Regione Emilia Romagna, within a Spinner PhD project coordinated by the University of Parma, and involving the universities of Bologna, Ferrara and Modena. The aim of the project was: - Production of polymorphs, solvates, hydrates and co-crystals of active pharmaceutical ingredients (APIs) and agrochemicals with green chemistry methods; - Optimization of molecular and crystalline forms of APIs and pesticides in relation to activity, bioavailability and patentability. In the last decades, a growing interest in the solid-state properties of drugs in addition to their solution chemistry has blossomed. The achievement of the desired and/or the more stable polymorph during the production process can be a challenge for the industry. The study of crystalline forms could be a valuable step to produce new polymorphs and/or co-crystals with better physical-chemical properties such as solubility, permeability, thermal stability, habit, bulk density, compressibility, friability, hygroscopicity and dissolution rate in order to have potential industrial applications. Selected APIs (active pharmaceutical ingredients) were studied and their relationship between crystal structure and properties investigated, both in the solid state and in solution. Polymorph screening and synthesis of solvates and molecular/ionic co-crystals were performed according to green chemistry principles. Part of this project was developed in collaboration with chemical/pharmaceutical companies such as BASF (Germany) and UCB (Belgium). We focused on on the optimization of conditions and parameters of crystallization processes (additives, concentration, temperature), and on the synthesis and characterization of ionic co-crystals. Moreover, during a four-months research period in the laboratories of Professor Nair Rodriguez-Hormedo (University of Michigan), the stability in aqueous solution at the equilibrium of ionic co-crystals (ICCs) of the API piracetam was investigated, to understand the relationship between their solid-state and solution properties, in view of future design of new crystalline drugs with predefined solid and solution properties.

Fundamental Dimensions of Social Judgment: Sociability and Morality as Distinct Characteristics of Social Warmth

The present dissertation focuses on the two basic dimensions of social judgment, i.e., warmth and competence. Previous research has shown that warmth and competence emerge as fundamental dimensions both at the interpersonal level and at the group level. Moreover, warmth judgments appear to be primary, reflecting the importance of first assessing others’ intentions before determining the other’s ability to carry out those intentions. Finally, it has been shown that warmth and competence judgments are predicted by perceived economic competition and status, respectively (for a review, see Cuddy, Fiske, & Glick, 2008). Building on this evidence, the present work intends to further explore the role of warmth and competence in social judgment, adopting a finer-grained level of analysis. Specifically, we consider warmth to be a dimension of evaluation that encompasses two distinct characteristics (i.e., sociability and morality) rather than as an undifferentiated dimension (see Leach, Ellemers, & Barreto, 2007). In a similar vein, both economic competition and symbolic competition are taken into account (see Stephan, Ybarra, & Morrison, 2009). In order to highlight the relevance of our empirical research, the first chapter reviews the literature in social psychology that has studied the warmth and competence dimensions. In the second chapter, across two studies, we examine the role of realistic and symbolic threats (akin economic and symbolic competition, respectively) in predicting the perception of sociability and morality of social groups. In study 1, we measure perceived realistic threat, symbolic threat, sociability, and morality with respect to 8 social groups. In study 2, we manipulate the level and type of threat of a fictitious group and measure perceived sociability and morality. The findings show that realistic threat and symbolic threat are differentially related to the sociability and morality components of warmth. Specifically, whereas realistic threat seems to be a stronger predictor of sociability than symbolic threat, symbolic threat emerges as better predictor of morality than realistic threat. Thus, extending prior research, we show that the types of threat are linked to different warmth stereotypes. In the third and the fourth chapter, we examine whether the sociability and morality components of warmth play distinct roles at different stages of group impression formation. More specifically, the third chapter focuses on the information-gathering process. Two studies experimentally investigate which traits are mostly selected when forming impressions about either ingroup or outgroup members. The results clearly show that perceivers are more interested in obtaining information about morality than about sociability when asked to form a global impression about others. The fourth chapter considers more properly the formulation of an evaluative impression. Thus, in the first study participants rate real groups on sociability, morality, and competence. In the second study, participants read an immigration scenario depicting an unfamiliar social group in terms of high (vs. low) morality, sociability, and competence. In both studies, participants are also asked to report their global impression of the group. The results show that global evaluations are better predicted by morality than by sociability and competence trait ascriptions. Taken together the third and the fourth chapters show that the dominance of warmth suggested by previous studies on impression formation might be better explained in terms of a greater effect of one of the two subcomponents (i.e., morality) over the other (i.e., sociability). In the general discussion, we discuss the relevance of our findings for intergroup relation and group perception, as well as for impression formation.