Biomechanical modelling of human knee during living activities

The knee joint is a key structure of the human locomotor system. The knowledge of how each single anatomical structure of the knee contributes to determine the physiological function of the knee, is of fundamental importance for the development of new prostheses and novel clinical, surgical, and rehabilitative procedures. In this context, a modelling approach is necessary to estimate the biomechanic function of each anatomical structure during daily living activities. The main aim of this study was to obtain a subject-specific model of the knee joint of a selected healthy subject. In particular, 3D models of the cruciate ligaments and of the tibio-femoral articular contact were proposed and developed using accurate bony geometries and kinematics reliably recorded by means of nuclear magnetic resonance and 3D video-fluoroscopy from the selected subject. Regarding the model of the cruciate ligaments, each ligament was modelled with 25 linear-elastic elements paying particular attention to the anatomical twisting of the fibres. The devised model was as subject-specific as possible. The geometrical parameters were directly estimated from the experimental measurements, whereas the only mechanical parameter of the model, the elastic modulus, had to be considered from the literature because of the invasiveness of the needed measurements. Thus, the developed model was employed for simulations of stability tests and during living activities. Physiologically meaningful results were always obtained. Nevertheless, the lack of subject-specific mechanical characterization induced to design and partially develop a novel experimental method to characterize the mechanics of the human cruciate ligaments in living healthy subjects. Moreover, using the same subject-specific data, the tibio-femoral articular interaction was modelled investigating the location of the contact point during the execution of daily motor tasks and the contact area at the full extension with and without the whole body weight of the subject. Two different approaches were implemented and their efficiency was evaluated. Thus, pros and cons of each approach were discussed in order to suggest future improvements of this methodologies. The final results of this study will contribute to produce useful methodologies for the investigation of the in-vivo function and pathology of the knee joint during the execution of daily living activities. Thus, the developed methodologies will be useful tools for the development of new prostheses, tools and procedures both in research field and in diagnostic, surgical and rehabilitative fields.

Vibration as a method for muscular stimulation

Human reactions to vibration have been extensively investigated in the past. Vibration, as well as whole-body vibration (WBV), has been commonly considered as an occupational hazard for its detrimental effects on human condition and comfort. Although long term exposure to vibrations may produce undesirable side-effects, a great part of the literature is dedicated to the positive effects of WBV when used as method for muscular stimulation and as an exercise intervention. Whole body vibration training (WBVT) aims to mechanically activate muscles by eliciting neuromuscular activity (muscle reflexes) via the use of vibrations delivered to the whole body. The most mentioned mechanism to explain the neuromuscular outcomes of vibration is the elicited neuromuscular activation. Local tendon vibrations induce activity of the muscle spindle Ia fibers, mediated by monosynaptic and polysynaptic pathways: a reflex muscle contraction known as the Tonic Vibration Reflex (TVR) arises in response to such vibratory stimulus. In WBVT mechanical vibrations, in a range from 10 to 80 Hz and peak to peak displacements from 1 to 10 mm, are usually transmitted to the patient body by the use of oscillating platforms. Vibrations are then transferred from the platform to a specific muscle group through the subject body. To customize WBV treatments, surface electromyography (SEMG) signals are often used to reveal the best stimulation frequency for each subject. Use of SEMG concise parameters, such as root mean square values of the recordings, is also a common practice; frequently a preliminary session can take place in order to discover the more appropriate stimulation frequency. Soft tissues act as wobbling masses vibrating in a damped manner in response to mechanical excitation; Muscle Tuning hypothesis suggest that neuromuscular system works to damp the soft tissue oscillation that occurs in response to vibrations; muscles alters their activity to dampen the vibrations, preventing any resonance phenomenon. Muscle response to vibration is however a complex phenomenon as it depends on different parameters, like muscle-tension, muscle or segment-stiffness, amplitude and frequency of the mechanical vibration. Additionally, while in the TVR study the applied vibratory stimulus and the muscle conditions are completely characterised (a known vibration source is applied directly to a stretched/shortened muscle or tendon), in WBV study only the stimulus applied to a distal part of the body is known. Moreover, mechanical response changes in relation to the posture. The transmissibility of vibratory stimulus along the body segment strongly depends on the position held by the subject. The aim of this work was the investigation on the effects that the use of vibrations, in particular the effects of whole body vibrations, may have on muscular activity. A new approach to discover the more appropriate stimulus frequency, by the use of accelerometers, was also explored. Different subjects, not affected by any known neurological or musculoskeletal disorders, were voluntarily involved in the study and gave their informed, written consent to participate. The device used to deliver vibration to the subjects was a vibrating platform. Vibrations impressed by the platform were exclusively vertical; platform displacement was sinusoidal with an intensity (peak-to-peak displacement) set to 1.2 mm and with a frequency ranging from 10 to 80 Hz. All the subjects familiarized with the device and the proper positioning. Two different posture were explored in this study: position 1 - hack squat; position 2 - subject standing on toes with heels raised. SEMG signals from the Rectus Femoris (RF), Vastus Lateralis (VL) and Vastus medialis (VM) were recorded. SEMG signals were amplified using a multi-channel, isolated biomedical signal amplifier The gain was set to 1000 V/V and a band pass filter (-3dB frequency 10 - 500 Hz) was applied; no notch filters were used to suppress line interference. Tiny and lightweight (less than 10 g) three-axial MEMS accelerometers (Freescale semiconductors) were used to measure accelerations of onto patient’s skin, at EMG electrodes level. Accelerations signals provided information related to individuals’ RF, Biceps Femoris (BF) and Gastrocnemius Lateralis (GL) muscle belly oscillation; they were pre-processed in order to exclude influence of gravity. As demonstrated by our results, vibrations generate peculiar, not negligible motion artifact on skin electrodes. Artifact amplitude is generally unpredictable; it appeared in all the quadriceps muscles analysed, but in different amounts. Artifact harmonics extend throughout the EMG spectrum, making classic high-pass filters ineffective; however, their contribution was easy to filter out from the raw EMG signal with a series of sharp notch filters centred at the vibration frequency and its superior harmonics (1.5 Hz wide). However, use of these simple filters prevents the revelation of EMG power potential variation in the mentioned filtered bands. Moreover our experience suggests that the possibility of reducing motion artefact, by using particular electrodes and by accurately preparing the subject’s skin, is not easily viable; even though some small improvements were obtained, it was not possible to substantially decrease the artifact. Anyway, getting rid of those artifacts lead to some true EMG signal loss. Nevertheless, our preliminary results suggest that the use of notch filters at vibration frequency and its harmonics is suitable for motion artifacts filtering. In RF SEMG recordings during vibratory stimulation only a little EMG power increment should be contained in the mentioned filtered bands due to synchronous electromyographic activity of the muscle. Moreover, it is better to remove the artifact that, in our experience, was found to be more than 40% of the total signal power. In summary, many variables have to be taken into account: in addition to amplitude, frequency and duration of vibration treatment, other fundamental variables were found to be subject anatomy, individual physiological condition and subject’s positioning on the platform. Studies on WBV treatments that include surface EMG analysis to asses muscular activity during vibratory stimulation should take into account the presence of motion artifacts. Appropriate filtering of artifacts, to reveal the actual effect on muscle contraction elicited by vibration stimulus, is mandatory. However as a result of our preliminary study, a simple multi-band notch filtering may help to reduce randomness of the results. Muscle tuning hypothesis seemed to be confirmed. Our results suggested that the effects of WBV are linked to the actual muscle motion (displacement). The greater was the muscle belly displacement the higher was found the muscle activity. The maximum muscle activity has been found in correspondence with the local mechanical resonance, suggesting a more effective stimulation at the specific system resonance frequency. Holding the hypothesis that muscle activation is proportional to muscle displacement, treatment optimization could be obtained by simply monitoring local acceleration (resonance). However, our study revealed some short term effects of vibratory stimulus; prolonged studies should be assembled in order to consider the long term effectiveness of these results. Since local stimulus depends on the kinematic chain involved, WBV muscle stimulation has to take into account the transmissibility of the stimulus along the body segment in order to ensure that vibratory stimulation effectively reaches the target muscle. Combination of local resonance and muscle response should also be further investigated to prevent hazards to individuals undergoing WBV treatments.

Biomeccanica del servizio e del diritto nel tennis: confronto fra atleti di livello di gioco differente

Il tennis è uno sport molto diffuso che negli ultimi trent’anni ha subito molti cambiamenti. Con l’avvento di nuovi materiali più leggeri e maneggevoli la velocità della palla è aumentata notevolmente, rendendo così necessario una modifica a livello tecnico dei colpi fondamentali. Dalla ricerca bibliografica sono emerse interessanti indicazioni su angoli e posizioni corporee ideali da mantenere durante le varie fasi dei colpi, confrontando i giocatori di altissimo livello. Non vi sono invece indicazioni per i maestri di tennis su quali siano i parametri più importanti da allenare a seconda del livello di gioco del proprio atleta. Lo scopo di questa tesi è quello di individuare quali siano le variabili tecniche che influenzano i colpi del diritto e del servizio confrontando atleti di genere differente, giocatori di livello di gioco diverso (esperti, intermedi, principianti) e dopo un anno di attività programmata. Confrontando giocatori adulti di genere diverso, è emerso che le principali differenze sono legate alle variabili di prestazione (velocità della palla e della racchetta) per entrambi i colpi. Questi dati sono simili a quelli riscontrati nel test del lancio della palla, un gesto non influenzato dalla tecnica del colpo. Le differenze tecniche di genere sono poco rilevanti ed attribuibili alla diversa interpretazione dei soggetti. Nel confronto di atleti di vario livello di gioco le variabili di prestazione presentano evidenti differenze, che possono essere messe in relazione con alcune differenze tecniche rilevate nei gesti specifici. Nel servizio i principianti tendono a direzionare l’arto superiore dominante verso la zona bersaglio, abducendo maggiormente la spalla ed avendo il centro della racchetta più a destra rispetto al polso. Inoltre, effettuano un caricamento minore degli arti inferiori, del tronco e del gomito. Per quanto riguarda il diritto si possono evidenziare queste differenze: l’arto superiore è sempre maggiormente esteso per il gruppo dei principianti; il tronco, nei giocatori più abili viene utilizzato in maniera più marcata, durante la fase di caricamento, in movimenti di torsione e di inclinazione laterale. Gli altri due gruppi hanno maggior difficoltà nell’eseguire queste azioni preparatorie, in particolare gli atleti principianti. Dopo un anno di attività programmata sono stati evidenziati miglioramenti prestativi. Anche dal punto di vista tecnico sono state notate delle differenze che possono spiegare il miglioramento della performance nei colpi. Nel servizio l’arto superiore si estende maggiormente per colpire la palla più in alto possibile. Nel diritto sono da sottolineare soprattutto i miglioramenti dei movimenti del tronco in torsione ed in inclinazione laterale. Quindi l’atleta si avvicina progressivamente ad un’esecuzione tecnica corretta. In conclusione, dal punto di vista tecnico non sono state rilevate grosse differenze tra i due generi che possano spiegare le differenze di performance. Perciò questa è legata più ad un fattore di forza che dovrà essere allenata con un programma specifico. Nel confronto fra i vari livelli di gioco e gli effetti di un anno di pratica si possono individuare variabili tecniche che mostrano differenze significative tra i gruppi sperimentali. Gli evoluti utilizzano tutto il corpo per effettuare dei colpi più potenti, utilizzando in maniera tecnicamente più valida gli arti inferiori, il tronco e l’arto superiore. I principianti utilizzano prevalentemente l’arto superiore con contributi meno evidenti degli altri segmenti. Dopo un anno di attività i soggetti esaminati hanno dimostrato di saper utilizzare meglio il tronco e l’arto superiore e ciò può spiegare il miglioramento della performance. Si può ipotizzare che, per il corretto utilizzo degli arti inferiori, sia necessario un tempo più lungo di apprendimento oppure un allenamento più specifico.

Bioengineering of exercise: biomechanical and metabolic aspects

The field of research of this dissertation concerns the bioengineering of exercise, in particular the relationship between biomechanical and metabolic knowledge. This relationship can allow to evaluate exercise in many different circumstances: optimizing athlete performance, understanding and helping compensation in prosthetic patients and prescribing exercise with high caloric consumption and minimal joint loading to obese subjects. Furthermore, it can have technical application in fitness and rehabilitation machine design, predicting energy consumption and joint loads for the subjects who will use the machine. The aim of this dissertation was to further understand how mechanical work and metabolic energy cost are related during movement using interpretative models. Musculoskeletal models, when including muscle energy expenditure description, can be useful to address this issue, allowing to evaluate human movement in terms of both mechanical and metabolic energy expenditure. A whole body muscle-skeletal model that could describe both biomechanical and metabolic aspects during movement was identified in literature and then was applied and validated using an EMG-driven approach. The advantage of using EMG driven approach was to avoid the use of arbitrary defined optimization functions to solve the indeterminate problem of muscle activations. A sensitivity analysis was conducted in order to know how much changes in model parameters could affect model outputs: the results showed that changing parameters in between physiological ranges did not influence model outputs largely. In order to evaluate its predicting capacity, the musculoskeletal model was applied to experimental data: first the model was applied in a simple exercise (unilateral leg press exercise) and then in a more complete exercise (elliptical exercise). In these studies, energy consumption predicted by the model resulted to be close to energy consumption estimated by indirect calorimetry for different intensity levels at low frequencies of movement. The use of muscle skeletal models for predicting energy consumption resulted to be promising and the use of EMG driven approach permitted to avoid the introduction of optimization functions. Even though many aspects of this approach have still to be investigated and these results are preliminary, the conclusions of this dissertation suggest that musculoskeletal modelling can be a useful tool for addressing issues about efficiency of movement in healthy and pathologic subjects.

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.

Genetic Manipulation, Gene Silencing and Biomarker Development in Multiple Experimental Models.

The thesis is set in three different parts, according to the relative experimental models. First, the domestic pig (Sus scrofa) is part of the study on reproductive biotechnologies: the transgenesis technique of Sperm Mediated Gene Transfer is widely studied starting from the quality of the semen, through the study of multiple uptakes of exogenous DNA and lastly used in the production of multi-transgenic blastocysts. Finally we managed to couple the transgenesis pipeline with sperm sorting and therefore produced transgenic embryos of predetermined sex. In the second part of the thesis the attention is on the fruit fly (Drosophila melanogaster) and on its derived cell line: the S2 cells. The in vitro and in vivo models are used to develop and validate an efficient way to knock down the myc gene. First an efficient in vitro protocol is described, than we demonstrate how the decrease in myc transcript remarkably affects the ribosome biogenesis through the study of Polysome gradients, rRNA content and qPCR. In vivo we identified two optimal drivers for the conditional silencing of myc, once the flies are fed with RU486: the first one is throughout the whole body (Tubulin), while the second is a head fat body driver (S32). With these results we present a very efficient model to study the role of myc in multiple aspects of translation. In the third and last part, the focus is on human derived lung fibroblasts (hLF-1), mouse tail fibroblasts and mouse tissues. We developed an efficient assay to quantify the total protein content of the nucleus on a single cell level via fluorescence. We coupled the protocol with classical immunofluorescence so to have at the same time general and particular information, demonstrating that during senescence nuclear proteins increase by 1.8 fold either in human cells, mouse cells and mouse tissues.

Analisi dell’influenza dei moti rigidi di una trattrice agricola sulle vibrazioni a corpo intero trasmesse all’operatore

L’esposizione degli operatori in campo agricolo alle vibrazioni trasmesse al corpo intero, produce effetti dannosi alla salute nel breve e nel lungo termine. Le vibrazioni che si generano sulle trattrici agricole hanno una elevata intensità e una bassa frequenza. Le componenti orizzontali, amplificate dalla posizione elevata della postazione di guida dall’asse di rollio, presentano maggiori criticità per quanto riguarda i sistemi di smorzamento rispetto alle componenti verticali. Queste caratteristiche rendono difficoltosa la progettazione dei sistemi dedicati alla riduzione del livello vibrazionale per questa categoria di macchine agricole. Nonostante l’installazione di diversi sistemi di smorzamento, il livello di vibrazioni a cui è sottoposto l’operatore può superare, in diverse condizioni di impiego, i livelli massimi imposti dalla legge per la salvaguardia della salute. L’obiettivo di questo lavoro è quello di valutare l’influenza dei moti rigidi di una trattrice (beccheggio, rollio e saltellamento) dotata di sospensione assale anteriore, sospensione cabina e sospensione sedile, sul livello vibrazionale trasmesso all’operatore.E’ stata pertanto strumenta una trattrice con accelerometri e inclinometri installati su telaio, cabina e sedile e utilizzata in diverse condizioni di lavoro in campo e di trasporto su strada. Dall’analisi delle prove effettuate emerge che durante il trasporto su strada è predominante l’accelerazione longitudinale, a causa dell’elevata influenza del beccheggio. La sospensione riduce notevolmente il moto rigido di beccheggio mentre l’effetto della sospensione della cabina è quello di incrementare, in ogni condizione di lavoro, il livello di accelerazione trasmesso dal telaio della macchina.

Assessment of vibratory stimulation on neuromuscular system

The thesis analyze a subject of renewed interest in bioengineering, the research and analysis of exercise parameters that maximize the neuromuscular and cardiovascular involvement in vibration treatment. The research activity was inspired by the increasing use of device able to provide localized or whole body vibration (WBV). In particular, the focus was placed on the vibrating platform and the effect that the vibrations have on the neuromuscular system and cardiovascular system. The aim of the thesis is to evaluate the effectiveness and efficiency of vibration applied to the entire body, in particular, it was investigated the effect of WBV on: 1) Oxygen consumption during static and dynamic squat; 2) Resonant frequency of the muscle groups of the lower limbs; 3) Oxygen consumption and electromyographic signals during static and dynamic squat. In the first three chapters are explained the state of the art concerning vibration treatments, the effects of vibration applied to the entire body, with the explanation of the basic mechanisms (Tonic Vibration Reflex, TVR) and the neuromuscular system, with particular attention to the skeletal muscles and the stretch reflex. In the fourth chapter is illustrated the set-up used for the experiments and the software, implemented in LabWindows in order to control the platform and acquire the electromyographic signal. In the fifth chapter were exposed experiments undertaken during the PhD years. In particular, the analysis of Whole Body Vibration effect on neurological and cardiovascular systems showed interesting results. The results indicate that the static squat with WBV produced higher neuromuscular and cardiorespiratory system activation for exercise duration <60 sec. Otherwise, if the single bout duration was higher than 60 sec, the greater cardiorespiratory system activation was achieved during the dynamic squat with WBV while higher neuromuscular activation was still obtained with the static exercise.

Gait analysis using a single wearable inertial measurement unit

Procedures for quantitative walking analysis include the assessment of body segment movements within defined gait cycles. Recently, methods to track human body motion using inertial measurement units have been suggested. It is not known if these techniques can be readily transferred to clinical measurement situations. This work investigates the aspects necessary for one inertial measurement unit mounted on the lower back to track orientation, and determine spatio-temporal features of gait outside the confines of a conventional gait laboratory. Apparent limitations of different inertial sensors can be overcome by fusing data using methods such as a Kalman filter. The benefits of optimizing such a filter for the type of motion are unknown. 3D accelerations and 3D angular velocities were collected for 18 healthy subjects while treadmill walking. Optimization of Kalman filter parameters improved pitch and roll angle estimates when compared to angles derived using stereophotogrammetry. A Weighted Fourier Linear Combiner method for estimating 3D orientation angles by constructing an analytical representation of angular velocities and allowing drift free integration is also presented. When tested this method provided accurate estimates of 3D orientation when compared to stereophotogrammetry. Methods to determine spatio-temporal features from lower trunk accelerations generally require knowledge of sensor alignment. A method was developed to estimate the instants of initial and final ground contact from accelerations measured by a waist mounted inertial device without rigorous alignment. A continuous wavelet transform method was used to filter and differentiate the signal and derive estimates of initial and final contact times. The technique was tested with data recorded for both healthy and pathologic (hemiplegia and Parkinson’s disease) subjects and validated using an instrumented mat. The results show that a single inertial measurement unit can assist whole body gait assessment however further investigation is required to understand altered gait timing in some pathological subjects.