Analisi della dinamica in-vitro della 3-Iodiotironamina (T1AM) utilizzando modellistica compartimentale

La 3-Iodiotironamina (T1AM) è un composto endogeno, derivato della tiroide, ottenuto da una serie di degradazioni successive della Tiroxina. L’interesse per le iodotironamine si è acceso a partire dal 2004, in seguito alla dimostrazione che il composto T1AM (3-iodotironamina) deve essere considerato come un ormone vero e proprio. Questa conclusione deriva dalle seguenti considerazioni: • T1AM è un composto endogeno; • T1AM interagisce con specifici recettori, detti TAARs (TraceAmine Associated Receptors), che sono totalmente distinti dai TAR ed appartengono alla classe dei recettori di membrana accoppiati a proteine G (GPCR = G Protein Coupled Receptors); • T1AM produce effetti funzionali su diversi organi ed apparati, in particolare i principali effetti sono una riduzione della temperatura corpore, un aumento del metabolismo dei lipidi a spese di quello dei carboidrati, una modulazione della secrezione di insulina, una risposta cardiaca inotropa e cronotropa negativa ( cioè una riduzione della contrattilità e della frequenza cardiaca). • La sostanza viene degradata, da parte dell’azione in cascata di enzimi quali monoaminossidasi (MAO) e aldeide deidrogenasi, in acido 3-iodiotiroacetico, chiamato comunemente TA1. In particolare, però, i meccanismi che regolano la dinamica di assorbimento- conversione- rilascio della sostanza non sono stati ancora chiariti o semplicemente data una stima quantitativa, sia in-vivo che ex-vivo che in-vitro. • In questo lavoro di tesi mi sono occupato di analizzare la dinamica in-vitro del T1AM, in particolare in colture cellulari statiche di cellule cardiomiocitiche di ratto della linea immortalizzata H9C2. Gli esperimenti eseguiti sono stati campionamenti di T1AM e TA1 sia nel mezzo di coltura che nell’interno delle cellule, per 1440 minuti. Vi sono stati vari esperimenti anche con inibitori della conversione e del trasporto della sostanza. Le concentrazioni sono state misurate utilizzando il tandem HPLC (High Performance Liquid Chromatography) e MS (Mass Spectrometry). In particolare il mio lavoro è stato lo sviluppo di modelli matematici atti a descrivere e correlare i dati sperimentali ottenuti. Nel primo capitolo di questa tesi vi è riassunta la letteratura riguardante il T1AM, così da fornire un quadro completo sulla fisiologia fino ad ora scoperta. Il capitolo due prevede un analisi statistica sui dati sperimentali, in particolare dell’incertezza dovuta al fatto che la misura di concentrazione non è diretta, ma deriva da un modello matematico di misura. Per il calcolo dell’incertezza finale (e quindi del peso) da applicare ad ogni dato sono state usate tecniche derivanti dalla teoria degli errori. Questa parte è stata implementata utilizzando fogli di calcolo in ambiente Microsoft Excel. Sempre nel capitolo due è stato provato ad approssimare i dati tramite modelli parametrici non-compartimentali (principalmente serie di esponenziali), ed ho provato a vedere se da queste semplici descrizioni era possibile trovare parametri macroscopici che descrivevano sia la dinamica della sostanza, che l’effetto indotto su di essi da sostanze aggiuntive quali ad esempio gli inibitori della conversione, e tentare una prima stima delle funzioni di trasferimento e dei tempi caratteristici del fenomeno. Il confronto di questi parametri ha poi avuto atto. Il tutto è stato eseguito con degli script in ambiente MATLAB, utilizzando funzioni provenienti principalmente dalle toolbox: Curve Fitting, Control System, Symbolic Math. Il capitolo 3 raccoglie invece i modelli compartimentali che simulano il sistema e la sua fisiologia. In particolare inizialmente sono stati provati modelli compartimentali di tipo LTI (Lineari Tempo Invarianti), per via del ricco arsenale di strumenti atti alla loro analisi, ed in seguito modelli non-lineari più accurati che più fedelmente simulano la realtà fisiologica. Il software utilizzati in questo caso sono MATLAB e SAAM II. Alla fine del capitolo vengono esposti i risultati ottenuti e vengono proposti sviluppi futuri e proseguimenti naturali del lavoro

Study, development and realisation of microfluidic devices able to generate nonlinear concentration gradients

Biomolecular gradients are an important, evolutionarily-conserved signalling mechanism for guiding growth, migration, and differentia- tion of cells within dynamic, three-dimensional environments of living tissues. Gradients play essential roles in many phenomena including development, inflammation, wound healing, and cancer. Interest in elucidating these phenomena has led to the development of numerous in vitro methods for exposing cells to chemical gradients. The core of this research was to develop and to realise engineered devices able to generate nonlinear concentration gradients of soluble species in cell culture chambers, in order to study cell response. During this PhD the 2D concentration gradient generator paradigm was first analysed in all its aspect, from theory (using fluid-electrical analogies) to simulation (CFD simulation showing accurately the species distribution within the device), ending with the realisation and experimental validation of the developed device. While advancing with the work, practical issues proper of those systems were faced, critically analysed and finally engineered solutions were proposed and applied, in order to make possible a reliable cell perfusion within the device. Concentration gradient concept was extended from common 2D to 3D, because soluble gradients in-vivo act in a 3D environment, thus stimulating a 3D scaffold with a 3D concentration gradient could lead in mimicking a more physiological environment. Gradients of me- chanical properties could lead cell migration and differentiation, and also them are in 3D in living tissues, i.e in the cartilage-subchondral bone system. For this reason a novel 3D concentration gradient maker, able to generate steady three-dimensional concentration gradients, was developed and realised.The device was applied as an hydrogel maker, fabricating hydrogel matrixes with a 3D gradient of mechanical properties. Those construct could be used as smart scaffolds. It is also important to guarantee the possibility to monitor environmental variables such as pH either into microfluidic devices and smart scaffolds, with not invasive/destructive methods. For this reason we developed 2D pH sensitive surfaces, with the perspective to be integrated into micro devices. To create a biocompatible 2D pH sensor, sensitive nanoparticles could be immobilised inside a hydrogel matrix, in order to guarantee a proper fluorescence Signal to Noise Ratio. Using extended range pH sensitive nanoparticles developed by the University of Nottingham, we developed sensitive surfaces. The idea of sensitive surfaces was then extended using inkjet printing coupled with Sol-gel method, using as polymeric ma- trix a Gelatine/Glycidoxypropyltrimethoxysilane (GPTMS) system, because the picoliter-size ink droplets evaporate quickly, thus allow- ing quick sol-gel transitions on the printed surface. An Ink with the pH sensitive nanoparticles was successfully printed with a modified Inkjet printer.Thanks to inkjet printing is also possible to think at 3D smart scaffolds of Gelatine/GPTMS, which inner behaviour could be investigated by optical fluorescence techniques, such as confocal microscopy, in order to have in-situ measurements. Devices and techniques developed during this PhD could have applications in biological research, as integrated tools for simultaneously stimulating and monitoring live cells, in order to gain knowledge about their behaviour. Also pharmacological profiling with ”one-shot” experiments, so drawing the dose-response curve of a compound in a single experimental run, could be an application of interest

Mixing of two miscible liquids in T-shaped microdevices

Numerical simulations were performed to study the flow fields and mixing characteristics of liquid flows converging in a T-shaped micromixer, when the two inlet fluids are both water or water and ethanol. We showed that at smaller Reynolds number, Re < 100, mixing is controlled by transverse diffusion, and therefore by the residence times of each fluids. Accordingly, mixing ethanol and water is slightly easier than mixing water with water, due to the fact that, as ethanol is slightly more viscous than water and therefore it is slower, the residence time of water-ethanol mixtures is larger than that of the water-water case. On the other hand, at larger Reynolds number, mixing water and ethanol may take considerably longer, as the onset of engulfment is retarded and occurs at larger Reynolds number, namely increasing from Re ≅ 140 in the water-water case to Re ≅ 230 in the water-ethanol case. This is due to the fact that a water-ethanol mixture has a viscosity that is up to almost three times larger than that of water; therefore, at the confluence of the T-mixer, the water and the ethanol streams are separated by a quite viscous layer of a water-ethanol mixture, that hampers any vortex formation, thus retarding mixing

Volume of Mixing Effect on Fluid Counter-Diffusion

The counter-current diffusion-driven mixing process of two miscible fluids is studied in the absence of gravity, assuming that the mixture is non regular, that is its volume is smaller than the sum of the initial volumes of the two components. Two competing effects are present in the mixing region: on one hand, the mass flow rate of each species increases, due to the larger density of the fluid; on the other hand, though, the volumetric flux is retarded by the inward convection due to volume disappearance, which opposes the outward velocity field due to diffusion. This intuition is confirmed by the analytical result of a 1D non-ideal mixing process, showing that, in the presence of the convection induced by a volume decrease, a) the process is self-similar; b) the mass flux of each species at the interface increases by approximately 0.8 e, where e is the maximum relative volume decrease; c) the volume flux of each species decreases by approximately a 0.2 e amount. This result is further confirmed by a perturbation analysis for small e

A new 3D concentration gradient maker and its application in building hydrogels with a 3D stiffness gradient

For a deeper knowledge of phenomena at cell and tissue level, for understanding the role on bimolecular signalling and for the development of new drugs it is important to recreate in vitro environments that mimic the physiological one. Spatial gradients of soluble species guide the cells' morphogenesis, and they range in a three-dimensional (3D) environment. Gradients of mechanical properties, which have a 3D pattern, could lead cell migration and differentiation. In this work, a new 3D Concentration Gradient Maker able to generate 3D concentration gradients of soluble species was developed, which could be used for differential perfusion of scaffolds. The same device can be applied to build hydrogel matrixes with a 3D gradient of mechanical properties. Computational dynamic fluid analysis was used to develop the gradient generator; the validation of the 3D gradient of stiffness was carried out using finite elements analysis and experimental studies. The device and its application could bring improvements in studying phenomena related to cell chemotaxis and mechanotaxis, but also to differentiation in the simultaneous presence of gradients in both soluble chemical species and substrate stiffnes

Genipin diffusion and reaction into a gelatin matrix for tissue engineering applications

Genipin is a natural low-toxic cross-linker for molecules with primary amino groups, and its use with collagen and gelatin has shown a great potential in tissue engineering applications. The fabrication of scaffolds with a well-organized micro and macro topology using additive manufacturing systems requires an accurate control of working parameters, such as reaction rate, gelling time, and diffusion constant. A polymeric system of 5% w/v gelatin in PBS with 2 mg/mL collagen solutions in a 1:1 weight ratio was used as template to perform measurements varying genipin concentration in a range of 0.1-1.5% w/w with respect to gelatin. In the first part of this work, the reaction rate of the polymeric system was estimated using a new colorimetric analysis of the reaction. Then its workability time, closely related to the gelling time, was evaluated thanks to rheological analysis: finally, the quantification of static and dynamic diffusion constants of genipin across nonreacting and reacting membranes, made respectively by agarose and gelatin, was performed. It was shown that the colorimetric analysis is a good indicator of the reaction progress. The gelling time depends on the genipin concentration, but a workability window of 40 min guaranteed up to 0.5% w/w genipin. The dynamic diffusion constant of genipin in the proposed polymeric system is in the order of magnitude of 10(-7) . The obtained results indicated the possibility to use the genipin, gelatin, and collagen, in the proposed concentrations, to build well-defined hydrogel scaffolds with both extrusion-based and 3D ink-jet system. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2015

Chemotherapy effects on brain glucose metabolism at rest

Background: A growing number of studies reports that chemotherapy may impair brain functions inducing cognitive changes which can persist in a subset of cancer survivors. Aims: To investigate the neural basis of the chemotherapy-induced neurobehavioral changes by means of metabolic imaging and voxel-based statistical parametric mapping analyses. Methods: We studied the resting brain [18]FDG-PET/CT images of 43 adult cancer patients with solid (n=12, 28%) or hematologic malignancies (n=31, 72%); 12 patients were studied prior to chemotherapy (No chemotherapy) while treated patients were divided into two matched subgroups: Early High (6 chemotherapy cycles, n=10), and Late Low (>9 months after chemotherapy, <6 chemotherapy cycles, n=21). Findings: Compared to No chemotherapy, the Early High subgroup showed a significant bilateral (p<0.05) lower regional cerebral metabolic rate of glucose metabolism in both the prefrontal cortices and white matter, cerebellum, posterior medial cortices and limbic regions. A similar pattern emerged in the Early High versus Low Late comparison, while no significant result was obtained in the Low Late versus No chemotherapy comparison. The number of cycles and the post-chemotherapy time were negatively and positively correlated, respectively, with a set of these same brain regions. Interpretation: The present study shows that chemotherapy induces significant transient changes in the glucose metabolism of multiple cerebral cortical and white matter regions with a prevailing involvement of the prefrontal cortex. The severity of these changes are significantly related with the number of chemotherapy cycles and a subset of brain regions seems to present longer lasting, but more subtle, metabolic changes

Clinical evaluation of two different protein content formulas fed to full-term healthy infants: A randomized controlled trial

Background: A high early protein intake is associated with rapid postnatal weight gain and altered body composition. We aimed to evaluate the safety of a low-protein formula in healthy full-term infants. Methods: A randomized controlled trial was conducted. A total of 118 infants were randomized to receive two different protein content formulas (formula A or formula B (protein content: 1.2 vs. 1.7g/100mL, respectively)) for the first 4 months of life. Anthropometry and body composition by air displacement plethysmography were assessed at enrolment and at two and 4 months. The reference group comprised 50 healthy, exclusively breastfed, full-term infants. Results: Weight gain (g/day) throughout the study was similar between the formula groups (32.5\ub16.1 vs. 32.8\ub16.8) and in the reference group (30.4\ub15.4). The formula groups showed similar body composition but a different fat-free mass content from breastfed infants at two and 4 months. However, the formula A group showed a fat-free mass increase more similar to that of the breastfed infants. The occurrence of gastrointestinal symptoms or adverse events was similar between the formula groups. Conclusions: Feeding a low-protein content formula appears to be safe and to promote adequate growth, although determination of the long-term effect on body composition requires further study

Developing Literacy Learning Model Based on Multi Literacy, Integrated, and Differentiated Concept at Primary School

The main issue addressed in this research is the low writing skills of primary school students. One of the reasons for this condition is that the existing model of writing literacy learning is not appropriate. The purpose of this study is to explain MID-based literacy teaching model and the impact of the model in increasing primary school students\u27 writing skills. This study used combined methods of exploratory type. The samples were elementary school students coming from six schools with three different characteristics. Based on the data analysis, it can be concluded that the implementation of MID-based literacy learning model has proven to signi cantly contribute to the improvement of students\u27 writing skills. Taking place in all sample schools, the improvement may suggest that the model ts not only to students with high- ability but also those with low-ability. Therefore, the MID-based literacy learning model is needed to improve the ability to write various text types appropriately

Volume of mixing effect on fluid counter-diffusion Additional information on Phys. Fluids Volume of mixing effect on fluid counter-diffusion

The counter-current diffusion-driven mixing process of two miscible fluids is studied in the absence of gravity, assuming that the mixture is non-regular, that is its volume is smaller than the sum of the initial volumes of the two components. Two competing effects are present in the mixing region: on one hand, the mass flow rate of each species increases, due to the larger density of the fluid; on the other hand, though, the volumetric flux is retarded by the inward convection due to volume disappearance, which opposes the outward velocity field due to diffusion. This intuition is confirmed by the analytical result of a 1D non-ideal mixing process, showing that, in the presence of the convection induced by a volume decrease: (a) the process is self-similar; (b) the mass flux of each species at the interface increases by approximately 0.8 , where is the maximum relative volume decrease; and (c) the volume flux of each species decreases by approximately a 0.2 amount. This result is further confirmed by a perturbation analysis for small . C 2013 AIP Publishing LLC