Dissertations / Theses on the topic 'Fluidi Biologici'

The PhD thesis is focused on the evaluation of different Mass Spectrometry approaches for the study of the proteome of biological fluids and tissues. In detail, the ClinProt technology has been applied to amniotic fluids and urines respectively, to evaluate potential biomarkers for the preterm premature rupture of the membranes (pPROM) and to invetsigate molecular mechanisms of kidney adaptation to hypobaric hypoxia conditions at high and very high altitude. MALDI Imaging Mass Spectrometry (IMS) has been applied for the study of tissues. In detail, this part of the work evaluated the use of the detergents to enhance sensitivity and number of peaks detected for protein IMS analysis.

Questo lavoro di tesi si articola in due parti: nella prima si riporta la ricerca bibliografica realizzata per lo sviluppo di un nuovo metodo analitico volto alla determinazione di acidi organici, in particolare ossalati e citrati, nelle urine mediante la formazione di complessi con metalli di transizione; la seconda invece riguarda le prove realizzate per ottimizzare un metodo in cromatografia liquida per la caratterizzazione di alcuni composti presenti nell'estratto della cannabis. La ricerca bibliografica iniziale si è focalizzata principalmente sulla stabilita dei complessi dei metalli di transizione con acidi carbossilici a diversi valori di pH, in particolare in ambiente acido, visto lo scopo di estendere il metodo all'analisi di matrici urinarie. In particolare è stato studiato il comportamento di ferro, rame e cromo con citrato e ossalato: sono stati registrati gli spettri di assorbimento UV-Vis prima di soluzioni contenti il metallo non legato poi di soluzioni del metallo legato agli acidi oggetto di indagine. La seconda parte invece riporta nel dettaglio tutte le prove realizzate su uno standard contente otto principali composti della cannabis ( CBDA CBD CNBG THCV THCA CBN THC CNBC) , variando le condizioni di temperatura, flusso, pH,, gradiente della fase mobile e colonna, prima è stata usata una C-18 poi una C-8. Ottimizzate le condizioni, è stata realizzata una retta di calibrazione e il metodo è stato poi esteso a campioni oleofiti e reali di fluidi biologici.

Il presente progetto di tesi è stato dedicato alla progettazione di un sensore indossabile, passivo e wireless in grado di rilevare fluidi biologici a contatto con la pelle. Il sistema di sensing consiste in un filtro a microonde (nella banda di 2,45 GHz) progettato con la tecnologia delle linee accoppiate con terminazioni di circuito aperto in cui una delle terminazioni della sezione centrale è sostituita da uno stub aperto la cui risonanza stabilita proprio dal contenuto del canale micro-fluidico integrato nell stub.In questo modo il comportamento del filtro è strettamente legato al fluido contenuto nel canale e si avvicina a quello ideale solo alla frequenza di risonanza dello stub che include il canale micro-fluidico contenente il liquido in esame. Uno dei punti chiave del lavoro è l’aver definito un modello circuitale equivalente del componente a microonde che include il canale: questo modello è stato ricavato da simulazioni EM per garantire una progettazione accurata ed efficiente dell’intero sistema. Al fine di rendere il sistema indossabile e passivo è stata progettata un’antenna a banda stretta connessa alla porta d’ingresso del filtro, la quale è in grado di ricevere la potenza necessaria per il sensing dei diversi fluidi presenti nel canale.

Nell’era dell’Internet of Things i sensori chimici indossabili rappresentano una frontiera avanzata nel campo delle analisi cliniche. La possibilità di integrare questo tipo di sensori su una vasta gamma di substrati tessili, in tandem a dei sistemi elettronici di lettura wireless, rende tali dispositivi estremamente versatili, permettendo all’utente finale di monitorare in tempo reale le concentrazioni degli analiti di interesse biologico in fluidi corporei, consentendo la diagnosi di potenziali patologie, la corretta somministrazione di farmaci o più semplicemente il controllo dello stato di buona salute. In questo elaborato sono stati dunque sviluppati dei sensori chimici indossabili, basati sul polimero conduttore PEDOT:PSS (applicato sotto forma di inchiostro per stampa serigrafica) in sinergia con materiali tessili per uso medico (forniti dall’azienda Plastod S.p.A. di Bologna), per la determinazione della concentrazione di ione cloruro nel sudore e del pH in essudato di ferita, attraverso la funzionalizzazione del PEDOT:PSS con particelle di Ag/AgCl e particelle di iridio ossido (IrOx). L’architettura del dispositivo, che è ispirata ai transistor elettrochimici organici (OECTs - Organic ElectroChemical Transistors), è in grado di generare un effetto di “gate” elettrochimico che permette un’amplificazione intrinseca del segnale. Inoltre, la struttura è notevolmente semplificata grazie alla presenza di soli due terminali sul dispositivo rispetto ai classici tre degli OECT. La fabbricazione dei dispositivi è stata ottimizzata attraverso lo studio della produzione dei contatti elettrici tessili e della formulazione dell’inchiostro per stampa serigrafica, mentre le prestazioni sensoristiche sono state valutate tramite applicazione di potenziali costanti durante analisi tradizionali nelle quali il dispositivo era immerso nella soluzione investigata ed analisi in flusso ideate in modo da simulare la sudorazione o la produzione di essudato da parte di una ferita.

The research project is focused on the evaluation of proteome in biological fluids from patients with renal cell carcinoma and diabetic nephropathy by a ClinProt-MALDI-TOF approach. Currently there aren’t specific biomarkers for an earlier diagnosis of RCC and nephropathic development especially in T1D patients. The most promising MS-based screening methods for the discovery of multiple biomarkers in body fluids is ClinProt, which couple a prefractionation chromatographic step with MS analysis. Renal Cell Carcinoma (RCC) is the most common kidney malignancy and its incidence is increasing worldwide each year. Since patients have a poor prognosis due to a lack of sensitivity to radio- and chemio-therapies biomarkers for early diagnosis and monitoring the state of disease are urgently needed. Concerning the diabetic nephropathy, it is the major vascular complication of type 1 diabetes and it is the leading cause of the end stage renal disease worldwide. The earliest diagnostic sign for the presence of diabetic renal damage is an increase in the albumin excretion rate, but unfortunately it isn’t an accurate predictor. So the study was aimed to perform profiling studies on urine/serum of healthy subjects and patients (RCC and diabetics with nephropathy) linking solid phase extraction technique using functionalized magnetic beads to MS analysis. In addition the proteome investigation was extended on patients with a non-RCC tumor allowing to distinguish different renal diseases. These results provided basis for the development of clinically valid proteins/peptides patterns for RCC and diabetic nephropathy diagnosis, improving the knowledge of the pathological processes in disease development.

Il carcinoma a cellule squamose è il più frequente tumore maligno del cavo orale (1, 2) ed è la sesta causa di mortalità legata a tumore nel mondo. Esso è caratterizzato da un basso tasso di sopravvivenza, dovuto principalmente alla diagnosi tardiva ed all’elevata frequenza di recidive e/o metastasi. Nel mio studio si è pertanto cercato di individuare dei marcatori a livello salivare che potessero facilitare la diagnosi precoce, utilizzando la metodica SELDI, e verificare le già note potenzialità della Survivina come marcatore biologico, al fine di rendere quindi possibile, in futuro, l’impiego della saliva come mezzo diagnostico, sia sfruttando i vantaggi che la caratterizzano, quali semplicità e rapidità di raccolta, assenza di rischi per l’operatore e minimo disagio per il paziente al momento del prelievo ,sia cercando di risolvere le problematiche legate al suo utilizzo in ambito clinico e non solo laboratoristico. Essendo riusciti nell’intento di evidenziare un nuovo marcatore, auspichiamo che nei prossimi anni si possano continuare le ricerche in modo tale da poterne evidenziare altri e standardizzarne le variazioni in condizioni patologiche.

The present thesis describes the potentials that the volatile organic compounds (VOCs) have in diagnostics as well as the results achieved by using innovative analytical systems (such as SPME-GC/MS spectrometer and electronic nose instruments) to reveal pathologies, inflammatory processes, metabolic disorders, infections and degenerative cellular processes, also occurring in the early stages. Therefore, in collaboration with both clinicians and research groups of medicine and biology, chemistry and electronic engineering, three different experiments were carried out and here described. The first one was related to the application of both a homemade electronic nose (Department of Electronic Engineering of “Tor Vergata” University), based quartz crystal microbalances (QCM) differently coated with metal porphyrins (synthesized in the Laboratory of Chemistry of “Tor Vergata” University), and a SPME-GC/MS spectrometer to investigate the pattern of the volatile compounds of synovial fluids coming from several patients suffering from two different knee‟s rheumatic diseases (“Tor Vergata” General Hospital, Medicine Department). In this case a greater abundance of volatile compounds in OA than RA. The biomarkers for differential diagnosis between OA and RA are still defective. Through the analysis of the head space of the synovial fluid by NE, good discrimination was obtained between the different pathologies considered the results indicate that the VOC mixture in the synovial fluid of patients with OA is different from that of patients with RA. The second one involved a similar equipment, whereas the electronic nose contained an increased number of sensors, to investigate VOCs from living mice (i.e. breath, skin, urine, faeces) infected with erythrocytes containing different strains of plasmodium (Higher Health Institute, ISS). The results by Plasmodium berghei infection alters the profile of VOCs released, NE analysis of the entire murine volatiloma has led to a good discrimination between a group of healthy mice and one of malaria-affected rats. The GC/MS was detected by a set of VOCs whose concentration was statistically different between infected and uninfected mice. The last one provided an intriguing investigation, using the same instrumentation, on the pattern of volatile compounds from stem cell (specifically Induced Pluripotent Stem cell) in the different stages of differentiation (Department of Genetics). In each application, a suitable measurement protocol was arranged. All the results were treated with statistical analysis and multicomponent analysis. The results support the hypothesis that the volatile fraction of the metabolic profile changes along the differentiation process as a reflection of the variations occurring in the cells. The GC/MS shows a number of compounds that can discriminate between the various stages of differentiation. The NE shows good ability to discriminate between different cellular stages, from pluripotency to differentiated cells.

Dans cette thèse, trois modèles mathématiques ont été proposés, avec l’objectif de modéliser autant d’aspects complexes de la biomédecine, dans lesquels la dynamique des fluides du système joue un rôle fondamental: i) les interactions fluide-structure entre la pulsatilité du liquide céphalo-rachidien et la moelle épinière (modélisation analytique); ii) dispersion efficace d’un médicament dans l’espace sous-arachnoïdien (modélisation numérique); et iii) la formation et l’évolution d’un thrombus au sein du système cardiovasculaire (modélisation numérique).Le liquide céphalorachidien est un fluide aqueux qui entoure le cerveau et la moelle épinière afin de les protéger. Une connaissance détaillée de la circulation du liquide céphalorachidien et de son interaction avec les tissus peut être importante dans l’étude de la pathogenèse de maladies neurologiques graves, telles que la syringomyélie, un trouble qui implique la formation de cavités remplies de liquide (seringues) dans la moelle épinière.Par ailleurs, dans certains cas, des analgésiques - ainsi que des médicaments pour le traitement de maladies graves telles que les tumeurs et les infections du liquide céphalorachidien - doivent être administrés directement dans le liquide céphalorachidien. L’importance de connaître et de décrire l’écoulement du liquide céphalorachidien, ses interactions avec les tissus environnants et les phénomènes de transport qui y sont liés devient claire. Dans ce contexte, nous avons proposé: un modèle capable de décrire les interactions du liquide céphalo-rachidien avec la moelle épinière, considérant cela, pour la première fois, comme un milieu poreux imprégné de différents fluides (sang capillaire et veineux et liquide céphalo-rachidien); et un modèle capable d’évaluer le transport d’un médicament dans l’espace sousarachnoïdien, une cavité annulaire remplie de liquide céphalo-rachidien qui entoure la moelle épinière.Avec le troisième modèle proposé, nous entrons dans le système cardiovasculaire.Dans le monde entière, les maladies cardiovasculaires sont la cause principale de mortalité. Parmi ceux-ci, nous trouvons la thrombose, une condition qui implique la formation d’un caillot à l’intérieur d’un vaisseau sanguin, qui peut causer sa occlusion. À cet égard, un modèle numérique a été développé qui étudie la formation et l’évolution des thrombus, en considérant simultanément les aspects chimico-biomécaniques et dynamiques des fluides du problème. Dans le modèle proposé pour la première fois, l'importance du rôle joué par les gradients de contrainte de cisaillement dans le processus de thrombogenèse est pris en compte.Les modèles sélectionnés ont fourni des résultats intéressants. Tout d’abord, l’étude des interactions fluide-structure entre le liquide céphalo-rachidien et la moelle épinière a mis en évidence es conditions pouvant induire l’apparition de la syringomyélie. Il a été observé comment la déviation des valeurs physiologiques du module d’Young de la moelle épinière, les pressions capillaires dans l’interface moelle-espace sousarachnoïdien et la perméabilité des compartiments capillaire et veineux, conduisent à la formation de seringues.Le modèle de calcul pour l’évaluation de la dispersion pharmacologique dans l’espace sousarachnoïdien a permis une estimation quantitatif de la diffusivité effective du médicament, une quantité qui peut aider à l’optimisation des protocoles d’injections intrathécales.Le modèle de thrombogenèse a fourni un instrument capable d’étudier quantitativement l’évolution des dépôts de plaquettes dans la circulation sanguine. En particulier, les résultats ont fourni des informations importantes sur la nécessité de considérer le rôle de l’activation mécanique et de l’agrégation des plaquettes aux côtés de la substance chimique
In the present thesis, three mathematical models are described. Three different biomedical issues, where fluid dynamical aspects are of paramount importance, are modeled: i) Fluid-structure interactions between cerebro-spinal fluid pulsatility and the spinal cord (analytical modeling); ii) Enhanced dispersion of a drug in the subarachnoid space (numerical modeling); and iii) Thrombus formation and evolution in the cardiovascular system (numerical modeling).The cerebrospinal fluid (CSF) is a liquid that surrounds and protects the brain and the spinal cord. Insights into the functioning of cerebrospinal fluid are expected to reveal the pathogenesis of severe neurological diseases, such as syringomyelia that involves the formation of fluid-filled cavities (syrinxes) in the spinal cord.Furthermore, in some cases, analgesic drugs -- as well drugs for treatments of serious diseases such as cancers and cerebrospinal fluid infections -- need to be delivered directly into the cerebrospinal fluid. This underscores the importance of knowing and describing cerebrospinal fluid flow, its interactions with the surrounding tissues and the transport phenomena related to it. In this framework, we have proposed: a model that describes the interactions of the cerebrospinal fluid with the spinal cord that is considered, for the first time, as a porous medium permeated by different fluids (capillary and venous blood and cerebrospinal fluid); and a model that evaluates drug transport within the cerebrospinal fluid-filled space around the spinal cord --namely the subarachnoid space--.The third model deals with the cardiovascular system. Cardiovascular diseases are the leading cause of death worldwide, among these diseases, thrombosis is a condition that involves the formation of a blood clot inside a blood vessel. A computational model that studies thrombus formation and evolution is developed, considering the chemical, bio-mechanical and fluid dynamical aspects of the problem in the same computational framework. In this model, the primary novelty is the introduction of the role of shear micro-gradients into the process of thrombogenesis.The developed models have provided several outcomes. First, the study of the fluid-structure interactions between cerebro-spinal fluid and the spinal cord has shed light on scenarios that may induce the occurrence of Syringomyelia. It was seen how the deviation from the physiological values of the Young modulus of the spinal cord, the capillary pressures at the SC-SAS interface and the permeability of blood networks can lead to syrinx formation.The computational model of the drug dispersion has allowed to quantitatively estimate the drug effective diffusivity, a feature that can aid the tuning of intrathecal delivery protocols.The comprehensive thrombus formation model has provided a quantification tool of the thrombotic deposition evolution in a blood vessel. In particular, the results have given insight into the importance of considering both mechanical and chemical activation and aggregation of platelets

Since Poiseuille times, several techniques has been developed to measure the viscosity of blood. During the 60's and 70's, with the appearance of the first rheomethers, the rheological properties of blood were accurately measured and a behavior dependent of the velocity gradient of the fluid flow was determined. As well, it was observed that blood had shear thinning properties, meaning, that if the velocity of the blood flow increases, the viscosity of blood decreases. From a medical point of view, blood and its blood plasma are the most effective fluids to detect global pathologies in human and animals. These pathologies may be related to their viscosities, their plasma proteins, or the properties of its red blood cells, as their aggregation, deformability or the elastic properties of its cellular membrane. Lately, with the birth of microfluidics at the beginnings of the 90’s, new techniques for the diagnostic of diseases has been developed. The avantages in the use of microfluidic devices for diagnostics are: the low amount of sample required to perform a measure, their portability, that they are easy to use and the low cost of its fabrication. The aim of this thesis project was to extend the study of front microrheology through the development of a device and method that describes accurately the non-linear rheology of biofluids, mainly blood, by means of a simple optical detection method based on tracking the fluid-air interface moving inside a microchannel. We centered in the fluid front (interface fluid-air) since is a direct, easy and cheap method to study fluid flows. In order to achieve this, we first had to developed a microfluidic device and method which would allow us to obtain a clear image of the fluid front. This was made, using a microscope and a high speed camera. The images obtained with the camera were analyzed by means of a computational code developed in Wolfram Mathematica©. The thesis work was mainly experimental comprinsing: fabrication of microfluidic devices and experiments with Newtonian and non-Newtonian fluids. Our results have been compared with theoretical and bibliographical results. The original results from this thesis are separated in two parts. The first part of the research was dedicated to the study the interface fluid-air of flows of Newtonian fluids. In order, to achieve reliable viscosity results with our device and method, and prove our system as a viscometer, we tested several fluids, including blood plasma which is known to be Newtonian. Our results were compared with the results obtained with a different viscometer to prove their reliability. This part of the study states our microfluidic device and method as a viscometer. The second part of this thesis was dedicated to extend the results for Newtonian fluid to non-linear hemorheology and comprises all the results for blood. Blood is essentially a difficult fluid to manipulate and study. In general, it presents non-Newtonian properties as shear thinning, meaning, that its viscosity decreases as the stress or the shear rate increases. This non-Newtonian properties are due to plasma proteins and especial characteristics of its red blood cells. With our device and method we were able to observe the non-Newtonian behavior of blood and to obtain its viscosity at different shear rates and stresses. As well, we related its viscosity to some of its red blood cells properties, as their tendency to form aggregates and the flexibility of their cellular membrane. The studies of blood were developed at different hematocrits, different dates from the extraction of the sample and with anemic blood and blood with alphathalessemia. In general, our device and method is usefull as a viscometer and rheometer, as well as, it enables to establish a relation between blood viscosity and its red blood cells characteristics.
Desde los tiempos de Poiseuille, se han desarrollado variadas técnicas para medir la viscosidad de la sangre. Durante las décadas de los 60’s y 70’s con la aparición de los primeros reómetros las propiedades reológicas de la sangre fueron medidas y se determinó su comportamiento dependiente del gradiente de velocidad. Además se observó que posee un comportamiento pseudoplástico, es decir, que a medida que aumenta su velocidad su viscosidad disminuye. Desde un punto de vista médico, la sangre y su plasma sanguíneo son los fluidos más eficaces para la detección de patologías globales. Estas patologías pueden estar relacionadas con su viscosidad, con las proteínas presentes en el plasma o con las propiedades de sus glóbulos rojos, como su agregación, deformabilidad o la capacidad elástica de su membrana celular. En los últimos años, con el nacimiento de la microfluídica a principio de los 90’s, nuevas técnicas para el diagnóstico de enfermedades se han desarrollado. La ventaja del uso de la microfluídica en el diagnóstico de enfermedades viene dada por: el bajo requerimiento de muestra para realizar la detección, su portabilidad, la facilidad de uso y el bajo costo de su fabricación. El objetivo de esta tesis ha sido el estudio de la interfase fluido-aire, por medio del desarrollo de un dispositivo microfluídico y método sencillo que permite obtener la viscosidad tanto de fluidos newtonianos e.g. plasma sanguíneo y sangre con un error no superior al 10%. Además de ser capaces de observar el comportamiento no-Newtoniano de la sangre, y a su vez, relacionar su viscosidad con características específicas de sus células rojas como la agregación y la flexibilidad de su membrana. Los estudios de sangre se realizaron a distintos hematocritos, distintos días desde la extracción de la muestra y muestras de anemia y alfa-talasemia. La tesis ha sido desarrollada principalmente desde un punto de vista experimental y está separada en 2 partes. La primera contempla los resultados obtenidos en el estudio de frentes de fluidos Newtonianos. La segunda parte se centra en los resultados obtenidos para la sangre y su relación con las propiedades de sus células rojas.

Fluid interactions are ubiquitous in the natural world; all organisms must find strategies to generate, utilise or resist flow in order to be successful. A process fundamental to all life on earth is reproduction, which in many cases entails the swimming of sperm cells. Cell swimming arises from coupled interactions between physical and biological processes. We will focus on the effects of changing fluid rheology on microscopic swimmers, with a particular application to the study of internal mammalian fertilisation. To reach the egg, mammalian sperm must navigate the convoluted geometry of the female reproductive tract, actively bending their flagella in order to propel themselves through cervical mucus: a suspension of polymer chains that twist, tangle and align with flow, giving it complex properties. Whilst recent work has examined the effects of fluid viscoelasticity on sperm-like swimmers, relatively less attention has been given to the shear-thinning property. We develop a new finite element technique to simulate free swimmers with prescribed beat kinematics in shear-thinning fluids with nonlinear governing equations. This technique is then applied to three qualitatively different viscous swimmers in order to examine the different phenomena that arise from swimmer interactions with of shear-thinning fluid.

Biological matter is driven far from thermodynamic equilibrium by active processes on the molecular scale. These processes are usually driven by the chemical reaction of a fuel and generate spontaneous movements and mechanical stresses in the system, even in the absence of external forces or torques. Moreover these active stresses effectively fluidify the material. The cell cytoskeleton, suspensions of swimming microorganisms or tissues are prominent examples of active fluids. Active processes in biological systems often exhibit chiral asymmetries. Examples are the chirality of cytoskeletal filaments which interact with motor proteins, the chirality of the beat of cilia and flagella as well as the helical trajectories of many biological micro-swimmers. Moreover, large scale chiral flows have been observed in the cell cortex of C. elegans and Xenopus embryos. Active force generation induces force and torque dipoles in the material. If all forces are internal the total force and torque vanish as required by the conservation of momentum and angular momentum. The density of force dipoles is an active stress in the material. In addition, active chiral processes allow for the existence of active torque dipoles which enter the conservation of angular momentum and generate an active antisymmetric stress and active angular momentum fluxes. We developed a generic description of active fluids that takes into account active chiral processes and explicitly keeps track of spin and orbital angular momentum densities. We derived constitutive equations for an active chiral fluid based on identifying the entropy production rate from the rate of change of the free energy and linearly expanding thermodynamic fluxes in terms of thermodynamic forces. We identified four elementary chiral motors that correspond to localized distributions of chiral force and torque dipoles that differ by their symmetry and produce different chiral fluid flows and intrinsic rotation fields. We employ our theory to analyze different active chiral processes. We first show that chiral flows can occur spontaneously in an active fluid even in the absence of chiral processes. For this we investigate the Taylor-Couette motor, that is an active fluid confined between two concentric cylinders. For sufficiently high active stresses the fluid generates spontaneous rotations of the two cylinders with respect to each other thus breaking the chiral symmetry of the system spontaneously. We then investigate cases where active chiral processes on the molecular scale break the chiral symmetry of the whole system. We show that chiral flows occur in films of chiral motors and derive a generic theory for thin films of active fluids. We discuss our results in the context of carpets of beating cilia or E. coli swimming close to a surface. Finally, we discuss chiral flows that are observed in the cellular cortex of the nematode C. elegans at the one cell stage. Two distinct chiral flow events are observed. The first chiral flow event (i) is a screw like chiral rotation of the two cell halves with respect to each other and occurs around 15min after fertilization. This event coincides with the establishment of cortical cell polarity. The second chiral flow event (ii) is a chiral rotation of the entire cell cortex around the anterior posterior axis of the whole cell and occurs around 30min after fertilization. Measuring densities of molecular motors during episode (i) we fit the flow patterns observed using only two fit parameters: the hydrodynamic length and cortical chirality. The flows during (ii) can be understood assuming an increase of the hydrodynamic length. We hypothesize that the cell actively regulates the cortical viscosity and the friction of the cortex with the eggshell and cytosol. We show that active chiral processes in soft biological matter give rise to interesting new physics and are essential to understand the material properties of many biological systems, such as the cell cortex.

The objectives of the current study were: 1) to examine serum bPAG concentration during pregnancy, and in the post-partum period in relation to the overall nutritional and metabolic status and post-partum anoestrous period; 2) to investigate bPAG decay models for postulating distribution and metabolism of bPAG in the blood circulation using half-life (t1/2) and to compare the t1/2 values between breeds and nutritional treatments for the clearance of bPAG; 3) to examine the suitability of the radioimmunoassay (RIA) for measuring bPAG in urine, saliva and milk, to investigate temporal relationships between bPAG in these fluids and that in the serum, and to assess the potential of bPAG in these fluids for pregnancy diagnosis in cows. Estimation of total bPAG disappearance from plasma per day during week 1 post-partum did not differ between the breeds. Urinary bPAG, over the same period of time, was lower for Welsh Black than Simmental and Charolais cows (0.45 vs 0.53 and 0.59 nmol (s.e.d 0.03) p<0.05) but not for Aberdeen Angus cows (0.48 nmol); bPAG in saliva was lower for Welsh Black than Charolais and Aberdeen Angus cows (1.65 vs 2.42 and 2.49 nmol (s.e.d 0.11) p<0.05) but not for Simmental cows (1.44 nmol/1), and bPAG measured in milk (whole) was lower for Charolais than Simmental cows (0.26 vs 0.47 nmol (s.e.d 0.06) p<0.05) but not for Aberdeen Angus (0.38 nmol/1) and Welsh Black cows (0.37 nmol/1). Total body elimination of bPAG in urine, saliva and milk accounted only for 5.28 % of the total plasma bPAG. Total body elimination of bPAG in urine, saliva and milk accounted for only 5.28 % of the total plasma bPAG. The remaining 94.72 % of bPAG may have been metabolished and was undetectable by the current RIA technique. A theoretical assessment for potential use of bPAG RIA for pregnancy diagnosis using urine, saliva and milk showed that the minimum detection limits of measuring bPAG in these fluids were reached and / or exceeded by Day 200 of gestation, or later. Consequently, assay of bPAG in these fluids for early pregnancy diagnosis would not be appropriate unless the sensitivity of the assays is improved considerably, or prior assay sample extraction/concentration is carried out.

The herein presented work aims to develop and expand Mars geological exploration in a search for life prospective and, accordingly, water resurgence features and possible degassing centers have been given a central role in the target selection and process investigation. Hydrothermal fluid circulation in the Martian crust is among the natural processes characterized by the combined involvement of fluids such water and methane so defining a potential set of environments prone to biosphere growth and flourish. Subsurface fluid flow is a key area of planetary science research because fluids affect almost every physical, chemical, mechanical and thermal property of the upper crust. Hydrothermal systems are closely bond to the transport of mass, heat, nutrients and chemical species in hydrogeological systems making these mechanisms central in fields such as volcano-tectonic, deep-biosphere and water/ice cycle. To step forward toward a new generation of planetary exploration that aims, not only to analyse and map the surfaces of planetary bodies other than Earth, but also to push the survey down in depth, in the first chapter we successfully test the efficiency of a rising technique that allows to probe the subsurface starting from surface case studies: fractal analysis. This method was firstly applied on many different study cases on Earth to investigate the location at depth of magma chambers and sediment source layers beneath volcanic vent and mud volcano fields. We thus took this technique and applied it to many different well-known morphologically convergent features on Mars, but with very different inferred formation process, in order to test if fractal analysis were an efficient methodology to identify spatial patterns linked to system of percolating connected fractures and drained material source depth by outputting the expected outcomes for the different cases. Thanks to the successfully obtained results we fostered the implementation of such method in the planetary exploration research field. In the second chapter is reported the produced work concerning the exploration and investigation aimed to identify new regions on Mars with a high astrobiological potential through the usage of classic and fractal analyses. Since the main objective of the herein presented work is to spot emission centers linked to water and methane release, we set our starting point on the search for fields of pitted mounds, which are good candidate morphologies for our purposes. Many different areas, with large coverages and very different geological context showed a relationship with systems of connected fractures extending many kilometres beneath the surface. We were not just able to profitably analyse different areas and locate several interesting vast regions, but we observed a systematic linkage between large fields of pitted mounds on the surface and the shallowest interface between gas hydrate-rich cryosphere and hydrosphere hypothesised for the Martian subsurface, so discovering the potential key role of clathrates on a, geologically speaking, recent Mars. The intriguing results produced and displayed in the first two chapters of this work led to a spectrum of unsolved questions concerning the processes that could be involved in such kind of phenomena. We thus choose to approach this topic from the structural side aiming to produce structural asset interpretations based on fluid circulation evidence, where information is available. In the third chapter, we hence face a propaedeutic explorative study which has the objective to compare sulfate vein networks on several locations on Earth with sulfate veins outcropping in the Gale crater (Curiosity Rover landing site, Mars), that represent the only case of close up acquisitions of Martian features that surely experienced fluid circulation. A better understanding of the structural asset on portions of the Martian surface will progressively lead to a contextualisation of the forces that could have contributed to drive the fluid flows in the upper Martian crust and again pushing the exploration toward the subsurface realm and to the identification of outgassing and water related environments. In the fourth chapter are exposed preliminary works that further pursue the aim of identify and investigate environments that experienced fluid circulation, backbone of this thesis. On one side, we moved on in exploring the Martian surface throughout the observation of the freshly acquired four-colours images of the CaSSIS camera we are involved in, with remarkable outcomes thanks to the location of light-toned ridges possibly linked to hydrothermal fluid percolation and connected rocks alteration. Contextually, we also approached the question from the compositional side by enhancing spectral libraries with the production of spectral signatures, on ultraviolet- far infrared wavelength span, of minerals belonging to environments that, on Earth, are linked to low temperature hydrothermal circulation and of rare bio-mineralisation features that are siliceous stromatolites.
Il lavoro presentato ha lo scopo di sviluppare ed espandere l'esplorazione geologica di Marte nell’ottica di ricerca di ambienti adatti allo sviluppo della vita e, di conseguenza, centri di risalita di acqua e centri di degassamento hanno avuto un ruolo centrale nella selezione degli obiettivi di indagine. La circolazione idrotermale nella crosta marziana è tra i processi naturali caratterizzati dal coinvolgimento combinato di fluidi quali acqua e metano, definendo così un potenziale insieme di ambienti inclini alla crescita e allo sviluppo della biosfera. La circolazione di fluidi nel sottosuolo è un'area chiave nel contesto delle scienze planetarie perché essi influenzano quasi ogni proprietà fisica, chimica, meccanica e termica della crosta superiore. I sistemi idrotermali sono strettamente legati al trasporto di massa, calore, sostanze nutritive e specie chimiche nei sistemi idrogeologici, rendendo questi meccanismi centrali in campi quali il ciclo vulcano-tettonico, la biosfera profonda e il ciclo acqua / ghiaccio. Per sviluppare una nuova generazione di esplorazione planetaria che mira non solo ad analizzare e mappare le superfici dei corpi planetari diversi dalla Terra, ma anche a sondarne le profondità, nel primo capitolo testiamo con successo l'efficienza di una nuova tecnica che permette di investigare il sottosuolo partendo dalle osservazioni di superficie: l’analisi frattale. Questo metodo è stato applicato per la prima volta sulla Terra per indagare la profondità delle camere magmatiche e degli strati sorgente che alimentano vulcanesimo magmatico e vulcani di fango. Abbiamo quindi applicato questa tecnica a diverse strutture di superficie su Marte con caratteristiche morfologicamente convergenti, ma con processi di formazione molto diversi, al fine di verificare se l'analisi frattale fosse una metodologia efficiente per identificare la presenza di un sistema percolante di fratture connesse e la profondità della sorgente del materiale drenato. I risultati sono stati positivi promuovendone così l'implementazione nel processo di esplorazione planetaria. Nel secondo capitolo viene riportato il lavoro prodotto relativo all'esplorazione volto a identificare nuove regioni ad alto potenziale su Marte attraverso l'uso di analisi classiche e frattali. Poiché l'obiettivo principale del presente lavoro presentato è quello di individuare i centri di emissione legati al rilascio di acqua e metano, poniamo il nostro punto di partenza nella ricerca di campi di pitted mounds, che sono ottimi candidati per i nostri scopi. Varie aree, con grandi coperture e un contesto geologico molto diverso, hanno mostrato una relazione con sistemi di fratture connesse con estensioni fino svariati chilometri di profondità. Non solo siamo stati in grado di analizzare proficuamente aree diverse e localizzare vaste regioni ad alto interesse, ma abbiamo osservato un collegamento sistematico tra grandi campi di pitted mounds sulla superficie e l'interfaccia più superficiale tra la criosfera ricca in clatrati e l'idrosfera ipotizzata per il sottosuolo marziano, scoprendo così il ruolo chiave che i clatrati potrebbero aver avuto su Marte i un passato geologicamente recente. I risultati promettenti prodotti e mostrati nei primi due capitoli di questo lavoro hanno portato a uno spettro di domande riguardanti i processi che potrebbero essere coinvolti in questo tipo di fenomeni. Scegliamo quindi di affrontare questo argomento tramite l’interpretazione dell’assetto strutturale basato su evidenze di circolazione di fluidi, in aree in cui tali informazioni sono disponibili. Nel terzo capitolo, quindi, affrontiamo uno studio esplorativo propedeutico che ha l'obiettivo di confrontare sistemi di vene a solfati in diverse località sulla Terra con le vene a solfati affioranti nel Gale crater, che rappresentano l'unico caso di acquisizioni ravvicinate di strutture marziane che sicuramente hanno sperimentato circolazione di fluidi. Una migliore comprensione dell’assetto strutturale su porzioni della superficie marziana può portare progressivamente ad una contestualizzazione delle forze che potrebbero aver contribuito a guidare i flussi di fluido nella crosta superiore marziana e inoltre a migliorare la corrente conoscenza del sottosuolo marziano nonché all’identificazione di ambienti legati all'acqua. Nel quarto capitolo sono esposti i lavori preliminari che hanno come obiettivo quello di identificare e indagare ambienti che hanno subito la circolazione di fluidi, spina dorsale di questa tesi. Da un lato, siamo andati avanti nell'esplorazione della superficie marziana attraverso l'osservazione delle immagini a quattro colori appena acquisite della camera CaSSIS, con esiti notevoli grazie all’individuazione di creste probabilmente legate alla percolazione di fluido idrotermale e all'alterazione delle rocce incassanti. Contestualmente, abbiamo anche affrontato la questione dal lato composizionale migliorando le librerie spettrali con la produzione di firme spettrali, in lunghezze d'onda dall'ultravioletto al lontano infrarosso, di minerali appartenenti ad ambienti che, sulla Terra, sono legati alla circolazione idrotermale a bassa temperatura e di rare bio-mineralizzazioni quali le stromatoliti silicee.

Microfluidics miniaturizes many benchtop processes and provides advantages of low cost, reduced reagent usage, process integration, and faster analyses. Microfluidic devices have been fabricated from a wide variety of materials and methods for many applications. This dissertation describes four such examples, each employing different features and fabrication methods or materials in order to achieve their respective goals. In the first example of microfluidic applications in this dissertation, thermoplastics are hot embossed to form t-shaped channels for microchip electrophoresis. These devices are used to separate six preterm birth (PTB) biomarkers and establish a limit of detection for each. The next chapter describes 3D printed devices with reversed-phase monoliths for solid-phase extraction and on-chip fluorescent labeling of PTB biomarkers. I demonstrate the optimization of the monolith and selective retention of nine PTB biomarkers, the first microchip study to perform an analysis on this entire panel. The third project describes the iterative design and fabrication of glass/polydimethylsiloxane (PDMS) devices with gold and nickel electrodes for the self-assembly of DNA nanotubes for site-selective placement of nanowires. Simple flow channels and “patch electrode” devices were successfully used, and DNA seeding was achieved on gold electrodes. Finally, a 3D printed device for cancer drug screening was developed as a replacement for one previously fabricated in PDMS. Devices of increasing complexity were fabricated, and those tested found to give good control over fluid flow for multiple inlets and valves. Although the applications and methods of these projects are varied, the work in this dissertation demonstrates the potential of microfluidics in several fields, particularly for diagnostics, therapeutics, and nanoelectronics. Furthermore, it demonstrates the importance of applying appropriate tools to each problem to gain specific advantages. Each of the described devices has the potential for increased complexity and integration, which further emphasizes the advantages of miniaturized analyses and the potential for microfluidics for analytical testing in years to come.

Le processus d'étalement et d'évaporation d'une goutte de suspensions de particules sur une surface solide est très intéressant permettant la formation de motifs. Une étude expérimentale à été effectuée avec du sang total humain et avec des fluides purs dans une chambre sous atmosphère contrôlée en humidité relative. Pour des angles de contact faible, le processus d'étalement/évaporation peut être divisé en deux régimes. Un premier régime rapide gouverné par un équilibre entre les forces visqueuses et les forces capillaires et un deuxième régime plus lent dominé par la cinétique d'évaporation. Nous montrons que les bio-colloïdes jouent un rôle significatif sur la dynamique de la ligne de contact. La vitesse moyenne de la ligne de contact suit la même dynamique d'étalement que le modèle de Tanner, où le temps d'étalement et les paramètres géométriques de la goutte sont fonctions de l'humidité. Dans cette étude, nous montrons que l'humidité relative influence les paramètres géométriques de la goutte et par conséquent le motif a la fin du processus d'évaporation. Un modèle purement diffusif pur a été obtenu dont le diamètre de mouillage et l'angle de contact sont fonction de l'humidité. Pour l'analyse morphologique des motifs de craquelures, une méthode de segmentation manuelle a été utilisée comme une méthode de référence pour la validation de la méthode de segmentation automatique développée dans "iBlood". Par cette méthode, nous montrons que la cinétique d'évaporation influence la distribution structurelle et morphologique des cellules de forme trapézoïdale, et par conséquent, l'espacement des fractures moyenne finale
Spreading/evaporation process of droplets over solid surfaces is a fundamental process and a wide research field because of number of applications in printing, micro-electronics, DNA analysis and even in biomedical. This experimental work aims to investigate the effect of relative humidity on the contact line dynamics, on the evaporation dynamics and on the final pattern of a drop of whole human blood. The spreading of a pure fluid model that has the same physical properties as human blood was studied and compared to the blood. We showed that bio-colloids play significant effect on the dynamics of contact line and the pinning effect of the drop. For low contact angles, we showed that the spreading/evaporation process could be divided into two regimes. A fast first regime determined by a balance between viscous forces and capillary forces and a second slower regime dominated by the evaporation rate. Physical mechanisms that are responsible for the spreading enhancement are proposed and discussed. The average velocity of the contact line was found to follow the same behaviour as Tanner's model, where the spreading dynamics and geometrical parameters of the droplet are function of relative humidity. The experimental measurements are in a good agreement with the purely diffusive model where the equilibrium wetting radius and contact angle are function of relative humidity. For the morphological analysis of crack patterns, a manual segmentation method was used as a reference for the validation of the automatic developed segmentation method. We showed that the evaporation rate influences structural distribution of plaques in the corona region and hence, the mean crack spacing

This doctoral thesis has integrated biological models (activated sludge models “ASM”) with simulation tools of computational fluid dynamics “CFD”, and has applied them to the design of two advanced technologies for wastewater treatment: “Microbial Fuel Cells” and the “Anammox” process. Wastewater treatments based on Microbial Fuel Cells use the catalytic bioelectrochemical activity of certain microorganisms to oxidise organic compounds and produce electricity without needing additional chemical compounds. In Anammox process (Anaerobic Ammonium Oxidation) microorganisms also play a main role in the autotrophic removal of nitrogen compounds.
Aquesta tesi doctoral ha integrat models biològics - en concret, models de fangs activats “ASM” amb eines de simulació de dinàmica de fluids computacional “CFD”, i les ha aplicat al disseny de dues tecnologies avançades per al tractament de les aigües residuals: les “Microbial Fuel Cells” o piles bioelectroquímiques i el procés “Anammox”. El sistemes de tractament d’aigües residuals basats en Microbial Fuel Cells aprofiten l’activitat catalítica bioelectroquímica de determinats microorganismes per oxidar compostos orgànics i produir electricitat sense necessitat d’addicionar compostos químics. També en el procés Anammox (Anaerobic Ammonium Oxidation) els protagonistes són els microorganismes, en aquest cas per eliminar de manera autotròfica compostos de nitrogen, i reduir així la quantitat de fangs generats i les necessitats d’aeració.

Orientadores: Theo Guenter Kieckbusch, Koichi Fujie
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Quimica
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Resumo: O controle do lodo em excesso, proveniente dos processos de tratamento de águas residuais industrial e doméstica é, cada vez mais, um assunto de considerável preocupação, pois o descarte destes resíduos pode causar sérios danos ao meio ambiente. Em vista disto, o desenvolvimento de novas tecnologias que permitam uma diminuição na quantidade de lodo produzido torna-se indispensável. Um outro problema encontrado no tratamento de águas residuais é o descarte de alguns nutrientes provenientes do processo, como o fosfato, em cursos d¿água, o que tem aumentado a eutrofização em rios. Um processo chamado Remoção Biológica Aprimorada de Fosfato ¿ EBPR (Enhanced Biological Phosphate Removal), é, atualmente, considerado um dos meios mais econômicos para remoção de fosfato. O presente trabalho teve como objetivo investigar a viabilidade técnica de tratar o lodo em excesso em condições sub e supercríticas da água e reutiliza-lo como fonte de carbono biodegradável no processo EBPR. Lodo em excesso de uma indústria de processamento de peixes (Toyohasshi ¿ Japão) foi submetido às reações hidrotérmicas em um reator em batelada, a uma faixa de temperatura de 200 '400 GRAUS¿C, pressões variando de 1,8 a 30 Mpa e tempo de reação de 10 min. Ensaios de liberação de fosfato foram conduzidos com o objetivo de analisar a capacidade dos microorganismos em liberar fosfato intracelular em meio anaeróbio, utilizando o lodo tratado como fonte de carbono... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital
Abstract: the control of excess sludge produced in the industrial and domestic wastewater treatment plants has been a matter of considerable concern. Discharge of excess sludge poses significant risks to the environment and the development of new technologies that are able to suppress the pollution have challenged many researches. On the other hand, the release of some nutrients like phosphorus in wastewater streams increases the eutrophication problem in many rivers throughout the world with the consequent growth of algae induced by high levels of phosphate. Enhanced Biological Phosphate Removal (EBPR) process has been currently considered one of the most economical ways to remove phosphate from the wastewaters. The objective of this work is to investigate the feasibility of reusing excess sludge from a fish processing industry located in Japan was treated by hydrothermal reactions, at reaction temperature ranging from 200 to '400 DEGREES¿, pressure of 1,8 to 30 MPa and fixed reaction time of 10 min. Experiments on phosphorus release under anaerobic conditions were also carried out using the treated excess sludge as carbon source. For the hydrothermal conditions tested, the results showed that the solubilization and the biodegradability were improved as evidenced by its content change after hydrothermal reactions... Note: The complete abstract is available with the full electronic digital thesis or dissertations
Mestrado
Engenharia de Processos
Mestre em Engenharia Química

Lactate is a key metabolite of glycolytic activity and as such, can be used as an indicator of the energy production of the whole organism, for the assessment of tissue perfusion and oxidative capacity. Estimating lactate levels in biological fluids allows the determination of anaerobic threshold during physical exercise. Likewise, lactate is of significant importance in several clinical situations, where a rapid and easy method is needed for diagnostic assessment and survival rate increase of the patient.
To achieve this objective, the potential of Near Infrared Spectroscopy (NIRS) to quantify lactate in biological fluids and tissues was evaluated. Initially, the project focused on quantifying of lactate in plasma samples taken from exercising humans. Using Partial Least Squares (PLS) and a leave-N-out cross validation routine, it was found that lactate concentration in human plasma could be estimated with a standard error of cross validation of 0.51 mmol/L.
To minimize sample preparation and reduce the time of analysis, NIRS was then evaluated as a technique for rapid analysis of lactate in whole blood from exercising rats and humans. Furthermore, standard addition method was used to expand the lactate concentration range and therefore cover a greater part of the physiological lactate concentration range. Regression analysis provided standard errors of cross validation of 0.29 mmol/L and 0.65 mmol/L for rats and humans respectively.
To improve precision, referenced lactate measurements were calculated. In this method, baseline spectra of subjects were subtracted from all collected spectra before chemometric routines were used. An improvement of the standard error of cross validation to 0.21 mmol/L was found by applying this procedure.
In vivo measurement of lactate during exercise in humans by NIRS was also evaluated. Using diffuse reflectance and 2D correlation spectroscopy, lactate was identified as the primary constituent monitored by in vivo measurements. Regression analysis resulted in a substantial error of 2.21 mmol/L for absolute measurements. However, results for referenced lactate measurements provided a significant improvement of the standard error of cross validation to 0.76 mmol/L. This finding suggests that NIRS may provide a valuable tool to assess in vivo physiological status for both research and clinical needs.

This thesis describes the development of an assay for free radical scavenging (antioxidant) activity in biological fluids based on an enhanced chemiluminescent reaction. Light emission from the reaction depends on the constant production of free radical intermediates and is therefore sensitive to interference by antioxidant compounds. The time period of light suppression is directly related to the amount of antioxidant added to the reaction. In this way the antioxidant activity of biological samples can be related to a standard antioxidant solution of the vitamin E analogue, trolox. The effect of a variety of pure compounds upon light emiSSion are described. Based on these observations a mathematical model for the reaction kinetics in response to the addition of antioxidants and other compounds is developed. The impact of more complex biological fluids on the reaction is described with particular reference to serum. An assessment of the contribution of individual antioxidants to serum total antioxidant activity suggests that urate accounts for 70% while ascorbate and vitamin E each account for a further 10%. The use of the assay is extended to the measurement of antioxidant activity in solutions of plasma lipoproteins isolated by density gradient ultracentrifugation. These studies suggest that vitamin E is the major (but not exclusive) contributor to lipoprotein antioxidant activity. The distribution of antioxidant activity across different lipoprotein fractions and co-operative interaction of antioxidant activity in lipoproteins are described.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2006.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. [137]-143).
Tracking the Brownian motion of colloids was first used about a hundred years ago to demonstrate the molecular nature of matter. Today's colloidal scientists perform particle tracking experiments to assess the structural and mechanical properties of complex materials at a micron length scale. Indeed, the dynamics of micron sized probe particles embedded in a material can be related to the local mechanical response of the system. This probing technique, called microrheology, has received much interest in the last few decades due to the importance of a materials local properties in its function and its macro-scale characteristics. These new assessments are especially relevant in soft matter sciences such as biophysics. Video microscopy particle tracking is an easy technique to implement experimentally. Movies of the fluctuating particles in a sample are recorded and analyzed off-line using custom algorithms. For this reason, it is widely used in studies of soft matter properties and in fluid dynamics. However, behind this apparent simplicity lie a number of subtle limitations that can alter significantly the validity of the measurements. The focus of the parts of this thesis is an exhaustive characterization of the errors incurred in the standard video microscopy particle tracking setup.
(cont.) Detailed understanding of these errors led to new methods to circumvent some of the intrinsic limitations. The trajectories extracted from particle tracking are used to compute the means-squared displacement that characterizes the dynamics of the probe particles. This measurement suffers from two kinds of limitations: the finite spatial resolution in the particle localization and statistical uncertainties. The source of localization errors was separated into two separate contributions. A "static error" arises in the position measurements of immobilized particles. A "dynamic error" comes from the particle motion during the finite exposure time that is required for visualization. We calculated the propagation of these errors on the mean-squared displacement and examined the impact of our analysis on theoretical model fluids used in biorheology. These theoretical predictions were verified for purely viscous fluids using simulations and a multiple particle tracking technique performed with video microscopy. We showed that the static contribution could be confidently eling the sampling design, we derived estimators for the mean and variance of particle's dynamics that are independent, under well-efined conditions, of the peculiar statistics of the measurement output.
(cont.) These estimators serve to quantify a material heterogeneity. Having gained a full characterization of the technique, we applied video multiple particle tracking to study a complex time-evolving system of self-assembling peptides. This material undergoes a transition from a purely viscous solution to an elastic hydrogel through the molecular assembly of the peptides into a fibrous network. We used the oligo-peptide KFE8 as a model self-assembling peptide and assessed the dependency of the gelation kinetics with the pH of the solution. We were able to develop a theoretical model for this dependency by using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory for the interaction between the peptides.
by Thierry Savin.
Ph.D.

The objective of this research was to develop new sample preparation procedures for the isolation of sulfonamides, as well as, to determine the applicability of employing on-line nitrogen selective and mass spectrometric detection methods. The first phase of this research investigated the effect of temperature and pressure on the supercritical fluid extraction (SFE) of sulfonamides from a spiked sand matrix. Temperature effects were either positive or negative with respect to extraction rate and total recovery, depending on the pressure and extraction fluid employed. The second portion of this research compared trifluoromethane (CHF3) and carbon dioxide (CO2) as fluids for the extraction of sulfonamides from spiked non-fat dry milk, beef liver, and egg yolk were found to be more selective using CHF3 than CO2. The polar trifluoromethane improved the extraction efficiency of the polar sulfonamides from the biological matrices and also reduced the amount of co-extractives. The next phase of this research considered the effect of organic modifier and CO2 in the SFE of sulfonamides from chicken liver, beef liver and egg yolk. Methanol, ethanol, acetone, acetonitrile were compared to determine optimum conditions. A SFE method employing 20% acetonitrile modified CO2 yielded quantitative recovery of sulfonamides from chicken liver, but 20% acetone modified CO2 was required to obtain quantitative recovery from beef liver. Either 20% acetone or 20% acetonitrile yielded quantitative recovery from egg yolk. The last phase of this research focused on the evaluation of selective detection methods for sulfonamide analysis. Chemiluminescence nitrogen detection (CLND) parameters were optimized for use with packed column supercritical fluid chromatography (SFC) yielding a minimum detectable quantity (MDQ) of 5 ng of sulfamethazine, on column. Improvements in the detector design decreased the MDQ to 0.5 ng, while, decreasing the column diameter further reduced the MDQ to 125 pg. The second part of this phase evaluated PLC/Atmospheric pressure chemical ionization (APCI) mass spectrometry for the detection of sulfonamides. Sensitivity in selective ion mode was found to be as low as 50 pg on column for sulfamethazine. Supercritical fluid extracts of sulfonamides spiked at 100μg/kg in chicken liver were found to be readily detected by this method.
Ph. D.

Background: Collection of biologic fluid for laboratory analysis requires relatively large samples, often with additives, and transport in fragile tubes. The analytes or matrices may be unstable so testing needs to be carried out quickly. Collection of these biologic fluids and drying them on filter paper can lower the cost of transporting the sample to the laboratory, avoid instability of the matrix, and degradation of the analytes. Aim: The aim of this project was to develop an inexpensive, convenient, comprehensive and reproducible patient sample collection system which ensures integrity and ease of transport of small-scale samples at room temperature, as well as ensuring convenient long-term storage for subsequent analysis. Methods: Samples (blood, buffy coat, serum, plasma and urine) were collected into various tubes and spotted onto filter paper cards. Concentrations of total cholesterol, triglyceride, phospholipids, glucose, lactate, and protein were measured in the original sample and dried plasma spots (DPS) and the concentration of creatinine was measured in urine and dried urine spots (DUS). Determination of oxidation of lipids by measurement of conjugated dienes (CD) and thiobarbituric acid reactive substances (TBARS) on dried serum spots (DSS) was carried out. Determination of salicylate on serum and dried serum spots and cyanide on whole blood and dried blood spots was carried out. Values obtained from original samples and dried spots were compared. In addition, DNA extracted from a dried buffy coat spot (DBCS) from a familial hypercholesterolemia patient was analysed after spotting. Results: The total cholesterol, triglyceride, phospholipid, glucose, lactate and protein concentration values of 14 samples were compared in whole plasma and DPS stored at different temperatures. These were highly correlated after 1 week and 3 months of collection and storage. Plasma cholesterol, glucose and lactate concentration values for DPS as well as urinary creatinine for DUS at 1 week were not significantly different to that at both 3 and 7 months’ analyses (p>0.05). Plasma triglyceride and phospholipid concentrations were significantly different (p blood vs DBS respectively) for cyanide. Salicylate in DSS and cyanide in DBS were not significantly different to the original samples (paired t-test, p>0.05). Conclusion: Dried filter spots may be used to transport and store biologic fluid samples for analyses of a number of water-soluble and water-insoluble analytes. To protect lipids from being oxidised, the filter paper should be pre-treated with BHT.

Le travail de cette thèse a pour objectif de développer un solveur dédié aux problèmes d'interaction fluide-structure (IFS), en particulier ceux rencontré en biologie, tels que la dynamique d'un écoulement sanguin à travers des tronçons veineux munis de valves. La circulation du sang est étudiée à l'aide de modèles pertinents sur les plans anatomique et physique. Le premier aspect des problèmes d'IFS concerne la gestion de la stabilité. Une formulation monolithique eulérienne basée sur la méthode des caractéristiques assure la stabilité inconditionnelle et introduit une approximation du premier ordre en temps avec deux modèles distincts de matériaux hyper élastiques. Le second aspect est relatif au contact entre deux parties du domaine solide, tel celui apparaissant entre deux valvules au cours de la fermeture de la valve et à l'état fermé sur un surface valvulaire relativement importante. Un algorithme de contact est proposé et validé à l'aide de tests de référence. L'étude computationnelle de l'écoulement sanguin à travers des tronçons veineux munis de valves est mené, une fois le solveur IFS vérifié et validé. Le domaine computationnel bidimensionnel est soit constitué d'une simple unité de base, soit du modèle de circuit veineux en forme d'échelle avec une veine superficielle et une profonde, communicant par une série de veines perforantes. Un maillage tridimensionnel de l'unité de base a été construit. Les simulations dans ce domaine tridimensionnel nécessite le recours au calcul haute performance. La dynamique de l'écoulement sanguin est fortement couplée à la mécanique de la paroi vasculaire. La paroi déformable des veines et artères de gros calibre est composée de trois couches principales (l'intima, la media, et l'adventitia) constituées de matériaux composites ayant une composition spécifique dans chaque couche. Dans ce travail, la rhéologie de la paroi est supposée être représentée par un matériau du type Mooney-Rivlin
The present work aims at developing a numerical solver for fluid-structure interaction (FSI) problems, especially those encountered in biology such as blood circulation in valved veins. Blood flow is investigated using anatomically and physically relevant models. The first aspect of FSI problems is related to management of algorithm stability. An Eulerian monolithic formulation based on the characteristic method unconditionally achieves stability and introduce a first order in time approximation with two distinct hyperelastic material models. The second aspect deals with between-solid domain contact such as that between valve leaflets during closure and in the closed state over a finite surface, which avoid vcusp tilting and back flow. A contact algorithm is proposed and validated using benchmarks. Computational study of blood flow in valved veins is investigated, once the solver was verified and validated. The 2D computational domain comprises a single basic unit or the ladder-like model of a deep and superficial veins communicating by a set of perforating veins. A 3D mesh of the basic unit was also built. Three-dimensional computation relies on high performance computing. Blood flow dynamics is strongly coupled to vessel wall mechanics. Deformable vascular walls of large veins and arteries are composed of three main layers (intima, media, and adventitia) that consist of composite material with a composition specific to each layer. In the present work, the wall rheology is assumed to be a Mooney-Rivlin material

Metalworking fluids (MWFs) are coolants and lubricants, which are widely employed in metal cutting works. They are designed to be a long lasting product. Manufacturers have designed MWFs with lack of awareness of end-of-life disposal by including biocides, which make biological treatment challenging. Here, Syntilo 9913 was used as a case study to develop a cradle-to-grave product that was biologically stable in use but amenable to sustainable hybrid biological treatment at end-of-life. The product was reverse engineered employing factorial design approach based on a priori knowledge of the product components. From the combinatorial work, it was observed that chemical interactions can results in synergistic and antagonistic effects in terms of the toxicity and biodegradability. One of the major components of most MWFs are amines such as Triethanolamine (TEA). TEA does not biodeteriorate in single compound screening, but in combination with many other components TEA was found to cause "softening" of MWF formulations. Octylamine was found to be best for "bio-hardening" but it was not economically sustainable. Hence, the modified biocide-free synthetic MWF, Syntilo 1601, was reformulated with TEA, isononanoic acid, neodecnoic acid, Cobratec TT50S, and pluronic 17R40, which were resistant to biological treatment. Although, no change in the overall oxidation state of the MWF, metabolic activity did occur as breakdown products were observed. This suggested that both raw materials and metabolic breakdown products were recalcitrant. Thus, immobilisation agents were applied to aid further biodegradation by removing toxic bottleneck compounds. It was found that hybrid nano-iron and kaffir lime leaf performed similarly in removing chemical oxygen demand and ammonium from the system. Work in this Thesis demonstrated that the combined use of biological treatment and immobilisation agents effectively overcome the limitations of biological treatment alone by removing bottleneck compounds, which allowed greater COD reduction. This laboratory scale is a proof of principle, which needs to be tested at full scale.

Orientador: Isabel Cristina Sales Fontes Jardim
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Doutorado

Sample preparation as the first step in an analytical procedure has an important role, particularly in bioanalysis, because of the complexity of biological samples (blood plasma and urine). Biological matrix such as plasma and blood contains proteins, organic and inorganic salts, acids, bases and various organic compounds with similar chemistry to the analytes of interest. Thus the basic concept of a sample preparation method is to convert a real matrix into a format that is suitable for analysis by an analytical technique. Therefore the choice of an appropriate sample preparation method greatly influences the reliability and accuracy of the analysis results. The aim of this thesis was to develop and validate of microextraction by packed syringe (MEPS) as a fast, selective, accurate and fully automated sample preparation technique for determination of BAM peptides in human plasma and local anaesthtics in human plasma and urine samples using silica and polymer sorbents. First work presents use of MEPS technique online with LC-MS/MS as a tool for the quantification of BAM peptide in plasma samples. MEPS technique provides significant advantages such as the speed and the simplicity of the sample-preparation process. Compared with other extraction techniques, such as protein precipitation and ultrafiltration, MEPS gave cleaner samples and higher recovery. In the second work, MEPS technique was developed by using synthesized molecularly imprinted polymer (MIP) as a sorbent for selective quantification of a homologous series of local anaesthetics, containing lidocaine, ropivacaine, mepivacaine, and bupivacaine in human plasma and urine samples. Compared with other conventional sorbent, the use of MIP provides high selectivity of the extraction and decrease the matrix effect.

After more than a century, there are no analytical solutions for the Navier-Stokes equations to describe complex fluid behavior, and we often resort to different computational methods to find solutions under specific conditions. In particular, to address many biological questions, we need to use techniques which are accurate at the mesoscale regime and computationally efficient, since atomistic simulations are still incredibly computationally costly, and continuum methods based on Navier-Stokes present challenges with complicated moving boundaries, in the presence of fluctuations. Here, we use a novel particle-based coarse-grained method, known as MPCD, to study ciliated swimmers. Using experimentally measured beating patterns, we show how we recapitulate the emergence of metachronal waves (MCW) on planar surfaces, and present new results on curved surfaces. To quantitatively study these waves, we also analyzed their effect on beating intervals, energy fluctuations, and fluid motion. We then extended our model to realistic cellular geometries, using experimentally obtained Basal Bodies locations.\par In the second part of our study, we focused on the intracellular fluid motion, neglecting hydrodynamic interactions. We developed the Digital Confocal Microscopy Suite (DCMS) that can run on multiple platforms using GPUs and can input realistic cell shapes and optical properties of the confocal microscope. It has this ability to simulate both (Fluorescence Recovery After Photobleaching) FRAP and Fluorescence Correlation Spectroscopy (FCS) experiments, as well as the capability to model photo-switching of fluorophores, acquisition photo-bleaching, and reaction-diffusion systems. With this platform, in collaboration with the Vidali Lab, we were able to elucidate the role of boundaries in interpreting FRAP experiments in \textit{moss} and estimate the binding rates of myosin XI.

Prostate cancer is the second-most diagnosed and fatal carcinoma for males in the United States, and better diagnostic markers and potential therapies are needed. microRNAs are small, single-stranded RNA molecules that affect protein expression at the translational level, and dysregulation can dramatically affect cell metabolism. Comparison of 736 microRNA expression levels between the poorly metastatic SV40T immortalized prostate epithelial cell line P69 to its highly tumorigenic and metastatic subline M12 identified 231 miRs that were overexpressed and 150 miRs that showed loss of expression in the M12 cell line. Further evaluation of fourteen identified miRs was accomplished using other prostate cell lines as well as laser-capture microdissected prostate samples. Inhibition of miR-147b was found to affect proliferative, migratory and invasive capabilities of M12 cells, and reduced tumour growth in nude athymic mice. AATF, an activator of the cell-cycle inhibitor p21, was identified as a target. Overexpression of miR-9 was found to affect the epithelial to mesenchymal transition through suppression of e-cadherin, a protein characterized as lost in EMT, as well as suppression of SOCS5, an attenuator of JAK-STAT signaling. Inhibition of miR-9 resulted in reduction of migratory and invasive potential, and significant reduction of tumorigenesis and metastases in male nude athymic mice. miR-17-3p was previously identified as down-regulated in prostate cancer and loss of miR-17-3p shown to cause vimentin transcriptional activation. Reverse phase microarray analysis (RPMA) identified c-KIT as a potential second mRNA target for miR-17-3p. miR-17-3p was shown to modulate not only protein levels, but also messenger RNA levels of c-KIT. Four miR-17-3p binding sites in the c-KIT mRNA were identified. Thus, a number of microRNAs involved in prostate cancer were identified, and their targets found to be highly relevant to tumour progression and could potentially be used as targets for therapy or diagnostics. Stability of microRNAs in forensically relevant biological fluids was evaluated through heat treatment, ultraviolet radiation, and chemical treatment. The dried body fluids showed some susceptibility to harsh treatment, but in most cases microRNAs were still detectable in the samples. microRNAs could represent a highly stable species for body fluid identification methods in forensic science.

Nanomedicine, the application of nanotechnology for medical purposes, has been widely identified as a potential solution for today‟s healthcare problems. Nanomedicine uses the "bottom-up‟ principles of nanoscale engineering to improve areas of medicine which have previously been considered undevelopable. One of the enduring challenges for medicine is the design of innovative devices able to overcome biological barriers, allowing drugs and therapeutics to effectively reach their correct location of action. Biological barriers are a defence mechanism of the body which are extremely well-evolved to protect the body from foreign and harmful particles. Therapeutic drugs and devices, which are not harmful, are often identified by the body as dangerous because their composition differs from native and accepted entities. The traversal of these biological barriers, such as mucus, remains a bottleneck in the progress of drug delivery and gene therapy. The mucus barrier physically limits the motion of particles due to its complicated mesh structure which obstructs the particles' traversal path. Mucus fibres can also adhere to the particles, entrapping them and restricting their motion. Particle traversal of mucus is carried out by passive diffusion. As diffusion has traditionally been defined by the Stokes-Einstein equation as inversely proportional to particle radius, it follows that reducing particle sizes into the nanoscale would result in increased diffusive ability. These predictions, however, do not consider the obstructive effects of the complicated mesh structure for the case of mucus. The exact effect of reducing particle size into the nanoscale for diffusion through mucus is therefore unknown. Multiple Particle Tracking was used to obtain real-time movies of the diffusion of nanoparticles, ranging from 12nm – 220nm in diameter, through mucus samples. The experimental data generated was used to systematically correlate the relationship between particle size and diffusivity through mucus. This study reveals that nanoparticles, smaller than the average pore size in the mucus mesh structure, can diffuse through lower viscosity pores which pose less resistance to diffusive motion, allowing nanoparticles to travel at up to four times the speed expected from the bulk viscosity of the mucus. This type of information can help researchers understand the importance of size for therapeutic nanoparticles, allowing researchers to decide whether attempts to decrease nanoparticle size at the expense of other functionality are worthwhile.

Background: Occupational exposure to used metalworking fluid (MWF) mists is a risk factor in the development of allergic and irritant respiratory disease. Respiratory disease "outbreaks" have prompted further investigation into possible causative factors. These might include sensitizing agents accumulating in used MWF. However, there is no clear evidence that shows whether levels of biologicals and chemicals detected within the sump are representative of what is found in the mist. Method: Samples of used MWF and mist samples were obtained from UK sites. Analysis of biological contaminants was conducted using a combination of 16S rRNA PCR-DGGE, qPCR, zymography, fluorescence based assays and NanoLC-ESI-MSe. Metals particulates and dissolved metals were analysed using ICP-MS. Results: Bacteria were detected in both used MWF sump and mist samples. These included Ochrobactrum and Proprionibacterium at site visit one, and Methylobacterium at site visit two. Other potentially pathogenic bacterium detected within the MWF sump sample included organisms from the Mycobacterium chelonae-Mycobacterium abscessus complex, and Wautersiella Falsenii. Bacterial toxins in the form of “serine-like” proteases were detected within 76% of the MWF samples and in two of the mist samples. Potentially sensitizing metals such as zinc, aluminium, manganese, chromium and nickel were detected at different levels within both the used MWF sumps and the mist samples taken. Conclusions: This study demonstrates that it is likely contaminants and constituents of MWF become airborne during machining processes. However, further research is required to determine the quantities of such contaminants in the mist to determine whether they would meet the threshold to initiate the development of allergic respiratory diseases seen in machine operators.

Orientadores: Denise Vaz de Macedo, Armindo Antonio Alves
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O objetivo central deste trabalho foi analisar a variação da capacidade antioxidante total, conferida por antioxidantes de baixo peso molecular (ABPM) em fluidos biológicos e tecidos, através do uso da voltametria de pulso diferencial (VPO). O capítulo 1 corresponde a um artigo de revisão sobre as possíveis fontes de geração de espécies reativas de oxigênio no músculo esquelético durante o exercício físico. O capítulo 2 é um artigo de divulgação, cujo objetivo é fornecer subsídios para a análise da capacidade antioxidante total de amostras biológicas conferida por ABPM para acompanhar a modulação do sistema de defesa antioxidante em função do treinamento físico. Para tanto, são discutidos o uso da voltametria cíclica e a possibilidade do uso de outras técnicas voltamétricas, como a VPO. O artigo contido no capítulo 3 apresenta dados da concentração de ABPM no plasma e na saliva de atletas submetidos a um período de treinamento físico. Observou-se, nesse estudo, uma diminuição da capacidade antioxidante total do plasma após 6 semanas de treinamento (879.28 :t 197.92 j..Imol Trolox equiv. L-1 na segunda semana versus 787.82 :t 154.71 j..Imol Trolox equiv. L-1 na sétima semana). Essa diminuição foi associada a um aumento no desempenho em testes físicos específicos e à manutenção das concentrações de marcadores de lesão muscular e de processo inflamatório, sugerindo que uma diminuição na capacidade antioxidante plasmática faz parte do estágio inicial de adaptação ao treino. As capacidades antioxidante da saliva e do plasma apresentaram correlação entre si (r=O.5871, pAbstract: The objective of this study was to analyze the changes in total antioxidant capacity conferred by low molecular weight antioxidants (LMWA) in biological fluids and tissues by the use of differential pulse voltammetry (DPV). Chapter 1 is a review article about the possible fonts of reactive oxygen species generation in skeletal muscle during exerci se. Chapter 2 is a divulgation article that deals with antioxidant defense systems, LMWA detection methods, cyclic voltametry and DPV. The paper in chapter 3 presents the results about LMWA concentration in plasma and saliva of athletes submitted to physical training. It was observed a decrease in plasma total antioxidant capacity after 6 weeks of training (879.28 :t 197.92 !-Imol Trolox equiv. L-1 in second week versus 787.82:t 154.71 !-Imol Trolox equiv. L-1 in seventh week) associated to an enhanced physical performance in specific tests and maintained indices of muscle damage and inflammation. The data suggests that a small decrease in the plasma antioxidant capacity is part of a normal answer in the initial stages of a physical training programo It was also shown in this study a significant correlation between plasma and saliva antioxidant capacity (r=0.5871, p<0.01) and a significant correlation between this samples antioxidant capacity and their respective urate concentrations. In chapter 4 the kinetics of liver and skeletal muscle hydrophilic and lipophilic LMWA and enzymatic antioxidants from rats submitted to exhaustive exerci se preceded by 24h of fasting, are shown. Liver and muscle hydrophilic extractions and liver lipophilic extraction presented two classes of LMWA while muscle lipophilic extraction presented only one class. It was observed a significant decrease in the antioxidant capacity conferred by hydrophilic and lipophilic LMWA and enzymatic antioxidants in ali analyzed moments (30 min, 1, 6 e 24 h). Only one class of muscle hydrophilic LMWA showed to be reestablished 24h after exerci se. Liver lipophilic first and second classes of LMWA were increased in 57% and 42% respectively after fasting. Enzymatic antioxidants activities were also reduced during the whole analyzed period, presenting a strong correlation with the antioxidant capacity conferred by LMWA. These is the first report of the use of differential pulse voltammetry for total antioxidant capacity determination conferred by LMWA of biological fluids as plasma and saliva and tissues like liver and skeletal muscle
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.
This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 137-143).
Many gastropods, such as snails and slugs, crawl using adhesive locomotion, a technique that allows the organisms to climb walls and walk across ceilings. These animals stick to the crawling surface by excreting a thin layer of biopolymer mucin gel, known as pedal mucus, and their acrobatic ability is due in large part to the theological properties of this slime. The primary application of the present research is to enable a mechanical crawler to climb walls and walk across ceilings using adhesive locomotion. A properly selected slime simulant will enable a mechanical crawler to optimally perform while climbing in the horizontal, inclined, and inverted positions. To this end, the rheology of gastropod pedal mucus is examined in greater detail than any previously published work. The linear rheological response of pedal mucus is examined with flow, oscillation, and creep tests. Nonlinear rheology is examined with large amplitude oscillatory shear (LAOS), and analyzed with Lissajous curves, Fourier transform rheology, and a new measure of non-linear elasticity. In addition, pedal mucus is examined with a flexure-based microgap rheometer, which can test the sample at the biologically relevant gap of 10-20lim, the measured thickness of pedal mucus under a crawling slug.
(Cont.) Adhesive locomotion of a mechanical crawler is modeled in order to find the criteria for an optimal slime simulant. After developing the selection criteria for the ideal simulant, a range of candidate materials are examined including polymeric gels, particulate gels, emulsions, composites, and field-responsive fluids. Two promising simulants are examined in detail and compared with native gastropod pedal mucus.
by Randy H. Ewoldt.
S.M.

The aim of this thesis was to develop analytical methods for measuring antimalarial drugs in biological fluids. Solid phase extraction (SPE) was used for the enrichment and purification of the drugs. Automatic extraction procedures using a SPE robot were developed to reduce the workload for the analyst and to minimize variations in the extraction procedure. Liquid chromatography (LC) with either UV or mass spectrometric (MS) detection was used to determine sample concentrations. Determination of Pyronaridine in whole blood utilised a weak cation exchanger to extract Pyronaridine from blood. To improve LC separation between Pyronaridine and the internal standard, ion-pairing was utilized. For the simultaneous quantification of the highly lipophilic Atovaquone and the strong basic drug Proguanil with metabolites, a novel mixed mode solid phase extraction column was used. It combines the properties of a carboxylic acid (CBA) column and a non-polar octyl-silica (C8) column to extract the compounds from plasma; it also required a gradient LC separation. Stability is an important factor when developing new methods. A new approach was used to evaluate the stability of Amodiaquine in blood and plasma. This included the use of a stability marker, a stable compound which was added together with Amodiaquine when preparing the stability samples. This eliminated between-run variations and variations associated with preparation of new stock solutions. Lumefantrine (LF) is one of the active components in a new drug combination recommended by the World Health Organization as a replacement for older drugs which have lost their effect. The first of the two methods described for this compound is the determination of LF and a possible metabolite in plasma with a calibration range suitable for pharmacokinetic studies. In the second method, a capillary sampling technique is used where the blood is dried on a sampling paper and sent to the laboratory where the extraction and determination of LF concentrations take place. This method facilitates sample collection and will enable drug efficacy studies conducted in rural settings. To monitor a current change in treatment policy and self medication, a screening assay was developed. Its purpose is to be a complement to interviewing patients about their previous medication (in the previous few weeks) and to detect some of the more common drugs which might have been used.
Paper 6. as Manuscript