Disorders of arousal: a physiopathological window to explore the mechanisms regulating sleep arousal

Disorders of Arousal (DoA) belong to NREM parasomnias and are characterized by motor and emotional episodes arising from incomplete awakenings from NREM sleep. DoA episodes embody at the same time the double nature of the arousal process, that is preserving sleep as well as respond to sleep perturbations, thus being an ideal model to study sleep arousal. In the first part of this work, we performed a spectral whole scalp EEG analysis exploring the neurophysiologic correlates of the pre-motor onset of the episodes in a large sample of patients with DoA, disclosing the co-existence of both slow and fast EEG frequencies over overlapping areas before DoA episodes, suggesting an alteration of local sleep mechanisms. Episodes of different complexity were preceded by a similar EEG activation, implying that they possibly share a similar pathophysiology. In the second part of this work, we performed a spectral whole scalp EEG analysis comparing the pre-motor onset of the episodes and normal arousals from healthy sleepers, disclosing the persistence of slow frequencies as well as sigma band (expression of sleep spindles) in DoA episodes. Overall, these results might subtend a higher tendence to preserve sleep and a more defective mechanism toward developing a complete arousal in patients with DoA. In the last part of our work, we evaluated 15 patients with DoA with 15 controls in a functional MRI study during wakefulness in addition to a proton magnetic resonance spectroscopy (1H-MRS) focused on cingulate cortex. We disclosed subtle alterations on posterior cingulate cortex as well as an increased connectivity in sensory-motor network, possibly representing a trait-functional feature responsible for the dysfunctional arousal process in DoA patients

Hypertensive disorders of pregnancy and long-term cardiovascular health and outcomes

Introduction The maternal vasculature undergoes significant adaptations during pregnancy to meet the increased metabolic demands of the developing fetus. These adaptations include increased cardiac output and blood volume, as well as reduced systemic vascular resistance. In Hypertensive disorders of pregnancy (HDP) there is an impaired cardiovascular adaptation to pregnancy with effects extending beyond pregnancy. In the present study we aimed to characterize long-term cardiovascular status of women who suffered from HDP. Methods Fifty-eight women who attended at least one post-partum visit and a follow-up visit after at least 5 years from delivery were enrolled in the study. Exclusion criteria included multiple pregnancy, fetal genetic or congenital abnormalities, maternal history of organ transplantation, or chronic renal failure (eGFR≤45ml/min/1.73m2). In the follow-up visit participants underwent a complete cardiovascular assessment including echocardiography and multiparametric vascular function assessment. Results and Discussion Two major cardiovascular events, one stroke and one myocardial infarction, occurred both in women with index-pregnancy complicated by preeclampsia (PE). While not statistically significant, women with HDP-non-PE and PE displayed a trend towards an increased risk of developing composite cardiovascular outcome, and women with PE tended to experience it sooner. Nearly half of the women with a history of HDP, whether PE or HDP-non-PE, developed chronic hypertension. Some women also developed hyperuricemia, chronic kidney disease (CKD), and type 2 diabetes at follow- up, most of them had a previous history of PE. Structural and functional cardiac changes were observed in a few cases, especially among women with PE, and vascular dysfunction was more common in women with a history of HDP compared to those with normotensive pregnancies. Results of the present study adds on literature on long-term cardiovascular impact of HDP and further emphasize the importance of a timely follow-up of women who suffered from HDP and particularly PE.

Role of organic acids and phytonutrients as natural alternatives to antibiotics in supporting intestinal functionality

The gastrointestinal tract (GIT) represents the major portion of the body that interfaces with the external environment, with the double function of food processing and line of defense of the body. Numerous components support and regulate the barrier function of the GIT, such as tight junctions (TJs), cytokines, commensal and pathogenic microorganisms, and other systems of the organism, as the endocannabinoid system (ECS). The ECS can control several gastrointestinal functions, as well as the regulation of intestinal inflammation. Failure of the intestinal barrier function triggers an increase of the concentration of pro-inflammatory cytokines and leads to a reduction in intestinal functionality. This thesis aimed to explore the potential of natural compounds as a new alternative approach to antibiotics not only as antimicrobial, but also supporting intestinal maturation and integrity, and as immune-boosting agents. Different experiments were performed to evaluate the potential of nature-identical compounds (NICs), organic acids (OAs), and essential oils (EOs) to support and fight various stressful stimuli. In vitro, a well characterized blend of NICs and OAs were able to improve TJs and transepithelial electrical resistance (TEER) in an intestinal cell line, exerting an anti-inflammatory potential. EOs enhanced TEER and TJs mRNA levels, with a reduction of paracellular permeability, showing antioxidant and antimicrobial properties. In vivo, thymol modulates the gene expression of ECS and gut chemosensing in the GIT of piglets, where the precise localization of the cannabinoid receptors was immunohistochemically confirmed, suggesting an anti-inflammatory potential. In conclusion, natural alternative molecules represent an effective alternative to support or replace the classical pharmacological prophylaxis. These alternative molecules act not only as antimicrobial agents, but also exerted a crucial role in supporting the intestinal barrier function, preventing oxidative stress, and reducing inflammation. Moreover, thymol seems able to modulate the ECS, representing a novel frontier to support animal health and productivity.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Technical innovations for the diagnosis and the rehabilitation of motor and perceptive impairments of the child with Cerebral Palsy

The treatment of the Cerebral Palsy (CP) is considered as the “core problem” for the whole field of the pediatric rehabilitation. The reason why this pathology has such a primary role, can be ascribed to two main aspects. First of all CP is the form of disability most frequent in childhood (one new case per 500 birth alive, (1)), secondarily the functional recovery of the “spastic” child is, historically, the clinical field in which the majority of the therapeutic methods and techniques (physiotherapy, orthotic, pharmacologic, orthopedic-surgical, neurosurgical) were first applied and tested. The currently accepted definition of CP – Group of disorders of the development of movement and posture causing activity limitation (2) – is the result of a recent update by the World Health Organization to the language of the International Classification of Functioning Disability and Health, from the original proposal of Ingram – A persistent but not unchangeable disorder of posture and movement – dated 1955 (3). This definition considers CP as a permanent ailment, i.e. a “fixed” condition, that however can be modified both functionally and structurally by means of child spontaneous evolution and treatments carried out during childhood. The lesion that causes the palsy, happens in a structurally immature brain in the pre-, peri- or post-birth period (but only during the firsts months of life). The most frequent causes of CP are: prematurity, insufficient cerebral perfusion, arterial haemorrhage, venous infarction, hypoxia caused by various origin (for example from the ingestion of amniotic liquid), malnutrition, infection and maternal or fetal poisoning. In addition to these causes, traumas and malformations have to be included. The lesion, whether focused or spread over the nervous system, impairs the whole functioning of the Central Nervous System (CNS). As a consequence, they affect the construction of the adaptive functions (4), first of all posture control, locomotion and manipulation. The palsy itself does not vary over time, however it assumes an unavoidable “evolutionary” feature when during growth the child is requested to meet new and different needs through the construction of new and different functions. It is essential to consider that clinically CP is not only a direct expression of structural impairment, that is of etiology, pathogenesis and lesion timing, but it is mainly the manifestation of the path followed by the CNS to “re”-construct the adaptive functions “despite” the presence of the damage. “Palsy” is “the form of the function that is implemented by an individual whose CNS has been damaged in order to satisfy the demands coming from the environment” (4). Therefore it is only possible to establish general relations between lesion site, nature and size, and palsy and recovery processes. It is quite common to observe that children with very similar neuroimaging can have very different clinical manifestations of CP and, on the other hand, children with very similar motor behaviors can have completely different lesion histories. A very clear example of this is represented by hemiplegic forms, which show bilateral hemispheric lesions in a high percentage of cases. The first section of this thesis is aimed at guiding the interpretation of CP. First of all the issue of the detection of the palsy is treated from historical viewpoint. Consequently, an extended analysis of the current definition of CP, as internationally accepted, is provided. The definition is then outlined in terms of a space dimension and then of a time dimension, hence it is highlighted where this definition is unacceptably lacking. The last part of the first section further stresses the importance of shifting from the traditional concept of CP as a palsy of development (defect analysis) towards the notion of development of palsy, i.e., as the product of the relationship that the individual however tries to dynamically build with the surrounding environment (resource semeiotics) starting and growing from a different availability of resources, needs, dreams, rights and duties (4). In the scientific and clinic community no common classification system of CP has so far been universally accepted. Besides, no standard operative method or technique have been acknowledged to effectively assess the different disabilities and impairments exhibited by children with CP. CP is still “an artificial concept, comprising several causes and clinical syndromes that have been grouped together for a convenience of management” (5). The lack of standard and common protocols able to effectively diagnose the palsy, and as a consequence to establish specific treatments and prognosis, is mainly because of the difficulty to elevate this field to a level based on scientific evidence. A solution aimed at overcoming the current incomplete treatment of CP children is represented by the clinical systematic adoption of objective tools able to measure motor defects and movement impairments. A widespread application of reliable instruments and techniques able to objectively evaluate both the form of the palsy (diagnosis) and the efficacy of the treatments provided (prognosis), constitutes a valuable method able to validate care protocols, establish the efficacy of classification systems and assess the validity of definitions. Since the ‘80s, instruments specifically oriented to the analysis of the human movement have been advantageously designed and applied in the context of CP with the aim of measuring motor deficits and, especially, gait deviations. The gait analysis (GA) technique has been increasingly used over the years to assess, analyze, classify, and support the process of clinical decisions making, allowing for a complete investigation of gait with an increased temporal and spatial resolution. GA has provided a basis for improving the outcome of surgical and nonsurgical treatments and for introducing a new modus operandi in the identification of defects and functional adaptations to the musculoskeletal disorders. Historically, the first laboratories set up for gait analysis developed their own protocol (set of procedures for data collection and for data reduction) independently, according to performances of the technologies available at that time. In particular, the stereophotogrammetric systems mainly based on optoelectronic technology, soon became a gold-standard for motion analysis. They have been successfully applied especially for scientific purposes. Nowadays the optoelectronic systems have significantly improved their performances in term of spatial and temporal resolution, however many laboratories continue to use the protocols designed on the technology available in the ‘70s and now out-of-date. Furthermore, these protocols are not coherent both for the biomechanical models and for the adopted collection procedures. In spite of these differences, GA data are shared, exchanged and interpreted irrespectively to the adopted protocol without a full awareness to what extent these protocols are compatible and comparable with each other. Following the extraordinary advances in computer science and electronics, new systems for GA no longer based on optoelectronic technology, are now becoming available. They are the Inertial and Magnetic Measurement Systems (IMMSs), based on miniature MEMS (Microelectromechanical systems) inertial sensor technology. These systems are cost effective, wearable and fully portable motion analysis systems, these features gives IMMSs the potential to be used both outside specialized laboratories and to consecutive collect series of tens of gait cycles. The recognition and selection of the most representative gait cycle is then easier and more reliable especially in CP children, considering their relevant gait cycle variability. The second section of this thesis is focused on GA. In particular, it is firstly aimed at examining the differences among five most representative GA protocols in order to assess the state of the art with respect to the inter-protocol variability. The design of a new protocol is then proposed and presented with the aim of achieving gait analysis on CP children by means of IMMS. The protocol, named ‘Outwalk’, contains original and innovative solutions oriented at obtaining joint kinematic with calibration procedures extremely comfortable for the patients. The results of a first in-vivo validation of Outwalk on healthy subjects are then provided. In particular, this study was carried out by comparing Outwalk used in combination with an IMMS with respect to a reference protocol and an optoelectronic system. In order to set a more accurate and precise comparison of the systems and the protocols, ad hoc methods were designed and an original formulation of the statistical parameter coefficient of multiple correlation was developed and effectively applied. On the basis of the experimental design proposed for the validation on healthy subjects, a first assessment of Outwalk, together with an IMMS, was also carried out on CP children. The third section of this thesis is dedicated to the treatment of walking in CP children. Commonly prescribed treatments in addressing gait abnormalities in CP children include physical therapy, surgery (orthopedic and rhizotomy), and orthoses. The orthotic approach is conservative, being reversible, and widespread in many therapeutic regimes. Orthoses are used to improve the gait of children with CP, by preventing deformities, controlling joint position, and offering an effective lever for the ankle joint. Orthoses are prescribed for the additional aims of increasing walking speed, improving stability, preventing stumbling, and decreasing muscular fatigue. The ankle-foot orthosis (AFO), with a rigid ankle, are primarily designed to prevent equinus and other foot deformities with a positive effect also on more proximal joints. However, AFOs prevent the natural excursion of the tibio-tarsic joint during the second rocker, hence hampering the natural leaning progression of the whole body under the effect of the inertia (6). A new modular (submalleolar) astragalus-calcanear orthosis, named OMAC, has recently been proposed with the intention of substituting the prescription of AFOs in those CP children exhibiting a flat and valgus-pronated foot. The aim of this section is thus to present the mechanical and technical features of the OMAC by means of an accurate description of the device. In particular, the integral document of the deposited Italian patent, is provided. A preliminary validation of OMAC with respect to AFO is also reported as resulted from an experimental campaign on diplegic CP children, during a three month period, aimed at quantitatively assessing the benefit provided by the two orthoses on walking and at qualitatively evaluating the changes in the quality of life and motor abilities. As already stated, CP is universally considered as a persistent but not unchangeable disorder of posture and movement. Conversely to this definition, some clinicians (4) have recently pointed out that movement disorders may be primarily caused by the presence of perceptive disorders, where perception is not merely the acquisition of sensory information, but an active process aimed at guiding the execution of movements through the integration of sensory information properly representing the state of one’s body and of the environment. Children with perceptive impairments show an overall fear of moving and the onset of strongly unnatural walking schemes directly caused by the presence of perceptive system disorders. The fourth section of the thesis thus deals with accurately defining the perceptive impairment exhibited by diplegic CP children. A detailed description of the clinical signs revealing the presence of the perceptive impairment, and a classification scheme of the clinical aspects of perceptual disorders is provided. In the end, a functional reaching test is proposed as an instrumental test able to disclosure the perceptive impairment. References 1. Prevalence and characteristics of children with cerebral palsy in Europe. Dev Med Child Neurol. 2002 Set;44(9):633-640. 2. Bax M, Goldstein M, Rosenbaum P, Leviton A, Paneth N, Dan B, et al. Proposed definition and classification of cerebral palsy, April 2005. Dev Med Child Neurol. 2005 Ago;47(8):571-576. 3. Ingram TT. A study of cerebral palsy in the childhood population of Edinburgh. Arch. Dis. Child. 1955 Apr;30(150):85-98. 4. Ferrari A, Cioni G. The spastic forms of cerebral palsy : a guide to the assessment of adaptive functions. Milan: Springer; 2009. 5. Olney SJ, Wright MJ. Cerebral Palsy. Campbell S et al. Physical Therapy for Children. 2nd Ed. Philadelphia: Saunders. 2000;:533-570. 6. Desloovere K, Molenaers G, Van Gestel L, Huenaerts C, Van Campenhout A, Callewaert B, et al. How can push-off be preserved during use of an ankle foot orthosis in children with hemiplegia? A prospective controlled study. Gait Posture. 2006 Ott;24(2):142-151.

Hepatobiliary diseases in small animals: a comparison of ultrasonography and multidetector-row computed tomography

Ultrasonography (US) is an essential imaging tool for identifying abnormalities of the liver parenchyma, biliary tract and vascular system. US has replaced radiography as the initial imaging procedure in screening for liver disease in small animals. There are few reports of the use of conventional and helical computed tomography (CT) to assess canine or feline parenchymal and neoplastic liver disease and biliary disorders. In human medicine the development of multidetector- row helical computed tomography (MDCT), with its superior spatial and temporal resolution, has resulted in improved detection and characterization of diffuse and focal liver lesions. The increased availability of MDCT in veterinary practice provides incentive to develop MDCT protocols for liver imaging in small animals. The purpose of this study is to assess the rule of MDCT in the characterization of hepatobiliary diseases in small animals; and to compare this method with conventional US. Candidates for this prospective study were 175 consecutive patients (dogs and cats) referred for evaluation of hepatobiliary disease. The patients underwent liver US and MDCT. Percutaneous needle biopsy was performed on all liver lesions or alterations encountered. As for gallbladder, histopatological evaluation was obtained from cholecystectomy specimens. Ultrasonographic findings in this study agreed well with those of previous reports. A protocol for dual-phase liver MDCT in small animals has been described. MDCT findings in parenchymal disorders of the liver, hepatic neoplasia and biliary disorders are here first described in dogs and cats and compared with the corresponding features in human medicine. The ability of MDCT in detection and characterization of hepatobiliary diseases in small animals is overall superior to conventional US. Ultrasonography and MDCT scanning, however, play complementary rules in the evaluation of these diseases. Many conditions have distinctive imaging features that may permit diagnosis. In most instances biopsy is required for definitive diagnosis.

Magnetic fields around radio galaxies from Faraday rotation measure analysis

The purpose of this thesis is to investigate the strength and structure of the magnetized medium surrounding radio galaxies via observations of the Faraday effect. This study is based on an analysis of the polarization properties of radio galaxies selected to have a range of morphologies (elongated tails, or lobes with small axial ratios) and to be located in a variety of environments (from rich cluster core to small group). The targets include famous objects like M84 and M87. A key aspect of this work is the combination of accurate radio imaging with high-quality X-ray data for the gas surrounding the sources. Although the focus of this thesis is primarily observational, I developed analytical models and performed two- and three-dimensional numerical simulations of magnetic fields. The steps of the thesis are: (a) to analyze new and archival observations of Faraday rotation measure (RM) across radio galaxies and (b) to interpret these and existing RM images using sophisticated two and three-dimensional Monte Carlo simulations. The approach has been to select a few bright, very extended and highly polarized radio galaxies. This is essential to have high signal-to-noise in polarization over large enough areas to allow computation of spatial statistics such as the structure function (and hence the power spectrum) of rotation measure, which requires a large number of independent measurements. New and archival Very Large Array observations of the target sources have been analyzed in combination with high-quality X-ray data from the Chandra, XMM-Newton and ROSAT satellites. The work has been carried out by making use of: 1) Analytical predictions of the RM structure functions to quantify the RM statistics and to constrain the power spectra of the RM and magnetic field. 2) Two-dimensional Monte Carlo simulations to address the effect of an incomplete sampling of RM distribution and so to determine errors for the power spectra. 3) Methods to combine measurements of RM and depolarization in order to constrain the magnetic-field power spectrum on small scales. 4) Three-dimensional models of the group/cluster environments, including different magnetic field power spectra and gas density distributions. This thesis has shown that the magnetized medium surrounding radio galaxies appears more complicated than was apparent from earlier work. Three distinct types of magnetic-field structure are identified: an isotropic component with large-scale fluctuations, plausibly associated with the intergalactic medium not affected by the presence of a radio source; a well-ordered field draped around the front ends of the radio lobes and a field with small-scale fluctuations in rims of compressed gas surrounding the inner lobes, perhaps associated with a mixing layer.

Bifidobacterium - Human Host Interaction: Role of Human Plasminogen

Bifidobacterium is an important genus of the human gastrointestinal microbiota, affecting several host physiological features. Despite the numerous Bifidobacterium related health-promoting activities, there is still a dearth of information about the molecular mechanisms at the basis of the interaction between this microorganism and the host. Bacterial surface associated proteins may play an important role in this interaction because of their ability to intervene with host molecules, as recently reported for the host protein plasminogen. Plasminogen is the zymogen of the trypsin-like serine protease plasmin, an enzyme with a broad substrate specificity. Aim of this thesis is to deepen the knowledge about the interaction between Bifidobacterium and the human plasminogen system and its role in the Bifidobacterium-host interaction process. As a bifidobacterial model, B. animalis subsp. lactis BI07 has been used because of its large usage in dairy and pharmaceutical preparations. We started from the molecular characterization of the interaction between plasminogen and one bifidobacterial plasminogen receptor, DnaK, a cell wall protein showing high affinity for plasminogen, and went on with the study of the impact of intestinal environmental factors, such as bile salts and inflammation, on the plasminogen-mediated Bifidobacterium-host interaction. According to our in vitro findings, by enhancing the activation of the bifidobacterial bound plasminogen to plasmin, the host inflammatory response results in the decrease of the bifidobacterial adhesion to the host enterocytes, favouring bacterial migration to the luminal compartment. Conversely, in the absence of inflammation, plasminogen acts as a molecular bridge between host enterocytes and bifidobacteria, enhancing Bifidobacterium adhesion. Furthermore, adaptation to physiological concentrations of bile salts enhances the capability of this microorganism to interact with the host plasminogen system. The host plasminogen system thus represents an important and flexible tool used by bifidobacteria in the cross-talk with the host.

Ecografia bidimensionale e con mezzo di contrasto nello studio delle patologie gastro-intestinali del gatto

L’ecografia del tratto gastroenterico è una delle metodiche d’elezione nella valutazione diagnostica delle patologie gastrointestinali nel gatto. In questa tesi dottorale sono presentati i risultati di tre studi in cui l’ecografia convenzionale e con mezzo di contrasto è stata impiegata in gatti sani o con patologie gastroenteriche. Lo scopo del primo studio, prospettico, è stato quello di determinare lo spessore ecografico dei singoli strati di parete nell’intestino tenue in una popolazione di gatti sani. Lo strato mucoso è risultato significativamente più spesso nel duodeno e nel digiuno, per la maggiore grandezza dei villi in queste porzioni dell’intestino tenue. A livello dell’ileo, gli strati di maggior spessore sono risultati quello sottomucoso, per l’abbondante presenza di aggregati linfoidi, e quello muscolare, a causa delle caratteristiche anatomo-funzionali di sfintere che questo tratto intestinale svolge. Il secondo progetto, retrospettivo, nasce dalla collaborazione tra due centri universitari, uno italiano e uno americano, con l’obiettivo di confrontare lo spessore della tonaca muscolare intestinale in gatti affetti da Inflammatory Bowel disease (IBD) o da neoplasie intestinali. In questo studio, l’ipertrofia della tonaca muscolare (ITM) è stato maggiormente osservato in gatti con IBD rispetto a gatti con neoplasie intestinali, ma non sono state evidenziate differenze di spessore della tonaca muscolare tali da poter differenziare le due patologie. Lo scopo del terzo progetto, prospettico, è stato quello di descrivere il pattern di perfusione parietale del piccolo intestino, valutato mediante uso di mezzo di contrasto ecografico, in gatti con ITM associata a IBD. In tutti gli animali studiati, l’ITM si è associato a una modesta assunzione del mezzo di contrasto rispetto agli altri strati della parete intestinale. Questi risultati confermano che l’ITM che si osserva in gatti con IBD non è associato a significativi aumenti della vascolarizzazione di tale strato parietale.

Diversity of the proximal femur in humans: morphological variations of the head-neck junction

The proximal femur is a high-diversity region of the human skeleton, especially at the anterior junction between head and neck, where various bony morphologies have been recognized since mid nineteenth century. Classical literature on this topic is chaotic and contradictory, making almost impossible the comparison of data from different researches. Starting from an extensive bibliographic review, the first standardized method to score these traits has been created. This method allows representing both the anatomical diversity of the region already described in literature and a part of variability not considered before, giving few and univocal definitions and allowing to collect comparable data. The method has been applied to three identified and five archaeological European skeletal collections, with the aim of investigating the distribution of these features by sex, age and side, in different places and time periods. It has also been applied to 3D digital reconstructions of femurs from CT scan files of coxo-femoral joints from fresh cadavers. In addition to the osseous traits described in the standardized method, the presence and frequency of some features known as herniation pits have been scored both on bones and on CT scans. The various osseous traits of the proximal femur are present at similar frequencies in skeletal samples from different countries and different historical periods, even if with clear local differentiation. Some of the features examined show significant trends related to their distribution by gender and age. Some hypotheses are proposed about the etiology of these morphologies and their possible implication with the acquisition of bipedalism in Humans. It is therefore highlighted the possible relation of some of these traits with the development of disorders of the hip joint. Moreover, it is not recommended the use of any of these features as a specific activity-related marker.

In vitro characterisation and expansion of human regulatory T cells for their in vivo application in the induction of tolerance in haematopoietic stem cell and solid organ transplantation

Solid organ transplantation (SOT) is considered the treatment of choice for many end-stage organ diseases. Thus far, short term results are excellent, with patient survival rates greater than 90% one year post-surgery, but there are several problems with the long term acceptance and use of immunosuppressive drugs. Hematopoietic Stem Cells Transplantation (HSCT) concerns the infusion of haematopoietic stem cells to re-establish acquired and congenital disorders of the hematopoietic system. The main side effect is the Graft versus Host Disease (GvHD) where donor T cells can cause pathology involving the damage of host tissues. Patients undergoing acute or chronic GvHD receive immunosuppressive regimen that is responsible for several side effects. The use of immunosuppressive drugs in the setting of SOT and GvHD has markedly reduced the incidence of acute rejection and the tissue damage in GvHD however, the numerous adverse side effects observed boost the development of alternative strategies to improve the long-term outcome. To this effect, the use of CD4+CD25+FOXP3+ regulatory T cells (Treg) as a cellular therapy is an attractive approach for autoimmunity disease, GvHD and limiting immune responses to allograft after transplantation. Treg have a pivotal role in maintaining peripheral immunological tolerance, by preventing autoimmunity and chronic inflammation. Results of my thesis provide the characterization and cell processing of Tregs from healthy controls and patients in waiting list for liver transplantation, followed by the development of an efficient expansion-protocol and the investigation of the impact of the main immunosuppressive drugs on viability, proliferative capacity and function of expanded cells after expansion. The conclusion is that ex vivo expansion is necessary to infuse a high Treg dose and although many other factors in vivo can contribute to the success of Treg therapy, the infusion of Tregs during the administration of the highest dose of immunosuppressants should be carefully considered.

Cognitive Assessment and Rehabilitation of subjects with Traumatic Brain Injury

This thesis regards the study and the development of new cognitive assessment and rehabilitation techniques of subjects with traumatic brain injury (TBI). In particular, this thesis i) provides an overview about the state of art of this new assessment and rehabilitation technologies, ii) suggests new methods for the assessment and rehabilitation and iii) contributes to the explanation of the neurophysiological mechanism that is involved in a rehabilitation treatment. Some chapters provide useful information to contextualize TBI and its outcome; they describe the methods used for its assessment/rehabilitation. The other chapters illustrate a series of experimental studies conducted in healthy subjects and TBI patients that suggest new approaches to assessment and rehabilitation. The new proposed approaches have in common the use of electroencefalografy (EEG). EEG was used in all the experimental studies with a different purpose, such as diagnostic tool, signal to command a BCI-system, outcome measure to evaluate the effects of a treatment, etc. The main achieved results are about: i) the study and the development of a system for the communication with patients with disorders of consciousness. It was possible to identify a paradigm of reliable activation during two imagery task using EEG signal or EEG and NIRS signal; ii) the study of the effects of a neuromodulation technique (tDCS) on EEG pattern. This topic is of great importance and interest. The emerged founding showed that the tDCS can manipulate the cortical network activity and through the research of optimal stimulation parameters, it is possible move the working point of a neural network and bring it in a condition of maximum learning. In this way could be possible improved the performance of a BCI system or to improve the efficacy of a rehabilitation treatment, like neurofeedback.

Novel developments of atropisomeric organic molecules. Design of atropisomeric drugs, fluorescent compounds and investigation of reaction mechanisms.

In recent years, the study of restricted rotation bonds in organic compounds has aroused increasing interest. The reason is that this characteristic can lead to obtaining new properties in organic compounds. In this research thesis, an intense investigation was carried out using DFT calculations and experimental evaluation of the barriers to rotational energies, in order to discover new properties deriving from the restricted rotation bonds. Research has been developed in various fields of organic chemistry, ranging from drugs (the atropisomeric atorvastatin in Chapter 3) to luminescent compounds (aryls amino borane in Chapter 4). Furthermore, an organocatalytic central to axial conversion mechanism was investigated through DFT calculations, finding out interesting outcomes (Chapter 5). Finally, a project in collaboration with Dr. Farran and Prof. Vanthuyne of the Aix-Marseille University was done to investigate the interactions in transition states of rotational barriers.

Intestinal microbiome in chronic intestinal failure and associations with intestinal failure associated liver disease

Background and Aims: Intestinal dysbiosis has been described in children with chronic intestinal failure (CIF) and in adults with short bowel syndrome (SBS), mostly with jejunocolic anastomosis (SBS-2) and jejuno-ileal anastomosis (SBS-3), linked to generic data with the pathogenesis of Intestinal Failure Associated Liver Disease (IFALD). Little is known about gut microbiome of adults with end-jejunostomy (SBS-1) and in CIF other than SBS and any specific associations with the onset of IFALD. We aimed to describe the fecal microbiome of adult patients with different mechanisms of CIF and any possible associations with the development of IFALD. Material and methods: Fecal samples from 61 patients with benign CIF. Phylogenetic characterization of the microbiome by amplification of the hypervariable regions V3 and V4 of the bacterial gene encoding 16S rRNA, and subsequent grouping of sequences in amplicon sequence variants (ASVs). Patient samples comparison to microbiome sequences from 61 healthy subjects, matched for sex and age, selected from the healthy subjects library of the Laboratory of the Microbial Ecology of Health Unit, Department of Pharmacy and Biotechnology, of the University of Bologna. IFALD was assessed by the diagnostic criteria of IFALD-cholestasis, IFALD-steatosis, IFALD-fibrosis. Results: Decreased bacterial α-diversity in CIF patients (increase of Proteobacteria and Actinobacteria and decrease in Bacteroidetes). Identification of microbial family-level signatures specific for CIF mechanisms (increase in Actinomycetaceae and Streptococcaceae in SBS-1, Bifidobacteriaceae and Lactobacillaceae in SBS-2, Bacteroidaceae and Porphyromonadaceae in dysmotility). Abundance of Lactobacillus and Lactobacillaceae strongly associated with IFALD-cholestasis and IFALD–fibrosis for SBS-1; Peptostreptococcus, Prevotellaceae (Prevotella) and Pasteurellaceae (Haemophilus) significantly increased in IFALD-fibrosis for other CIF mechanisms. Conclusions: CIF patients had a marked intestinal dysbiosis with microbial family-level signatures specific to the pathophysiological mechanism. Specific characteristics of microbiome may contribute to the pathogenesis of IFALD. Intestinal microbiome could become a therapeutic target in patients with CIF.