Дисертації з теми "Bio medicine"

In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications. In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications. In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects. In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.

In the last decades mesenchymal stromal cells (MSC), intriguing for their multilineage plasticity and their proliferation activity in vitro, have been intensively studied for innovative therapeutic applications. In the first project, a new method to expand in vitro adipose derived-MSC (ASC) while maintaining their progenitor properties have been investigated. ASC are cultured in the same flask for 28 days in order to allow cell-extracellular matrix and cell-cell interactions and to mimic in vivo niche. ASC cultured with this method (Unpass cells) were compared with ASC cultured under classic condition (Pass cells). Unpass and Pass cells were characterized in terms of clonogenicity, proliferation, stemness gene expression, differentiation in vitro and in vivo and results obtained showed that Unpass cells preserve their stemness and phenotypic properties suggesting a fundamental role of the niche in the maintenance of ASC progenitor features. Our data suggests alternative culture conditions for the expansion of ASC ex vivo which could increase the performance of ASC in regenerative applications. In vivo MSC tracking is essential in order to assess their homing and migration. Super-paramagnetic iron oxide nanoparticles (SPION) have been used to track MSC in vivo due to their biocompatibility and traceability by MRI. In the second project a new generation of magnetic nanoparticles (MNP) used to label MSC were tested. These MNP have been functionalized with hyperbranched poly(epsilon-lysine)dendrons (G3CB) in order to interact with membrane glycocalix of the cells avoiding their internalization and preventing any cytotoxic effects. In literature it is reported that labeling of MSC with SPION takes long time of incubation. In our experiments after 15min of incubation with G3CB-MNP more then 80% of MSC were labeled. The data obtained from cytotoxic, proliferation and differentiation assay showed that labeling does not affect MSC properties suggesting a potential application of G3CB nano-particles in regenerative medicine.

Among all the disciplines in which local medicine and spirituality are still intricately entangled, there exists one, namely Tibetan medicine. These medical knowledge and practices are also known as Sowa Rigpa gso ba rig pa, which can literally translate to the “science of healing” or the “Tibetan knowledge of the field of healing”. It is based on two essential elements: local medicine and Buddhism. These two coexist together and are both pivotal to treating the patient’s imbalance or illness. Inspired by Tibetan medical tradition, this thesis proposes research on some aspects concerning the connection between medicine and religion. Despite the abundant scientific literature available about Sowa Rigpa and other medicines and religions such as Ayurvedic medicine and Indian Buddhism, there are no studies investigating the relationship between biomedicine and Sowa Rigpa that took into consideration the medical dimension and religious contexts of each practice, i.e. Buddhism and Christianity. This experimental and interdisciplinary thesis aims at investigating the dialogue between these two coexisting worlds in order to highlight the contribution of spirituality as a complementary and supportive element to accept and confront an illness. During hundreds of years, Christianity as well as Buddhism had a strong influence on the development of medicine. Although this is no longer the case, it is yet possible for biomedicine and Christianity to keep interacting together, as demonstrated by Tibetan medicine and Buddhism. Therefore, I ultimately decided to examine the connection between biomedicine and religion in the Trompone complex in Moncrivello, in the province of Vercelli (in Piedmont), which brings together both the medical facilities and the Catholic Sanctuary devoted to “Beata Vergine del Trompone”. Apart from standard pharmacological treatments, patients can benefit from religious services and spiritual support. Most of the investigative work with patients, health and social professionals and spiritual caregivers was conducted in this medical center. By adopting an anthropological approach, this essay attempted to contribute to the dialogue between biomedicine and Sowa Rigpa via Christianity and Buddhism.

This thesis aims to develop rapid, quantitative point of care sensing systems that exploit molecular wire platforms to enable the electrochemical detection of multiple target biomarkers, thereby empowering new technologies for the diagnosis of various medical conditions. Accordingly, the first chapter provides an overview of clinically important biomarkers for pregnancy and cardiovascular disease. The second chapter of this thesis details the electrochemical concepts underpinning subsequent chapters, with the third chapter providing an experimental overview. The first two research chapters develop a nano-structured, molecular wire platform based on diazonium salt electrochemistry coupled with immobilisation of antibodies conjugated to a suitable electroactive label. Here, the abundance of amine functionalities present at the antibody paratope enable the statistically large number of redox tags to be present at the antibody-antigen binding site, empowering the exquisitely selective and strong affinity of the antibody for a suitable antigen to be monitored quantitatively, through assessing the extent with which the redox labels are partially blocked in the presence of the antigen. In Chapter 4, experiments are contrasted with bespoke theory developed in order to unravel the thermodynamic and kinetic factors that empower this methodology to be singularly sensitive for the pregnancy biomarker human chorionic gonadotropin (hCG). It is demonstrated that quantitative analysis of hCG detection in artificial urine does not suffer interference. Chapter 5 adapts this approach to survey other biomarkers including β-hCG and brain natriuretic peptide. Combinatorial immunoassays are also investigated through the use of two types of antibodies, each tagged with a different redox label. Chapter 6 exploits the electroactive spin-trapping molecule TEMPO for the detection of biomolecules that have been damaged through oxidative stress. Last, Chapter 7 presents an overall conclusion to the work presented in this thesis.

L'étude des plantes utilisées en médecine traditionnelle au Togo est compliquée à cause de l’absence de matériel de pointe. L'identification assistée par ordinateur de produits basés sur des utilisations en médecine traditionnelle (CAPITURE) a été évaluée dans le cadre d'une enquête ethnobotanique sur le traitement traditionnel des maladies fongiques dans la Préfecture de Tchamba (Togo). Cette méthode a prédit et identifié les plantes les plus biologiquement actives parmi les 43 espèces recensées au cours de l’enquête : Pterocarpus erinaceus prédit être plus actif contre les champignons, et Daniellia oliveri contre les bactéries. Les plantes ont ensuite été testées contre les champignons, les bactéries et les cellules cancéreuses. Comme prédit par CAPITURE, P. erinaceus était plus actif contre les champignons et D. oliveri contre les bactéries. Fait intéressant, les deux plantes ont présenté une activité sur les cellules cancéreuses sans être toxique pour les cellules humaines normales. Dans une troisième étape, en utilisant la chimie analytique, les composés responsables des activités biologiques ont été identifiés. La plupart de ces composés n'ont jamais été signalés dans les espèces végétales ou dans la nature, avec une activité biologique dans la gamme micro-molaire. Enfin, en réduisant la poudre des organes végétaux à la taille de nano-particules, une meilleure activité biologique a été observée par rapport à celle de l'extrait organique. En conclusion, cette recherche a mené à la découverte de nouvelles molécules avec une activité biologique intéressante, molécules qui nécessiteront une étude approfondie et détaillée
The investigation of plants used for traditional medicine in Togo is complicated as modern techniques are not available. Computer-aided product identification from traditional usage records (CAPITURE) was evaluated in the context of an ethnobotanical survey on the traditional treatment of fungal diseases in Tchamba District (Togo). This method predicted and identified the most biologically active plants out of the 43 species survey-recorded: Pterocarpus erinaceus predicted to be more active against fungi and Daniellia oliveri against bacteria. The plants were then tested against fungi, bacteria and cancer cells. As predicted with CAPITURE, P. erinaceus was more active against fungi and D. oliveri against bacteria. Interestingly, both plants presented activity on cancer cells without being toxic to normal human cells. In a third step, using analytical chemistry, the compounds responsible for the biological activities were identified. Most of those compounds have never been reported in the plant species or in nature at all, with biological activity in the micromolar range. Finally, pharmaceutical technology was used: by nanosizing the powder of the plant organs, a better biological activity was observed in comparison to that of the organic extract. In conclusion, this research led to the discovery of new molecules with an interesting biological activity that will need further and more detailed investigation

Proteomics involves the systematic study of proteins in order to provide a comprehensive view of the structure, function and regulation of biological systems. Advances in instrumentation and methodologies have fueled an expansion of the scope of biological studies from simple biochemical analysis of single proteins to measurements of complex protein mixtures. Proteomics is rapidly becoming an essential component of biological research. Coupled with advances in bioinformatics, this approach to comprehensively describing biological systems will undoubtedly have a major impact on our understanding of the phenotypes of both normal and diseased cells. Initially, proteomics was focused on the generation of protein maps using two-dimensional polyacrylamide gel electrophoresis. Protein expression, or the quantitative measurement of the global levels of proteins, may still be done with two-dimensional gels, however, mass spectrometry has been incorporated to increase sensitivity, specificity and to provide results in a high-throughput format. Mass spectrometry applied to proteins offers many advantages: in addition to calculating the molecular weight with high accuracy, this technique allows to analyze and characterize the amino acid sequence. It can also be used in the study of post-translational modifications and to monitor the formation of complexes in solution. Finally it can be applied with different purposes such as the conformational analysis, analysis of the kinetics of refolding and studies on the catalytic activities of proteins. During my Ph.D. course my efforts have been mainly devoted on the use of this technique in combination with methods of protein chemistry such as the one- and two-dimensional electrophoresis, differents liquid chromatographies, solid phase peptide synthesis and use of proteolytic enzymes. Herein reported in my Ph.D. Thesis, the different treated subjects are divided into independent chapters each containing a single case study. Briefly in chapter 2 it has been proposed the study on protease nexin-1 (PN-1), the main inhibitor of thrombin in the brain, aimed at clarifying the role of the carbohydrate portion on the conformation, stability and function, through studies on recombinant protein produced in E.coli. In chapter 3 it has been showed the work on purification and chemical characterization, including de novo identification of amino acid sequence, of a similar inhibitor of phospholipase A2 extracted from the Python sebae serum, which demonstrated an effect cytotoxic pro-apoptotic and that could be exploited for the development of new anticancer strategies. In chapter 4 it has been reported the molecular dynamics that lead to the development of primary hyperoxaluria type I by studying the G41R mutant enzyme alanine: glyoxylate aminotransferase (AGT). In particular, were investigated the mechanisms leading to G41R be more susceptible to degradation and aggregation than wild type protein. Finally, chapter 5 deals with the effect of oxidative stress on the metabolism of von Willebrand Factor (VWF). The von Willebrand Factor is a very complex plasma glycoprotein whose dimensions help to regulate the hemostatic balance. Specifically, in the study was observed as the oxidation of a methionine residue located in the A2 domain of glycoprotein prevents proteolytic cleavage by ADAMTS-13, while not going to influence or, in some cases, promote proteolysis of VWF by proteases released from polymorphonuclear leukocytes in inflammatory conditions.
La proteomica riguarda lo studio sistematico delle proteine al fine di fornire una visione completa della funzione, della struttura e della regolazione dei sistemi biologici. I progressi avvenuti negli ultimi decenni, sia per quanto riguarda la strumentazione sia le metodologie utilizzate, hanno permesso di ampliare il campo di studi biologici passando dall’analisi di proteine purificate all’analisi di miscele complesse. La proteomica sta rapidamente diventando una componente essenziale della ricerca biologica ed associato ai progressi della bioinformatica, questo approccio alla descrizione dei sistemi biologici avrà indubbiamente un impatto notevole sulla nostra comprensione dei fenotipi sia delle cellule normali e malate. Inizialmente la proteomica era focalizzata principalmente sulla generazione di mappe proteiche bidimensionali utilizzando elettroforesi su gel di poliacrilammide. La verifica dell’espressione o la misurazione quantitativa dei livelli globali di proteine può ancora essere fatta sulla base dei gel bidimensionali, tuttavia oramai questi compiti sono affidati alla spettrometria di massa la quale può contare su di un’elevata sensibilità e specificità. La spettrometria di massa applicata alle proteine offre molti vantaggi: oltre a calcolare il peso molecolare con elevata precisione, questa tecnica permette di analizzare e caratterizzare la sequenza aminoacidica. Può anche essere utilizzata nello studio delle modificazioni post-traduzionali e per monitorare la formazione di complessi in soluzione. Infine può essere applicata con differenti scopi, quali l'analisi conformazionale, l'analisi della cinetica di ripiegamento e di studi sulle attività catalitiche delle proteine. Durante il dottorato di ricerca la mia attenzione è stata focalizzata soprattutto sull’utilizzo di tale tecnica abbinata a metodologie di chimica delle proteine quali ad esempio l’elettroforesi mono e bidimensionali, differenti cromatografie in fase liquida, la sintesi peptidica in fase solida e l’utilizzo di proteasi enzimatiche. In particolare in questa Tesi di Dottorato gli argomenti di studio sono stati trattati singolarmente, distinguendo i principali progetti in cui sono stato coinvolto in capitoli indipendenti. Brevemente, nel capitolo 2 è proposto lo studio di protease nexin-1 (PN-1), il principale inibitore della trombina a livello cerebrale, volto a chiarire la funzione della porzione glucidica sulla conformazione, stabilità e funzione della proteina mediante lo studio della proteina ricombinante prodotta in E. coli. Nel capitolo 3 è riportato il lavoro concernente la purificazione e la caratterizzazione chimica, in particolare dell’identificazione de novo della sequenza amminoacidica, di un analogo dell’inibitore della fosfolipasi A2 estratto dal siero di Python sebae, il quale ha dimostrato di possedere un effetto citotossico pro-apoptotico e che potrebbe essere sfruttato per lo sviluppo di nuove strategie antitumorali. Nel capitolo 4 l’attenzione è stata concentrata a chiarire le dinamiche molecolari che portano allo sviluppo di iperossaluria primaria di tipo I mediante lo studio del mutante G41R dell’enzima alanina:gliossilato amminotransferasi (AGT) analizzando in particolar modo i meccanismi che portano G41R ad essere maggiormente soggetto a degradazione e aggregazione rispetto alla proteina WT. Infine, il capitolo 5 tratta dell’effetto dello stress ossidativo sul metabolismo del fattore di von Willebrand (VWF). Il fattore di von Willebrand è una glicoproteina plasmatica estremamente complessa le cui dimensioni contribuiscono a regolare l’equilibrio emostatico. Nello specifico, è stato osservato come l’ossidazione di un residuo di metionina situato nel dominio A2 della glicoproteina impedisca il taglio proteolitico da parte di ADAMTS-13, mentre non vada ad influenzare o in alcuni casi addirittura favorisca la proteolisi di VWF da parte di proteasi leucocitarie liberate dai polimorfonucleati in seguito a stati infiammatori.

Parkinson’s disease (PD) is the second most common neurodegenerative disease, after Alzheimer’s disease, and the most common movement disorder. Drug treatment and deep brain stimulation can ameliorate symptoms, but the progressive degeneration of dopaminergic neurons in the substantia nigra eventually leads to severe motor dysfunction. While some effective treatments for patients with PD exist, these treatment strategies are mainly symptomatic and aimed at increasing dopamine levels in the degenerating nigrostriatal system. Existing drugs are limited in their relief and decrease in effectiveness as PD progresses.The transplantation of stem cells has emerged as a promising approach to replace lost neurons in order to restore dopamine levels in the striatum and reactivate functional circuits. Post mortem neural precursor cells (PM-NPCs) are a subclass of sub ventricular zone (SVZ)-derived neural progenitors, capable of surviving many hours (16 hours) after donor death. The in vitro differentiation yields more neurons (about 30-40%) compared to regular NPCs (Marfia et al., 2011). Recently from the SVZ of a transgenic mouse strain expressing green fluorescent protein (GFP) under the promoter C of the ubiquitin gene [(C57BL/6-Tg(UBC-GFP)30Scha/J)] we isolated GFP PM-NPCs, from mice at 6 hours after the donor death. These cells were characterized and their potential of in terms of replacement therapy was investigated in a mouse model of Parkinson disease. The degeneration of dopaminergic neurons was obtained through the administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at the dosage of 36 mg/kg intraperiteoneally (i.p.). After 1 week the lesion was stabilized by a second administration (i.p.) of the neurotoxin at the dosage of 20 mg/kg. 1x 105 of PM-PCs-GFP were injected unilaterally into the striatum of C57/BL mice by using specific stereotaxic coordinates 3 days after the second MPTP administration. The effects of transplanted cells were determined by means of performance tests aimed at detecting behavioral improvements. Moreover, the neurochemical changes were also studied by high performance liquid chromatography (HPLC). In order to study the in vivo fate of grafted GFP PM-NPCs animals were perfused 2 weeks after transplantation and immunohistochemistry studies were performed. Our results show that animals grafted with GFP PM-NPCs determined a remarkable improvement of behavioral parameters measured by means of both horizontal and vertical grid tests (forepaw fault and time required to grab on the grids while turning and climbing down) since the third day after transplantation. These improvements were very significant and the average values were close to control animals. This was maintained during all the two weeks of experimental observation. By means of immunofluorescence staining we observed that the majority of transplanted GFP-PM-NPCs were vital and able to migrate ventrally and caudally from the injection site lengths as far as 1000 microns into the striatum, and could reach the ipsilateral and contralateral substantia nigra pars compacta. Moreover, morphological analyses revealed that transplanted cells in the striatum are able to differentiate into tyrosine hydroxylase (40%), cholinergic (40%), and gabaergic neurons (25%). This study provides new evidences that PM-NPCs will be useful for developing cellular PD therapies. Future studies should further explore the clinical potential role of the investigated post mortem neural precursors cells in order to provide new perspectives for PD treatment.

The incapacity of injured adult central nervous system to restore damaged neuronal circuitry and the large peripheral nervous system nerve defect inability to be naturally regenerated are a critical medical and social issue. An emerging approach in neuronal regenerative medicine is the use of native extracellular stimuli at nano-scale level influencing cell growth, differentiation and regeneration. Our biomimetic nanosystems mimic as much as possible the nanotopographic, conductive features and guidance cues of the neuronal extracellular environment. They are made of a freestanding and biocompatible nanocomposite scaffold, combining conductive, mechanical and topographical feature of carbon-based nanomaterials with the biocompatible properties of the poly-L-lactic acid (PLLA) matrix. Moreover, biomimetic peptides have been developed deriving them from neuronal proteins involved in the control of neurite outgrowth and axon pathfinding. In recent work from our team, the combination of the nanocomposite scaffold and the peptides proved to enhance neuronal differentiation of a human neuroblastoma cell line and to promote per se neural differentiation of human multipotent stem cells, even in the absence of exogenously added neurotrophins. In my PhD project I further developed such biomimetic nanosystems. About the scaffold, we checked the biocompatibility and effect on neuronal differentiation of varying types and concentration of nanofiller. We increased from 0.25 to 5% CNTs dispersed in the PLLA-matrix to improve electrical conductivity and nanoroughness of our nanocomposite scaffold. The enhanced CNTs concentration doesn’t affect cell proliferation, viability and adhesion while promoting neurite elongation. Moreover, we tested the same range of carbon nanohorns (CNHs) and reduced graphene oxide (RGO) dispersed in the PLLA matrix and proved they are as biocompatible as CNTs. Interestingly, 5%RGO has an inductive effect on neuronal differentiation. In last months, 3D printing has been used for patterned scaffold that allow to control the cell growth direction. About biomimetic peptides, we focused on the characterization of novel peptides sharing a conserved motif to better reproduce neuronal biochemical cues. These peptides are derived from the Ig-like domain of a number of proteins playing important roles in neuronal differentiation and axon elongation: CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, Contactin 1, 2 and 5. All such peptides were able to promote neuritogenesis and neuronal differentiation of SH-SY5Y cells, with efficacy similar to previously tested peptides. In order to shed light on the mechanism by which our peptides act, we studied L1-A peptide in comparison to L1CAM extracellular domain it is derived from. As negative controls we used a scrambled and mutant version of the L1-A peptide. In silico simulations and in vitro evidence suggest an agonist-antagonist mechanism for our peptides: L1-A peptide binds L1CAM and exerts the same neuritogenic effect of the protein acting as L1CAM’s agonist; scrambled and mutant peptides bind the protein and inhibit the L1CAM homophilic binding, but they are not able to activate the signalling intracellular pathway leading to neuronal differentiation, acting as antagonists of L1CAM. In conclusion, our new nanocomposite scaffold and biomimetic peptides are potential tools for neuronal regenerative medicine, even if further investigations are needed to check their effect in combination.
The incapacity of injured adult central nervous system to restore damaged neuronal circuitry and the large peripheral nervous system nerve defect inability to be naturally regenerated are a critical medical and social issue. An emerging approach in neuronal regenerative medicine is the use of native extracellular stimuli at nano-scale level influencing cell growth, differentiation and regeneration. Our biomimetic nanosystems mimic as much as possible the nanotopographic, conductive features and guidance cues of the neuronal extracellular environment. They are made of a freestanding and biocompatible nanocomposite scaffold, combining conductive, mechanical and topographical feature of carbon-based nanomaterials with the biocompatible properties of the poly-L-lactic acid (PLLA) matrix. Moreover, biomimetic peptides have been developed deriving them from neuronal proteins involved in the control of neurite outgrowth and axon pathfinding. In recent work from our team, the combination of the nanocomposite scaffold and the peptides proved to enhance neuronal differentiation of a human neuroblastoma cell line and to promote per se neural differentiation of human multipotent stem cells, even in the absence of exogenously added neurotrophins. In my PhD project I further developed such biomimetic nanosystems. About the scaffold, we checked the biocompatibility and effect on neuronal differentiation of varying types and concentration of nanofiller. We increased from 0.25 to 5% CNTs dispersed in the PLLA-matrix to improve electrical conductivity and nanoroughness of our nanocomposite scaffold. The enhanced CNTs concentration doesn’t affect cell proliferation, viability and adhesion while promoting neurite elongation. Moreover, we tested the same range of carbon nanohorns (CNHs) and reduced graphene oxide (RGO) dispersed in the PLLA matrix and proved they are as biocompatible as CNTs. Interestingly, 5%RGO has an inductive effect on neuronal differentiation. In last months, 3D printing has been used for patterned scaffold that allow to control the cell growth direction. About biomimetic peptides, we focused on the characterization of novel peptides sharing a conserved motif to better reproduce neuronal biochemical cues. These peptides are derived from the Ig-like domain of a number of proteins playing important roles in neuronal differentiation and axon elongation: CHL1, Neurofascin, NrCAM, DCC, ROBO2 and 3, Contactin 1, 2 and 5. All such peptides were able to promote neuritogenesis and neuronal differentiation of SH-SY5Y cells, with efficacy similar to previously tested peptides. In order to shed light on the mechanism by which our peptides act, we studied L1-A peptide in comparison to L1CAM extracellular domain it is derived from. As negative controls we used a scrambled and mutant version of the L1-A peptide. In silico simulations and in vitro evidence suggest an agonist-antagonist mechanism for our peptides: L1-A peptide binds L1CAM and exerts the same neuritogenic effect of the protein acting as L1CAM’s agonist; scrambled and mutant peptides bind the protein and inhibit the L1CAM homophilic binding, but they are not able to activate the signalling intracellular pathway leading to neuronal differentiation, acting as antagonists of L1CAM. In conclusion, our new nanocomposite scaffold and biomimetic peptides are potential tools for neuronal regenerative medicine, even if further investigations are needed to check their effect in combination.

Objective tests of motor function suitable for older people in epidemiological studies and community settings are lacking. The current study aimed to establish non-invasive biomarkers using conventional and novel tests that do not rely entirely on volition, and identify suitable analysis techniques for complex data. It was hypothesised that novel technologies would improve the discriminant validity of motor function testing. In 138 self-reported healthy males and females (65 young, mean age±SD = 25.7±4.8 years; 73 older, 74.9±5.9 years), nine tests (25 parameters) included: hand grip and quadriceps strength; respiratory muscle strength (peak flow); thigh composition (ultrasound imaging); muscle mechanical properties (Myoton technology); upper limb kinematics (Motor Task Manager); timed up and go; stair climbing; balance. Three questionnaires and one mobility assessment were administered including the health related quality of life (SF36). Four experiments tested hypotheses regarding the influence of recording conditions on mechanical properties to validate the novel MyotonPRO device. Reliability of all tests was confirmed and, as expected, data indicated reduced function with ageing (all p<0.05), with the majority showing gender differences. Some mechanical properties were significantly influenced by testing site, muscle length, contractile status and prior activity. Seven of the 25 parameters (5novel) had high discriminant ability for classifying healthy adults into age/gender groups analysed by linear discriminant function using a stepwise approach. Novel technologies, notably mechanical properties of muscle and thigh composition (relative contribution of muscle and subcutaneous fat on ultrasound scans), improved classification accuracy (from 75% to 89%) when combined with conventional tests, supporting the hypothesis and providing potential screening tools independent of participant effort. High discriminant ability (73 to 80%) was also found for classification based on functional measures. This research has advanced the approach to functional assessment and analysis by producing a comprehensive battery of non-invasive biomarkers with high discriminant ability for indicating musculoskeletal health, providing reference data for comparison with clinical populations. The most sensitive novel biomarkers did not require volition, highlighting potential powerful tests for vulnerable older people with pain or cognitive impairment.

Les anomalies chromosomiques sont une cause majeure de morbidité et de mortalité périnatales. Le dépistage de ces affections a toujours été crucial pour une prise en charge optimale des femmes enceintes.Initialement, le dépistage de trisomie 21 était uniquement basé selon le risque lié à l’âge maternel. L’ajout de différents marqueurs biochimiques sériques constituant successivement les double, triple et quadruple tests a pu améliorer le taux de détection. Néanmoins, c’est en 1997 qu’apparaît un point tournant de l’histoire de dépistage des trisomies :l’introduction de la mesure de la clarté nucale à l’échographie du premier trimestre.Depuis 2011, de nouvelles recherches se sont concentrées sur le dépistage prénatal non invasif (DPNI) utilisant l'ADN fœtal libre circulant dans le sang maternel. Cette technique a suggéré directement une supériorité marquée pour la détection de la trisomie 21 comparée à toutes les autres méthodes de dépistage connues. Rapidement, ce test a été introduit en pratique clinique dans le monde entier sans une évaluation préalable et approfondie, que ce soit au niveau scientifique, technique ou éthique.Les objectifs de cette thèse étaient de fournir des réponses aux diverses questions persistantes concernant l’utilisation clinique des tests de dépistages basés sur ADN fœtal libre dans la circulation maternelle.À notre connaissance, nous sommes la première équipe à essayer d'évaluer le taux d'échec et la performance du DPNI effectué par différents laboratoires utilisant différentes méthodes analytiques. Nous avons démontré que les approches « DANSR » (approche ciblée sur les chromosomes d’intérêt) et « GW-MPS » (approche globale sur les séquences géniques reparties sur la totalité du génome par un séquençage à haut débit) offraient toutes les deux un taux échec bas avec une bonne performance dans la détection des trisomies 21, 13 et 18. Cependant, le niveau de fraction fœtale semble varier d'un laboratoire à l'autre et n’est, par conséquent, pas comparable. Nous avons également observé que le taux d'échec des laboratoires avec un test « home-brew » était 4 fois supérieur à celui des tests commerciaux développés par les laboratoires en interne. De plus, aucune pertinence clinique de la divulgation des aneuploïdies autres que les 3 trisomies communes décelées par les DPNI « GW-MPS » n’a pu être démontrée.Ensuite, nous nous sommes intéressés au groupe particulier des grossesses gémellaires. Dans ce groupe, le DPNI était faisable mais il était associé à un taux d'échec supérieur aux grossesses uniques, tout en fournissant un taux de détection moindre des trisomies communes. Un poids maternel élevé et la conception par fécondation in vitro étaient des facteurs indépendants associés à l’échec du test. De plus, il faut souligner l’importance de développer des normes de contrôle de qualité pour les analyses faites sur l’ADN fœtal libre. Nous avons aussi étudié les modifications de l’ADN fœtal libre après une mort fœtale en raison d’une aneuploïdie chez l’un des deux fœtus lors d’une grossesse gémellaire. Après le décès du fœtus atteint, la fraction fœtale de l’ADN libre circulant dans le sang maternel a montré une évolution imprévisible, pouvant augmenter, diminuer ou rester stable dans le temps. Par conséquent, ces résultats déconseillent l’utilisation du DPNI en cas de décès d’un des deux fœtus, même après un intervalle de temps de plusieurs semaines.Notre attention s’est ensuite portée vers la performance du DPNI pour le dépistage des anomalies autres que les 3 trisomies communes. Nous avons d’abord étudié la performance du DPNI pour les anomalies des chromosomes sexuels ainsi que les caractéristiques des patientes optant pour ce test. Plus de la moitié des patientes ayant eu un DPNI ont également souhaité le dépistage des anomalies des chromosomes sexuels. Les valeurs prédictives positives suivantes ont été observées :24% pour 45 X et 47 XXX et environ 71% pour 47 XXY et 47 XYY. Ainsi, la recherche d’anomalies des chromosomes sexuels peut causer la détection accidentelle d’anomalies chromosomiques sans conséquence clinique. Par conséquent, un conseil génétique est obligatoire dans toutes ces situations, de même qu’un examen invasif pour un caryotype fœtal de confirmation.Ensuite, nous avons montré que le test à ADN fœtal libre utilisant une technologie ciblée basée sur le microarray permettait d'identifier les grossesses à risque accru de délétions 22q11.2. Cependant, des données fiables sur les performances du DPNI pour le syndrome 22q11.2 sont encore manquantes, et des recherches plus poussées sont nécessaires. Depuis 2015, nous participons à une étude prospective, multicentrique et en aveugle, qui évalue cliniquement un test d’ADN fœtal libre pour la détection de délétions ou de duplications dans la région 22q11. Le recrutement s’est terminé le 1er novembre 2019.Enfin, en décrivant le profil et le choix des patientes belges soumises à un test à ADN fœtal libre, nous avons observé un changement vers une population à faible risque, ce qui peut entrainer une réduction de la valeur prédictive positive du test. Il est donc primordial que cet effet soit connu des professionnels de la santé qui conseillent, prescrivent et interprètent ces tests.En conclusion, notre recherche a démontré la complexité des tests à ADN fœtal libre circulant dans le sang maternel, non seulement du point de vue technique, mais également en termes de conseils aux patients avant et après le test, ce qui requière des connaissances et compétences spécifiques des médecins proposant ces tests.L’ère du test à ADN fœtal libre circulant dans le sang maternel n’en est qu’à ses débuts. Notre travail n’a exploré qu’une petite partie de l’énorme potentiel de ce nouvel outil de dépistage des aneuploïdies.L’intégration responsable des innovations dans la pratique clinique reste, aujourd’hui, l’un des défis majeurs.
Doctorat en Sciences médicales (Médecine)
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Alterations of CDKL5 give rise to several forms of neurological disorders generally characterised by epileptic encephalopathy, severe developmental delay, hypotonia and RTT-like features. To date no cure exists and only secondary symptoms can be treated. About 15% of CDKL5 patients carry a nonsense mutation and might benefit of a readthrough strategy as “personalised” medicine approach. The read-through process occurs when a near-cognate aminoacyl-tRNA binds a premature stop codon (PTC), allowing its suppression and the subsequent protein elongation. This mispairing event can rarely occur, but can be facilitated using a wide range of drugs. In order to test PTC suppression, we have chosen some human pathogenic CDKL5 nonsense mutations located in the two main domains of the protein: the catalytic N-terminus (R59X, R134X) or the C-terminal tail (Q347X, E364X, R550X, S855X). We then evaluated the read-through process using aminoglycoside and non-aminoglycoside drugs in cells transfected with the mutagenized constructs. In this study, we have demonstrated that tested CDKL5 PTCs can be suppressed by gentamicin and geneticin (G418) in a dose-dependent manner and that PTC position can be critical for read-through. In particular, G418 was found to be more effective than gentamicin. Considering the known aminoglycosides toxicity, we evaluated the activity of PTC124 and GJ072 but no PTC suppression was detectable in our experimental conditions. Finally, in order to understand whether the full-length derivatives may maintain the proper function of WT CDKL5, we analysed some features of read-through products compared to the WT protein. In particular, while premature truncated proteins showed an altered subcellular localisation, read-through products demonstrated a nucleo-cytoplasmic distribution more similar to the WT one. Moreover, by evaluating the auto-phosphorylation of the TEY motif, the read-through derivatives demonstrated to recover some catalytic activity, although remaining highly hypomorphic. Nevertheless, preliminary studies on Cdkl5-null neurons transfected with R134X construct suggested that G418 treatment can ameliorate impaired neuronal morphology. Collectively, our results indicate that: (i) aminoglycosides are able to induce read-through of different CDKL5 PTCs; (ii) the read-through derivatives recover some features characterizing the WT protein; (iii) PTC position can be crucial for read-though and for rescue of a proper function and (iv) neuronal morphological defects might be rescued by small amount of a possible hypomorphic CDKL5, therefore supporting the potential validity of a read-through therapy.

An acceptable agreement permits interchangeability of the instruments. For this purpose, we have investigated the agreement of several clinical instruments frequently used in clinical practice with their laboratory counterpart. We have estimated the agreement between a point-of-care blood gas analyzer (i-Stat, Abaxis) and a bench-top blood gas analyzer (Nova, Biomedical) in venous samples from Hermann’s tortoises. We have estimated the agreement between a point-of-care chemistry analyzer (VetScan VS2, Abaxis) and a laboratory analyzer (Olympus AU400, Olympus Co.) in venous samples from Hermann’s tortoises. We have estimated the agreement between portable blood glucose meters (Accu-Chek, Aviva; AlphaTrak 2, Abbott) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. We have estimated the agreement between point-of-care bench-top glucose measurement (VetScan VS2, Abaxis) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. Beyond method comparison and validation, reference interval determination for common laboratory testing is required to allow the clinician to discriminate individuals that are different from the remaining population for a certain parameter. We have calculated reference intervals for blood gas in Hermann’s tortoises. We have calculated reference intervals for protein electrophoresis in Hermann’s tortoises. We have described normal hematology in Hermann’s tortoises. We have calculated reference intervals for clinical chemistry in Hermann’s tortoises. We have calculated reference intervals for aldosterone in ferrets. Based on our results, animal species requires individual validation of laboratory methods and reference intervals. Lack of consideration of these findings may result in clinical misdiagnosis and improper treatment of animals.

An acceptable agreement permits interchangeability of the instruments. For this purpose, we have investigated the agreement of several clinical instruments frequently used in clinical practice with their laboratory counterpart. We have estimated the agreement between a point-of-care blood gas analyzer (i-Stat, Abaxis) and a bench-top blood gas analyzer (Nova, Biomedical) in venous samples from Hermann’s tortoises. We have estimated the agreement between a point-of-care chemistry analyzer (VetScan VS2, Abaxis) and a laboratory analyzer (Olympus AU400, Olympus Co.) in venous samples from Hermann’s tortoises. We have estimated the agreement between portable blood glucose meters (Accu-Chek, Aviva; AlphaTrak 2, Abbott) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. We have estimated the agreement between point-of-care bench-top glucose measurement (VetScan VS2, Abaxis) and a laboratory analyzer (Dimension EXL, Siemens) in venous samples from client-owned rabbits. Beyond method comparison and validation, reference interval determination for common laboratory testing is required to allow the clinician to discriminate individuals that are different from the remaining population for a certain parameter. We have calculated reference intervals for blood gas in Hermann’s tortoises. We have calculated reference intervals for protein electrophoresis in Hermann’s tortoises. We have described normal hematology in Hermann’s tortoises. We have calculated reference intervals for clinical chemistry in Hermann’s tortoises. We have calculated reference intervals for aldosterone in ferrets. Based on our results, animal species requires individual validation of laboratory methods and reference intervals. Lack of consideration of these findings may result in clinical misdiagnosis and improper treatment of animals.

Two Amerindian populations from the Peruvian Amazon (Yanesha) and from rural lowlands of the Argentinean Gran Chaco (Wichi) were analyzed. They represent two case study of the South American genetic variability. The Yanesha represent a model of population isolated for long-time in the Amazon rainforest, characterized by environmental and altitudinal stratifications. The Wichi represent a model of population living in an area recently colonized by European populations (the Criollos are the population of the admixed descendents), whose aim is to depict the native ancestral gene pool and the degree of admixture, in relation to the very high prevalence of Chagas disease. The methods used for the genotyping are common, concerning the Y chromosome markers (male lineage) and the mitochondrial markers (maternal lineage). The determination of the phylogeographic diagnostic polymorphisms was carried out by the classical techniques of PCR, restriction enzymes, sequencing and specific mini-sequencing. New method for the detection of the protozoa Trypanosoma cruzi was developed by means of the nested PCR. The main results show patterns of genetic stratification in Yanesha forest communities, referable to different migrations at different times, estimated by Bayesian analyses. In particular Yanesha were considered as a population of transition between the Amazon basin and the Andean Cordillera, evaluating the potential migration routes and the separation of clusters of community in relation to different genetic bio-ancestry. As the Wichi, the gene pool analyzed appears clearly differentiated by the admixed sympatric Criollos, due to strict social practices (deeply analyzed with the support of cultural anthropological tools) that have preserved the native identity at a diachronic level. A pattern of distribution of the seropositivity in relation to the different phylogenetic lineages (the adaptation in evolutionary terms) does not appear, neither Amerindian nor European, but in relation to environmental and living conditions of the two distinct subpopulations.

Two Amerindian populations from the Peruvian Amazon (Yanesha) and from rural lowlands of the Argentinean Gran Chaco (Wichi) were analyzed. They represent two case study of the South American genetic variability. The Yanesha represent a model of population isolated for long-time in the Amazon rainforest, characterized by environmental and altitudinal stratifications. The Wichi represent a model of population living in an area recently colonized by European populations (the Criollos are the population of the admixed descendents), whose aim is to depict the native ancestral gene pool and the degree of admixture, in relation to the very high prevalence of Chagas disease. The methods used for the genotyping are common, concerning the Y chromosome markers (male lineage) and the mitochondrial markers (maternal lineage). The determination of the phylogeographic diagnostic polymorphisms was carried out by the classical techniques of PCR, restriction enzymes, sequencing and specific mini-sequencing. New method for the detection of the protozoa Trypanosoma cruzi was developed by means of the nested PCR. The main results show patterns of genetic stratification in Yanesha forest communities, referable to different migrations at different times, estimated by Bayesian analyses. In particular Yanesha were considered as a population of transition between the Amazon basin and the Andean Cordillera, evaluating the potential migration routes and the separation of clusters of community in relation to different genetic bio-ancestry. As the Wichi, the gene pool analyzed appears clearly differentiated by the admixed sympatric Criollos, due to strict social practices (deeply analyzed with the support of cultural anthropological tools) that have preserved the native identity at a diachronic level. A pattern of distribution of the seropositivity in relation to the different phylogenetic lineages (the adaptation in evolutionary terms) does not appear, neither Amerindian nor European, but in relation to environmental and living conditions of the two distinct subpopulations.

Stem Cells are rare cells with the crucial ability to self-renew and to generate mature cells of any tissue through differentiation. Adult stem cells hold great promise for regenerative medicine, tissue repair, and gene therapy. Adult bone marrow cells (BMCs) include two populations of bone marrow stem cells (BMCs): hematopoietic stem cells (HSCs), which give rise to all mature lineages of blood, and mesenchymal stem cells (MSCs), which can differentiate into osteoblasts, chondrocytes, adipocytes, myocytes, tenocytes, and haematopoiesis supporting stromal cells. Under normal condition these stem cells are tightly regulated by both intrinsic and extrinsic signals and malfunctioning in this balance can result in cancer. In this thesis we focused on two different aspects of stem cells: the leukemia stem/initiating cells in acute myeloid leukemia (AML) and the usage of stem cells in regenerative medicine. In the first part we focused on the molecular mechanism of AML-ETO, a results from the t(8:21) translocation which has been associated with leukemic transformation. Acute myeloid leukaemia (AML) is defined as a heterogeneous group of clonal disorders caused by malignant transformation of a bone marrow-derived self-renewing stem or progenitor cell, which demonstrates an enhanced proliferation as well as aberrant differentiation resulting in haematopoietic insufficiency (i.e. granulocytopenia, thrombocytopenia or anaemia). These leukaemias are suggested to result from the acquisition of chromosomal rearrangements and multiple gene mutations in either a hematopoietic multipotent cell or a more differentiated, lineage-restricted progenitor cell that is transformed in a so-called leukaemic stem or initiating cell, which keeps the ability to self-renewal. AML is generally regarded as a stem cell disease and is commonly altered both at the epigenetic as well as the genetic level. AML is the most common acute leukemia affecting adults, and its incidence increases with age. Therapies based on the current knowledge target the bulk leukemic population and spare the leukemic stem cells. It is therefore critical to determine and characterize the exact molecular mechanism involved in leukemic transformation for the development of novel therapeutic targets. AML patients harboring the t(8:21) translocation has intermediate prognosis and the identification of genome wide events in this subset of AML is clinically relevant and would lead to the understanding of molecular mechanism of disease progression. To this end we analyzed the DNA binding pattern of AML1-ETO in AML cell lines and in primary AML blasts. We demonstrate that AML1-ETO preferentially binds regions that contain RUNX1/AML1 and ETS core consensus sequences and that the AML1-ETO binding sites invariably consist of HEB and partially CBFβ, RUNX1/AML1 as well as of ETS factors such as ERG and FLI1. Subsequent analysis in t(8;21) and t(15;17) (another AML associated translocation) cells revealed cell type specific ETS factor binding and preferential AML1-ETO binding to the cell type specific ETS factor binding sites. In addition, we uncovered that binding of the ETS factor ERG correlates with the ‘active’ histone acetylation mark. Together our results suggest that ETS factors demarcate hematopoietic regulatory sites that provide a target for (aberrant) epigenetic regulation by oncofusion proteins. In the second part we attempted to evaluate the possibility to obtain in vitro an implantable tissue-engineered esophagus composed of acellular esophageal matrix and Mesenchymal stem cells (MSCs). Mesenchymal Stem Cells (MSCs) are multipotent precursors to many mesodermal cell lineages in vertebrate animals and are most often obtained from bone marrow. Certain attributes of MSCs, including migration toward sites of inflammation, ease of transduction, and lack of immunogenicity, suggest these cells may be potentially useful for regenerative medicine. Putative therapeutic uses include regeneration of damaged tissue, acting as a vessel for delivering a therapeutic transgene, support of other cell types for tissue repair, and modulating the immune reaction to co-transplanted cells or tissues. The use of MSCs in tissue engineering approaches avoids the moral and technical issues associated with the use of those from embryonic source and MSCs have already demonstrated their efficacy in preliminary tissue engineering application. Artificial materials and autologous tissues used for esophageal reconstruction often induce complications like stenosis and leakage at long-term follow-up. In the present study we attempted to evaluate the adhesion of MSCs on acellular esophageal matrix for esophagus tissue engineering. MSCs were isolated from rabbit bone marrow, characterized, expanded in vitro, and seeded onto rabbit acellular esophageal matrix. Acellular matrices obtained by detergent-enzymatic method did not present any major histocompatibility complex marker. Moreover, they supported cell adhesion, and in as much as just after 24 h from seeding, the scaffold appeared completely covered by MSCs in static as well as in bioreactor. Collectively, these results suggest that patches composed of homologous esophageal acellular matrix and autologous MSCs may represent a promising tissue engineering approach for the repair of esophageal injuries
Le cellule staminali sono una popolazione cellulare con la particolare capacità di moltiplicarsi indefinitamente autorinnovandosi e di differenziarsi in cellule mature di qualsiasi altro tessuto attraverso il processo di differenziazione. In particolare l'utilizzo delle cellule staminali adulte costituisce una promettente applicazione nel campo della medicina rigenerativa, la riparazione dei tessuti e la terapia genica. Le cellule staminali adulte da midollo osseo (BMCs) comprendono due popolazioni cellulari: le cellule staminali ematopoietiche (HSCs), dalle quali originano tutte le cellule mature del sangue, e le cellule staminali mesenchimali (MSCs) che possono differenziare in osteoblasti, condrociti, adipociti, miociti, tenociti e cellule stromali di supporto per l'ematopoiesi. In condizioni normali l'autorinnovamento della popolazione staminale è strettamente regolato sia da segnali estrinseci che intrinseci ed un'alterazione di questo equilibrio può portare all'instaurarsi di un cancro. In questa tesi abbiamo analizzato due differenti aspetti delle cellule staminali: le cellule staminali che danno origine a leucemia nella leucemia mieloide acuta (AML) e l'utilizzo delle cellule staminali nella medicina rigenerativa. Nella prima parte del lavoro abbiamo approfondito il meccanismo molecolare dell' AML-ETO, risultato della traslocazione genica t(8:21) che viene associata alla trasformazione leucemica. La leucemia mieloide acuta (AML) è definita come un gruppo eterogeneo di disordini clonali causati dalla trasformazione maligna di cellule staminali o progenitori staminali di derivazione midollare, che mostrano un aumento della capacità proliferativa così come un differenziamento aberrante che porta ad una insufficienza ematopoietica (per esempio: granulocitopenia, trombocitopenia o anemia). Questi tipi di leucemia sembrano essere il risultato dell'acquisizione di riarrangiamenti cromosomici e mutazioni geniche multiple da parte delle cellule ematopoietiche multipotenti o di progenitori cellulari più differenziati e indirizzati verso una linea cellulare specifica, che risultano così trasformati in cellule staminali leucemiche o cellule inizianti la leucemia, che mantengono la capacità di autorinnovamento. L' AML è solitamente considerata una malattia delle cellule staminali e comunemente presenta alterazioni sia a livello genetico che epigenetico. L' AML è la forma più comune di leucemia acuta che colpisce soprattutto la popolazione adulta e la sua incidenza aumenta con l'età. Gli attuali approcci terapeutici hanno come target le cellule staminali leucemiche e la popolazione leucemica per intero. E' quindi di cruciale importanza riuscire a determinare e caratterizzare l'esatto meccanismo molecolare coinvolto nella trasformazione leucemica per lo sviluppo di nuovi bersagli terapeutici. I pazienti affetti da AML che manifestano la traslocazione t(8:21) hanno una prognosi intermedia e l'identificazione di ampi eventi genici in questo subset delle AML è clinicamente rilevante in quanto potrebbe portare alla comprensione dei meccanismi molecolari della progressione della malattia. A questo scopo sono stati analizzati i pattern di legame al DNA di AML1-ETO nelle cellule di linea AML e nei blasti di AML. Abbiamo dimostrato che AML1-ETO lega preferenzialmente le regioni che contengono le sequenze di consenso RUNX1/AML1 e ETS e che i siti di legame di AML1-ETO si sovrappongono invariabilmente a quelli di HEB e parzialmente a quelli di CBFβ, RUNX1/AML1 così come accade per i fattori ETS, quali ERG e FLI1. Le successive analisi sulle cellule t(8;21) e t(15;17) (un'altra traslocazione associata con l' AML) hanno evidenziato il legame di fattori ETS specifici per questi tipi cellulari e il legame preferenziale di AML1-ETO ai siti di legame per i fattori ETS specifici per il tipo cellulare. Inoltre è stato anche scoperto che il legame di un fattore ETS, ERG, correla con un segnale di acetilazione istonica "attiva". Presi insieme questi risultati suggeriscono che i fattori ETS demarcano i siti regolatori ematopoietici che forniscono un target per la regolazione epigenetica (aberrante) da parte delle proteine di oncofusione. Nella seconda parte di questa tesi è stata testata la possibilità di ottenere in vitro un esofago ingegnerizzato composto da matrice acellulare esofagea e cellule staminali mesenchimali (MSCs) che potesse essere impiantato in vivo. Le cellule staminali mesenchimali (MSCs) nei vertebrati sono precursori multipotenti di molte linee cellulari di origine mesodermica e vengono ottenute per la maggior parte dal midollo osseo. Alcune caratteristiche delle MSCs, inclusa la capacità di migrare verso i siti di infiammazione, la facilità di trasduzione e la perdita di immunogenicità, suggeriscono che queste cellule possano essere potenzialmente utilizzabili nella medicina rigenerativa. I probabili usi terapeutici includono la possibilità di rigenerare un tessuto danneggiato, agendo come veicolo per il trasporto di transgeni terapeutici, di supportare altri tipi cellulari per il riparo tessutale, e di modulare la reazione immunitaria dell'ospite nei confronti delle cellule o dei tessuti co-trapiantati. L'uso delle MSCs permette di evitare i problemi di natura etica e morale associati all'utilizzo delle cellule staminali di origine embrionale; inoltre le MSCs hanno già dimostrato la loro efficacia in studi preliminari che prevedevano la loro applicazione in ingegneria tessutale. I materiali artificiali e i tessuti autologhi utilizzati per la ricostruzione dell'esofago spesso comportano complicazioni come stenosi e rottura dell'impianto nei follow-up a lungo termine. Nel presente studio è stata valutata l'adesione delle MSCs ad una matrice acellulare di esofago per la costruzione di un tessuto esofageo ingegnerizzato. Le MSCs sono state isolate da midollo osseo di coniglio, caratterizzate, espanse in vitro e seminate su una matrice esofagea di coniglio. Le matrici acellulari ottenute attraverso un metodo detergente-enzimatico non presentavano marker per il complesso maggiore di istocompatibilità. Inoltre supportavano l'adesione cellulare e in non più di 24 ore dalla semina lo scaffold appariva completamente coperto dalle MSCs sia in condizione statica che in bioreattore. Complessivamente questi risultati suggeriscono che i tessuti ingegnerizzati composti da matrice acellulare omologa e MSCs autologhe possono rappresentare un promettente approccio per il riparo di danni all'esofago

Achieving complete health requires a deep understanding of complementary cultural competency sensitivity between physician and patient. This may include but is not limited to access to preventative health care resources, access to health educational resources and access to cultural healing resources, for example, shamans, Ayurvedic physicians, and herbal healers. Advocates of cultural competency emphasize great importance on knowledge of the patients' cultural background; however, the transcendence of this knowledge can be explained further through complementary cultural competency sensitivity. This is when the cultures of the physician and patient complement each other in terms of understanding what is in the patients' best interest in the overall goal of healing and complete health for the patient. The explanation of this concept revolves around the idea that health is not just found within body wellness physically, but also mentally and emotionally. The tragedies of poor health outcomes we face have psychological repercussions with a significant social determinant that bio-medical medication cannot and should not solve. The purpose of research includes theoretical discussions that address questions of: What roles do Evidence Based Results play for Medical Anthropologists? How will having knowledge of socioeconomic status, cultural practices and determinants of environmental insult and structural violence as experienced by the individual patient influence the facilitation of the process of creating a positive health outcome for the patient? How can "End of Life" issues be better addressed? How does language influence health? Does a positive dialogue between health professionals and patients contribute to better health outcomes? Research will emphasize the idea that Ethnomedicine (traditional medicine) and Western Bio-medicine complement each other within the model of complementary cultural competency sensitivity. The Holistic Complementary Structure of Western Bio-medicine and Traditional Healing is a multifaceted mean by which the manifestations of complete and positive health results occur. The methods of research used in the research include ethnographic interview content discussions, primary and secondary literature sources, and research of bio-statistical data. The interview discussions consist of dialogue with Medical Anthropologists, a Nurse Practitioner, a Global Health Studies Ph.D. professor and an Africana Studies Ph.D. professor. In order to prove the hypothesis, explanations through examples of Ethnomedicine (traditional medicine) and Western Bio-medicine working together, show how the combination of the two modalities along with the factors of complementary cultural competency sensitivity between patient and physician contribute to positive health outcomes.

The master's thesis is devoted to extracting the bioactive fractions of Speranskia tuberculata (Bunge) Baill and purifying the extracts by isolation. The extracts obtained are used in cellular experiments to explore their positive effects in preventing and treating atherosclerosis, liver cancer, cervical cancer, and melanoma. This research verifies that the active compounds in Speranskia tuberculata (Bunge) Baill have antiatherosclerotic and therapeutic effects on some cancers. The crude extracts of ethyl acetate had a significant ability to kill tumour cells. It was determined that the crude extracts of petroleum ether (PE) effectively inhibited the transition from macrophages to foam cells by the traditional method of labelling foam cells with neutral fat. The uptake and efflux of cholesterol by macrophages were characterized by fluorescently labelling cholesterol flow. The results showed that PE minimized cholesterol deposition in macrophages by inhibiting macrophage phagocytosis of cholesterol while promoting the dual effect of intracellular cholesterol efflux.
Магістерська робота присвячена екстрагуванню біоактивних фракцій Speranskia tuberculata (Bunge) Baill та очищенню екстрактів шляхом виділення. Отримані екстракти використовуються в клітинних експериментах для дослідження їх позитивного впливу на профілактику та лікування атеросклерозу, раку печінки, раку шийки матки та меланоми. Це дослідження підтверджує, що активні сполуки Speranskia tuberculata (Bunge) Baill мають антиатеросклеротичний та терапевтичний вплив на деякі види раку. Неочищені екстракти етилацетату мали значну здатність вбивати пухлинні клітини. Встановлено, що неочищені екстракти петролейного ефіру ефективно інгібують перехід від макрофагів до пінистих клітин традиційним методом мічення пінистих клітин нейтральним жиром. Поглинання та відтік холестерину макрофагами характеризувався флуоресцентним міченим потоком холестерину. Результати показали, що екстракт у петролейному ефірі мінімізував відкладення холестерину в макрофагах, пригнічуючи макрофагальний фагоцитоз холестерину, одночасно сприяючи подвійному ефекту внутрішньоклітинного витоку холестерину.
Магистерская работа посвящена экстрагированию биоактивных фракций Speranskia tuberculata (Bunge) Baill и очистке экстрактов путем выделения. Полученные экстракты используются в клеточных экспериментах для исследования их положительного воздействия на профилактику и лечение атеросклероза, рака печени, рака шейки матки и меланомы. Это исследование подтверждает, что активные соединения Speranskia tuberculata (Bunge) Baill оказывают антиатеросклеротическое и терапевтическое влияние на некоторые виды рака. Неочищенные экстракты этилацетата обладали значительной способностью убивать опухолевые клетки. Установлено, что неочищенные экстракты петролейного эфира эффективно ингибируют переход от макрофагов к пенистым клеткам традиционным методом мечения пенистых клеток нейтральным жиром. Поглощение и отток холестерина макрофагами характеризовался флуоресцентным меченым потоком холестерина. Результаты показали, что экстракт в петролейном эфире минимизировал отложения холестерина в макрофагах, подавляя макрофагальный фагоцитоз холестерина, одновременно способствуя двойному эффекту внутриклеточной утечки холестерина.

In this dissertation, we aimed to bring together a team of clinical experts, translational researchers, biostaticians and bioinformaticians to develop and implement innovative scientific methodologies in precision medicine applied to High Grade Serous Ovarian Cancer (HGS OvCa). We used a variety of translational and computational methods in order to generate impactful outcomes. These pipelines produced statistically robust results, with particular emphasis on drawing clinical and biological correlations. The results presented here contribute to the body of evidence necessary to substantiate these findings in a clinical setting. Bioassays, PDX models and ancillary specimen evaluation of previous clinical trials will help to validate our candidate biomarkers. Enhanced understanding of the molecular pathology of disease grounded in acquisition of genomic knowledge will facilitate the development of targeted treatment in cancer. Because clinical trials must be developed with correct metrics, patient selection and drug efficacy should incorporate adaptive designs.
Doctorat en Sciences médicales (Santé Publique)
info:eu-repo/semantics/nonPublished

The goal of this thesis is the discovery of a bioinformatics solution for network-based predictive analysis of NGS data, in which network structures can substitute gene lists as a more rich and complex signature of disease. I have focused on methods for network stability, network inference and network comparison, as additional components of the pipeline and as methods to detects outliers in high-throughput datasets. Besides a first work on GEO datasets, the main application of my pipeline has been on original data from the FDA SEQC (Sequencing Quality Control)project. Here I will report some initial findings to which I have contributed with methods and analysis: as the corresponding papers are being submitted. My goal is to provide a comprehensive tool for network reconstruction and network comparison as an R package and user-friendly web service interface available on-line at https://renette.fbk.eu The goal of this thesis is the discovery of a bioinformatics solution for network-based predictive analysis of NGS data, in which network structures can substitute gene lists as a more rich and complex signature of disease. I have focused on methods for network stability, network inference and network comparison, as additional components of the pipeline and as methods to detects outliers in high-throughput datasets. Besides a first work on GEO datasets, the main application of my pipeline has been on original data from the FDA SEQC (Sequencing Quality Control)project. Here I will report some initial findings to which I have contributed with methods and analysis: as the corresponding papers are being submitted. My goal is to provide a comprehensive tool for network reconstruction and network comparison as an R package and user-friendly web service interface available on-line at https://renette.fbk.eu.

My research activity, carried out during the period of the doctorate, concerned the development of specific bio-sensing platforms to be used for applications in sport medicine and in real-time environmental monitoring. During the first year of my doctorate I developed a disposable biosensor for lactate determination in saliva. The biosensor has been evaluated in terms of analytical performance and then tested with real saliva specimens. I also proposed a new, simple and effective protocol for saliva sample pretreatment which could be used for measurements outside the laboratory. The lactate biosensor was also compared to a commercial portable lactate analyzer, demonstrating a good correlation. This study was published in SENSORS AND ACTUATORS B, CHEMICAL, ISSN: 0925-4005. Throughout the second year I was engaged in the development of a biosensing assay for the detection of microcystins (MCs). This study makes part of a national project called "Acquasense" (Ministero per lo Sviluppo Economico, Industria 2015, Bando Nuove Tecnologie per il Made in Italy), which deals with the determination of chemical pollutants in drinking water, including MCs. Based on the purposes of the project for a simple and sensitive assay, I proposed an enzyme inhibition colorimetric method that uses the protein phosphatase type 2A (PP2A) as recognition element. The colorimetric assay has been applied in surface water, drinking water and a culture medium selective for cyanobacterial growth, with consequent release of MCs. The results obtained were compared to a standard chromatographic technique (UHPLC-DAD). This research, made in collaboration with the Departments of Biology and Inorganic Chemistry of the University of Rome, “Tor Vergata”, was the subject of two publications: BIOSENSORS & BIOELECTRONICS, ISSN: 0956-5663 and PROCEDIA ENGINEERING, ISSN: 1877-7058. From the half of the second year until the end of the third year of my doctorate, I have also been involved in a European project entitled “Sensing toxicants research in Marine waters make Sense using biosensors”, whose acronym is SMS and deals with the determination of marine water contaminants in coastal areas, including algal toxins. My concern was to develop novel sensing platforms, to be integrated in sensing devices, for the determination of two marine toxins, Okadaic Acid (OA) and Domoic Acid (DA), directly in marine water. My original idea was to develop an aptamer-based sensor for OA detection. To do this, I functionalized and tested different specific aptamers for OA. Unfortunately, none of these aptameric sequences demonstrated high affinity for its target. For this reason, I had to relinquish the idea to use aptamers as recognition elements to detect low levels of the mentioned toxins. Considering that OA is a potent inhibitor of the enzyme PP2A, I proposed the same colorimetric method, mentioned above, for its determination. After applying the assay in different seawater samples, I have been actively participated in its integration within a miniaturized automated apparatus (prototype colorimetric device) able to perform real-time monitoring of the toxin. The prototype device was tested and compared to the non-automated enzymatic assay. The results correlate well. As for the detection of DA, I focused my attention on the development of a competitive ELIMC (Enzyme-Linked Immuno-Magnetic Colorimetric) assay that uses antibodies as recognition elements. I proposed a format based on a single step competition procedure, which guarantees a short incubation time, combined with a colorimetric detection. The integration of the ELIMC assay in an automated apparatus and recovery experiments are still in progress.

2008/2009
La medicina rigenerativa applicata al campo ortopedico è considerata una possibile opzione terapeutica per la riparazione del tessuto osseo danneggiato. Si stanno studiano e sviluppando una gran varietà di sostituti ossei sintetici come valida alternativa agli innesti di tipo tissutale. Lo scopo di questo lavoro di tesi è la progettazione e lo sviluppo di un materiale iniettabile per il riempimento dei difetti ossei. In particolare abbiamo sviluppato un riempitivo composito iniettabile usando materiali biodegradabili di tipo polisaccaridico funzionalizzati con elementi bioattivi come mediatori dell’adesione cellulare, fattori di crescita osteoinduttivi e peptidi di tipo antimicrobico. Il costrutto finale è un composito a due fasi, le cui parti, inizialmente, sono state sviluppate separatamente. La struttura è stata progettata come composta da una parte bioattiva, costituita da microsfere disidratate di alginato e idrossiapatite recanti peptidi, immersa in una matrice veicolante rappresentata da una soluzione concentrata di acido ialuronico. Con lo scopo di ottenere le microsfere bioattive, sono state esplorate diverse tecniche per la coniugazione peptide-polisaccaride e sono stati presi in considerazione svariati sistemi di rilascio di sequenze peptidiche. In particolare sono stati considerati tre peptidi noti per la loro bioattività: peptidi di tipo RGD, sequenza favorente l’adesione cellulare, un frammento della proteina BMP-2 (bone morphogenetic protein-2) in grado di promuovere il differenziamento di cellule mesenchimali ad osteoblasti e LL-37 peptide antimicrobico umano. Per ottenere un costrutto con proprietà bioadesive, gli sforzi sono stati volti ad un miglioramento dell’interfaccia fra le sfere di alginato/idrossiapatite e il tessuto osseo. Questo è stato possibile immobilizzando sulla superficie delle sfere dei peptidi contenti la sequenza RGD e assicurandone il gusto orientamento ed un’alta resa di immobilizzazione. Dopo aver testato diverse strategie chimiche, l’immobilizzazione effettuata sfruttando la formazione di un ponte disolfuro fra ChitLac preventivamente modificato con gruppi tiolici e un peptide contenente un residuo di cisteina, è stata valutata come la miglior strategia in termini di resa di reazione; allo stesso tempo le microsfere funzionalizzate in questo modo hanno dimostrato un’alta capacità di promuovere l’adesione e la crescita di osteoblasti in esperimenti effettuati in vitro. Un altro importante tema ha riguardato l’incorporazione di un frammento della proteina BMP-2 nel costrutto al fine di promuovere il differenziamento e quindi la proliferazione cellulare. La sequenza temporale degli eventi fisiologici, ha suggerito lo sviluppo di un sistema che risulta essere la somma di due diverse strategie di rilascio: la prima caratterizzata dal semplice intrappolamento del peptide in un sistema di sfere di alginato capace di un rilascio veloce, ed il secondo caratterizzato da un rilascio lento e costante ottenuto grazie all’azione idrolitica di esterasi. Questo è stato possibile inserendo un legame enzimaticamente idrolizzabile nella struttura contenente il frammento di BMP. Questa seconda strategia ha sfruttato una chimica altamente selettiva quale la “click chemistry”. La sintesi della molecola spaziatrice recante un legame enzimaticamente idrolizzabile è stata effettuata partendo da un γ-valerolattone. Questo spaziatore è stato inoltre progettato per recare un gruppo terminale funzionale adeguato per le reazioni di click chemistry ed è stato legato al ChitLac. La reazione di cicloaddizone è stata poi effettuata fra il ChitLac funzionalizzato con lo spaziatore e il frammento di BMP anch’esso opportunamente funzionalizzato. L’idrolisi enzimatica è stata verificata, inoltre è stata eseguita un’esaustiva caratterizzazione tramite NMR ed elettroforesi capillare. E’ stata poi presa in considerazione l’incorporazione di peptidi antimicrobici nel costrutto. Partendo da dati di dicroismo circolare riguardanti l’influenza che alcuni polisaccaridi hanno sulla conformazione del peptide LL-37 in soluzione, è stata riconosciuta alla miscela LL- 37/alginato la capacità di modulare la citotossicità del peptide. La miscela ha inoltre dimostrato la capacità di mantenere l’attività antimicrobica su batteri Gram negativi e questo ci ha spinto a considerarne l’applicazione su costrutti solidi con finalità ortopediche. In quest’ambito è stato escluso, da esperimenti in vitro, un effetto causato dal semplice contatto fra cellule o batteri e una superficie di alginato caricato col peptide. L’unico possibile meccanismo di rilascio riscontrato in un costrutto di alginato/LL-37 è stato tramite la degradazione di questo. Infine, il costrutto è stato assemblato incorporando le microsfere disidratate in una soluzione concentrata di acido ialuronico, ottenendo un costrutto dall’aspetto simile ad una pasta che è in grado di facilitare il processo di iniezione del riempitivo. Misure di tipo reologico hanno indicato in incremento della componente viscoelastica aggiungendo il particolato nella soluzione di acido ialuronico, questo generalmente è associato ad una buona capacità di recuperare l’elasticità dopo l’iniezione il che è un requisito richiesto ai biomateriali usati come riempitivi ossei. I risultati ottenuti hanno dimostrato l’applicabilità del composito come sostituto osseo dotato di proprietà bioattivite (osteoconduzione, osteoinduzione, bioadesività ed effetto antimicrobico su ceppi di tipo Gram negativo).
Bone regenerative medicine is considered a potential therapeutic option for the healing of damaged bone tissue. A variety of synthetic bone graft substitutes have been investigated as alternative to current tissue based bone graft materials. In this study efforts have been made to achieve an injectable material to fill bone defects. We have designed composite injectable filler by using polysaccharides as biodegradable materials, and by functionalizing them with bioactive elements such as adhesion mediators, osteoinductive growth factors and anti-microbial peptides. The final construct is represented by a two-phase composite, whose parts have been initially built separately. The structure was thought as composed by bioactive alginate/hydroxyapatite dried microbeads functionalized with peptides, suspended in a concentrate hyaluronic acid solution as the matrix vehicle. With the aim to obtain bioactive alginate/HAp beads, different techniques of peptides-polysaccharides conjugation were considered and different peptide delivery systems were explored. In detail the peptides considered were three, the RGD active sequence with bioadhesive properties, a fragment of BMP-2 (bone morphogenetic protein-2) that promotes the differentiation of MSCs into osteoblasts, and LL-37 a human antimicrobial peptide. In order to obtain a construct with bioadhesive properties, efforts have been made to improve the tissue-alginate/HAp beads interface by immobilizing RGD peptide sequence in a manner that assure the right motif orientation and high yields. After having tested various chemical strategies, the immobilization by disulfide bridge formation between a ChitLac previously modified with a thiol group and a peptide containing a cysteine residue was evaluated to be the best in terms of yield; at the same time μbeads functionalized in that way exhibit a very high osteoblast adhesion and growth promotion in in vitro experiments. Another important topic regarded the incorporation of BMP-2 epitope fragment into the construct, to enhance differentiation and proliferation. Time-tuned physiological functionality suggested the development of a system being the sum of two delivery strategies: the first characterized by simple entrapment in alginate beads for a fast burst release and the second one giving a slow and constant release, obtained by the action of bone esterases on an enzymatically cleavable linker. This second strategy exploited a high selective chemistry such as that of click reactions. Starting by a γ-valerolactone the synthesis of the enzymatically cleavable spacer was performed. The linker was designed to contain a final functional group for click chemistry. It was bound to ChitLac and then to a modified BMP fragment by a click reaction. Enzymatic cleavage was verified and an exhaustively characterization by means of NMR and capillary electrophoresis was performed. The incorporation of antimicrobial peptides was also taken into account. Starting from the information gained by circular dichroism data on the influence on LL-37 conformation given by several polysaccharides, an optimal modulation activity of the cytotoxic effect of LL-37 was found using the peptide/alginate mixture. The demonstrated maintenance of antimicrobial activity on Gram negative strains drove to an application on solid constructs for orthopaedic applications. An effect caused by the simple contact between cells or bacteria and alginate surface loaded with the peptide, was excluded by in vitro experiments. The only possible release mechanism that an LL-37/alginate construct was able to show was related to the degradation of this one. Finally, the whole construct was assembled by incorporating the dried microbeads in a concentrate hyaluronic acid solution, obtaining a paste-like construct that can facilitate the injectability of the particulate. Rheological measurements indicated an increment of the viscoelastic component by adding particulate into hyaluronate solution; this, generally, is associated to a good capacity to regain the elasticity after the injection, as expected for a biomaterial for bone filler applications. The results collected demonstrate the applicability of the obtained composite as bone graft substitute with bioactive properties (osteoinduction, osteoconduction, bioadhesivity and antibiotic effect on Gram negative strains).
XXII Ciclo
1978

The goal of this thesis is the discovery of a bioinformatics solution for network-based predictive analysis of NGS data, in which network structures can substitute gene lists as a more rich and complex signature of disease. I have focused on methods for network stability, network inference and network comparison, as additional components of the pipeline and as methods to detects outliers in high-throughput datasets. Besides a first work on GEO datasets, the main application of my pipeline has been on original data from the FDA SEQC (Sequencing Quality Control)project. Here I will report some initial findings to which I have contributed with methods and analysis: as the corresponding papers are being submitted. My goal is to provide a comprehensive tool for network reconstruction and network comparison as an R package and user-friendly web service interface available on-line at https://renette.fbk.eu The goal of this thesis is the discovery of a bioinformatics solution for network-based predictive analysis of NGS data, in which network structures can substitute gene lists as a more rich and complex signature of disease. I have focused on methods for network stability, network inference and network comparison, as additional components of the pipeline and as methods to detects outliers in high-throughput datasets. Besides a first work on GEO datasets, the main application of my pipeline has been on original data from the FDA SEQC (Sequencing Quality Control)project. Here I will report some initial findings to which I have contributed with methods and analysis: as the corresponding papers are being submitted. My goal is to provide a comprehensive tool for network reconstruction and network comparison as an R package and user-friendly web service interface available on-line at https://renette.fbk.eu.

L’obiettivo di questo lavoro è stato quello di studiare l’efficacia di trattamenti biologici alternativi per la cura dell’endometrite nelle specie equina e bovina. Per endometrite si intende un processo infiammatorio che coinvolge l’endometrio. L’endometrite costituisce una delle più frequenti cause di ridotta fertilità. In particolare, i trattamenti biologici che abbiamo studiato includono l’uso di terreno condizionato da cellule staminali mesenchimali isolate dalla membrana amniotica equina (CM-AMCs) e il plasma arricchito di piastrine (PRP), che è un concentrato di fattori bioattivi derivato dalle piastrine isolate da sangue bovino. è stato ipotizzato che questi trattamenti siano capaci di modulare la risposta infiammatoria facendo così aumentare il tasso di guarigione e di conseguenza il tasso di gravidanza. Sulla base di questa ipotesi, è stato studiato l’effetto diretto di AMCs equine sulla capacità proliferativa di cellule isolate dallo stroma dell’endometrio equino in fase di diestro (EDCs) attraverso il ricorso a i) sistemi transwell per la co-cultura delle EDCs con AMCs e a ii) colture di EDCs in presenza CM-AMCs. Studi comparativi tra amnios in toto (AM) e tessuto endometriale (in fase di diestro) in toto (ED) e tra AMCs e EDCs relativi all’espressione di geni associati alla fase inziale della gravidanza (Hoxa9, Wnt4a-7a, ERα, ERβ, PGR, PGRMC1 e mPR) hanno permesso di identificare similarità tra le popolazioni cellulari e i tessuti da cui derivano. Inoltre, si è pensato di ricreare in vitro una condizione di coltura per le AMCs più vicina possibile alle condizioni in vivo durante la gravidanza ed è per questo che le AMCs sono state coltivate anche in presenza di progesterone. Dai risultati è emerso che sia l’AM sia le AMCs mostrano caratteristiche molecolari comparabili rispettivamente con l’ED e l’EDCs. Inoltre è stato dimostrato che i segnali solubili prodotti dalle AMCs promuovono la proliferazione delle EDCs sia quando queste due linee cellulari sono co-coltivate in transwell sia quando le EDCs sono coltivate in presenza di CM-AMCs. Per esplorare il potenziale del PRP sulla rigenerazione dell’endometrio bovino, abbiamo valutato il suo effetto sulla proliferazione delle cellule endometriali e sull’espressione dei geni coinvolti nella regolazione del ciclo estrale e nell’interazione materno-fetale (ERα, ERβ, PR, TP53 e PTGS2). Le cellule isolate da endometrio bovino sono state coltivate in vitro fino al passaggio (P) 10 in presenza di due diverse concentrazioni di PRP (5% e 10%). Le cellule di controllo sono state coltivate in presenza del 10% di siero fetale bovino (FBS). I risultati ottenuti indicano che il PRP al 5%, a P5, aumenta il tasso di proliferazione delle cellule endometriali e induce un aumento significativo dell’espressione dei geni studiati rispetto al 10% di PRP o FBS. Le cellule endometriali sono state poi trattate in vitro con due diverse concentrazioni dell’endotossina batterica lipopolisaccaride (LPS) (10 e 100 ng/ml) e successivamente con PRP al 5% per 1, 6, 12, 24 e 48 ore. La capacità del PRP di contrastare in vitro l’infiammazione delle cellule endometriali indotta da LPS è stata valutata studiando l’espressione di alcuni geni pro-infiammatori (IL-1β, IL-8, iNOS, PTGS2 e CXCL5). È stata osservata una significativa diminuzione dell’espressione dei geni pro-infiammatori nelle cellule trattate con PRP rispetto alle cellule che non hanno subito nessun trattamento. In generale, i risultati ottenuti dimostrano che le AMCs, i loro prodotti solubili e il PRP rappresentano candidati ottimali per il trattamento dell’endometrite nei grandi animali.
The aim of this work was to study the efficacy of alternative biological treatments for endometritis in the equine and bovine species. Endometritis is an inflammation of the uterine lining that causes decreased fertility. The biological treatments studied include the conditioned medium derived from equine amniotic membrane-derived mesenchymal stem cells (AMCs) (AMCs-CM), and bovine platelet-rich plasma (PRP). PRP is a preparation of concentrated platelet-derived bio-active factors isolated from blood. Both treatments have the potential to modulate the inflammatory response thus increasing the cure rate and consequently pregnancy rate. Based on this, AMCs and cells isolated from endometrium (EDCs) at diestrus were studied for their proliferative capacity. EDCs were then co-cultured with AMCs in a transwell system or grown in the presence of AMCs-CM to understand whether AMCs could induce endometrial cells to proliferate through the release of paracrine agents. Lastly, the amniotic membrane in toto and AMCs (at different passages) have been compared with the endometrial tissue (at diestrus stage) in toto and cells isolated from it, respectively, for the expression of early pregnancy-associated genes (Hoxa9, Wnt7a, Wnt4a, PGR, ERα, ERβ, PGRMC1 and mPR). In addition, since in vivo amniotic cells within the amniotic membrane are probably influenced by progesterone during pregnancy, AMCs were cultured also in the presence of progesterone. This is also important because the phenotype of the isolated AMCs should be more possibly similar to the endometrial cells for their use in replacement therapy. This study highlights a substantial overlap between equine amnion and endometrium based on gene expression profiling. Furthermore the present study demonstrates that soluble signals produced by AMCs promote EDCs proliferation. To explore the potential of PRP in bovine endometrial regeneration, its effect on the proliferation of endometrial cells and on the expression of genes involved in the regulation of oestrus cycle and fetal-maternal interaction (PTGS2, TP53, ERα, ERβ and PR) were evaluated. Bovine endometrial-derived cells were cultured in vitro until passage (P) 10 with two different concentrations of PRP (5% and 10%). These data were compared to control cells, cultured with 10% fetal bovine serum (FBS) only. We observed that 5% PRP at passage 5 increased proliferation rate of endometrial cells and induce a significant increase in the expression of all studied genes compared to 10% PRP or 10% FBS. In a second step, endometrial cells were treated in vitro with two different concentrations of bacterial endotoxin lipopolysaccharide (LPS) (nominally 10 and 100 ng/ml) and subsequently with 5% PRP for 1, 6, 12, 24 and 48 h. The ability of PRP to counteract in vitro inflammation of endometrial cells induced by LPS was studied by evaluating the expression of pro-inflammatory genes (IL-1β, IL-8, iNOS, PTGS2 and CXCL5). PRP treatment had a significant effect on the endometrial expression of pro-inflammatory genes. In fact, the expression of all genes studied was found significantly decreased in cells treated with PRP compared to untreated cells. These findings make AMCs, its conditioned medium and PRP suitable candidates for the treatment of endometritis in large animals.

Nell'ultimo decennio, uno dei messaggi chiave derivante dalle ricerche scientifiche sul cancro è la necessità di comprendere meglio il metabolismo delle cellule tumorali per lo sviluppo di una terapia personalizzata migliore e più efficace. Le cellule tumorali attuano un riarrangiamento metabolico che coinvolge diversi processi per supportare la loro natura proliferativa ed invasiva. Per meglio comprendere l’entità del cambiamento metabolico, in questo studio abbiamo utilizzato un approccio systems level impiegando la metabolomica untargeted e la flussomica mediante isotopi stabili del carbonio (13C) in cellule tumorali esprimenti un K-Ras oncogenico. Abbiamo testato gli effetti di farmaci inibitori del metabolismo di glucosio e glutammina per indagare eventuali vie metaboliche alternative attivate per la sopravvivenza delle cellule tumorali. Inoltre abbiamo investigato il ruolo del metabolismo cellulare nello sviluppo della resistenza alla terapia endocrina nel carcinoma mammario ERα positivo. I dati ottenuti hanno permesso di identificare specifici meccanismi metabolici di utilizzo della glutammina in cellule resistenti alla terapia, suggerendo l’utilizzo del farmaco metformina come adiuvante nel trattamento dei tumori resistenti alla terapia ormonale. Infine, abbiamo contribuito alla comprensione del metabolismo delle cellule tumorali nel guidare la crescita e la proliferazione, esplorando il ruolo della glutammina oltre la nota funzione di fonte di carbonio e azoto; infatti sostituendo la glutammina con fonti alternative di carbonio e azoto, si osserva un fenotipo reverse Warburg. I risultati di questa tesi aprono strade di ricerca per l'identificazione di nuovi potenziali obiettivi terapeutici e ci portano verso la progettazione di una strategia terapeutica migliore e più efficace per il trattamento dei pazienti oncologici.
In the recent decade, one of the important keynote message derived through the summation of our global efforts against cancer is the need to better understand cancer cell metabolism for the development of better and efficacious personalized therapy. Cancer cells undertake a multifaceted rewiring of metabolic pathways in order to support their proliferative and invasive nature, which requires a systems level investigation to fully comprehend the scale of metabolic deregulation. In this study, we systematically investigated the metabolic differences using untargeted metabolomics and 13C flux omics approach in oncogenic K-Ras driven tumours. We tested the effects of drug inhibitors targeting glucose and glutamine metabolism to unravel the alternative metabolic pathways required for cancer cell survival. We further expanded our research towards understanding the role of cellular metabolism in driving resistance to endocrine therapeutic drugs in ERα positive breast cancer. We identified specific metabolic mechanisms of utilization of glutamine in resistant cells while also providing further basis for the use of metformin as an adjuvant in the treatment of endocrine therapyresistant cancers. Finally, we contributed to current understanding about cancer cell metabolism by exploring the role of glutamine beyond its role as a carbon and nitrogen source in driving growth and proliferation of cancer cells. Upon substitution of glutamine with appropriateiv nitrogen and carbon sources, cancer cells exhibited reverse Warburg phenotype. The findings from this thesis open up new avenues of research through the identification of new putative targets and bring us one step closer towards designing much better and efficacious therapeutic strategy for the treatment of cancer patients.

Influenza epidemics have become a major public health concern worldwide. According to the World Health Organization, the annual epidemics results in about three to five million cases of severe illness, and about 250 to 500 thousand deaths. Recurring emergence of new influenza viruses and viruses that are resistant to currently approved antiviral medications pose a critical need to explore new or alternative medications. A classical Traditional Chinese Medicine (TCM) decoction consisting of nine herbs, named Yinqiaosan (YQS, 銀翹散), has a long history for treating respiratory diseases in China. However, the efficacy of YQS has not been investigated mechanistically. In the present study, the effectiveness of YQS in treating influenza virus infection was examined. The potential mechanisms of action of two active compounds present in one of the component herbs of YQS were also investigated. Results showed that YQS increased the survival rate of the mice in an in vivo influenza virus infection model with significant reduction in lung viral titers. In order to further delineate the mechanisms of action of YQS, compounds present in a principal ingredient of YQS were examined for antiviral and immunomodulatory effects. In screening a panel of fractions extracted from YQS, forsythoside A was demonstrated to suppress the viral titers of a wide range of influenza viruses including the oseltamivir-resistant and the 2009 pandemic H1N1 viruses. Through electron microscopy, slow or abnormal viral budding events were observed upon forsythoside A treatment during influenza virus infection. Western blot analysis revealed a reduced influenza virus M1 protein expression. As previous report showed that assembly of viral components into an infectious particle required a threshold level of M1 protein, reduced M1 expression in the cells treated with forsythoside A may contribute to the virus replication suppression. On the other hand, innate immune responses provide first line protection against influenza virus infections. However, excessive responses often result in tissue damage. Cyclooxygenase (COX)-2 is an immunomodulatory factor that has been shown to play a role in the pathogenesis of influenza viruses. A previous COX-2 knock-out mice model showed that COX-2 deficiency is beneficial to the host during influenza viral infection, in which mortality was significantly reduced. Furthermore, during H5N1 infection, it has been shown that COX-2 level significantly increased and it played an essential role in coordinating the productions of inflammatory cytokines, while in another study, pharmacological inhibition of COX-2 suppressed H5N1 virus replication in primary human macrophages. In view of the roles of COX-2 during influenza virus infection, the presence of compound in YQS that reduces the influenza virus-induced COX-2 level was examined. Present results showed that jacaranone not only reduced the influenza virus-induced cyclooxygenase (COX)-2 mRNA level, it also suppressed the subsequent production of prostaglandin E2 level in primary human macrophages. At the same time, jacaranone inhibited the virus induced-activations of ERK1/2 and Akt, which are involved in the COX-2 induction. Jacaranone also suppressed, at least in part, the COX-2 mRNA level at the transcriptional level by inhibiting the nuclear translocation of NF-κB. To conclude, TCM has been recognized as an important part in complementary and alternative medicine and it is an ample source of antimicrobial drugs. The use of a mixture of herbs is the major therapeutic approach of TCM in which, the principal ingredients provide the main therapeutic actions while the others enhance the effects or diminish the side effects of the principal ones. Some components act mainly for symptomatic control. The present study not only supports the efficacy of YQS, but also gives evidences to an active antiviral compound and an immunomodulatory compound found in YQS. They may act as either principle or supporting components depending on the purpose of application. This study provides new insights on future novel drug development from the existing wisdom of TCM.
published_or_final_version
Paediatrics and Adolescent Medicine
Doctoral
Doctor of Philosophy

Inflammatory bowel disease (IBD) is a lifelong chronic inflammatory condition of the gastrointestinal tract (GIT), with incidence and prevalence increasing worldwide. It is considered a complex, multifactorial disease with no cure. Even though large progress has been made in recent years, current therapies are far from satisfactory, and show extreme variability of outcomes due to patient heterogeneity. The traditional therapy consists of anti-inflammatories, corticosteroids, antibiotics, and immunomodulatory drugs. This non-specific immunosuppression guarantees disease-control in some patients although the long-term use of these drugs is correlated with a significant number of therapy-associated complications and side-effects. A dramatic improvement in disease management was achieved by the introduction of biological agents targeting pro-inflammatory cytokines such as anti-TNF-α. Despite the revolutionary impact of these agents in IBD disease management, treatments such as anti-TNF-α do show several drawbacks – for example, up to 50% of patients do not respond at all or eventually lose response. This variability in clinical outcome is reflecting the variability of individuals due to different genetics, life style and inflammatory state. Therefore, there is a need to define the specific inflammatory state of a given patient, considering individual complications and develop new in-vitro systems and biomarkers that predict drug responsiveness and allow developing patient-specific treatment In this thesis, different in-vitro models were developed addressing different aspects and compartments of IBD pathology including the enteric nervous system, the ECM component fibrillin-1, as well as patient-derived, three dimensional short-term and long-term cultures that will bring us a step closer towards personalized medicine.
Le malattie infiammatorie croniche intestinali (MICI) sono un gruppo di patologie complesse ad eziologia multifattoriale, caratterizzate da uno stato infiammatorio cronico del tratto gastrointestinale, la cui incidenza a livello mondiale è in continuo aumento. Nonostante nel corso degli ultimi anni siano stati fatti numerosi progressi nel controllo della malattia, l’attuale approccio terapeutico rimane ancora lontano dall’essere soddisfacente e l’esito clinico che ne deriva è estremamente variabile a causa della vasta eterogeneità tra i pazienti. La terapia tradizionale, che consiste nella somministrazione di farmaci antinfiammatori, corticosteroidi, antibiotici o farmaci immunomodulatori, garantisce il controllo della malattia in alcuni pazienti, ma, a causa della non-specificità e del lungo periodo di utilizzo, è correlata all’insorgenza di numerose complicanze ed effetti collaterali. Un significativo miglioramento nella gestione della malattia è stato raggiunto attraverso l’introduzione di farmaci biologici, il cui target è rappresentato principalmente dalle citochine pro-infiammatorie implicate nella patologia, come ad esempio il TNF-α. Nonostante il forte impatto clinico, l’utilizzo di farmaci biologici, come l’anti-TNF-α ha mostrato diversi svantaggi, tra cui un’alta percentuale di non-responsività al trattamento oppure la perdita di risposta nel corso del tempo. La grande variabilità che si riscontra nella risposta clinica riflette di fatto la variabilità che sussiste tra i diversi individui, ed è dovuta principalmente a differenze a livello genetico, nello stile di vita e nello stato infiammatorio. Di conseguenza, cresce sempre più la necessità di definire nello specifico lo stato infiammatorio e le complicanze caratteristiche di ciascun paziente e di sviluppare nuovi sistemi di screening in-vitro che possano predire la risposta al trattamento e quindi consentire un approccio terapeutico specifico per ciascun individuo. Nell’ottica di una medicina predittiva e della terapia personalizzata, in questa tesi sono stati sviluppati differenti modelli in-vitro, che prendono in considerazione diversi aspetti e compartimenti implicati nelle malattie infiammatorie croniche intestinali, quali il sistema nervoso enterico, la fibrillina-1, componente della matrice extracellulare, così come colture tridimensionali a breve e lungo termine derivate da campioni bioptici umani. Questi modelli sperimentali di infiammazione cronica intestinale potranno costituire uno strumento clinico utile per applicazioni di medicina personalizzata.

Production of recombinant FVIII, the protein that is missing or dysfunctional in haemophilia A patients, is highly inefficient compared to other recombinant clotting factors such as FIX. This is predominantly due to complex intracellular trafficking, short half-life and protein instability. This study aimed to increase the amount of functional FVIII produced in mammalian cells by co-expression with anti-FVIII Camelid antibody fragments (VHH). Three VHH ligands were supplied by BAC BV (as DNA constructs), two of which when expressed in yeast are known to bind recombinant FVIII (ligands 2 and 7) and are used commercially as FVIII purification tools. From these three constructs, nine new VHH plasmids constructs were designed and transiently expressed in a stable BHK-human FVIII-expressing cell line. Of the nine VHH fragments that were co-expressed in the BHK FVIII cell line, four of these had a statistically significant impact on the ‘clotting time’ of the cell media as demonstrated by the activated partial thromboplastin time assay (aPTT). Two ligand 2 constructs (L2C1 and L2C2) prolonged the coagulation time by 4 seconds (P-value 0.0001, 95% confidence intervals 38.5-43.5), and 3.4 seconds (P-value 0.0072, 95% CI 36.5-40.3) respectively, indicating a decrease in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Two ligand 7 constructs (L7C1 and L7C3) caused a decrease in coagulation time of 3.2 seconds (P=0.0057, 95% CI 30.5-33.3), and 4 seconds (P=0.0002, 95% CI 29.1-32.9) respectively, indicating an increase in functional FVIII activity versus media from the untransfected and null transfected BHK-FVIII cell line. Ligand 7 and ligand 2 both bind to the FVIII light chain, albeit in different regions and with different affinities (data confidential to BAC BV). BAC studies showed that ligand 7 competes with vWF on the FVIII light chain, which is known to increase stability of FVIII in vivo, whereas ligand 2 does not compete for this binding site. The opposing effects of ligand 7 and ligand 2 on FVIII clotting times seen in this study could be due to their differences in FVIII binding properties, since it is known that binding location of FVIII ligands can have an impact on FVIII clotting activity.

Type 1 diabetes affects one in every 400-600 children and adolescents in the US. Standard therapy with exogenous insulin is burdensome, associated with a significant risk of dangerous hypoglycemia, and only partially efficacious in preventing the long term complications of diabetes. Pancreatic islet transplantation has emerged as a promising therapy for type 1 diabetes. However, this cell-based therapy is significantly limited by inadequate islet supply (more than one donor pancreas is needed per recipient), instant blood-mediated inflammatory reaction, and loss of islet viability/function during isolation and following implantation. In particular, inadequate revascularization of transplanted islets results in reduced islet viability, function, and engraftment. Delivery of pro-vascularization factors has been shown to improve vascularization and islet function, but these strategies are hindered by insufficient and/or complex release pharmacokinetics and inadequate delivery matrices as well as technical and safety considerations. We hypothesized that controlled presentation of angiogenic cues within a bioartificial matrix could enhance the vascularization, viability, and function of transplanted islets. The primary objective of this dissertation was to enhance allogenic islet engraftment, survival and function by utilizing synthetic hydrogels as engineered delivery matrices. Polyethylene glycol (PEG)-maleimide hydrogels presenting cell adhesive motifs and vascular endothelial growth factor (VEGF) were designed to support islet activities and promote vascularization in vivo. We analyzed the material properties and cyto-compatibility of these engineered materials, islet engraftment in a transplantation model, and glycemic control in diabetic subjects. The rationale for this project is to establish novel biomaterial strategies for islet delivery that support islet viability and function via the induction of local vascularization.

This thesis develops an Artificial Intelligence (AI) approach intended for accurate patient stratification and precise diagnostics/prognostics in clinical and preclinical applications. The rapid advance in high throughput technologies and bioinformatics tools is still far from linking precisely the genome-phenotype interactions with the biological mechanisms that underlie pathophysiological conditions. In practice, the incomplete knowledge on individual heterogeneity in complex diseases keeps forcing clinicians to settle for surrogate endpoints and therapies based on a generic one-size-fits-all approach. The working hypothesis is that AI can add new tools to elaborate and integrate together in new features or structures the rich information now available from high-throughput omics and bioimaging data, and that such re- structured information can be applied through predictive models for the precision medicine paradigm, thus favoring the creation of safer tailored treatments for specific patient subgroups. The computational techniques in this thesis are based on the combination of dimensionality reduction methods with Deep Learning (DL) architectures to learn meaningful transformations between the input and the predictive endpoint space. The rationale is that such transformations can introduce intermediate spaces offering more succinct representations, where data from different sources are summarized. The research goal was attacked at increasing levels of complexity, starting from single input modalities (omics and bioimaging of different types and scales), to their multimodal integration. The approach also deals with the key challenges for machine learning (ML) on biomedical data, i.e. reproducibility, stability, and interpretability of the models. Along this path, the thesis contribution is thus the development of a set of specialized AI models and a core framework of three tools of general applicability: i. A Data Analysis Plan (DAP) for model selection and evaluation of classifiers on omics and imaging data to avoid selection bias. ii. The histolab Python package that standardizes the reproducible pre-processing of Whole Slide Images (WSIs), supported by automated testing and easily integrable in DL pipelines for Digital Pathology. iii. Unsupervised and dimensionality reduction techniques based on the UMAP and TDA frameworks for patient subtyping. The framework has been successfully applied on public as well as original data in precision oncology and predictive toxicology. In the clinical setting, this thesis has developed1: 1. (DAPPER) A deep learning framework for evaluation of predictive models in Digital Pathology that controls for selection bias through properly designed data partitioning schemes. 2. (RADLER) A unified deep learning framework that combines radiomics fea- tures and imaging on PET-CT images for prognostic biomarker development in head and neck squamous cell carcinoma. The mixed deep learning/radiomics approach is more accurate than using only one feature type. 3. An ML framework for automated quantification tumor infiltrating lymphocytes (TILs) in onco-immunology, validated on original pathology Neuroblastoma data of the Bambino Gesu’ Children’s Hospital, with high agreement with trained pathologists. The network-based INF pipeline, which applies machine learning models over the combination of multiple omics layers, also providing compact biomarker signatures. INF was validated on three TCGA oncogenomic datasets. In the preclinical setting the framework has been applied for: 1. Deep and machine learning algorithms to predict DILI status from gene expression (GE) data derived from cancer cell lines on the CMap Drug Safety dataset. 2. (ML4TOX) Deep Learning and Support Vector Machine models to predict potential endocrine disruption of environmental chemicals on the CERAPP dataset. 3. (PathologAI) A deep learning pipeline combining generative and convolutional models for preclinical digital pathology. Developed as an internal project within the FDA/NCTR AIRForce initiative and applied to predict necrosis on images from the TG-GATEs project, PathologAI aims to improve accuracy and reduce labor in the identification of lesions in predictive toxicology. Furthermore, GE microarray data were integrated with histology features in a unified multi-modal scheme combining imaging and omics data. The solutions were developed in collaboration with domain experts and considered promising for application.

Age-dependent changes in murine cardiac pacemaker activity. Cardiovascular diseases (CDVs) are the main causes of death. The incidence of morbidity and mortality associated with CDVs increases exponentially in the elderly population due to the increase of both the number of old people and of the average lifespan (for example it is estimated that in the United States there will be 70 million people over the age of 65 by the year 2030, representing almost 25% of the population, 6 . Amongst the age-dependent cardiovascular diseases, the sinoatrial node dysfunction has a primary role 7 ; for this reason, there is a high interest in the study of the pathophysiological mechanisms that are at the basis of cardiac aging. It is known that in humans during ageing there is a decline in maximum heart rate (MHR) and a reduction in the intrinsic heart rate (IHR) 8,9 ; however basal heart rate (BHR) remains the same between the adults and the elderly 10 . We thus decided to better understand the age-associated mechanisms responsible for the changes in intrinsic and maximum heart rate but not in the basal heart rate, and we chose the mouse as a study model. We therefore first confirmed that in our murine model the IHR, but not the basal HR, decreases with ageing; and we also noticed that in adult mice the IHR and the BHR were similar. We focused our studies on the funny current since it is a main contributor to pacemaking generation and modulation and we observed a reduction of the current density and a negative shift of the activation curve in the old animals. We then evaluated the influence of the autonomic system and we found that in adult mice both branches of the autonomic system contribute similarly to HR, though in opposite directions; old mice instead had a lower vagal tone, leading to a reduction in the overall HRV as assessed by spectral analysis, and an increased sympathetic tone, leading to a larger vulnerability to spontaneous arrhythmias compared to adult. Our data highlight that this model recapitulates well the HR features observed in aged humans and can help understanding the mechanisms of the ageing-associated changes in heart chronotropism. The shift of the sympatho-vagal balance toward a sympathetic prevalence in the old animals may explain why, despite the difference in the intrinsic heart rate, the basal heart rate is similar between the two groups. While the current view is that the sympathetic increase is necessary to ensure the homeostatic balance of heart rate during ageing, it is also possible that the age-dependent increase of the sympathetic tone is the cause of the reduction of the intrinsic heart rate which therefore ultimately represents the compensatory effect.
Mode of action of the Traditional Chinese Medicine drug TMYX on pacemaker activity in freely-moving mice and in isolated SAN cells. The identification of novel pharmacological agents able to selectively reduce sinus rate has a strong interest in the clinic because of their potential use in the treatment of ischemic heart disease such as angina pectoris. Despite longstanding and intense investigation, at present there is only one such agent (Ivabradine) that has reached therapeutic application, since all other compounds tested had additional undesired side-effects 11,12 . Our laboratory has started few years ago a collaboration with the University of Tianjin (China) to investigate the efficacy of the cardioactive compound Tongmai Yangxin (TMYX) which is currently used in the Traditional Chinese Medicine as a cardiac regulator 13 . Electrophysiological experiments performed on rabbit SA node cells have shown a dose-dependent slowing effect of TMYX on pacemaking action potential rate. The investigation of the effects of TMYX on the I f current, the major contributor of diastolic depolarization phase, has revealed that at physiological potential the drug exerts a bradycardic action, thus confirming the effect on the spontaneous automaticity observed in SA node cells. During my experimental work, I continued the characterization of the mechanism of action of this Traditional Chinese Medicine drug, working on two parallel aspects: 1) the evaluation of the systemic effect of TMYX and 2) the identification of the molecular mechanisms of the drug. 1) The systemic effect of TMYX was evaluated in freely-moving mice implanted with ECG transmitters. When TMYX was delivered during pharmacological blockade of either sympathetic alone or in combination with parasympathetic block, a deep bradycardia was observed, confirming the in-vitro bradycardic effect. However, quite surprisingly, when the drug was delivered (i.p. injection) during basal heart rate condition, the experiments show an increment of heart rate. 2) Our previous in-vitro experiments had revealed that TMYX acts directly on pacemaker SAN cells and blocks the pacemaker current. We therefore started a series of experiments aiming at the identification of the site of action of TMYX. Our experiments suggest that TMYX shares the modulatory pathway of ACh, but they also exclude that TMYX can bind to the M2 receptor because atropine does not alter the action of TMYX. We then performed inside-out experiments and demonstrated that TMYX does not have any action on the channel when cAMP is either completely absent or it is present at saturating high levels, while TMYX decreases the pacemaker current when cAMP is present at non-saturating levels. Taken together our results demonstrate the presence of at least two molecules that exert different actions: one is a bradycardic agent, whose effect is observed in isolated SAN cells and in the in-vivo model, and the other one is a systemic tachycardic agent, whose effect is observed in the absence of autonomic block. The bradycardic agent appears to behave as an antagonist of the sympathetic stimulation of heart rate. The antagonist action on the sympathetic activity is a key pharmacologic mechanism used by the so-called β-blocker drugs. These drugs are largely used to treat a large number of cardiac diseases including Coronary Artery Disease (CAD) and Heart Failure (HF). However, the often-undesired side effect of β-blockers is a decreased strength of contraction (inotropism) of the heart. According to our data it appears that the active molecule in the TMYX acts directly on the pacemaker channel by antagonizing the stimulating action of cAMP, while β-blockers slow heart rate by decreasing the cAMP levels. This direct antagonistic effect, rather than a block of cAMP production should decrease the heart rate leaving unaltered the strength of contraction. We thus intend to identify and isolate the specific tachycardic/bradycardic molecules since they could have a strong impact in the clinical use.

Introduction: Chronic myeloid leukemia (CML) is a white blood cells cancer, which is characterized by a BCR-ABL fusion gene. The disease it is caused by a reciprocal translocation between chromosome 9 and 22, t(9; 22)(q34; 11) commonly known as the Philadelphia chromosome (Ph), resulting in an abnormally BCR-ABL tyrosine kinase, which is responsible for the pathogenesis of CML. The high efficacy of the tyrosine kinase inhibitors (TKIs) in the treatment of CML has caused the need for sensitive methods to monitor the course of therapy. Quantification of BCR-ABL transcripts with qRT-PCR Real-Time has demonstrated to be the most accurate method available. Following the European LeukemiaNet recommendations, the lack of initial response can be detected only after 3-6 months from the diagnosis. The ability to understand how patients respond to the different TKIs available as first-line treatment at the moment of diagnosis would help clinicians to prescribe more patient-tailored therapy, decreasing the onset of future drug resistance and decreasing treatment cost. Materials and Methods: We have developed and validated two devices (RQ-BCR-ABL p210 One-Step and RQ-BCR- ABL p190 One-Step) for monitoring therapy of CML. The RQ-BCR-ABL p210 One-Step kit has undergone a further external validation in three reference laboratories, belonging to LabNet network.?For what concern the prevision of therapy’s response the University of Verona has developed LeukoPredict, an in-vitro device to screen the inhibitory potential of several BCR- ABL-targeting drugs and to obtain the percentage of inhibition compared to the same non- treated samples. We took part to this project in the framework of industrial planning, performing a Freedom to Operate analysis and a Cost-Effectiveness analysis. Results: Both kits based in qRT-PCR Real-Time One-Step have showed high reproducibility and high sensitivity in quantification of BCR-ABL transcripts, proving to be suitable for CE-IVD mark. Moreover, RQ-BCR-ABL p210 One-Step kit has been verified by the LabNet network as a suitable device for the monitoring of CML, improving the reproducibility regarding the current system used in routine.?The Freedom to Operate analysis of LeukoPredict has found some close prior patents documents, but none could hinder entry into the market. The Cost-Effectiveness analysis has demonstrated that LeukoPredict is either cost saving or very cost-effective, depending on the scenario analyzed, generating significant savings for health systems. Conclusions: This project is able to connect actual principal issues in CML. On one side we have developed and validated two devices that completely satisfy actual request of therapeutic monitoring in pharmaceutical market making a complete panel to track CML. The develop of devices as LeukoPredict helps to decrease the risk of disease’s progression to more aggressive phase, personalizing the therapy and obtaining the maximum effectiveness of therapeutical choices. This can help physicians in an evidence based decisional therapeutic process, avoiding potential conflict of interest and giving a rational explanation to other kind of treatment when the risk of failure is too high. Finally, it has been considered as a technology that can be affordable and that could contain the cost of healthcare.
Introduzione: La Leucemia Mieloide Cronica (LMC) è un disordine mieloproliferativo delle cellule staminali pluripotenti caratterizzato per la presenza del gene di fusione BCR-ABL. Questo disturbo è causato da una traslocazione reciproca di materiale genetico tra il cromosoma 9 e 22 t(9; 22)(q34; 11) comunemente riconosciuta come cromosoma Philadelphia (Ph), che porta alla formazione di una proteina tirosinchinasica con un’attività alterata, causante della patogenesi della LMC. L’elevata efficacia degli inibitori della tirosinchinasa (TKIs) nel trattamento della LMC ha originato la necessità di metodi molto sensibili per monitorare il corso terapeutico. La quantificazione dei trascritti BCR-ABL con qRT-PCR Real Time si è dimostrato il metodo disponibile più accurato al giorno di oggi. Secondo le raccomandazioni degli esperti appartenenti alla European LeukemiaNet, la mancanza di risposta iniziale può essere rilevata solo dopo 3-6 mesi dal momento della diagnosi. La capacità di comprendere come i pazienti rispondono ai diversi TKIs disponibili nel momento della diagnosi aiuterebbe ai medici a prescrivere la terapia di prima linea più conveniente in ogni caso, riducendo l’insorgere di una futura resistenza ai farmaci e riducendo così i costi del trattamento. Materiali e Metodi: Abbiamo sviluppato e validato due dispositivi (RQ-BCR-ABL p210 One-Step e RQ-BCR- ABL p190 One-Step) per il monitoraggio terapeutico della LMC. Il kit RQ-BCR-ABL p210 One-Step ha subito un’ulteriore validazione esterna in tre diversi centri di riferimento per la LMC appartenenti alla rete italiana LabNet. Per quanto riguarda alla previsione della risposta terapeutica, l’Università degli Studi di Verona ha sviluppato LeukoPredict, un dispositivo in-vitro per individuare il potenziale inibitorio di diversi farmaci che hanno BCR-ABL come bersaglio, ottenendo il percentile d’inibizione rispetto agli stessi campioni non trattati. Abbiamo partecipato a questo progetto nell’ambito della pianificazione industriale, eseguendo un’analisi Freedom to Operate e un’analisi costo-efficacia sul prodotto. Risultati: Entrambi I kit basati sulla tecnologia qRT-PCR Real-Time One-Step hanno mostrato una elevata riproducibilità e un’alta sensibilità nella quantificazione dei trascritti BCR-ABL, dimostrando di essere adatti alla marcatura CE-IVD. Inoltre, il kit RQ-BCR-ABL p210 One-Step è stato certificato dalla rete LabNet come un dispositivo adatto per il monitoraggio della LMC, migliorando notevolmente la riproducibilità dei sistemi attuali utilizzati nella routine. L’analisi Freedom to Operate di LeukoPredict ha rilevato alcuni brevetti relazionati con la tecnologia presente nel dispositivo, ma nessuno potrebbe ostacolare la sua uscita nel mercato in questo momento. L’analisi costo-efficacia ha dimostrato che LeukoPredict ha un costo ridotto ed è molto conveniente a livello economico secondo lo scenario analizzato, generando rilevanti risparmi per i sistemi sanitari. Conclusioni: Questo progetto è in grado di collegare le problematiche attuali della LMC. Da un lato abbiamo sviluppato e validato due dispositivi che soddisfano completamente le richieste del mercato farmaceutico per il monitoraggio terapeutico, proporzionando un panello completo per la gestione della LMC. Lo sviluppo di dispositivi come LeukoPredict aiuta a diminuire il rischio di progressione della malattia verso fasi più aggressive, personalizzando la terapia e ottenendo la massima efficacia delle diverse scelte terapeutiche. Ciò aiuterebbe ai medici nella decisione della miglior terapia basandosi sull’evidenza, evitando potenziali conflitti d’interesse e fornendo una spiegazione razionale ad altri tipi di trattamento quando il rischio di fallimento terapeutico è troppo alto. Infine, la sua tecnologia è stata definita come molto conveniente potendo contribuire a contenere il costo dell’assistenza sanitaria.

Next-generation sequencing (NGS) based on “sequencing by synthesis”, such as Illumina’s MiSeq, NextSeq500 or HiSeq instruments has reached a state where consistency, throughput and quality make it a mature technology to use for cancer genome research, such as single-cell sequencing or in clinical diagnostics. Within the scope of this thesis, we designed and tested three protocols to mobilise high-throughput sequencing for precision medicine to provide optimal solutions for doctors to help with understanding, diagnosing and determining states of human cancer cells. Targeted re-sequencing of clinically relevant cancer genes using NGS ensures an economical use of tissue, by only doing one experiment for testing many drug-resistance tests. In collaboration with oncologists, three protocols are introduced and discussed depending on the respective use case. First, a comprehensive cancer panel based on targeted bait capture technology was developed, for a quick and in-depth genetic variant detection of 69 commonly examined cancer genes. After designing, over 300 clinical formalin-fixed and paraffin-embedded (FFPE) samples from patients, with known mutations in KRAS, NRAS, BRAF and EGFR at varying rates were sequenced. Sequencing data was collected and analysed in-depth to define assay properties and to determine sensitivity, specificity and overall reliability. Input material was evaluated by measuring DNA integrity (DIN) scores for each sample and correlated with overall performance. In doing so, we were able to set precise input criteria to ensure experimental success. The second step was the development of a diagnostic cancer panel of nine genes, based on enzymatic digestion and target amplification for a rapid and cost-effective testing of multiple genetic markers, who have an immediate impact on doctor’s decision on cancer drug therapies. The method introduces a novel approach by adding a molecular barcode to each molecular fragment sequenced, which is supposed to increase sensitivity as it allows removal of PCR artefacts and sequencing errors. The new panel was tested on 48 clinical FFPE samples that were previously genotyped on common cancer mutation hotspots with validated pyrosequencing. Strengths and imitations of this library-preparation method could be identified and considered. The third protocol describes a method to quickly extract and prepare DNA from thousands of individual cells simultaneously by capturing each cell in water droplets in an emulsion. Each droplet contains a collection of barcoded primer pairs that allows not only-barcoding individual fragments, but entire cells. The system has been tested by mixing mouse NIH3T3 cells with human KRAS insert, which carries a heterozygous mutation and human K562 cells with no known mutation in KRAS in an 80:20 ratio. Cells were emulsified with barcoded primer, a polymerase chain reaction amplified exons two and three, added cell-specific barcodes and prepared a library for sequencing in one step. After emulsion-breaking and clean-up and a second PCR, library was ready for sequencing. Around ten percent of a standard MiSeq run is enough to genotype over 10,000 cells. A developed script extracts barcodes from the sequencing data, which is then aligned with a standard alignment software, such as Burroughs-Wheeler-Aligner (BWA). Subsequently the introduced single-cell barcodes clustered according to cellular sub-populations to give deep insight into heterogeneity of the prepared cell population. This will prove a valuable tool in the assessment of diversity amongst cells in a variety of disease backgrounds and contribute to the development of precision medicine through informing patient-specific, personalised diagnostic approaches.

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

L’utilizzo delle cellule iPSC–paziente specifiche costituiscono uno degli strumenti -di modello malattia- utilizzati per studiare i meccanismi biologici e patologici alla base di una specifica malattia. L'obiettivo principale di questa tesi di dottorato è stato quello di generare iPSC da cellule mononucleate del sangue periferico (PBMC) ottenute da pazienti con Sindrome di Cri-du-Chat (CdCS), poi differenziate in cellule staminali mesenchimali indotte (iMSC). CdCS è un malattia genetica causata da una delezione totale o parziale nel braccio corto del cromosoma 5, caratterizzata da un grido acuto, caratteristiche dismorfiche, scarsa crescita e ritardo dello sviluppo. L'aploinsufficienza dei geni situati all'interno di 5p contribuisce al fenotipo. Le cellule PBMC sono state riprogrammate in cellule staminali pluripotenti indotte (iPSC) mediante i fattori Yamanaka Oct4, Sox2, Klf4 e c-Myc utilizzando un vettore di riprogrammazione CytoTune-iPS 2.0 non integrato; le cellule iPSC ottenute sono state caratterizzate per la pluripotenza e la staminalità. Le cellule CdC-iPSC sono state differenziate in iMSC utilizzando MesenCult™-ACF Plus Medium e Culture Kit. Tali cellule CdCs-iMSC sono risultate aderenti alla plastica, positive per i marcatori delle cellule MSC e capaci di differenziarsi in osteociti, adipociti, e condroblasti. Tali proprietà hanno soddisfatto i criteri minimi delle MSC umane proposti dalla Società Internazionale di Terapia Cellulare. Nel periodo trascorso presso l’Università di Santiago De Compostela (Spagna), l’obiettivo dell’attività sperimentale è stato quello di isolare vescicole extracellulari da urina (uEV). L'urina infatti è stata ampiamente studiata per identificare biomarcatori di patologie renali; quindi riflette un quadro olistico dell'intero sistema urinario ed i campioni possono essere raccolti ripetutamente in modo non invasivo. L'analisi delle uEV, isolate appunto dalle urine, può fornire una soluzione interessante per il loro -cargo- (proteine, RNA, lipidi e glicani) che rimane protetto. L'ultracentrifugazione è la tecnica comune per isolare le EV da fluidi biologici. Tuttavia, tale tecnica è laboriosa, dispendiosa in termini di tempo e di solito richiede apparecchiature costose. In questa tesi di dottorato, per isolare le uEV da donatori umani sani, abbiamo utilizzato ExoGAG (NasasBiotech, Santiago de Compostela, Spagna) Kit ed il metodo convenzionale di ultracentrifugazione. Quindi, per esaminare l'internalizzazione delle uEV nelle cellule di topo mIMCD3 e la colocalizzazione delle cilia primarie in colture 2D di tali cellule del dotto di raccolta midollare interno 3 (mIMCD3) di topo, le uEV isolate sono state marcate con la colorazione dei globuli rossi Vybrant™ DiD e le ciglia primarie sono state indagate con anticorpi anti α -Tubulina acetilata ed analisi al microscopio confocale. In particolare abbiamo isolato con successo da donatori sani le uEV che esprimono le tetraspanine di superficie CD63 e marcatori proteici CD81 ed abbiamo valutato il loro contenuto di RNA con Spectrometry Nanodrop e Capillary Tapstation Electhrophoresis. I dati ottenuti dimostrano che sia le uEV isolate con il kit ExoGAG, sia le uEV isolate con ultracentrifugazione non sono state internalizzate dalle cellule mIMCD3 e la microscopia confocale ha rivelato la mancanza di colocalizzazione delle cilia primarie e delle uEV colorate con Vybrant™ DiD Cell-labeling solution. Questi risultati indicano che potrà essere utile utilizzare in questo tipo di esperimenti cellule umane.
Cellular disease modelling using patient-specific iPSCs is one of the tools used to study the biological and pathological mechanisms underlying specific disease. The main objective of this PhD thesis was to generate iPSCs from peripheral blood mononuclear cells (PBMCs) obtained from patients with Cri-du-Chat Syndrome (CdCS) and differentiation into mesenchymal stem cells (MSCs). CdCS is a genetic disease caused by a total or partial deletion in the short arm of chromosome 5, characterized by a high-pitched cry, dysmorphic features, poor growth, and developmental delay. Haploinsufficiency of the genes located within 5p contributed to the phenotype. PBMCs were reprogrammed into iPSCs by introducing the Yamanaka factors Oct4, Sox2, Klf4, and c-Myc using a nonintegrating CytoTune-iPS 2.0 reprogramming vectors and characterized for pluripotency and stemness and their ability to differentiate into the three germ layers. Then CdCs-iPSCs were differentiated into iMSCs using MesenCult™-ACF Plus Medium and Culture Kit. CdCs-iMSCs were plastic adherent, expressed MSC surface markers and could differentiate into osteocytes, adipocytes, and chondroblasts. These properties satisfy the minimal criteria of human MSCs proposed by the International Society of Cellular Therapy. Urine has been tremendously studied to discover biomarkers of distinct renal pathologies; hence it reflects a holistic picture of the entire urinary system. It could be collected repeatedly in a noninvasive manner. The analysis of urinary Extracellular Vesicles, uEVs, encountered in urine provides an attractive solution due to their cargo (Proteins, RNAs, lipids, and Glycans) which remains protected. Ultracentrifugation is the common technique for isolating EVs from biological fluids. However, this technique is laborious, time-consuming, and usually requires expensive equipment. In this PhD thesis, firstly, to isolate uEVs from healthy human donors, we utilized ExoGAG (NasasBiotech, Santiago de Compostela, Spain) Extracellular vesicles purification kit and conventional Ultracentrifugation method. Secondly, to assess uEVs internalization and primary cilia colocalization in 2D culture of mouse inner medullary-collecting duct 3 (mIMCD3) cells, the isolated uEVs were Labelled with Vybrant™ DiD Red Cell stain, and primary cilia were immunologically stained with anti-acetylated α -Tubulin. Confocal microscopy images were obtained for further analysis. We successfully isolated uEVs from healthy donors expressing the surface tetraspanins CD63 and CD81 protein markers and assessed their RNAs contents with Spectrometry Nanodrop and Capillary Tapstation Electrophoresis System. Isolated uEVs with ExoGAG commercial kit and Ultracentrifugation could not be internalized and uptaken by Mouse inner medullary-collecting duct 3 (mIMCD3), Confocal Microscopy images revealed the lack of colocalization of expressed primary cilia and uEVs stained with Vybrant™ DiD Cell-Labelling solution

Este ensaio inicia-se com um sucinto painel a respeito de uma pesquisa bibliográfica sobre Medicina Legal que começa com Ambroise Paré em 1532 e chega ao ano de 2008; pesquisa esta que muito nos ajudou no planejamento desta Tese. É preciso que se diga que o conceito de Medicina Legal só aparece em 1621 com Paolo Zacchia (Quaestiones Medico Legales...). Nosso objetivo neste trabalho envolve diferentes aspectos teóricos dessa ciência. O primeiro é o de demonstrar que a Medicina Legal pode ser a ciência de uma classe no sentido da Lógica e que ela não seria, portanto, a exemplo da Medicina Clínica, uma ciência do indivíduo, como diz Gilles Granger em sua obra sobre Epistemologia já tornada célebre. O segundo aspecto teórico seria o de propor a Medicina Legal como uma ciência do freqüente aristotélico (hòs epì tò polú ws epi to polu - termo cunhado pelo helenista Porchat Pereira, enquanto conceito filosófico) graças ao qual situamos nossa ciência entre o acidental e o necessário e universal do pensamento lógico-matemático. Em terceiro lugar discorreremos sobre o fato de que os laudos médico-legais estão normalmente restritos a constatação empírica e, então, iremos demonstrar a também indispensável consideração das condições a priori da possibilidade de se estabelecer o visum et repertum, ou seja, a consideração do papel do encéfalo na leitura da experiência possível ao ser humano, numa linguagem atual. No que diz respeito à prática, realizamos uma pesquisa na qual analisamos 996 laudos médico-legais, no Brasil, cujos problemas nos remeteram à questão do Método. Sobre o Método, consideramos as teorias de Aristóteles, Descartes, Kant, Popper, Piaget e Granger, deixando de lado os grandes empiristas como Francis Bacon, David Hume, Stuart Mill, na medida em que suas teorias, devido às crenças embutidas no próprio Empirismo, não há lugar para o cérebro como condição primeira de qualquer tipo de leitura da experiência no mundo sensível. Ora, muitos biólogos, inclusive no Brasil, a partir do Prêmio Nobel em Fisiologia ou Medicina, Konrad Lorenz, consideram que o a priori kantiano pode ser interpretado hoje, como o aspecto endógeno, orgânico, da possibilidade humana de conhecer o mundo (o encéfalo) necessário a toda e qualquer leitura da experiência vivida, sobretudo quando houver a necessidade de explicá-la e reportá-la a terceiros. No caso da Medicina Legal, reportá-la à Justiça, com muitíssimas implicações psico-sociais. Nós proporemos então, à Medicina Legal, um Método Dialético, procurando demonstrar suas vantagens teóricas e práticas.
This essay begins with an overview of a bibliographic research on Forensic Medicine, from its early onset with Ambroise Paré, in 1532, up to the present. This research played an important role on the planning of this thesis (it is important to emphasize that the proper concept of Forensic Medicine, however, appears only in 1621 with Paolo Zacchia,-Quaestiones-Medico-Legales...). Our purpose in this work includes many theoretical aspects of this science, first of all, to show that Forensic Medicine should be considered a science of a class in the sense of Logic, instead of, as in Clinical Medicine, \"a science of the individual\", as stated by Gilles Granger in his already classical work on epistemology. The second theoretical objective will be to propose Forensic Medicine as a science of the \"Aristotelian frequent (hòs epì tò polú term coined by the Hellenist Porchat Pereira as a philosophical concept), through which such science should find its place between the realm of the mere accidental, and that of the necessary and universal, proper to the logic-mathematical reasoning. Thirdly, we also address the problem that the forensic reports are usually limited to empirical observations, and, then, we will also demonstrate the necessity to take into account a priori conditions of the possibility of the making of Visum et repertum, that means, we aim to consider the role of the brain in the framing of the human beings accessible experience, through a contemporary approach. On the empirical side, we conducted an extensive research, analyzing 996 Brazilian forensic reports, whose problems led us to questioning the generally used method. On the method, we discussed the theories of Aristotle, Descartes, Kant, Popper, Piaget and Granger, leaving aside the great empiricists such as Francis Bacon, David Hume, Stuart Mill, as in their theories, due to the embedded beliefs of empiricism, there is no room for the brain as the first condition of the possibility of any kind of sensory experience of the world. However, many biologists worldwide (Brazil included), since the work of Konrad Lorenz (Nobel Prize, Physiology or Medicine, 1973) has begun to circulate amid the scientific community, moved to consider that the Kantian a priori can be interpreted as the endogenous aspect (organic) of the human ability to apprehend the world, necessary to any construction of actual experience, especially when there is an intent to explain and report it to others. In the case of Forensic Medicine, it means reporting the findings to the Judicial System, which entails many psycho-social effects. Finally, we propose to Forensic Medicine a dialectical method, aiming to demonstrate its theoretical and practical advantages.

Chronical kidney disease (CKD) is a worldwide health problem with increasing incidence, where the major part accounts for chronic glomerulonephrites (GN). It is a group of diseases of various aetiology and multiform clinical course, having various prognosis which is often hardly predictable as well as existing prognostic markers are not always certain. There is an urging need of new reliable and specific prognostic and therapeutic markers research. A modern proteomic technology - Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been employed in many GN studies showing promising results. We aimed to study several forms of primary and secondary glomerulonephrites (membranous nephropathy, IgA nephropathy, diabetic nephropathy) to enlighten possible molecular alterations significant of diseases’ progression. The studies were performed on renal tissue biopsies, analysing them with high spatial resolution MALDI-MSI to get better visualisation of signals’ co-localisation on tissue. We performed a comparison of molecular profiles of various forms of GN. As a result, we were able to generate and distinguish specific tryptic peptides profiles of different cell regions (tubules, glomeruli, interstitium, connective tissue) and detect proteins with an altered intensity, implicated in inflammatory and healing pathways. MALDI-MSI, being able to define renal structures, could provide additional diagnostic and prognostic information. Generation of collective diagnostic panels may fulfil our pathogenesis understanding and assist clinical prognostic assessment.
Chronical kidney disease (CKD) is a worldwide health problem with increasing incidence, where the major part accounts for chronic glomerulonephrites (GN). It is a group of diseases of various aetiology and multiform clinical course, having various prognosis which is often hardly predictable as well as existing prognostic markers are not always certain. There is an urging need of new reliable and specific prognostic and therapeutic markers research. A modern proteomic technology - Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has been employed in many GN studies showing promising results. We aimed to study several forms of primary and secondary glomerulonephrites (membranous nephropathy, IgA nephropathy, diabetic nephropathy) to enlighten possible molecular alterations significant of diseases’ progression. The studies were performed on renal tissue biopsies, analysing them with high spatial resolution MALDI-MSI to get better visualisation of signals’ co-localisation on tissue. We performed a comparison of molecular profiles of various forms of GN. As a result, we were able to generate and distinguish specific tryptic peptides profiles of different cell regions (tubules, glomeruli, interstitium, connective tissue) and detect proteins with an altered intensity, implicated in inflammatory and healing pathways. MALDI-MSI, being able to define renal structures, could provide additional diagnostic and prognostic information. Generation of collective diagnostic panels may fulfil our pathogenesis understanding and assist clinical prognostic assessment.

Obiettivo di questo lavoro è stato quello di mettere a punto protocolli per l’isolamento, l’espansione e la caratterizzazione in vitro di popolazioni omogenee di cellule staminali mesenchimali (MSC) isolate da tessuti extra-fetali equini (matrice cordonale e amnios), valutandone il possibile impiego nella terapia delle lesioni tendinee. Dai risultati ottenuti emerge che le popolazioni cellulari isolate sono caratterizzate dall’espressione di marker di mesenchimalità e pluripotenza, dall’assenza di espressione di marker di immunogenicità, da un’intensa capacità proliferativa e differenziativa nella linea mesodermica ed ectodermica e sono in grado di tollerare la crioconservazione senza perdere tali proprietà. Per la prima volta in campo equino, MSC allogeniche di derivazione amniotica sono state inoltre utilizzate per il trattamento di lesioni tendinee acute. L’assenza di reazioni al trapianto e la rapida evoluzione riscontrata nei soggetti trattati, probabilmente stimolata da un’intrinseca attività antinfiammatoria di questa popolazione cellulare, confermano l’interesse sempre crescente rivolto agli invogli fetali come fonte alternativa e facilmente accessibile di cellule staminali multipotenti. Durante il periodo di ricerca trascorso presso il Centro di Medicina Rigenerativa dell’Università di Edimburgo, è stata messa a punto un’innovativa tecnologia basata sull’impiego di nanoparticelle biodegradabili in grado di veicolare in maniera altamente specifica fattori di crescita utili al mantenimento delle caratteristiche di pluripotenza di cellule staminali embrionali di topo. Tale approccio, trasferibile ad altri tipi cellulari, si propone in prima istanza di ridurre significativamente il rischio di alterazioni fenotipiche o cromosomiche per le cellule che richiedono cicli ripetuti di espansione in vitro, per un loro impiego efficace nella medicina rigenerativa.
The aim of this work was to optimise protocols for the isolation, expansion and characterisation in vitro of homogeneous mesenchymal stem cell (MSC) populations obtained from extra-fetal tissues (umbilical cord matrix and amnion) to investigate their biological properties and to assess whether they can be used for the treatment of tendinopathies in the horse. Data obtained show that cells express MSC- and pluripotent-associated markers, have low immunogenicity but high prolificacy and plasticity, differentiating in vitro toward mesodermic and ectodermic lineages. Moreover, cryopreservation does not affect these properties. For the first time, amnion-derived MSCs have been allogeneically transplanted into horses with tendon injuries. All the clinical findings reported demonstrated that transplanted cells were well-tolerated by horses and provided compelling evidences to support the exertion of beneficial effects by the injected cells. Studies in vivo confirm the increasing interest in extra-fetal tissues, representing a promising alternative and easily accessible source of multipotent stem cells. At the Scottish Centre for Regenerative Medicine (University of Edinburgh), a new strategy to improve the efficiency of stem cell culture has been established. In particular, a spatial and temporal controlled release of bioactive factors sequestered in biodegradable nanoparticles specific for the cell type of interest has been achieved, in first instance in the context of supporting mouse embryonic stem cells self-renewal and pluripotency. This approach is adaptable to other stem cell systems, including those requiring in vitro extended cultures often challenged by phenotypic modifications and genetic changes in karyotype. It will contribute significantly to the scalable manufacture of stem cells and the clinical delivery of new advanced cell-based therapies for regenerative medicine.

Mesenchymal stem cells (MSCs) are uniquely capable of crossing germinative layers borders (these cell populations are able to differentiate towards ectoderm-, mesoderm- and endoderm- derived lineages) and are viewed as promising cells for regenerative medicine approaches in several diseases. Some undoubtedly limiting factors for the clinical use of MSCs, i.e. for the repair of bone defects, are related to many different problems, that only partially today can be overcome through ex-vivo expansion and cells modification strategies. Considering MSCs sources, the use of fetal annexes-derived MSCs, such as MSCs from Whartonʼs jelly of umbilical cord (WJMSCs), or the recruitment of MSCs from “stem cell niches” in adult discard tissues, like periodontal ligament (PDL) of extracted teeth (PDLMSCs), could be promising in compensating limits of MSCs traditional sources, i.e. Bone Marrow, like small cells number, high harvesting technique morbility, difficult cells commitment due to early senescence. Uniforming and investigating best cells culture conditions with in vitro different experimental strategies is useful in our job to understand and control differentiation mechanisms and finally to influence the yield and proliferation rate of these MSCs populations, together with their osteogenic potential. The aim of our study is briefly sumarized: - To isolate and culture MSCs cells from human Umbilical Cord Whartonʼs Jelly and Periodontal Ligament; - To compare characteristics between all samples recruited and to link them to clinical aspects of tissue donors; - To characterise both MSCs population in regard to their proliferation and differentiation potential; - To investigate their functional characteristics before and after alginate microbeads encapsulation; - To investigate effects of three-dimensional systems and microgravity on MSCs before and after differentiation. WJMSCs and PDLSCs were analyzed for the expression of MSC markers, and then committed to osteogenic differentiation. Before and after differentiation, alkaline phosphatase (ALP) activity, the expression level of a specific osteoblast transcription factor (Runx2), and mineralization status were evaluated. The performance of WJMSCs and PDLSCs was then compared in 3-D culture systems (alginate beads and bioreactor system) in terms of viability, proliferation, secretive profile, expression of markers and effectiveness of phenotype modulation. Characterization of MSCs population was succesfull for all samples investigated, and for largest samples cohort (WJMSCs) it was possible to correlate cells biochemical parameters to clinical donors features. These findings may help during selection of best donors to combine them with scaffolds and biomaterials to promote tissue regeneration. For both MSCs populations, we demonstrated that cells can live and grow in 3- D systems. All MSCs samples analyzed showed a substantial osteogenic potential, before and after encapsulation in alginate microbeads. Modulation of cells culture conditions, with the use of nanotechnologies strategies or the use of bioreactors, like Rotary Cell Culture SystemTM (RCCS-4TM bioreactor, SyntheconTM, Inc., Houston, TX, U.S.A.) with High Aspect Ratio Vessel (HARVTM) has been shown to be a useful and promising approach to investigate characteristics and environmental effects of cells/ biomaterials combinations, in order to predict their effect and potential for regenerative medicine strategies.

The purpose of this study was to examine isometric strength, pain threshold and perceived health responses to partial-body cryotherapy (PBC, 150s at a temperature range between -130 and -160°C). Three different populations of subjects were enrolled for this study: 200 healthy people were exposed to a single PBC session in order to assess the maximum handgrip isometric strength responses by means of an hydraulic dynamometer, 30 healthy female carried out a cycle of ten consecutive PBC sessions for evaluate the objective pain threshold in the low back region via the painpressure test and finally 28 female fibromyalgic patients were exposed to repeated exposure to PBC (ten sessions in a row) with the purpose to examine perceived health and quality of life responses utilizing two self-reported questionnaires. A single session of PBC lead to improve, immediately after the very-low temperatures exposure, muscle isometric strength in healthy people both in females and in males. Repeated exposures to PBC in healthy women was shown to be efficient in decreasing very rapidly the skin temperature in the lowback region, and thus to induce a significant increase in pain-pressure threshold values. The same protocol of repeated exposures to PBC induced significant improvements for all indexes and sub-indexes of the perceived health and quality of life questionnaires assessed in fibromyalgic patients.

Amniotic Fluid Stem Cells (AFSCs) can be isolated from the amniotic fluid after amniocentesis that pregnant women undergo during the second trimester of pregnancy, this population has already been characterised and share common features between embryonic stem cells and adult mesenchymal stem cells. AFSCs are identified by specific markers; this study – as already published – focuses on the CD117 (c-Kit) positive fraction of AFSCs. Those cells are of great interest for regenerative medicine purposes considering their potential of differentiation and the relative constant availability, moreover retrieving prenatal autologous cells can offer new strategies for new-borns with congenital malformations or diseases. This study was intended to explore the possibility of isolating AFSCs at term of pregnancy (so at the third trimester, retrieving the amniotic fluid during delivery). Comparison on phenotype and test of potential for different lineages differentiation has been investigated on cells from both trimesters. These cells have a great potential also because they can be kept in culture and their number significantly increased for successive applications. Hypoxia is also a well-known factor that influence culture of stem cells, cultivating cells at lower oxygen tension has already demonstrated various beneficial effects on other stem cells so it has been decided to try this approach to ameliorate the expansion of AFSCs. In vivo experiments were performed to verify in vitro results on angiogenic potential of AFSCs.
Le cellule staminali del liquido amniotico (AFSCs) possono essere isolate dal liquido amniotico in seguito ad amniocentesi cui le donne incinte si sottopongono durante il secondo trimestre della gravidanza, questa popolazione è già stata caratterizzata e condivide proprietà comuni fra le cellule staminali embrionali e le cellule staminali adulte mesenchimali. Le AFSCs possono essere identificate tramite specifici marcatori; in questo studio – com’è già stato pubblicato – si è puntata l’attenzione sulla frazione positiva per CD117 (c-Kit). Queste cellule sono di grande interesse ai fini della medicina rigenerativa, considerando il loro potenziale di differenziamento e la relativa costante disponibilità, inoltre il recupero di cellule prenatali di origine autologa offre la possibilità di nuove strategie per curare i neonati con malformazioni congenite o altre malattie. In questo studio si è voluto esplorare la possibilità di isolare le AFSCs al termine della gravidanza (quindi al terzo trimestre, recuperando il liquido amniotico durante il parto). Sono stati investigati i fenotipi e le potenzialità differenziative di entrambi i trimestri. Queste cellule hanno anche un grande potenziale poiché possono essere mantenute in cultura e il loro numero significativamente aumentato per successive applicazioni. L’ipossia è un ben conosciuto fattore che influenza la coltura delle cellule staminali; coltivare le cellule in basse tensioni di ossigeno è già stato dimostrato avere un effetto benefico su altre cellule staminali, è stato quindi deciso di provare quest’approccio per migliorare l’espansione delle AFSCs. Sono stati effettuati esperimenti in vivo per verificare il potenziale angiogenico delle AFSCs.

La medicina “rigenerativa” ha come scopo quello di riparare o ripristinare la funzionalità di organi o tessuti danneggiati da malattie, traumi o dal “semplice” invecchiamento mediante l’utilizzo di cellule staminali. Una linea cellulare staminale molto importante e molto utilizzata in approcci di medicina rigenerativa è costituita dalle staminali adulte o staminali mesenchimali. Il midollo osseo e il tessuto adiposo sono stati considerati, per lungo tempo, la principale fonte di staminali mesenchimali adulte. Tuttavia le staminali adulte prevedono tecniche di prelievo invasive, non prive di complicazioni, e la coltura richiede diverse settimane; inoltre, possiedono limitate capacità di proliferazione e differenziaamento che sono inversamente proporzionali all’età del donatore. Da qui l’interesse, per il clinico, di nuove fonti cellulari. La recente scoperta in campo umano dell’esistenza di staminali mesenchimali nei tessuti fetali ha reso queste cellule molto interessanti agli occhi dell’intera comunità scientifica. I tessuti fetali sono di facile accesso perchè solitamente eliminati alla nascita, non richiedono procedure invasive, il loro utilizzo non suscita controversie etico-morali e inoltre le staminali isolte da tali tessuti sono caratterizzate da proliferazione più rapida, una maggiore espansione in vitro e da un notevole potenziale differenziativo. Alla luce di queste considerazioni, l’obiettivo principale di questo studio è stato quello di recuperare cellule staminali mesenchimali da tessuti fetali umani extraembrionali (il villo coriale, il liquido amniotico e la membrana amniotica) durante 3 diverse epoche gestazionali e mantenerle in coltura per un lungo periodo. Allo scopo di verificare se l’età gestazionale influenzasse le proprietà di staminalità, le staminali isolate da tali tessuti sono state confrontate nel fenotipo, nella espressione del markers specifici di pluripotenza e mesenchimalità, nel potenziale proliferativo, differenziativo e immunosoppressivo e immunomodulatorio.
Regenerative medicine involves the use of living cells to repair, replace or restore normal function to injured tissues and organ. With the field of mesenchymal stem cell (MSCs) research taking off in the late 1980’s and the early 1990’s, scientists highlighted the importance of adult MSCs for regenerative medicine applications, and identified their presence in all adult tissues. Among MSCs, MSCs derived from fetal tissues (bone marrow, blood and liver) and extra-embryonic compartments (amniotic fluid, umbilical cord blood, amniotic membrane, chorion and placenta) are promising in cell-based therapies because of their beneficial properties in wound healing. Moreover, these tissues are ideal sources for studying the features of stem cell characteristics due to the possibility of harvesting large amount of tissue, without posing ethical debate, following prenatal diagnosis (as in the case of chorion from chorionic villi sampling and amniotic fluid from amniocentesis) or at birth (as in the case of amniotic membrane). In addition, MSCs isolated from fetal sources non only fulfill general characteristic of MSCs but exhibit features of embryonic stem cells including the expression of specific pluripotent markers. In fact, they possess higher proliferation rates, lower immunogenicity and wider differentiation capacity than their adult counterparts as well as immuno- suppressive and modulatory properties. Scarce information on the behavior of MSCs from different stages of human gestation are so far available. The first aim of this study was the isolation of MSCs from fetal (extra-embryonic) tissues during first- second- and third- gestation period and their long-term culturing; secondly, the detection of the common features between chorionic villi (CV), amniotic fluid (AF) and amniotic membrane (AM)-derived MSCs; thirdly, the observation of differences in phenotype, proliferative capacity, differentiation ability as well as in the immuno-suppressive/immuno-modulatory properties among them.

As natural environments have been identified as places for mental restoration and social development there is the potential to address a number of bio-psychosocial health inequalities by encouraging urban park use. The current research explores the link between people and nature within the urban context of Liverpool across 3 phases of research. The reconnaissance phase explored health inequalities, physical activity levels and park access in Liverpool. Analysis showed that Liverpool is one of the most socially and economically deprived areas within England, with less than 70% of the survey population not meeting recommended physical activity levels. Although Liverpool has an abundance of parks and urban green spaces, the high health inequalities and lack of physical activity correlation with environment features suggests residents might not access parks and urban green spaces for health benefits. The exploratory study adopted a multi-method approach to investigate bio-psychosocial responses to urban city and park environments. Using repeated measures, 18 participants walked on a treadmill for 20 minutes whilst viewing and listening to either a Liverpool urban park or city centre scene. A two-way ANOVA was conducted to compare means between data collection time points within each condition for heart rate and blood pressure. Analysis for mean arterial pressure found a significant reduction for the park condition post physical activity [F(2,18) 6.83, p=.02] with the same effect on systolic blood . pressure [F(2, 18)=8.61, p=.OO] in comparison to the city scene. Semi-structured interviews conducted after testing found that cultural and social experiences influenced how participants interact with the urban environment. In particular, opportunities to access parks and natural environments during childhood was attributed to a lifespan connection. Participants reported psychological benefits of stress reduction and attention restoration and social benefits including providing a place for family and friend interaction in a park setting. Social barriers included fear from crime and harassment from teenagers. While the city was associated with traffic, congestion and noise that could evoke negative emotions, the history and diversity of Liverpool aroused pride and enjoyment that could also promote psychosocial benefits. The intervention study adopted ethnographic principles to explore cultural and individual beliefs of a group of eleven teenagers engaged in a park based physical activity programme. Observations during the programme indicated that the practitioner's role and skill base was paramount to park and activity engagement. Changes to participants across the programme impacted negatively on group dynamics, with external pressures from family and friends contributing to low attendance rates, poor time keeping and low concentration during activities. The social intervention highlighted the need to fully engage participants in the planning process and provide an agreed structure and policy for behaviour. The research highlighted a number of organisational, cultural and social issues that need to be tackled before benefits from green spaces can be fully realised. Overall research findings suggest that potential bio-psychosocial benefits of physical activity in parks and urban green spaces may be influenced by complex social issues surrounding values, culture and tradition. Further investigation into the interrelationships between neighbourhood residents, parks and urban green spaces, activities of users, and potential restorative effects could provide beneficial insights for policy makers and practitioners who would look to use these spaces for bio-psychosocial wellbeing.

Skeletal muscle is an essential tissue for several vital functions. It displays an intrinsic regenerative ability in case of injury, thanks to the activation of satellite cells (SCs), the adult skeletal muscle stem cells. In presence of large defects, the renewing capacities of skeletal muscle are compromised. In such situations regenerative medicine may be a promising solution. This project is focused on a neonatal pathology known as congenital diaphragmatic hernia (CDH), in which the diaphragm fails to close during gestation. CDH is a severe anomaly with an incidence of 1 on 2,500-3,000 new-borns and high mortality rate. Currently, the most frequently used material for the surgical CDH repair is polytetrafluoroethylene (Gore-Tex[R]), but its application can lead to several drawbacks, as hernia recurrence and chest deformation. Great interest has been shown in alternative solutions based on tissue engineering approaches. In this regard, the use of decellularised extracellular matrix (ECM) revealed to be encouraging. When transplanted in vivo it can integrate with the native tissue, recruit host stem cells and influence their behaviour towards a regenerative process. The aim of this work is to characterise a novel tissue engineering approach based on the use of diaphragm decellularised ECM (dECM) as an alternative solution to the current CDH clinical options. The final purpose is to close the defect on the diaphragm and to induce its regeneration and functional recovery. In vivo, we created the first surgical CDH mouse model and we repaired the defect on the diaphragm using mouse dECM and expanded-polytetrafluoroethylene (ePTFE) as control. The transplantation of dECM patches did not cause any rejection effect nor hernia recurrence, differently from ePTFE treated mice. Moreover, ePTFE patches induced a foreign body reaction that was absent when dECM patches were used. We further considered three essential aspects of tissue regeneration: new muscle tissue formation, angiogenesis and re-innervation. In all the cases the biologic patch demonstrated to be better compared to ePTFE. The prolonged activation of muscle regeneration together with the angiogenic and re-innervation processes induced by dECM translated into an overall amelioration of diaphragmatic function compared to ePTFE-treated animals. Despite the positive clinical outcome, dECM patches did not activate complete regeneration of the defect. For this reason, we set up a tissue engineering technique to re-create in vitro diaphragmatic muscle tissues recellularising mouse diaphragm dECM and human MPCs cells. The aim was to obtain skeletal muscle-like substitutes for CDH capable to boost myofibers generation and further improve tissue functionality. We demonstrated that human MPCs not only were able to engraft the scaffold and repopulate the dECM in all its thickness, but most importantly, they differentiated giving rise to metabolic active myotubes. Moreover, a subpopulation of cells maintained SCs features, showing the ability to respond to in vitro injury. Given the positive outcomes obtained using dECM, the next step to get closer to clinic would be to use larger animal models. Moreover, the recellularisation could be improved by using mechanical stimulation, perfusion systems and by adding other cell types as endothelial and neural cells, in order to obtain a more complete in vitro construct for pre-clinical and clinical applications. Finally, the two parts of this project could be joined by closing the defect on the diaphragm using recellularised ECM, with the aim to favour tissue regeneration and reduce the drawbacks related to the use of current synthetic patches.
Il muscolo scheletrico ha un’intrinseca capacità rigenerativa grazie all’attività svolta dalle cellule satelliti. In presenza di danni estesi però tali capacità rigenerative possono essere compromesse. In queste situazioni un approccio di medicina rigenerativa può costituire una soluzione promettente. Questo progetto è focalizzato sull’ernia diaframmatica congenita, patologia neonatale caratterizzata da un’incompleta formazione del diaframma, con incidenza di 1 su 2,500-3,000 neonati e un alto tasso di mortalità. Attualmente, il materiale più usato per il riparo dell’ernia è il politetrafluoroetilene (Gore-Tex[R]), tuttavia il suo utilizzo può causare effetti collaterali, come la ricorrenza dell’ernia e malformazioni della cassa toracica. Grande interesse è stato rivolto a soluzioni di ingegneria tissutale, come l’uso di matrici extracellulari decellularizzate. Quando trapiantate in vivo esse riescono ad integrarsi in maniera fisiologica con il tessuto nativo e reclutano cellule staminali, modulando il loro comportamento verso un processo rigenerativo. Lo scopo di questo progetto è caratterizzare un approccio di ingegneria tissutale basato sull’uso di matrici decellularizzate come soluzione alternativa all’attuale metodo per il riparo l’ernia. L’obiettivo è chiudere il difetto sul diaframma ed indurne la rigenerazione. In vivo, abbiamo creato il primo modello murino di ernia diaframmatica e abbiamo riparato il difetto usando una matrice decellularizzata. Il politetrafluoroetilene espanso (ePTFE) è stato usato come controllo. Il trapianto di matrici decellularizzate non ha causato rigetto o ricorrenza dell’ernia, a differenza degli animali trattati con ePTFE. Inoltre, ePTFE ha indotto una reazione da corpo estraneo che era completamente assente negli animali trattati con la matrice biologica. Ci siamo poi concentrati su tre aspetti fondamentali della rigenerazione: la formazione di nuovo tessuto muscolare, angiogenesi e re-innervazione. In tutti i casi la matrice biologica ha dimostrato di essere migliore di quella sintetica. La prolungata attivazione della rigenerazione muscolare insieme ai processi angiogenici e di re-innervazione indotti dalla matrice extracellulare si sono tradotti in un generale miglioramento delle funzioni diaframmatiche rispetto a quanto ottenuto negli animali con ePTFE. Nonostante i risultati positivi, la matrice extracellulare non era in grado di indurre una completa rigenerazione del difetto. Perciò abbiamo messo a punto una tecnica di ingegneria tissutale per ricreare in vitro tessuti diaframmatici ricellularizzando matrici decellularizzate con precursori muscolari umani. Lo scopo era di ottenere dei possibili costrutti paragonabili al muscolo scheletrico da usare per il riapro dell’ernia in modo da stimolare maggiormente la generazione di nuove miofibre e migliorare la funzionalità tissutale. I precursori muscolari umani erano in grado di attecchire sulla matrice decellularizzata, di ripopolarla in tutto il suo spessore e di differenziare dando origine a miotubi attivi metabolicamente. Inoltre, una sottopopolazione di cellule manteneva le caratteristiche tipiche delle cellule satelliti, dimostrando di saper rispondere in vitro ad un danno. Visti i risultati positivi ottenuti usando la matrice decellularizzata, il passaggio successivo per avvicinarsi alla cinica è rappresentato dall’utilizzo di modelli animali più grandi. Inoltre, la ricellularizzazione potrebbe essere migliorata grazie a stimolazione meccanica, a sistemi di perfusione e all’aggiunta di altri tipi cellulari (cellule endoteliali e neurali) con lo scopo di ottenere un costrutto più completo per possibili applicazioni pre-cliniche e cliniche. Infine, le due parti di questo progetto potrebbero essere unite in futuro riparando il difetto sul diaframma usando matrici biologiche ricellularizzate al fine di favorire la rigenerazione e ridurre gli svantaggi legati all’uso delle matrici sintetiche.

2006/2007
The advent of molecular “-omics” technologies enabled an unprecedented view into the inner molecular mechanisms of cancer and enhanced optimism towards a patient-tailored vision of medicine. The successful application of these molecular approaches in the discovery of candidate biomarker has accelerated the shift towards personalization of medicine. Indeed, biomarkers hold great promise for refining our ability to establish early diagnosis and prognosis, and to predict response to therapy. The develoment of clinically useful biomarkers would be impossible without access to human biological specimens and associated patient data, since they complete the molecular information gained from laboratory research. Furthermore, with the advances of sensitive molecular technologies, human bio-specimens can be now successfully used for wide analysis at all molecular levels (DNA, RNA and proteins), in addition to conventional cytologic and histologic investigations. However, despite the hundreds of reports on tumor markers, only a few markers have proven clinically useful. The insufficient experience in clinical application of molecular methods combined with the high complexity of clinical material represent the major obstacles for the development of clinically useful biomarkers. Thanks to the possibility to have access to the fresh and archival samples from the hospital, our laboratory can investigate the potential of technological innovations and the current technical pitfalls directly on clinical material. The work in my thesis is strictly correlated to this activity. In particular, the first part is focused on the technical optimization of molecular methods for gene expression analysis in biological fluids and especially in urine samples. In this context we validated a new experimental kit for total RNA extraction from urine samples and tested the potential of a colorimetric approach for PCR product detection. The major part of the study is focused on the technical optimization of molecular methods for gene expression analysis in archival material. This activity is in step with one of the main objectives of the European project called “Archive tissues: improving molecular medicine research and clinical practice-IMPACTS”, in which my laboratory and other 20 European centres are directly involved. In this phase the comparison of the experiences between laboratories and their active collaboration are essential for a more rapid validation of protocols dedicated to RNA (but also DNA and protein) analysis. In particular, we investigated some molecular aspects involved in the pre-analytical phase (tissue fixation procedures) and analytical phase (RNA extraction, RNA quantification and integrity assessment, qRT-PCR) of tissue processing. The final objective of this activity will be the definition of common technical guidelines for a reliable quantification of molecular biomarkers for diagnosis, prognosis and therapy directly in human archival samples. Finally, my thesis includes the clinical application of molecular methods for the quantification of candidate biomarkers in two archival case studies (a breast cancer and an adrenal gland cancer case study). In the breast cancer case study we showed that a panel of seven genes (involved in different cell pathways) is associated to patients’ survival. The adrenal gland tumor case study is part of a preliminary study about the angiogenetic process in rare human cancers.
1979