Evolutionary genetics of lactase persistence in Eurasian human populations

Although ability to digest lactose generally declines after weaning in all mammals, in some human populations it persists also in adult individuals, a condition named lactase persistence (LP). Studies on the prevalence of the LP phenotype in worldwide human populations have shown that the frequency of this trait is highly variable in different ethnic groups, appearing to be positively correlated with the importance of milk in the diet. In particular, several single-nucleotide polymorphisms (SNPs) in the proximity of the LCT gene have been proved to be associated with LP. Nevertheless, few studies have till now analyzed genetic variation underlying LP in a wide set of Eurasian populations and, especially, in the Italian one. In the present study, we thus typed 40 SNPs surrounding the LCT gene in more than 1,000 samples from Italian and Arabic peninsulas to investigate patterns of LP-related genetic diversity in two regions which have played a pivotal role in the recent human evolutionary history according to their geographical position and historical/archaeological records. Our results underline a high and complex variability of the explored genomic region in both studied populations. In particular, a clear diversification of Northern Italian groups from the rest of the peninsula, was observed, with the formers being genetically more similar to Northern European populations than to Southern Italians. These observation are consistent with known decreasing pattern of LP from Northern to Southern Italy and suggest the possibility of an independent evolution of LP-associated genotypes in Northern Italy. A similar scenario was observed in the Arabian peninsula, with Dhofari Arabs from Southern Oman and Yemeni clustering together with respect to Arabs from Northern Oman and the subgroup of Omanis of Asian origin which appeared instead to be genetically closer to Europeans than to the rest of Arabic groups.

Evolutionary epigenetics of modern human populations

Epigenetic variability is a new mechanism for the study of human microevolution, because it creates both phenotypic diversity within an individual and within population. This mechanism constitutes an important reservoir for adaptation in response to new stimuli and recent studies have demonstrated that selective pressures shape not only the genetic code but also DNA methylation profiles. The aim of this thesis is the study of the role of DNA methylation changes in human adaptive processes, considering the Italian peninsula and macro-geographical areas. A whole-genome analysis of DNA methylation profile across the Italian penisula identified some genes whose methylation levels differ between individuals of different Italian districts (South, Centre and North of Italy). These genes are involved in nitrogen compound metabolism and genes involved in pathogens response. Considering individuals with different macro-geographical origins (individuals of Asians, European and African ancestry) more significant DMRs (differentially methylated regions) were identified and are located in genes involved in glucoronidation, in immune response as well as in cell comunication processes. A "profile" of each ancestry (African, Asian and European) was described. Moreover a deepen analysis of three candidate genes (KRTCAP3, MAD1L and BRSK2) in a cohort of individuals of different countries (Morocco, Nigeria, China and Philippines) living in Bologna, was performed in order to explore genetic and epigenetic diversity. Moreover this thesis have paved the way for the application of DNA methylation for the study of hystorical remains and in particular for the age-estimation of individuals starting from biological samples (such as teeth or blood). Noteworthy, a mathematical model that considered methylation values of DNA extracted from cementum and pulp of living individuals can estimate chronological age with high accuracy (median absolute difference between age estimated from DNA methylation and chronological age was 1.2 years).

Unlocking ecological history using fish remains: Eco-evolutionary consequences of exploitation in the Atlantic bluefin tuna

During recent decades, the health of ocean ecosystems and fish populations has been threatened by overexploitation, pollution, and anthropogenic-driven climate change. Due to a lack of long-term data, we have a poor understanding of when intensive exploitation began and what impact anthropogenic activities have had on the ecology and evolution of fishes. Such information is crucial to recover degraded and depleted marine ecosystems and fish populations, maximise their productivity in-line with historical levels, and predict their future dynamics. In this thesis, I evaluate anthropogenic impacts on the iconic Atlantic bluefin tuna (Thunnus thynnus; BFT), one of the longest and recently most intensely exploited marine fishes, with a tremendous cultural and economic importance. Using a long-time series of archaeological and archived faunal remains (bones) dating back to approximately two millennia ago, I apply morphological, isotopic, and genomic techniques to perform the first studies on long-term BFT size and growth, diet and habitat use, and demography and adaptation, and produce the first genome-wide data on this species. My findings suggest that exploitation had impacted BFT foraging behaviour by the ~16th century when coastal ecosystem degradation induced a pelagic shift in diet and habitat use. I reveal that BFT biomass began to decline much earlier than hitherto documented, by the 19th century, consistent with intensive tuna trap catches during this period and catch-at-size increasing. I find that BFT juvenile growth had increased by the early 1900s (and more dramatically by the 21st century) which may reflect an evolutionary response to size selective harvest–which I find putative genomic signatures of. Further, I observed that BFT foraging behaviours have been modified following overexploitation during the 20th century, which previously included a isotopically distinct, Black Sea niche. Finally, I show that despite biomass declining from centuries ago, BFT has retained genomic diversity.

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.

Development and characterization of micromachined devices for separation techniques

Nowadays microfluidic is becoming an important technology in many chemical and biological processes and analysis applications. The potential to replace large-scale conventional laboratory instrumentation with miniaturized and self-contained systems, (called lab-on-a-chip (LOC) or point-of-care-testing (POCT)), offers a variety of advantages such as low reagent consumption, faster analysis speeds, and the capability of operating in a massively parallel scale in order to achieve high-throughput. Micro-electro-mechanical-systems (MEMS) technologies enable both the fabrication of miniaturized system and the possibility of developing compact and portable systems. The work described in this dissertation is towards the development of micromachined separation devices for both high-speed gas chromatography (HSGC) and gravitational field-flow fractionation (GrFFF) using MEMS technologies. Concerning the HSGC, a complete platform of three MEMS-based GC core components (injector, separation column and detector) is designed, fabricated and characterized. The microinjector consists of a set of pneumatically driven microvalves, based on a polymeric actuating membrane. Experimental results demonstrate that the microinjector is able to guarantee low dead volumes, fast actuation time, a wide operating temperature range and high chemical inertness. The microcolumn consists of an all-silicon microcolumn having a nearly circular cross-section channel. The extensive characterization has produced separation performances very close to the theoretical ideal expectations. A thermal conductivity detector (TCD) is chosen as most proper detector to be miniaturized since the volume reduction of the detector chamber results in increased mass and reduced dead volumes. The microTDC shows a good sensitivity and a very wide dynamic range. Finally a feasibility study for miniaturizing a channel suited for GrFFF is performed. The proposed GrFFF microchannel is at early stage of development, but represents a first step for the realization of a highly portable and potentially low-cost POCT device for biomedical applications.