Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

Study of the thermal effects induced by magnetic resonance on endocardial leads: numerical models and experimental validation

Magnetic resonance imaging (MRI) is today precluded to patients bearing active implantable medical devices AIMDs). The great advantages related to this diagnostic modality, together with the increasing number of people benefiting from implantable devices, in particular pacemakers(PM)and carioverter/defibrillators (ICD), is prompting the scientific community the study the possibility to extend MRI also to implanted patients. The MRI induced specific absorption rate (SAR) and the consequent heating of biological tissues is one of the major concerns that makes patients bearing metallic structures contraindicated for MRI scans. To date, both in-vivo and in-vitro studies have demonstrated the potentially dangerous temperature increase caused by the radiofrequency (RF) field generated during MRI procedures in the tissues surrounding thin metallic implants. On the other side, the technical evolution of MRI scanners and of AIMDs together with published data on the lack of adverse events have reopened the interest in this field and suggest that, under given conditions, MRI can be safely performed also in implanted patients. With a better understanding of the hazards of performing MRI scans on implanted patients as well as the development of MRI safe devices, we may soon enter an era where the ability of this imaging modality may be more widely used to assist in the appropriate diagnosis of patients with devices. In this study both experimental measures and numerical analysis were performed. Aim of the study is to systematically investigate the effects of the MRI RF filed on implantable devices and to identify the elements that play a major role in the induced heating. Furthermore, we aimed at developing a realistic numerical model able to simulate the interactions between an RF coil for MRI and biological tissues implanted with a PM, and to predict the induced SAR as a function of the particular path of the PM lead. The methods developed and validated during the PhD program led to the design of an experimental framework for the accurate measure of PM lead heating induced by MRI systems. In addition, numerical models based on Finite-Differences Time-Domain (FDTD) simulations were validated to obtain a general tool for investigating the large number of parameters and factors involved in this complex phenomenon. The results obtained demonstrated that the MRI induced heating on metallic implants is a real risk that represents a contraindication in extending MRI scans also to patient bearing a PM, an ICD, or other thin metallic objects. On the other side, both experimental data and numerical results show that, under particular conditions, MRI procedures might be consider reasonably safe also for an implanted patient. The complexity and the large number of variables involved, make difficult to define a unique set of such conditions: when the benefits of a MRI investigation cannot be obtained using other imaging techniques, the possibility to perform the scan should not be immediately excluded, but some considerations are always needed.

Development of Clinical Decision Support Systems based on Mathematical Models of Physiological Systems

In the last years of research, I focused my studies on different physiological problems. Together with my supervisors, I developed/improved different mathematical models in order to create valid tools useful for a better understanding of important clinical issues. The aim of all this work is to develop tools for learning and understanding cardiac and cerebrovascular physiology as well as pathology, generating research questions and developing clinical decision support systems useful for intensive care unit patients. I. ICP-model Designed for Medical Education We developed a comprehensive cerebral blood flow and intracranial pressure model to simulate and study the complex interactions in cerebrovascular dynamics caused by multiple simultaneous alterations, including normal and abnormal functional states of auto-regulation of the brain. Individual published equations (derived from prior animal and human studies) were implemented into a comprehensive simulation program. Included in the normal physiological modelling was: intracranial pressure, cerebral blood flow, blood pressure, and carbon dioxide (CO2) partial pressure. We also added external and pathological perturbations, such as head up position and intracranial haemorrhage. The model performed clinically realistically given inputs of published traumatized patients, and cases encountered by clinicians. The pulsatile nature of the output graphics was easy for clinicians to interpret. The manoeuvres simulated include changes of basic physiological inputs (e.g. blood pressure, central venous pressure, CO2 tension, head up position, and respiratory effects on vascular pressures) as well as pathological inputs (e.g. acute intracranial bleeding, and obstruction of cerebrospinal outflow). Based on the results, we believe the model would be useful to teach complex relationships of brain haemodynamics and study clinical research questions such as the optimal head-up position, the effects of intracranial haemorrhage on cerebral haemodynamics, as well as the best CO2 concentration to reach the optimal compromise between intracranial pressure and perfusion. We believe this model would be useful for both beginners and advanced learners. It could be used by practicing clinicians to model individual patients (entering the effects of needed clinical manipulations, and then running the model to test for optimal combinations of therapeutic manoeuvres). II. A Heterogeneous Cerebrovascular Mathematical Model Cerebrovascular pathologies are extremely complex, due to the multitude of factors acting simultaneously on cerebral haemodynamics. In this work, the mathematical model of cerebral haemodynamics and intracranial pressure dynamics, described in the point I, is extended to account for heterogeneity in cerebral blood flow. The model includes the Circle of Willis, six regional districts independently regulated by autoregulation and CO2 reactivity, distal cortical anastomoses, venous circulation, the cerebrospinal fluid circulation, and the intracranial pressure-volume relationship. Results agree with data in the literature and highlight the existence of a monotonic relationship between transient hyperemic response and the autoregulation gain. During unilateral internal carotid artery stenosis, local blood flow regulation is progressively lost in the ipsilateral territory with the presence of a steal phenomenon, while the anterior communicating artery plays the major role to redistribute the available blood flow. Conversely, distal collateral circulation plays a major role during unilateral occlusion of the middle cerebral artery. In conclusion, the model is able to reproduce several different pathological conditions characterized by heterogeneity in cerebrovascular haemodynamics and can not only explain generalized results in terms of physiological mechanisms involved, but also, by individualizing parameters, may represent a valuable tool to help with difficult clinical decisions. III. Effect of Cushing Response on Systemic Arterial Pressure. During cerebral hypoxic conditions, the sympathetic system causes an increase in arterial pressure (Cushing response), creating a link between the cerebral and the systemic circulation. This work investigates the complex relationships among cerebrovascular dynamics, intracranial pressure, Cushing response, and short-term systemic regulation, during plateau waves, by means of an original mathematical model. The model incorporates the pulsating heart, the pulmonary circulation and the systemic circulation, with an accurate description of the cerebral circulation and the intracranial pressure dynamics (same model as in the first paragraph). Various regulatory mechanisms are included: cerebral autoregulation, local blood flow control by oxygen (O2) and/or CO2 changes, sympathetic and vagal regulation of cardiovascular parameters by several reflex mechanisms (chemoreceptors, lung-stretch receptors, baroreceptors). The Cushing response has been described assuming a dramatic increase in sympathetic activity to vessels during a fall in brain O2 delivery. With this assumption, the model is able to simulate the cardiovascular effects experimentally observed when intracranial pressure is artificially elevated and maintained at constant level (arterial pressure increase and bradicardia). According to the model, these effects arise from the interaction between the Cushing response and the baroreflex response (secondary to arterial pressure increase). Then, patients with severe head injury have been simulated by reducing intracranial compliance and cerebrospinal fluid reabsorption. With these changes, oscillations with plateau waves developed. In these conditions, model results indicate that the Cushing response may have both positive effects, reducing the duration of the plateau phase via an increase in cerebral perfusion pressure, and negative effects, increasing the intracranial pressure plateau level, with a risk of greater compression of the cerebral vessels. This model may be of value to assist clinicians in finding the balance between clinical benefits of the Cushing response and its shortcomings. IV. Comprehensive Cardiopulmonary Simulation Model for the Analysis of Hypercapnic Respiratory Failure We developed a new comprehensive cardiopulmonary model that takes into account the mutual interactions between the cardiovascular and the respiratory systems along with their short-term regulatory mechanisms. The model includes the heart, systemic and pulmonary circulations, lung mechanics, gas exchange and transport equations, and cardio-ventilatory control. Results show good agreement with published patient data in case of normoxic and hyperoxic hypercapnia simulations. In particular, simulations predict a moderate increase in mean systemic arterial pressure and heart rate, with almost no change in cardiac output, paralleled by a relevant increase in minute ventilation, tidal volume and respiratory rate. The model can represent a valid tool for clinical practice and medical research, providing an alternative way to experience-based clinical decisions. In conclusion, models are not only capable of summarizing current knowledge, but also identifying missing knowledge. In the former case they can serve as training aids for teaching the operation of complex systems, especially if the model can be used to demonstrate the outcome of experiments. In the latter case they generate experiments to be performed to gather the missing data.

Valutazione preclinica di nuovi potenziali bersagli terapeutici per la cura del medulloblastoma.

Il medulloblastoma (MB) è il tumore più comune del sistema nervoso centrale. Attualmente si riesce a curare solo il 50-60% dei pazienti che tuttavia presentano gravi effetti collaterali dovuti alle cure molto aggressive. Una recente classificazione molecolare ha ridistribuito le varianti più comuni di MB in quattro distinti gruppi. In particolare, il gruppo SHH e D sono caratterizzati da un’ alta espressione di MYCN ed associati a prognosi sfavorevole. MYCN è coinvolto nella regolazione della proliferazione cellulare, differenziazione, apoptosi, angiogenesi e metastasi. L’obiettivo di questo lavoro è la valutazione dell’attività antitumorale di oligonucleotidi diretti contro MYCN, sia in vitro su diverse linee cellulari di MB che in vivo in modelli murini xenograft ortotopici di MB. I risultati hanno dimostrato un’ottima inibizione della crescita in linee cellulari di MB, accompagnata da una riduzione della trascrizione genica e dei livelli proteici di MYCN. Inoltre, sono stati confermati tramite RT-PCR alcuni dei geni trovati significativamente variati nell’esperimento di microarray , dopo il trattamento. Molto interessanti sono stati geni quali BIRC5, che risulta down regolato mentre il gene p21 risulta up regolato in tutte le linee cellulari di MB utilizzate. Inoltre, sono stati generati modelli murini di MB utilizzando cellule precedentemente trasfettate per esprimere il gene della luciferasi e valutarne così la crescita tumorale tramite imaging bioluminescente in vivo (BLI), al fine di poter testare l’attività antitumorale data dagli oligonucleotidi. I risultati hanno dimostrato una buona risposta al trattamento come rilevato dalla tecnica di BLI. I dati preliminari prodotti, dimostrano che MYCN potrebbe esser un buon target per la terapia del MB nei casi in cui è overespresso. In particolare, una sua inibizione potrebbe presentare effetti indesiderati moderati in quanto è poco espresso dopo la nascita.

Generation and characterization of mouse models of Small cell lung cancer and Basal cell carcinoma for the preclinical evaluation of new therapies

Background. Human small cell lung cancer (SCLC) accounting for approximately 15-20% of all lung cancers, is an aggressive tumor with high propensity for early regional and distant metastases. Although the initial tumor rate response to chemotherapy is very high, SCLC relapses after approximately 4 months in ED and 12 months in LD. Basal cell carcinoma (BCC) is the most prevalent cancer in the western world, and its incidence is increasing worldwide. This type of cancer rarely metastasizes and the death rate is extraordinary low. Surgery is curative for most of the patients, but for those that develop locally advanced or metastatic BCC there is currently no effective treatment. Both types of cancer have been deeply investigated and genetic alterations, MYCN amplification (MA) among the most interesting, have been found. These could become targets of new pharmacological therapies. Procedures. We created and characterized novel BLI xenograft orthotopic mouse models of SCLC to evaluate the tumor onset and progression and the efficacy of new pharmacological strategies. We compared an in vitro model with a transgenic mouse model of BCC, to investigate and delineate the canonical HH signalling pathway and its connections with other molecular pathways. Results and conclusions. The orthotopic models showed latency and progression patterns similar to human disease. Chemotherapy treatments improved survival rates and validated the in vivo model. The presence of MA and overexpression were confirmed in each model and we tested the efficacy of a new MYCN inhibitor in vitro. Preliminar data of BCC models highlighted Hedgehog pathway role and underlined the importance of both in vitro and in vivo strategies to achieve a better understanding of the pathology and to evaluate the applicability of new therapeutic compounds

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Transposable elements dynamics in taxa with different reproductive strategies or speciation rate

In recent years the advances in genomics allowed to understand the importance of Transposable Elements (TE) in the evolution of eukaryotic genomes. In this thesis I face two aspects of the TE impact on the in the animal kingdom. The first part is a comparison of the dynamics of the TE dynamics in three species of stick-insects of the Genus Bacillus. I produced three random genomic libraries of 200 Kbps for the three parental species of the taxon: a gonochoric population of Bacillus rossius (facultative parthenogenetic), Bacillus grandii (gonochoric) and Bacillus atticus (obligate parthenogenetic). The unisexual taxon Bacillus atticus does not shows dramatic differences in TE total content and activity with respect to Bacillus grandii and Bacillus rossius. This datum does not confirm the trend observed in other animal models in which unisexual taxa tend to repress the activity of TE to escape the extinction by accumulation of harmful mutations. In the second part I tried to add a contribute to the debate initiated in recent years about the possibility that a high TE content is linked to a high rate of speciation. I designed an evolutionary framework to establish the different rate of speciation among two or more taxa, then I compared TE dynamics considering the different rates of speciation. The species dataset comprises: 29 mammals, four birds, two fish and two insects. On the whole the majority of comparisons confirms the expected trend. In particular the amount of species analyzed in Mammalia allowed me to get a statistical support (p<0,05) of the fact that the TE activity of recently mobilized elements is positively related with the rate of speciation.

Le cure oncologiche e la prospettiva del paziente. Revisione di letteratura e un caso di studio

Numerose ricerche indicano i modelli di cure integrate come la migliore soluzione per costruire un sistema più efficace ed efficiente nella risposta ai bisogni del paziente con tumore, spesso, però, l’integrazione è considerata da una prospettiva principalmente clinica, come l’adozione di linee guida nei percorsi della diagnosi e del trattamento assistenziale o la promozione di gruppi di lavoro per specifiche patologie, trascurando la prospettiva del paziente e la valutazione della sua esperienza nei servizi. Il presente lavoro si propone di esaminare la relazione tra l’integrazione delle cure oncologiche e l’esperienza del paziente; com'è rappresentato il suo coinvolgimento e quali siano i campi di partecipazione nel percorso oncologico, infine se sia possibile misurare l’esperienza vissuta. L’indagine è stata svolta sia attraverso la revisione e l’analisi della letteratura sia attraverso un caso di studio, condotto all'interno della Rete Oncologica di Area Vasta Romagna, tramite la somministrazione di un questionario a 310 pazienti con neoplasia al colon retto o alla mammella. Dai risultati, emerge un quadro generale positivo della relazione tra l’organizzazione a rete dei servizi oncologici e l’esperienza del paziente. In particolare, è stato possibile evidenziare quattro principali nodi organizzativi che introducono la prospettiva del paziente: “individual care provider”,“team care provider”,“mixed approach”,“continuity and quality of care”. Inoltre, è stato possibile delineare un campo semantico coerente del concetto di coinvolgimento del paziente in oncologia e individuare quattro campi di applicazione, lungo tutte le fasi del percorso: “prevenzione”, “trattamento”,“cura”,“ricerca”. Infine, è stato possibile identificare nel concetto di continuità di cura il modo in cui i singoli pazienti sperimentano l’integrazione o il coordinamento delle cure e analizzare differenti aspetti del vissuto della persona e dell’organizzazione.

Incompressible limit and well-posedness of PDE models of tissue growth

Both compressible and incompressible porous medium models are used in the literature to describe the mechanical aspects of living tissues. Using a stiff pressure law, it is possible to build a link between these two different representations. In the incompressible limit, compressible models generate free boundary problems where saturation holds in the moving domain. Our work aims at investigating the stiff pressure limit of reaction-advection-porous medium equations motivated by tumor development. Our first study concerns the analysis and numerical simulation of a model including the effect of nutrients. A coupled system of equations describes the cell density and the nutrient concentration and the derivation of the pressure equation in the stiff limit was an open problem for which the strong compactness of the pressure gradient is needed. To establish it, we use two new ideas: an L3-version of the celebrated Aronson-Bénilan estimate, and a sharp uniform L4-bound on the pressure gradient. We further investigate the sharpness of this bound through a finite difference upwind scheme, which we prove to be stable and asymptotic preserving. Our second study is centered around porous medium equations including convective effects. We are able to extend the techniques developed for the nutrient case, hence finding the complementarity relation on the limit pressure. Moreover, we provide an estimate of the convergence rate at the incompressible limit. Finally, we study a multi-species system. In particular, we account for phenotypic heterogeneity, including a structured variable into the problem. In this case, a cross-(degenerate)-diffusion system describes the evolution of the phenotypic distributions. Adapting methods recently developed in the context of two-species systems, we prove existence of weak solutions and we pass to the incompressible limit. Furthermore, we prove new regularity results on the total pressure, which is related to the total density by a power law of state.