Дисертації з теми "Biological fluid analysis"

Amniotic fluid (AF) is a protective pool and a resource of amino acids for the growing fetus. In study 1, we investigated if any of these AF amino acids at mid gestation were associated with fetal development in humans. Nineteen amino acids differed across birth weight percentiles. Arginine, 3-methyl histidine and tryptophan were positive predictors of birth weight, while ornithine was a negative predictor. In study 2, we used a diet induced model of IUGR to see if specific AF amino acids were predictive of fetal weight near term. Methionine and phenylalanine were modified by diet, and 12 amino acids were independently modified by gestational age, respectively. Cysteine, lysine, methionine and tyrosine were predictors of fetal weight. Thus, the AF amino acid pool is associated in animals and humans with fetal growth.

Studies of protein and peptide expression are vital in order to understand complex biological systems. As demonstrated in this thesis, on-line packed capillary liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR MS) is a useful analytical tool for such studies.

A proteomics method, based on global tryptic digestion and subsequent separation and detection of the peptides by LC-FTICR MS, was developed for qualitative analysis of body fluids. Initial experiments on cerebrospinal fluid (CSF) provided results that were comparable or superior to those achieved by more time- and sample-consuming techniques. The method was also successfully applied on plasma and amniotic fluid. One of the major challenges in proteomics is the broad dynamic range of proteins in biological matrices. The advantages of removing high-abundant components from CSF and plasma prior to MS were demonstrated.

In order to search for potential biomarkers, mass chromatograms of CSF from patients suffering from amyotrophic lateral sclerosis (ALS) and controls were compared using an in-house constructed pattern recognition program. ALS-specific patterns were observed, and four out of five unknown samples were correctly assigned. Alternative strategies to quantitatively compare two pools of samples rely on differential chemical labeling. The performance of one such method, quantification-using-enhanced-signal-tags, was investigated in complex sample analysis. The experimental intensity ratios were proven to be consistent with the prepared concentration ratios of abundant proteins in CSF.

Finally, the thesis reports on the first experiments where electron capture dissociation (ECD) was successfully incorporated in on-line LC-MS experiments. ECD and nozzle-skimmer fragmentation were applied to a sample of endocrine peptides extracted from mouse pancreatic islets. The two fragmentation methods provided complementary information. However, the method needs further optimization before it can be applied in the analysis of more complex samples, such as body fluids.

District heating has a long standing tradition in Sweden and today it is the most common way of producing and transporting heat. A District heating system (DH system) is divided into three parts: a production facility, distribution network (DH network) and one more heat stations. The heat produced in the facilities is distributed to the customers via a heat transfer medium, usually water (DH water), in piping networks that make up the DH network. The heat is transferred to the customers via the heat exchanger at which point they can use it as heated tap water or for heating purposes. The DH networks are often constructed in steel as it is cheap and a relatively resistant material. However it has the disadvantages of corrosion and expansions when it is exposed high temperatures which lead to damages in the DH network resulting in loss of the DH water, this is an unavoidable occurrence in any DH network. This results in addition of pollutants by leakages into the DH network or with the water that is used to compensate for the losses. The pollutants cause further corrosion, leading to metal contamination, and more damages on the DH network meaning there is a continuous degradation. Therefore various treatments are used to clean and ascertain an acceptable chemical environment in the DH systems. These treatments are effective but not at a level which is required so many chemicals are used to enhance the treatment of the water. Some of these are known to be toxic to humans and water ecosystems. As leakages are abundant and often end up in the WWTPs of the concerned municipality, which often have troubles with disturbances of the biological treatment, it was decided that an assessment of the toxic effects that DH water pose on activated sludge was to be investigated. This was done by testing water from two DH networks, Växjö and Kalmar, on the same activated sludge obtained from Tegelviken WWTP in Kalmar. A respirometric bioassay approach established by the Organization for Economic Co-operation and Development (OECD), OECD standard 209; OECD Guidelines for the Testing of Chemicals was used with changes made to exposure and measuring time as this decrease the risk of misinterpretation of the results. A dilution series using different concentrations (6.25%, 25% and 100%) of DH water was tested and compered to a blank control samples containing only activated sludge. Assessment of toxicity on total oxidation, oxidation carbon and oxidation of nitrogen was made. To get some idea of what might cause toxic effect samples of the waters was sent to outside laboratories for analyses of metals. The result from the bioassay and metal analysis was used to formulate risk factors associated with a DH water spill and exposure to WWTPs. It was found that both DH waters have a significant inhibition on nitrification in WWTPs. The DH water from Kalmar exhibited similar toxicity dynamics, roughly 20% inhibition, despite large differences in concentration. The DH water from Växjö showed a negative correlation between an increase in concentration of DH water and toxicity, 74% for the lowest concentration and 11% for the highest. The metal analysis concluded that there was no abundance of metal contamination which led to the inference that toxicity is probably caused by the chemicals used for treatment. This poses a great risk for the Baltic Ocean as many WWTPs release their treated water directly into water courses with a short detention time before reaching the sea.

Дисертація на здобуття наукового ступеня доктора технічних наук за спеціальністю 05.11.17 – біологічні та медичні прилади i системи. – Національний технічний університет "Харківський політехнічний інститут", Харків, 2018. Розв'язано комплекс задач, які вирішили науково-прикладну проблему створення теоретичних засад, методів і засобів багатопараметричного поляризаційного відтворення та об'єктивного аналізу структури фазово-неоднорідних біологічних об'єктів з підвищенням достовірності оцінювання патологічних станів в системах діагностики гістологічних зрізів парціальних і двошарових біологічних тканин і плівок біологічних рідин. Запропоновано модель відтворення та аналізу оптичної анізотропії багатошарових біологічних тканин і рідин із виділенням груп їх мюллер-матричних зображень при оцінюванні патологічних станів. Розроблено методи і системи з підвищеною достовірністю диференціації станів "норма – патологія" на основі прямого відтворення та аналізу координатних розподілів орієнтаційних та фазових параметрів оптично тонких біологічних шарів та мюллер-матричного відтворення "екранованих" зовні шарів двошарової біологічної тканини. Розроблена та апробована архітектура багатопараметричної системи поляризаційно-фазового відтворення та аналізу параметрів анізотропії біологічних шарів з розширеними функціональними можливостями та підвищеною достовірністю діагностування патологічних станів. Оцінено метрологічні характеристики запропонованих систем на основі статистичного, кореляційного та фрактального аналізу двомірних розподілів похибок вимірювання.
Dissertation for a Doctor`s of Science (Engineering) Degree on Specialty 05.11.17 – Biological and Medical Devices and Systems. – Vinnytsia National Technical University, National Technical University "Kharkiv Polytechnic Institute", Kharkiv, 2018. Dissertation is dedicated to the solution of the scientific-applied problem, aimed at elaboration of theoretical fundamentals, methods and means of multiparameter polarization reconstruction and unbiased analysis of the phaseheterogeneous biological objects structure that enabled to enhance the validity of pathological states assessment in the diagnostic systems of the histologic sections of fractional and multilayered biological tissues (BT) and films of biological fluids (BF). The model of reconstruction and analysis of optical anisotropy of multilayer BT and BF with the allocation of groups of their mueller-matrix images in the evaluation of pathological states is improved. Methods and systems with increased reliability of the differentiation of states "norm – pathology" on the basis of direct reproduction and analysis of coordinate distributions of orientation and phase parameters of optically thin biological layers (BL) and mueller-matrix reproduction of "shielded" exterior layers of two-layer BT are developed. The architecture of the multiparameter system of polarization-phase reproduction and analysis of parameters of anisotropy of BL with advanced functional capabilities and high reliability of diagnostics of pathological states was developed and tested. The metrological characteristics of the offered systems based on statistical, correlation and fractal analysis of two-dimensional distributions of measurement errors are estimated.

A health condition called “Oxidative Stress” (OS), resulting from an excessive level of Reactive Oxygen Species (ROS) is a “state harmful to the body, which arises when oxidative reactions exceed antioxidant reactions because the balance between them has been lost”[1] OS appears to be associated with and might be a cause of, many serious diseases such as cardio-vascular accidents, cancer, Parkinson’s and Alzheimer’s[2]. This is not surprising as ROS are free oxygen radicals that can attack lipids, proteins, cellular membranes, enzymes and even modify DNA. Extensive correlation studies have shown that the complex impedance spectrum of blood samples from patients diagnosed with an OS syndrome differs significantly from the spectra obtained from the blood of healthy people, which is quite normal as the presence of an excessive amount of ROS should affect the physico-chemical properties of a blood sample. Measuring the complex impedance spectrum of a blood sample can be done quickly by means of low-cost electronic devices, making possible and affordable the early detection of OS among a large population. In order to quantitatively evaluate the OS, the impedance spectra being insufficient, the concentration of oxidative stress markers such as hydrogen peroxyde, malondialdehyde or F2 isoprostanes needs to be measured. Such measurements can, for instance, be used for monitoring the severity of a disease during a treatment. These concentration measurements are traditionally based upon analytical techniques but recently biosensors acting as transducers transforming directly a specific biochemical reaction into a measurable signal have been developed. They are essentially obtained by modifying the surface of metal or carbon electrodes using biomaterials such as enzymes antibodies or DNA that allow bindings or catalytic reactions with other specific biomaterials to occur on the surface of the electrodes. The resulting modifications of the electrical properties of the medium separating the electrodes can be analyzed through ad-hoc electronic and signal processing systems to yield the desired concentration. Biosensors have the advantages of rapid analysis, low-ost and high-precision. They are widely used in various fields, such as medical care, disease diagnosis and food analysis [3]. Hydrogen peroxide (H2O2) generated by cellular processes directly via two-electron reduction of molecular oxygen or indirectly via dismutation of superoxide, is the most widely studied ROS and its overproduction results in OS. Therefore, an ability to quantify the level of hydrogen peroxide and by ricochet the assessment of oxidative stress can be useful in order to assess certain health conditions occurring inside the body and as a result, an integrated electrochemical biosensor coupled with the hydrogen peroxide quantification can become a practical solution as a point of care device at home[4] Most of the time, H2O2 biosensors are based on HRP (Horseradish peroxidase) which is the most commonly used enzyme in the design of biosensors that can supervise the activity of oxidases and determine in terms of concentration, oxidase substrate such as lactate oxidase, cholesterol oxidase, or glucose oxidase, which all induce the production of hydrogen peroxide (HRP’s substrate). In the first part of this research, we explore the development of low-cost and compact measurement systems aiming to determining the impedance of biological samples as they grant access to information from electrical cellular characteristics. It is indeed possible to measure capacitance or conductance that are dependent on the health state of cells. The development of such measurement systems allowing the portability of biological essays requires sensitive electronics. Afterward, in the second part of our work, we explore the design of an electrochemical biosensor by immobilizing an enzyme (HRP) onto the surface of golden electrodes in order to detect and assess the analyte, hydrogen peroxide (H2O2). We also discuss the design of a potentiostat readout circuit to measure and convert the biosensor’s current. The combined results of the two parts of this work can be considered as a first prototype of a low cost and robust instrument easy to use in the field, away from a biological laboratory, with the goal of reaching the so called “point of care diagnostic” [5] The present thesis is organized as follows: Chapter I, introduces the present thesis. In Chapter II, we provide an overview in the field of biosensing technology. Chapter III deals with the design of a portable EIS measurement system to investigate reactive oxygen species in blood. Chapter IV presents an improved version of the previously designed instrument. Moreover, it points out the significance of EIS-based blood analysis through relevant medical diagnosis parameters such as hematocrit and erythrocyte sedimentation rate, extracted from the measured impedance spectra. In Chapter V we discuss on one hand the design of the H2O2 biosensor, and on the other hand the realization of the front-end circuit of the amperometric sensor. Finally, in Chapter VI, a conclusion is drawn..

Дисертація на здобуття наукового ступеня доктора технічних наук за спеціальністю 05.11.17 – біологічні та медичні прилади i системи. – Національний технічний університет "Харківський політехнічний інститут", Харків, 2018. Розв'язано комплекс задач, які вирішили науково-прикладну проблему створення теоретичних засад, методів і засобів багатопараметричного поляризаційного відтворення та об'єктивного аналізу структури фазово-неоднорідних біологічних об'єктів з підвищенням достовірності оцінювання патологічних станів в системах діагностики гістологічних зрізів парціальних і двошарових біологічних тканин і плівок біологічних рідин. Запропоновано модель відтворення та аналізу оптичної анізотропії багатошарових біологічних тканин і рідин із виділенням груп їх мюллер-матричних зображень при оцінюванні патологічних станів. Розроблено методи і системи з підвищеною достовірністю диференціації станів "норма – патологія" на основі прямого відтворення та аналізу координатних розподілів орієнтаційних та фазових параметрів оптично тонких біологічних шарів та мюллер-матричного відтворення "екранованих" зовні шарів двошарової біологічної тканини. Розроблена та апробована архітектура багатопараметричної системи поляризаційно-фазового відтворення та аналізу параметрів анізотропії біологічних шарів з розширеними функціональними можливостями та підвищеною достовірністю діагностування патологічних станів. Оцінено метрологічні характеристики запропонованих систем на основі статистичного, кореляційного та фрактального аналізу двомірних розподілів похибок вимірювання.
Dissertation for a Doctor`s of Science (Engineering) Degree on Specialty 05.11.17 – Biological and Medical Devices and Systems. – Vinnytsia National Technical University, National Technical University "Kharkiv Polytechnic Institute", Kharkiv, 2018. Dissertation is dedicated to the solution of the scientific-applied problem, aimed at elaboration of theoretical fundamentals, methods and means of multiparameter polarization reconstruction and unbiased analysis of the phaseheterogeneous biological objects structure that enabled to enhance the validity of pathological states assessment in the diagnostic systems of the histologic sections of fractional and multilayered biological tissues (BT) and films of biological fluids (BF). The model of reconstruction and analysis of optical anisotropy of multilayer BT and BF with the allocation of groups of their mueller-matrix images in the evaluation of pathological states is improved. Methods and systems with increased reliability of the differentiation of states "norm – pathology" on the basis of direct reproduction and analysis of coordinate distributions of orientation and phase parameters of optically thin biological layers (BL) and mueller-matrix reproduction of "shielded" exterior layers of two-layer BT are developed. The architecture of the multiparameter system of polarization-phase reproduction and analysis of parameters of anisotropy of BL with advanced functional capabilities and high reliability of diagnostics of pathological states was developed and tested. The metrological characteristics of the offered systems based on statistical, correlation and fractal analysis of two-dimensional distributions of measurement errors are estimated.

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.

Ce manuscrit est divisé en trois parties : (I) Sont présentées deux études concernant les forces effectives entre objets plongés dans un milieu fluctuant. A l'équilibre thermodynamique, nous caractérisons complètement la distribution, non universelle, des forces ressenties par deux objets pour quelques cas simples. Nous étendons aussi le concept de forces induites par les fluctuations à des milieux maintenus hors équilibre. (II) Nous y étudions la réponse dynamique d'adhésifs moléculaires. Une première étude concerne l'interprétation d'expériences récentes sur molécule unique. Nous identifions et discutons, en particulier, l'ambiguïté des interprétations usuelles de ces expériences. Nous montrons ensuite comment la topologie des chemins vers la rupture se traduit dans la complexité de la réponse dynamique des adhesifs. (III) On y fournit quelques commentaires sur les interactions élastiques qui existent entre des inclusions rigides déformant localement une interface molle.

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

During recent years a consistent number of central nervous system (CNS) drugs have been approved and introduced on the market for the treatment of many psychiatric and neurological disorders, including psychosis, depression, Parkinson disease and epilepsy. Despite the great advancements obtained in the treatment of CNS diseases/disorders, partial response to therapy or treatment failure are frequent, at least in part due to poor compliance, but also genetic variability in the metabolism of psychotropic agents or polypharmacy, which may lead to sub-therapeutic or toxic plasma levels of the drugs, and finally inefficacy of the treatment or adverse/toxic effects. With the aim of improving the treatment, reducing toxic/side effects and patient hospitalisation, Therapeutic Drug Monitoring (TDM) is certainly useful, allowing for a personalisation of the therapy. Reliable analytical methods are required to determine the plasma levels of psychotropic drugs, which are often present at low concentrations (tens or hundreds of nanograms per millilitre). The present PhD Thesis has focused on the development of analytical methods for the determination of CNS drugs in biological fluids, including antidepressants (sertraline and duloxetine), antipsychotics (aripiprazole), antiepileptics (vigabatrin and topiramate) and antiparkinsons (pramipexole). Innovative methods based on liquid chromatography or capillary electrophoresis coupled to diode-array or laser-induced fluorescence detectors have been developed, together with the suitable sample pre-treatment for interference removal and fluorescent labelling in case of LIF detection. All methods have been validated according to official guidelines and applied to the analysis of real samples obtained from patients, resulting suitable for the TDM of psychotropic drugs.

2015 - 2016
Metabolomics and metabonomics encompass the comprehensive profiling of multiple metabolite concentrations and their cellular and systemic fluctuations in response to drugs, diet, lifestyle, environment, stimuli and genetic modulations, in order to characterize the beneficial and adverse effects of such interactions. In the context of biomedical applications, metabolomics will have a preferential role with respect to the other "Omics" sciences for its ability to detect in real time the response of the organisms to pathological stressors. The application of the NMR technique for the metabolomics analysis was applied to bio-fluids deriving from populations of patients respectively affected by salivary gland tumor, antiphospholipid autoimmune syndrome and altered lipid profile. This NMR metabolomic screening was aimed i) at the definition of a metabolomic profile that may be patognomonic of the disease under scrutiny and ii) at the identification of biomarkers to be used with diagnostic and prognostic scope. In the present work, we present a NMR-based metabolomic study of saliva of patients suffering of salivary gland tumors. Our data show that individuals suffering parotid tumor have a characteristic metabolomic profile with abnormalities associated to the metabolism of acetate, alanine, lactate, methanol, phenylalanine, propionate, succinate. We have identified for the first time the metabolomic fingerprint characterizing parotid tumor patients disease having potential application to improve timely diagnosis and appropriate therapeutic approaches. Salivary gland tumor, as many other cancers, is a complex disease, resulting from an interdependent series of biochemical alterations, rather than a single disruptive event. In this case our approach aimed at the identification of a panel of metabolite markers rather than a single biomarker, will improve the sensitivity and specificity for detection. Integrating the protocols of tumor grading and histological classification. Our NMR-based metabolomic study revealed different metabolomic profiles in saliva of male patients affected by salivary gland tumors compared with the profiles of age, gender, and sampling-date matched control individuals. Our approach provide preliminary data for the identification of metabolites that can be used as metabolomics fingerprint of salivary gland tumor. Determination of metabolomics fingerprint, rather than single metabolic biomarker, may fully reflect the multifactorial nature of oncogenesis and the heterogeneity of oncogenic pathways, providing precious elements to integrate diagnostic laboratory and clinical tests. Antiphospholipid syndrome (APS) is a rheumatic inflammatory chronic autoimmune disease inducing hypercoagulable state associated with vascular thrombosis and pregnancy loss in women. Cardiac, cerebral and vascular strokes in these patients are responsible for reduction in life expectancy. Timely diagnosis and accurate monitoring of disease is decisive to improve the accuracy of therapy. In the present work, we present a NMR-based metabolomic study of blood sera of APS patients. Our data show that individuals suffering APS have a characteristic metabolomic profile with abnormalities associated to the metabolism of methyl group donors, ketone bodies and amino acids. We have identified for the first time the metabolomic fingerprint characterizing APS disease having potential application to improve APS timely diagnosis and appropriate therapeutic approaches. The first stratification of APS patients according to the gender offers preliminary indications for the management of the disease according to the gender oriented medicinal approach. Human serum includes a large number of components which derive from endogenous metabolism and nutritional intake. Serum components vary in response to diet. Serum lipid composition is probably the most important benchmark in assessing cardiovascular risk and disease progression. Serum components, also derived from nutritional intake, can affect general metabolism and, more specifically, affect molecular mechanisms and pathways linking nutritional intake and chronic disease risk. To identify the effect exerted by altered lipid composition on the genome expression pattern, response of gene expression to serum samples from hypercholesterolemic and normocholesterolemic male subjects was previously studied. In the present part of my PhD thesis, using a NMR metabolomics approach I studied the metabolomics profile of the aforementioned hypercholesterolemic and normocholesterolemic sera to correlate the previously identified trascriptomic signature of human hepatoma cells to the relative metabolomics profile. Hypercholesterolemic sera previously proved to increase in human hepatoma cells, the mRNA expression of HMGCS2, an enzyme involved in the pathway of keton bodies. Our NMR based metabolomics analysis evidences abnormal concentrations of metabolites involved in the keton bodies pathway. This indicates a correlation between the trascriptomic profile of hepatoma cells treated with hypercholesterolemic sera, and the metabolomics profile of the same sera. [edited by author]
La metabolomica e la metabonomica comprendono il profilo completo di numerosi metaboliti con riferimento alle varie concentrazioni e fluttuazioni sia cellulari che sistemiche in risposta a farmaci, dieta, stile di vita, influenza dell'ambiente, stimoli e modulazioni genetiche, al fine di caratterizzare gli effetti benefici e negativi di tali interazioni. Nel contesto delle applicazioni biomediche, la metabolomica avrà in futuro un ruolo preferenziale rispetto alle altre scienze 'omiche' per la possibilità di rilevare in tempo reale la risposta degli organismi agli stress patologici. L' applicazione della tecnica NMR è stata utilizzata per l' analisi metabolomica di bio-fluidi derivanti da popolazioni di pazienti affetti rispettivamente da tumore delle ghiandole salivari; da sindrome da antifosfolipidi; pazineti con profilo lipidico alterato. Questo screening metabolomico NMR è mirato i) alla definizione di un profilo metabolomico che potrebbe essere patognomonico delle malatte monitorate e ii) l'identificazione di biomarcatori da utilizzare in ambito diagnostico e prognostico. In questo studio metabolomico basato su analisi NMR della saliva di pazienti affetti dai tumori delle ghiandole salivari i nostri dati mostrano caratteristiche anomalie nel profilo metabolomico connesse con il metabolismo di acetato , alanina, lattato, metanolo, fenilalanina, propionato, succinato. Abbiamo identificato per la prima volta l'impronta digitale metabolomica che caratterizza pazienti con tumori della parotide con una potenziale applicazione per migliorare la diagnosi tempestiva ed un approccio terapeutico adeguato. I tumori alle ghiandole salivari, come molti altri tipi di cancro, sono patologie complesse, risultanti da una serie interdipendente di alterazioni biochimiche, piuttosto che un singolo evento dirompente. In questo caso, con un approccio rivolto all'identificazione di un panel di metaboliti marcatori, piuttosto che ad un singolo biomarcatore, miglioreranno ed aumenteranno la sensibilità e la specificità per il rilevament, integrando i protocolli diagnostici classici e la classificazione istologica. Il nostro studio metabolomico NMR-based ha rivelato diversi profili nella saliva di pazienti affetti da tumori delle ghiandole salivari, confrontati in base all' età e al sesso, abbinati con i controlli. Il “finger print”, piuttosto che i singoli biomarkers, può riflettere in pieno la natura multifattoriale ed etrogenea della oncogenesi , fornendo preziosi elementi per integrare i test diagnostici clinici e di laboratorio. La sindrome antifosfolipidi (APS) è una malattia autoimmune, reumatica, infiammatoria cronica associata ad uno stato di ipercoagulabilità: inducendo trombosi vascolari ed aborti spontaeni nelle donne. Ictus cerebrali e vascolari in questi pazienti sono responsabili della riduzione della aspettativa di vita: una diagnosi tempestiva ed un accurato monitoraggio della malattia è determinante per migliorare la precisione della terapia. Nel presente lavoro, vi presentiamo uno studio di metabolomica NMR su siero di pazienti affetti da APS. I nostri dati mostrano che gli individui che soffrono di APS hanno un profilo metabolomico caratteristico con anomalie del metabolismo associate ai donatori di gruppi metilici, di aminoacidi e corpi chetonici. Abbiamo identificato per la prima volta il “finger print” della sindrome da APS con la potenziale applicazione di migliorare la diagnosi tempestiva e favorire un approccio terapeutico adeguato. La prima stratificazione di pazienti APS pazienti in base al sesso offre indicazioni per la gestione della malattia secondo un approccio medico gender oriented. Il siero umano comprende un gran numero di componenti derivanti sia dal metabolismo endogeno sia dall' apporto nutrizionale i quali variano in risposta alla dieta. La composizione lipidica del siero è probabilmente il punto di riferimento più importante nella valutazione del rischio cardiovascolare e della progressione della malattia. Inoltre la composizione lipidica può influenzare il metabolismo e più in particolare, i percorsi molecolari che collegano l' apporto nutrizionale ed rischio di malattia cronica. L'effetto esercitato dalla composizione lipidica modificata sul pattern genomico in risposta all' espressione su campioni di siero da sogetti maschi ipercolesterolemici, confrontati con normocholesterolemici è stato oggetto di un precedente studio. Nell' ultimaparte di questa tesi di dottorato, utilizzando l'approccio metabolomico NMR ho studiato il profilo dei supramenzionati ipercolesterolemici e normocholesterolemici per correlare il profilo trascrittomico ottenuto dalle cellule epatiche umane con il profilo metabolomico del siero umano utilizzato per la cultura, mostrando una aumentata espressione di mRNA di HMGCS2, un enzima coinvolto nel percorso di corpi chetonici. Dall' analisi NMR sono emerse concentrazioni alterate di metaboliti coinvolti della via biosintetica dei corpi chetonici. Questo indica una correlazione tra il profilo trascriptomico di cellule epatiche trattate con sieri ipercolesterolemici, e il profilo metabolomica dei sieri stessi. [a cura dell'autore]
XV n.s. (XXIX )

Le but de cette these est d'etudier les reponses immunes humorales intracerebrales lors de processus tumoraux cerebraux. Elle comprend donc une introduction sur les interactions entre les systemes immunitaire et nerveux central d'une part et sur les tumeurs cerebrales d'autre part. Puis une presentation du materiel biologique donne notamment les raisons du choix des liquides kystiques pour cette etude immunologique. Le troisieme chapitre decrit les etudes qualitative et quantitative des immunoglobulines des liquides kystiques des tumeurs cerebrales. Les deux chapitres suivants presentent la recherche des activites anticorps des liquides kystiques dirigees respectivement contre le tissu cerebral non tumoral et contre les tissus cerebraux tumoraux, par immunohistochimie et par immunotransfert. Les effets des immunoglobines de classe g des liquides kystiques, sur des lignees cellulaires, sont decrits dans le sixieme chapitre. Puis les protocoles developpes pour la purification des antigenes reconnus par les liquides kystiques sont exposes, avant une discussion generale sur l'ensemble des resultats

Nous avons étudié divers aspects de la morphogenèse dans des liquides et des
solides confinés - instabilités, singularités et phéomènes multi-échelles -
en combinant expériences, simulations numériques et outils analytiques. Dans
un premier temps, nous abordons le compactage et le flambage de tiges et de
plaques et nous relions le flambage à la croissance différentielle dans le
vivant. Ensuite, nous considérons des films liquides sur un substrat solide
et en particulier la dynamique d'une ligne de contact. Enfin, nous analysons
deux formes d'auto-organisation : des goutes rebondissant sur un liquide et
la coalescence élastocapillaire d'une assemblée de tiges.

The thesis exposes and develops the analysis by Micellar Liquid Chromatography (MLC) of different methods for the determination of compounds of medical and pharmaceutical interest, in biological fluids (plasma and urine), pharmaceutical compounds and food. The compounds analyzed are: antitumor group Tyrosine Kinase Inhibitors (TKIs: Afatinib, Axitinib, Dabrafenib, Lapatinib, Pazopanib, Regorafenib), Antituberculous (Rifampicin, Rifabutin, Isoniazide, B6), Oral Anticoagulant (Rivaroxaban), Antibiotic group Fluoroquinolone (Flumequine, Marbofloxacin, Difloxacin, Sarafloxacin, Oxolinic acid, Ciprofloxacin, Enrofloxacin, Sarafloxacin). All procedures have been validated following guidelines for validation of analytical methods established by official institutions (EMA, FDA, ICH, EC) depending on the type of sample and matrix analyzed for each compound, in order to guarantee the reliability and quality of the results . One of the advantages of MLC is that it allows the direct injection of physiological and food samples, which considerably reduces the stage of pretreatment of the same, as well as the loss of analytes. Another advantage is that the micellar mobile phases use a smaller amount of organic solvent than those used in conventional HPLC, which reduces the costs of analysis and favors the achievement of objectives set by the European Union in relation to the "Green Chemistry" . In any case, all the analysis methods included in this thesis show their validity and application for the control of the compounds in real samples analyzed.
La tesis expone y desarrolla el análisis mediante cromatografía Líquida Micelar (MLC) de diferentes métodos para la determinación de compuestos de interés médico y farmacéutico, en fluidos biológicos (plasma y orina), compuestos farmacéuticos y alimentos. Los compuestos analizados són: grupo antitumoral Inhibidores de Tirosin Kinasa (TKIs: Afatinib, Axitinib, Dabrafenib, Lapatinib, Pazopanib, Regorafenib), Antituberculosos (Rifampicina, Rifabutina, Isoniazida, B6), Anticoagulante Oral (Rivaroxaban), Antibióticos del grupo de las Fluoroquinolonas (Flumequine, Marbofloxacin, Difloxacin, Sarafloxacin, Oxolinic acid, Ciprofloxacin, Enrofloxacin, Sarafloxacin). Todos los procedimientos han sido validados siguiendo guías de validación de procesos analíticos establecidas por organismos oficiales (EMA, FDA, ICH, EC) dependiendo del tipo de muestra y matriz analizada para cada compuesto, con el objetivo de garantizar la fiabilidad y calidad de los resultados. Una de las ventajas del MLC es que permite la inyección directa de muestras fisiológicas y de alimentos, lo que reduce considerablemente la etapa de pretratamiento de las mismas, así como la pérdida de analitos. Otra ventaja es que las fases móviles micelares utilizan una menor cantidad de disolvente orgánico que las empleadas en la HPLC convencional, lo que reduce los costes de analísis y favorece la consecución de objetivos marcados por la Unión Europea en lo referente a la "Green Chemistry". En todo caso, todos los métodos de análisis incluidos en esta tesis muestran su validez y aplicacíón para el control de los compuestos en muestras reales analizadas.

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Dissertação (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

Ce travail de thèse a eu pour objectif de développer de nouvelles méthodes de dépôts et de formation de motifs pour mieux contrôler l'organisation de molécules d'intérêt biologique sur des substrats solides.
Dans la première partie du manuscrit, nous présentons une méthode originale, la micro-aspiration, permettant de réaliser des assemblages réversibles de canaux microfluidiques sur un substrat et servir à guider les liquides. Nous avons étudié les propriétés de ces systèmes avec des modèles physiques simples et appliqué ces phénomènes à la micromanipulation de liquides, le dépôt de protéines à diverses concentrations sur un substrat, la fabrication de motifs de polymères, nanoparticules, gels, etc.
Dans la seconde partie, nous avons exploré des nouvelles méthodes de dépôt de films de phospholipides multicouches sur des substrats solides et les avons appliqués à la fabrication de vésicules unilamellaires géantes de taille contrôlée. Tout d'abord, l'adaptation de techniques conventionnelles (micro-contact printing, moulage, etc.) a permis d'obtenir des motifs de phospholipides de taille micrométrique. Les dépôts ont ensuite été réalisés par retrait d'un ménisque en situation d'évaporation (assemblage capillaire). Nous avons identifié deux régimes de dépôt en fonction de l'importance relative des forces visqueuses et de l'évaporation, permettant un contrôle de l'épaisseur du film jusqu'à 200 nm à la bicouche près. L'émergence d'instabilités de mouillage ou le guidage sur micro-structures ont permis en outre de réaliser des motifs variés. En utilisant des substrats comme électrodes, ces différents niveaux d'organisation ont permis d'obtenir par électroformation des vésicules unilamellaires géantes de taille contrôlée. L'ensemble de ces travaux ouvre de nouvelles voies à la réalisation de surfaces et de motifs micrométriques d'intérêt biologique.

This thesis deals with the development of analytical methods for the determination of new antimalarial drugs in biological fluids. The goal was to develop methods that facilitate clinical studies performed in the field, such as capillary blood sampling onto sampling paper.

Methods for the determination of atovaquone (ATQ) in plasma, whole blood and capillary blood applied onto sampling paper were developed and validated.

Automated solid-phase extraction (SPE) and liquid chromatography (LC) with UV absorbance detection was used to quantify ATQ. Venous blood contained higher levels of ATQ than capillary blood after a single dose of Malarone (ATQ + proguanil).

Ion-pairing LC was used to separate amodiaquine (AQ), chloroquine (CQ) and their metabolites on a CN-column. A method for quantification of AQ, CQ and their metabolites in capillary blood applied onto sampling paper was developed and validated. Perchloric acid and acetonitrile were used to facilitate the extraction of the analytes from the sampling paper. The liquid extract was further cleaned by SPE.

Methods for the determination of piperaquine (PQ) in plasma and whole blood using SPE and LC were developed and validated. Addition of trichloroacetic acid (TCA) to the samples prior to injection into the LC-system significantly enhanced the efficiency for the PQ peak. Serum and whole blood contained higher levels (about 300 nM) of PQ than plasma (about 200 nM) after a single oral dose of 340 mg PQ. This indicates that PQ may be taken up in the leucocytes and thrombocytes.

We have recently characterized two distinct populations of Satellite Cells (SCs), defined as Low Proliferative Clones (LPC) and High Proliferative Clones (HPC), that differ for proliferation, egenerative potential and mitochondrial coupling efficiency. In here, we have deep investigated their cell biology and characterized features that remark their intrinsic differences retrievable also at the initial phases of their cloning. LPC and HPC can indeed be istinguished for characteristic mitochondrial membrane potential (ΔΨm) just after isolation from their parental fibre. This is merged by mitochondrial redox state measured via NAD+/NADH analysis- and alternative respiratory CO2 production in cloned cells, which are accountable for metabolic differences reflected by alternative expression of the glycolytic enzyme Pfkfb3. In addition also mitochondrial Ca2+ handling and the sensitivity to apoptosis triggered via the intrinsic pathway are modified as well as the size of the mitochondrial network. In conclusion, we were able to determine which clone represents the suitable stem cell within the SCs population. These further experimental observations report novel physiological features in the cell biology of SCs populations before and after cloning, highlighting an intrinsic heterogeneity on which the stemness of the satellite cell is likely to depend. In the second part of my work we have also investigated their potential to trans-differentiate into smooth muscle cells. Enteric Nervous System normally interacts with muscle cells to control the peristaltic and secretory activity of the gut wall. Incomplete gut colonization by neural crest cells causes Hirschsprung’s disease, characterized by aganglionosis of the distal bowel. Multipotent, self-renewing enteric precursor neurosphere-like bodies (NLBs) -capable of generating neurons and glia derived from the neural crest- can be isolated from the gut of mice, rats, and human and they are able to colonize the gut after transplantation. Our aim is to understand the relationship between satellite cells-derived muscle precursor cells (MPCs) and NLBs using an in vitro co-culture model: this will be useful in perspective of a tissue engineering approach for bowel regeneration and skeletal muscle. Our records highlighted that NLBs were able to form new myotubes in presence of MPCs. Co-cultures in myogenic medium showed a remarkable improvement of MPCs ifferentiation by NLBs, promoting the formation of sarcomeric striatures onto myotubes and increasing the desmin expression of MPCs. On the other side, using neurogenic medium MPCs-NLBs showed a neural-like phenotype. As future perspectives, we need to understand the relationship between MPCs and NLBs and if the synapses are involved in this process; to verify if the seeding on a biocompatible polymer influences the behaviour of neural cells; and we must confirm these data with an in vivo skeletal and smooth muscle differentiation. We have finally explored the possibility of deriving smooth muscle cells from a different source, taking in consideration the difficulties related to the expansion of both skeletal and smooth muscle progenitors. Therefore, we aim to derive functional smooth muscle cells (SMCs) from non-muscle cells, such as human Amniotic Fluid Stem (hAFSC) cells. hAFSC were transduced using vector encoding ZsGreen under the αSMA promoter. SMhAFSC expressed significantly higher level of smooth muscle genes (such as αSMA, desmin, calponin and smoothelin expression) after selective culture condition. These features were confirmed by immunofluorescence, demonstrating a single lineage commitment; TEM established increased intermediate filaments, dense bodies and glycogen deposits in SMhAFSC, similar pattern compared to SMCs; and sequential imaging analyses demonstrated that SMhAFSC have a higher contractile potential than hAFSC. Consecutive single cell sampling showed the presence of voltagedependent calcium activated potassium channels on differentiated SMhAFSC and showed a higher production of carbon dioxide. In conclusion, we were able to generate to functional SMCs starting from a non-muscle precursor; secondly the transduction process may represent a valuable tool to select SM committed population. This step may eventually overcome the well-known problem of expanding SM progenitors, making these cells amenable to tissue engineering.
Il nostro gruppo ha recentemente caratterizzato due distinte popolazioni di cellule satelliti, classificate come cloni a bassa proliferazione (LPC) e ad alta proliferazione (HPC), che si differenziano in termini di proliferazione, potenziale rigenerativo e metabolismo mitocondriale. Nel mio lavoro di dottorato, abbiamo valutato e caratterizzato la loro biologia cellulare con particolare attenzione a quelle differenze intrinseche presenti anche prima della loro clonazione. Infatti, ambo le tipologie clonali possono essere distinte mediante il potenziale di membrana mitocondriale (ΔΨm) subito dopo l’isolamento dalla fibra. Questo dato è in accordo con lo stato ossido riduttivo mitocondriale misurato tramite NAD+/NADH e la quantificazione della produzione di CO2. Questi risultati sono responsabili delle differenze metaboliche e possono essere spiegati dalla diversa espressione dell’enzima glicolitico Pfkfb3. Inoltre la concentrazione mitocondriale del Ca2+ e la sensibilità all’apoptosi sono modificate così come la dimensione della rete mitocondriale. In conclusione, siamo stati in grado di determinare quale clone rappresenta la cellula staminale all’interno della popolazione di cellule satelliti. Queste nuove osservazioni sperimentali rivelano caratteristiche fisiologiche della biologia delle popolazioni delle cellule satelliti prima e dopo la clonazione, mettendo in luce un’eterogeneità intrinseca della cellula satellite. Nella seconda parte della mia tesi abbiamo esplorato la possibilità che le cellule satelliti possano, se opportunamente stimolate, trans-differenziarsi in cellule muscolari lisce. Il sistema nervoso enterico normalmente interagisce con le cellule muscolari per controllare l’attività peristaltica e secretoria della parete intestinale. L’incompleta colonizzazione dell’intestino da parte delle cellule della cresta neurale provoca la malattia di Hirschsprung, caratterizzata da aganglionosi del colon distale. Le neurosfere (NLBs), precursori enterici in grado di auto-rinnovarsi, possono generare neuroni e glia; essere isolate dall’intestino di topi, ratti e umani e sono in grado di colonizzare l'intestino dopo il trapianto. Il nostro obiettivo è di capire la relazione tra i precursori di cellule satelliti (MPCs) e NLBs utilizzando un modello in vitro di co-coltura: questo sarà utile in prospettiva di un approccio di ingegneria tissutale per la rigenerazione intestinale e muscolo scheletrico. I nostri dati hanno evidenziato che NLBs, in presenza di MPCs, sono in grado di formare nuovi miotubi. L’uso di terreni di coltura miogenici ha evidenziato un notevole aumento della differenziazione in senso muscolare, promuovendo la formazione di striature ed aumentando l’espressione di desmina. Dall’altra parte, l’utilizzo di terreni di coltura neurogenici ha mostrato un fenotipo simil neurale. Come prospettive future, dobbiamo comprendere ulteriormente la relazione tra MPCs e NLBs e se le sinapsi sono coinvolte in questo processo; si deve verificare se un loro utilizzo su polimeri biocompatibili ne possa influenzare il comportamento, ed infine è necessaria una conferma dei suddetti dati tramite un’analisi di differenziazione in vivo in muscolo scheletrico e liscio. Nella terza ed ultima fase del mio lavoro, ci siamo focalizzati ad esplorare la possibilità che cellule non-muscolari possano, se opportunamente stimolate, differenziare in senso muscolare liscio. Il nostro obiettivo è stato quello di ottenere cellule muscolari lisce (SMCs) partendo da cellule staminali del fluido amniotico umano (hAFSC). hAFSC sono state trasdotte utilizzando un virus codificante per ZsGreen sotto il promotore αSMA. SMhAFSC così ottenute hanno evidenziate un alto livello d’espressione dei geni del muscolo liscio (come αSMA, desmina, calponina e smoothelin). Queste caratteristiche sono state confermate da molteplici analisi: di immunofluorescenza, dimostrando la positività a marcatori specifici per il muscolo liscio; microscopia a trasmissione elettronica (TEM), dove si verificava l’aumento della presenza di filamenti intermedi, di corpi densi e depositi di glicogeno, modello simile rispetto alle SMCs. Analisi in timelapse di SMhAFSC hanno dimostrato che queste possiedono un potenziale contrattile superiore rispetto hAFSC e studi su singola cellula hanno evidenziato la presenza di canali calcio voltaggio-dipendenti attivati da potassio solamente su SMhAFSC. In conclusione, siamo stati in grado di generare di cellule muscolari lisce funzionali da un precursore nonmuscolare ed in secondo luogo il processo di trasduzione può rappresentare un valido strumento per distinguere e selezionare differenti popolazioni. Questa fase può eventualmente superare il ben noto problema dell’espansione di progenitori di cellule muscolari lisce, rendendo queste cellule suscettibili per approcci d’ingegneria tessutale.

Après deux chapitres introductifs, ce manuscrit est divisé en trois parties independantes. (I) Nous étudions l'influence du cisaillement sur l'agrégation de particules paramagnétiques soumises à un champ magnétique parallèle à l'écoulement. Nous montrons que les chaines formées croissent linéairement suivant un modèle utilisant l'équation de Smoluchovsky. (II) Une étude théorique est menée afin d'étendre aux situations expérimentales rencontrées en microfluidique les relations décrivant les phénomènes électrocinétiques. Les symétries entre courant d'écoulement et électroosmose sont démontrées. L'approximation de couche de Debye fine est appliquée au courant d'écoulement puis utilisée pour décrire les structures des courants électriques et hydrodynamiques dans des géométries modèles. (III) Un système d'analyse de protéines est présenté, permettant l'identification d'un mélange de protéines. Il comporte un étage de séparation électrophorétique suivi d'une digestion enzymatique.

Electrospray ionization (ESI) mass spectrometry (MS) in combination with liquid chromatography (LC) is an excellent tool for the identification of drug metabolites. Utilizing this hyphenated technique in combination with proper sample pretreatment, the metabolic pathways of the analgesic drug ketobemidone were investigated in human urine and rat microdialysate from blood and brain. Two novel phase I metabolites (ketobemidone N-oxide and meta-hydroxymethoxyketobemidone) and three novel phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in human urine. Further, norketobemidone and ketobemidone N-oxide were identified in rat microdialysate from brain after regional distribution of ketobemidone in striatum. This indicates that the brain itself has the possibility to metabolize ketobemidone.

Synthetic ketobemidone metabolites were used for comparison of retention times and tandem MS spectra with the possible metabolites recovered from the biological samples. The conjugated metabolites were identified by accurate mass measurements and tandem MS spectra of the aglycones. The accuracy of the estimated masses was better than 2.1 ppm for two out of three conjugates in presence of internal standard.

On-line micro-SPE was successfully used for trapping and desalting of the microdialysates. The small SPE pre-column made it possible to inject approximately 100 times more sample on the analytical column compared to injection without pre-column. Selective trapping was demonstrated for the polar catechol amine metabolite, dihydroxyketobemidone, which forms covalent complexes with phenylboronic acid (PBA). A fluorinated silica type stationary phase was the only column out of several tested that was able to separate ketobemidone and all relevant phase I metabolites.

Liquid chromatography and mass spectrometry are independently valuable tools in the field of analytical pharmaceutical chemistry. The present study showed that the combination of LC-MS, with its excellent selectivity and sensitivity, offers an outstanding tool in the qualitative analysis of drugs and metabolites in biological fluids.

No presente trabalho são apresentadas análises de amostras de proteínas totais e aminoácidos empregando-se os reagentes p-benzoquinona e tetraamin cobre (II). No caso do reagente p-benzoquinona, as análises foram feitas através de um sistema inédito em fluxo, no qual a mistura reacional passava através de um reator composto de um tubo capilar de aço inoxidável aquecido num banho de glicerina. Os derivados formados passavam através de uma cela de fluxo acoplada a um espectrofotômetro, onde eram feitas as leituras das absorbâncias. Este método se mostrou mais rápido em relação àquele feito em banho-maria. A utilização de um sistema de análise por injeção em fluxo (FIA) com o reagente tetraamin cobre (II), permitiu que as análises fossem feitas de forma rápida, a baixo custo e com a vantagem de não necessitarem de aquecimento, como no caso da p-benzoquinona. Os resultados das análises de amostras de albumina humana obtidos em ambos os métodos acima apresentaram boa concordância quando comparados àqueles obtidos pelo método Kjeldahl para as mesmas amostras.
This work describes two methods for analysis of total proteins and aminoacids using both p-benzoquinone (PBQ) and tetraamin copper (II) reagents. With the p-benzoquinone reagent the method was performed by an original flow system made with stainless steel capillary tubing, conveniently heated into a glycerin bath, where the reactional mixture travels in its way to the detector. The derivate products are carried out to a flow cell adjusted to a spectrophotometer where the absorbances are measured. This method was efficient, with a fair cost, and providing results very faster than in the batch mode of operation. The second method, using the tetraamin copper (II) reagent, was performed by a common flow injection system (FIA). When compared with the PBQ method, the FIA tetramin copper (II) method was also rapid, efficient, with a fair cost and with the great advantage of simplicity, once it is performed at room temperature. The results obtained with human albumine samples using both methods are in agreement with those ones obtained by the Kjeldahl method for the same samples.

Određivanje koncentracije etanola u telesnim tečnostima, pre svega u krvi, neophodan je uslov da bi se ustanovio uticaj alkoholemije na psihomotorne sposobnosti. Poznavanje stabilnosti lekova, droga i metabolita u biološkim uzorcima je od ključne važnosti kada se ukaže potreba za ponovljenom analizom i evaluacijom rezultata u sudskom postupku. Osnovni ciljevi ovog rada su da se uz pomoć HS-GC metode (hedspejs gasna hromatografija) ustanovi da li postoji statistički značajna promena koncentracije etanola u uzorcima krvi dobijenih od živih osoba i u biološkim uzorcima uzorcima sa autopsijskog materijala. Na osnovu rezultata potrebno je bilo utvrditi u kojem tipu uzorka uzetog sa lešnog materijala postoji najmanja promena koncentracije tokom perioda čuvanja uzorka. Istraživanje je bilo otvoreno, randomizirano i prospektivnog tipa. Biološki uzorci krvi krvi živih osoba i lešnog materijala (krv, mokraća i staklasto telo) uzimani su metodom slučajnog izbora, u rasponu alkoholemije od 0,1 mg/ml do 5 mg/ml. Nakon inicijalne dvostruke analize, jedan biološki uzorak čuvan je u trajanju od 180 dana, dok je drugi otvaran i analiziran nakon 60, 120 i 180 dana. Ukupan broj analiza alkoholemije u krvi živih osoba iznosio je 500. Ukupan broj analiza koncentracije etanola u krvi, mokraći i staklastom telu sa leševa iznosio je 360. Etanol je u uzorcima krvi živih osoba, kao i u biološkim uzorcima sa autopsijskog materijala određivan metodom HS GC. Tokom čuvanja bioloških uzoraka u periodu od šest meseci ustanovljeno je da je došlo do značajnog smanjenja koncentracije etanola u svim analiziranim uzorcima, nezavisno od njegovog porekla. Promena koncentracije etanola tokom čuvanja u zavisnosti je od tkivne vrste uzorka, inicijalne alkoholemije, dužine čuvanja, integriteta vijala i čepova, temperature, odnosa tečne i gasne faze, prisustva konzervansa i potencijalnog intermitentnog otvaranja radi analiza.
Determination of ethanol concentration in body fluids, especially blood, is a necessary objective to establish the influence of alcohol on psychomotor skills. Knowing the stability of medicines, drugs and metabolites in biological samples is of crucial importance when there is a need for repeated analysis and result evaluation in court. The main objectives of this work were to determine whether there was a statistically significant change in ethanol concentration in blood samples obtained from living subjects and from autopsy material, by using HS-GC method (headspace gas chromatography). Based on the results it was necessary to determine which type of sample collected from autopsy showed the lowest change in concentration during the storage period. The study was open, randomized and prospective. Biological samples of living person's blood and autopsy biological samples (blood, urine and the vitreous humor) were taken at random, in the level range between 0.1 mg/ml and 5 mg/ml. After an initial duplicate analysis, one biological sample was stored for a period of 180 days, while the other was opened and analyzed after 60, 120 and 180 days. Total number of analysis of living person's blood samples was 500. The total number of analysis of autopsy biological samples was 360. All concentrations were determined by HS-GC method. During the storage, results showed that there has been a significant decrease in the concentration of ethanol in all of the analyzed samples, regardless of its origin. The level of this change was dependent on the type of tissue sample, initial alcohol concentration, duration of storage, integrity of the vials and stoppers, temperature, ratio of liquid and gas phases, presence of preservatives and intermittent opening for analysis.

Um estudo sistemático a respeito da utilização de padrão interno em determinações multielementares por espectrometria de absorção atômica (ETAAS) foi desenvolvido. O objetivo principal do presente trabalho foi verificar a possibilidade de melhorar a precisão e a exatidão dos resultados analíticos, que são obtidos na análise de fluidos biológicos. O pré-tratamento dessas amostras foi simplificado e reduzido a uma única etapa de diluição com surfactante (Triton X-100) e ácido (HNO3). Conseqüentemente, a complexidade da solução diluída de amostra, a ser introduzida no tubo de grafite, apresenta uma elevada quantidade de concomitantes que podem provocar interferências químicas. A seleção preliminar dos elementos a serem testados como padrão interno considerou a semelhança de parâmetros físico-químicos relacionados com o processo de atomização. Desta forma, Ag, Bi, In e Tl foram testados como padrão interno para a determinação simultânea de Cd/Pb em sangue e urina, enquanto Bi, Ge, In, Sb, Sn e Te foram os elementos selecionados para a determinação de Mn/Ni/Se em soro sangüíneo. A melhoria da qualidade dos resultados analíticos obtidos na determinação simultânea de Cd e Pb em sangue foi observada quando Ag foi utilizada como padrão interno, na presença de NH4H2PO4 como modificador químico. Verificou-se uma melhoria na exatidão dos resultados obtidos para Cd e Pb, após a correção com padrão interno. Por outro lado, os resultados obtidos na análise de urina não foram corrigidos por nenhum dos elementos testados. Os melhores resultados para a determinação simultânea de Mn, Ni e Se foram obtidos com a utilização de Bi, Sn e Te como padrão interno. Entretanto, verificou-se que a correção de todos os resultados não seria viável com o uso de um único padrão interno. O melhor desempenho nos testes realizados na presença de soro sangüíneo foi obtido com Bi, que melhorou discretamente a precisão dos resultados obtidos para Se. Desta forma, a padronização interna visando a determinação simultânea de Mn, Ni e Se não foi eficiente. A padronização interna em ETAAS, com a finalidade de melhorar a precisão e a exatidão dos resultados analíticos, é uma estratégia tão complexa, quanto os efeitos interferentes que se pretende corrigir: são necessários mais estudos para compreender melhor como a utilização de uma condição de compromisso afeta os processos de atomização, bem como mais informações a respeito das interferências físicas e químicas causadas por amostras complexas, analisadas por ETAAS após uma simples etapa de diluição. Deve-se considerar com especial atenção o modificador químico e as temperaturas das etapas de pirólise e de atomização empregadas, que são parâmetros críticos para o desempenho de um elemento como padrão interno.
A systematic study involving the use of internal standard for multielement determinations by electrothermal atomic absorption spectrometry was developed. The main objective of this work was evaluate the possibility of improving precision and accuracy of the analytical results for biological fluids. The sample pre-treatment was reduced to a single dilution step with surfactant (Triton X-100) and acid (HNO3), increasing the amount of concomitant introduced into the atomizer. The preliminary selection of the elements to be tested as internal standard considered the resemblance of physico-chemical parameters related with the atomization process. Thus, Ag, Bi, In and Tl were tested as internal standard for the simultaneous determination of Cd/Pb in blood and urine, and Bi, Ge, In, Sb, Sn and Te were the selected elements for the determination of Mn/Ni/Se in blood serum. The correction of the results obtained for the simultaneous determination of Cd and Pb in blood was achieved when Ag was used as internal standard, in presence of NH4H2PO4 as chemical modifier. An improvement for the accuracy of the results was observed for both analytes after their correction with the internal standard. On the other hand, the results obtained for the urine analysis were not corrected by using the tested elements. The best results for the simultaneous determination of Mn, Ni and Se were observed when Bi, Sn and Te were used as internal standard. However, the correction for the results for all analytes was not possible by using only one internal standard. The best performance in presence of the serum was obtained for Bi, which improves slightly the precision for the Se results. Thus, the internal standardization for the simultaneous determination of Mn, Ni and Se was not efficient. The internal standardization in ETAAS, aiming the improvement of precision and accuracy of the analytical results, is a strategy as complex as the interference effects to be corrected: more studies are required in order to better understand how the adoption of a compromised condition disturbs the atomization processes, as well as to get more information about the physical and chemical interference caused by complex samples, analyzed by ETAAS after a single dilution step. The chemical modifier and the selected temperatures for the pyrolysis and atomization steps are critical parameters for the performace of an internal standard and they should be carefully considered.

The boundary between biological cells and the external environment is represented by a lipid membrane that does not allow the passage of polar molecules such as ions and water. However, their orchestrated movements are fundamental for the function of vital organs. For this reason, there are transmembrane proteins called as “ion channels” that forming pores on the cells allow the ion to flow through these impermeable membranes. Normally, an ion channel has three functional states: the open state that is permeable to ions, the closed state where they can not pass, and the inactivated state that is structurally similar to the open state but functionally closed. The mechanisms by which an ion channel switches from the open to the closed state and vice versa are defined as “activation” and “deactivation” respectively, while the transition from the open to the inactivated state is defined as “inactivation”. All these transitions define the so-called “gating” mechanism. The main features of the biological channels are the selectivity, the sensing and the regulation of their gating mechanism. The characterization of these properties gives the possibility to extract the basic principles to design bioinspired artificial channels that can be used, for example, to study various molecules in confined spaces and in real time by current measurements. The biomimetic channels are becoming the focus of attention for bionanotechnology because they offer greater flexibility in terms of shape and size, robustness and surface properties boosting, in this way, their fields of application. In this context, we studied the human ether-à-go-go–related gene channel (hERG, KCNH2, or Kv11.1) that is a voltage-gated potassium channel involved in the heart contraction. hERG malfunctions are associated with severe pathologies such as long QT syndrome type 2 (LQTS2) that can be due to loss-of-function mutations (congenital LQTS2) or channel blockage induced by unspecific interactions with medications (acquired LQTS2). These conditions have been described to promote arrhythmia and sudden cardiac death. Based on the protein architecture, hERG is a member of the non-domain-swapped channel family where the sensor of the channel is always connected covalently to the pore of the same subunit. The gating mechanism of these channels is still not completely characterized. Due to its importance in human health, we decided to focus on hERG gating addressing the problem with a theoretical approach based on Molecular Dynamics simulations. Using a network analysis, we microscopically identified the kinematic chain of residues that couple the sensor of the channel to the pore. In general, our results could provide the basis for studying the coupling mechanisms between sensors and pores in ion channels to explain the origin of inherited and induced channelopathies such as LQTS2.

My research work, during my Ph.D. course, was mainly focused on the development of analytical methods based on the use of supercritical carbon dioxide (CO2) for the analysis of food and biological samples. In particular, the techniques used were: supercritical fluid chromatography coupled to mass spectrometry (SFC-MS) and supercritical fluid extraction online supercritical fluid chromatography coupled to mass spectrometry (SFE-SFC-MS). Specifically, the use CO2 under supercritical condition, has allowed the development of green analytical methods, in which the use of organic solvents, both for extraction and separation processes, is drastically reduced. The on-line SFE-SFC-MS system, has allowed the development of advanced extractive and separative methods potentially applicable in fields clinical, health and food fraud. Furthermore, the use of a mass spectrometer as a detector allowed to obtain detailed and precise information on the composition of the matrices analyzed for some classes of molecules with fundamental biological activities. The on-line SFE-SFC-MS it was used for the extraction and detection of carotenoids and it oxidative-cleavage derivates apo-carotenoids determination in food and biological samples. Moreover, the SFC-MS system was also employed for a detailed elucidation on limonoids in cold-pressed Citrus essential oils.

Dissertação de Mestrado em Química Forense apresentada à Faculdade de Ciências e Tecnologia
O desenvolvimento do presente estudo tornou-se pertinente devido ao consumo indevido de opióides e ao número de mortes por overdoses associadas ao seu consumo, tanto em Portugal como no resto do mundo. Por outro lado, sendo o recurso a matrizes biológicas postmortem alternativas ao sangue, uma área de crescente interesse em toxicologia forense estudámos a viabilidade do uso do líquido pericárdico na determinação das substâncias selecionadas.Assim, o objetivo deste trabalho foi o desenvolvimento, otimização e validação de uma metodologia analítica para a determinação qualitativa e quantitativa de alguns opióides em sangue e líquido pericárdico.Os opióides estudados foram: morfina, codeína, 6-acetilmorfina, 6-acetilcodeína, oxicodona, oximorfona e o fentanil.O estudo incluiu a otimização do procedimento analítico e do método cromatográfico. Otimizámos a extração em fase sólida (SPE), a derivatização com e sem recurso a hidroxilamina aquosa a 1% e o tempo de derivatização induzida por micro-ondas com o reagente químico MSTFA (n-metil-n-(trimetilsilil) trifluoroacetamida)+5% TMCS (trimetilclorosilano).O método mais eficiente e seletivo correspondeu ao seguinte procedimento: precipitação com acetonitrilo de volumes de 250 μL de amostras de sangue e de líquido pericárdico, derivatização das substâncias de interesse usando 1% hidroxilamina aquosa em PBS (1:2, v/v) promovida por irradiação de micro-ondas, durante 30 segundos com uma potência de 900 W a 50%. Procedeu-se à extração dos analitos de interesse por SPE. Após evaporação dos eluatos (sob corrente de azoto a 40 ᵒC) os extratos foram derivatizados com MSTFA+5% TMCS sob ação de micro-ondas durante 100 segundos com uma potência de 900 W a 100%. Seguidamente os extratos derivatizados foram injetados (2 μL, splitless) diretamente no sistema de cromatografia de gases associado à espectrometria de massas (GC-MS-EI) com monitorização dos iões selecionados (modo SIM) e com o forno à temperatura inicial de 50 ᵒC.Após a otimização, o método foi validado seguindo as normas da Scientific Working Group for Forensic Toxicology (SWGTOX) de forma a garantir que o método é adequado para os fins a que se destina e assim atestar a sua fiabilidade na interpretação dos resultados analíticos. O método apresentou linearidade no intervalo 5-1000 ng/mL com coeficientes de determinação superiores a 0.99 para todos os analitos. Os limites de deteção (LOD) variaram entre 3 e 4 ng/mL, dependendo da substância e/ou da matriz analisada e os limites de quantificação (LOQ) foram de 5 ng/mL para todas as substâncias. Em relação às precisões (intra-dia e intermédia) todos os níveis de concentração apresentaram valores de CV <20% e a exatidão situou-se dentro do intervalo ±20%.Verificou-se ainda que as substâncias apresentaram estabilidade sob as seguintes condições: nos extratos deixados no amostrador em condições ambientais por pelo menos 24 h; nas amostras de sangue e líquido pericárdico deixadas na bancada de trabalho durante 4 h e nas amostras de líquido pericárdico durante 3 ciclos de congelação e descongelação ao longo de pelo menos 4 semanas.Por fim, a metodologia analítica foi aplicada a amostras reais disponibilizadas pelo Serviço de Química e Toxicologia Forenses da Delegação do Centro do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P..De acordo com a revisão bibliográfica efetuada, este foi o primeiro método desenvolvido para a deteção e quantificação simultânea deste grupo de substâncias em sangue e líquido pericárdico com recurso à derivatização promovida por micro-ondas com os reagentes químicos hidroxilamina e MSTFA+5% TMCS.
The development of the present study became pertinent due to the misuse of opioids and the number of overdose deaths associated with its use, both in Portugal and in the rest of the world. On the other hand, being the use of alternative biological matrices to postmortem blood, an area of growing interest in forensic toxicology, we studied the feasibility of using pericardial fluid in the determination of the selected substances.Thus, the objective of this work was the development, optimization and validation of an analytical methodology for the qualitative and quantitative determination of some opioids in blood and pericardial fluid.The opioids studied were: morphine, codeine, 6-acetylmorphine, 6-acetylcodeine, oxycodone, oxymorphone and fentanyl.The study included the optimization of the analytical procedure and the chromatographic method. We have optimized solid phase extraction (SPE), derivatization with and without 1% aqueous hydroxylamine and microwave derivatization time with the chemical reagent MSTFA (n-methyl-n-(trimethylsilyl) trifluoroacetamide)+5% TMCS (trimethylchlorosilane).The most efficient and selective method was as follows: precipitation with acetonitrile of 250 μL volumes of blood and pericardial fluid samples, derivatization of the substances of interest using 1% aqueous hydroxylamine in PBS (1:2, v/v) with microwave action for 30 seconds with a power of 900 W at 50%. The samples were cooled and then the analytes of interest were extracted by SPE. After evaporation of the eluates (under nitrogen stream at 40 °C) the extracts were derivatized with MSTFA+5% TMCS under microwave action for 100 seconds with a power of 900 W at 100%. Then the derivatized extracts were injected (2 μL, splitless) directly into a gas chromatography mass spectrometry system (GC-MS-EI) with selective ion monitoring mode (SIM mode) and with the oven at the initial temperature of 50 °C.After optimization, the method was validated following the standards of the Scientific Working Group for Forensic Toxicology (SWGTOX) to ensure that the method is suitable for its intended purpose and thus attest to its reliability in interpreting the analytical results. The method presented linearity in the range 5-1000 ng/mL with coefficients of determination above 0.99 for all analytes. The limits of detection (LOD) ranged from 3 to 4 ng/mL, depending on the substance and/or matrix analysed and the limits of quantitation (LOQ) were 5 ng/mL for all substances. Regarding the precision (intra-day and intermediate) all concentration levels presented CV values <20% and the bias was within ±20%. It was also verified that the substances presented stability under the following conditions: in the extracts left in the autosampler under environmentalconditions for at least 24 h; blood and pericardial fluid samples on the workbench for 4 h; pericardial fluid samples for 3 freeze-thaw cycles for at least 4 weeks.Finally, the analytical methodology was applied to real samples provided by the Serviço de Química e Toxicologia Forenses da Delegação do Centro do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P. (Forensic Chemistry and Toxicology department of the Centre Branch of the National Institute of Legal Medicine and Forensic Sciences, I.P.).According to the literature review, this was the first method developed for the simultaneous detection and quantification of this group of substances in blood and pericardial fluid using microwave induced derivatization with the chemical reagent’s hydroxylamine and MSTFA+5% TMCS.

Following the intensification of agriculture and the promotion of agro-chemicals in low and middle income countries, acute pesticide poisoning has become a major public health problem with more than 300,000 deaths each year around the world. The easy availability of highly toxic pesticides in the homes of farming communities has made pesticides a preferred choice for suicide with an extremely high case fatality. In fact, the World Health Organization (WHO) indicates that there may be 1 million serious unintentional poisonings each year and in addition 2 million people hospitalized for suicide attempts with pesticides. The goal of this work was the detection and quantification of eight organophosphorous pesticides in blood samples using solid phase extraction and gas chromatography-mass spectrometry. The studied analytes were omethoate, dimethoate, diazinon, chlorpyrifos, parathion, clorfenvinphos, quinalphos and azinphos. Ethion was used as internal standard (IS). The analytes and IS were extracted by solid-phase extraction using Oasis® HLB extraction cartridges, and the extracts were analyzed by gas chromatography-electron ionisation-mass spectrometry (GC/EI-MS). Calibration curves were established using a weighed linear calibration model (except for omethoate, for which a power regression was used) between 0.05 and 25.00 μg/mL. The correlation coefficients were higher than 0.991. Precision (intraday and intermediate) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. Limits of quantification were 50 ng/mL for all compounds, except for ometoathe, for which 100 ng/mL were obtained. Because of its simplicity and speed, the proposed method can be applied in the determination of these compounds in post-mortem blood samples, and is suitable for application in toxicology routine analysis.

The complete set of lipids in an organism or a cell along with its interactions with other molecules, such as other lipids, proteins, and metabolites constitutes the lipidome. Lipidomics is the comprehensive and quantitative study of the lipidome. It involves identification and quantitation of thousands of biological pathways involving lipids and their interactions. Lipids are the essential metabolites in human body; their main biological functions are the energy storage, endocrine actions, morphogenesis, building blocks of cellular and subcellular membranes and signaling molecules. The dysregulation of lipids is related to various serious human diseases, such as cancer, Alzheimer, cardiovascular diseases, and lysosomal disorders. Using lipidomics approaches, it has become easier to study the lipids species in an organism. Lipidomics is an emerging field in the name of the ‘omics’ for system-level analysis of lipids and their interacting partners within a cell. Lipidomics aims to define and quantitate all of the molecular lipid species present in a cell. The lipid molecular species can be described by the eight known categories of lipids, numerous classes, and subclasses, such us fatty acids, glycerolipids, sphingolipids, prenols, sterols, glycerophospholipids, poliketides and saccharolipides. Current studies related to lipid identification and determination, or lipidomics in biological samples, are one of the most important issues in modern bioanalytical chemistry. There are many articles dedicated to specific analytical strategies and to the actual analytical methodologies used in lipidomics in various kinds of biological samples. The most important methods used to characterize the lipidomics in modern bioanalysis are: chromatography/separation methods (thin layer chromatography (TLC), (ultra) high-pressure liquid chromatography ((U)HPLC), gas chromatography (GC), (ultra-performance) supercritical fluid chromatography ((UHP)SFC), and capillary electrophoresis (CE)); spectroscopic methods (Raman spectroscopy (RS), Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR)); mass spectrometry and also hyphenated methods (matrix-assisted laser desorption/ionization (MALDI), hyphenated methods, which include liquid chromatography–mass spectrometry (LC-MS), gas chromatography–mass spectrometry (GC-MS) and also multidimensional techniques). These are being used to identify and quantify all the lipid species in order to understand their function in biological systems. MS technology has been proved to be highly efficient in the characterization and quantification of lipid molecular species in lipid extracts. One of the reasons behind this could be the ability of MS to characterize and separate each ionized particle according to their mass-to-charge (m/z) ratio. MS can also provide structural information by fragmenting the lipid ions which can be achieved by using tandem MS, or MS/MS. Basically, there are two different approaches for lipidomics analysis: - to apply some extraction protocols optimized for each lipid category, and then subject to LC to separate the present lipids molecular species optimally, then the LC eluate is coupled directly to the mass spectrometer for further analysis such as molecular fragmentation (MS/MS), ion scanning, etc. - another approach, also known as “shotgun lipidomics”, involves the offline extraction of lipids followed by MS analysis without LC separation. Several tools are available for lipidomics and some are emerging concerning the combination of genomics and lipidomics to identify clinically relevant biomarkers. For example, SimLipid is a high-throughput characterization tool for lipids. It analyzes lipid mass spectrometric data to profile them using LC coupled with MALDI-MS, MS/MS data, and also remove the overlapping isotopic peaks from multiple spectra in batch mode. "Lipidomics" applies to studying lipid metabolism on a broad scale and it may elucidate the biochemical mechanism(s) underlying specific changes in lipid metabolism. Advances in mass spectrometry have greatly accelerated the lipidomics field. Chemical derivatization has shown its broad use in improving analytical sensitivity and specificity in lipidomics. Lipidomics aims to quantitatively define lipid classes, including their molecular species, in biological systems and it has experienced rapid progress, mainly because of continuous technical advances in instrumentation that are now enabling quantitative lipid analyses with an unprecedented level of sensitivity and precision. The still-growing category of lipids includes a broad diversity of chemical structures with a wide range of physicochemical properties. Reflecting this diversity, different methods and strategies are being applied to the quantification of lipids. Since its advent, LC is being exploited by separation scientists and applied to a wider and wider range of sample matrices for the separation, identification and quantification of ever more compounds, particularly in lipidomic analysis. The unceasing progresses in column and stationary phases production, and the enormous developments in detection techniques have contributed to the outstanding success of chromatography, as an invaluable tool in analytical chemistry in many different fields including nutraceutical, food, environmental, clinical, forensic, and pharmaceutical applications. The great advantages to be gained by the use of LC are especially increased with the hyphenation to MS (LC-MS). Recent trends in the area of LC-MS and related techniques involve: (a) the shift from conventional HPLC-MS to ultra high pressure liquid chromatography (UHPLC)-MS or other fast LC-MS techniques (core-shell particles, high-temperature LC and monolithic columns) requiring fast MS analyzers (typically time-of-flight (TOF)-based systems); (b) the use of supercritical fluid chromatography (SFC) for fast and “green” separations, with reduction in solvents consumption; (c) the use of multidimensional liquid chromatography techniques (MDLC) for complex samples, and other dimension also in MS, such as ion mobility spectrometry (IMS)-MS, the coupling of two or more mass analyzer (tandem MS); (d) the shift from low-resolution to (ultra)high-resolution MS to allow accurate mass measurements. Each of these techniques will be described in detail in the following chapters, illustrating selected applications developed for the analysis of lipid and lipid-like molecules, as well as other bioactive compounds.

Tese de mestrado em Biologia Humana e Ambiente, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2017
A exposição a metais pesados, tais como o chumbo, arsénio e manganês, constitui uma potencial ameaça para a saúde humana. A persistência destes metais no meio ambiente, juntamente com o seu uso intensivo pelas sociedades modernas, tem ao longo dos anos vindo a aumentar significativamente. Esta exposição pode ocorrer através do ar, alimentos ou água contaminada, e varia com o tipo de metal e com o nível de exposição a que estamos sujeitos. A toxicidade dos metais pesados reside essencialmente na formação de espécies reactivas de oxigénio, que desempenham um papel determinante para o desenvolvimento de efeitos adversos, tais como efeitos neurotóxicos e cancerígenos. O desenvolvimento de biomarcadores, que possibilitem a detecção de exposições continuadas, e/ou elevadas a metais pesados, e que possam assinalar atempadamente o aparecimento de efeitos adversos na fase inicial, enquanto o efeito tóxico for ainda reversível, é essencial para a saúde humana. Alguns metais interferem em pontos-chave da via metabólica da síntese do heme, alterando a excreção de porfirinas e de ácido delta-aminolevulínico (ALA) na urina; e promovem a oxidação das porfirinas reduzidas (porfirinogénios), que irão acumular-se em tecidos alvo, causando efeitos tóxicos no organismo. Concentrações urinárias de ALA superiores a 15 mg/L urina, podem provocar danos cerebrais em trabalhadores expostos, alucinações e convulsões. Neste sentido, o perfil de porfirinas e a concentração de ALA na urina são considerados biomarcadores de exposição e efeito para metais pesados. As porfirinas são compostos macrocíclicos, que desempenham um papel preponderante no metabolismo dos organismos vivos, participando em processos como a fotossíntese e transporte de oxigénio. No organismo, uroporfirina (uro), heptaporfirina (hepta), hexaporfirina (hexa), pentaporfirina (penta) e coproporfirina (copro) são produzidas em excesso, e por isso excretadas normalmente pela urina e fezes, em determinadas concentrações. No entanto, a excreção de porfirinas urinárias pode ser anormalmente elevada devido a vários factores: doenças como as porfirias, ingestão de determinados fármacos, e exposição ambiental a químicos, em particular metais pesados. Considerando as características estruturais e químicas das porfirinas é possível desenvolver métodos que permitam a sua extracção dos fluidos biológicos, e a sua posterior identificação e quantificação, possibilitando obter o perfil de porfirinas de cada indivíduo. A detecção de porfirinas através de métodos espectrofotométricos é eficaz e frequentemente utilizada, uma vez que estas apresentam um espectro característico com bandas na região do visível (400 a 750 nm), correspondentes à banda de Soret e bandas Q. Posteriormente, através de cromatografia líquida de alta eficiência (HPLC), foram desenvolvidos métodos altamente sensíveis que permitem a separação e identificação das várias porfirinas e a determinação da sua concentração em amostras biológicas. Novos e mais eficientes métodos que permitam uma melhor extracção e quantificação das porfirinas em diferentes fluidos biológicos, são necessários. O desenvolvimento de condições e meios de conservação eficazes, sem exporem as porfirinas a ambientes que interfiram com a sua fluorescência e absorvência, são igualmente necessários. Estes métodos serão uteis para melhorar o diagnóstico diferencial das porfirias, e aumentar a sensibilidade e especificidade das porfirinas como biomarcadores. Este trabalho teve como objectivos determinar as melhores condições de preservação de porfirinas em amostras de urina e avaliar vários métodos de extracção e quantificação de porfirinas, tendo em vista a obtenção de resultados mais rigorosos. Também, através da análise do perfil de porfirinas e do nível de ALA na urina de cada indivíduo, propomos biomarcadores de exposição e efeito, em subpopulações expostas em diferentes contextos de exposição a metais Amostras de urina de voluntários saudáveis, foram recolhidas e guardadas nas mesmas condições de temperatura (4º, -20º e -80 ºC), armazenamento (em alíquotas ou pool) e sem ou com conservantes (Na2CO3 ou HCl) ao longo de 90 dias. O perfil urinário de porfirinas determinado por HPLC, de cada amostra armazenada na respectiva condição, foi obtido no dia de recolha (dia 0), ao fim de uma semana (dia 7), ao fim de um mês (dia 30) e três meses depois (dia 90). Ao comparar as concentrações de porfirinas nas mesmas amostras em diferentes condições de conservação, é possível identificar quando houve perdas/ganhos de concentração de uro, hepta, hexa, penta e copro, em relação ao dia 0. As condições que não revelem diferenças significativas (p>0.05) são consideradas condições óptimas de conservação. Copro conseguiu manter-se estável a -20ºC e hepta a -80 ºC durante 90 dias, sem a adição de conservantes às amostras de urina. Na presença de Na2CO3, a copro permaneceu estável a -20 ºC e -80ºC e a hexa a -80 ºC, ao longo de 90 dias. Quando se utilizou HCl, a fracção uro manteve-se estável a -80ºC durante 90 dias. As amostras conservadas com Na2CO3 e HCl apresentaram concentrações mais estáveis ao longo do tempo do que amostras sem conservante. As amostras armazenadas em alíquotas apresentaram menos diferenças significativas de concentração em comparação com as denominadas de pools de urina. O método de Soulsby (1974) permite quantificar a concentração total de copro em urina, enquanto que o método de Elder (2001) permite quantificar a concentração de porfirinas totais, ambos através de espectrofotometria UV-Vis. A principal diferença entre os métodos, é que o método Soulsby utiliza éter e vários passos de extracção, enquanto o método Elder apenas acidifica a urina com HCl, não havendo qualquer extração. De forma a estudar cada método, utilizámos padrões de copro I e copro III e uro I para obter os respectivos espectros em UV-Vis, quando aplicados em matriz de água e urina. Também analisámos uma mistura equimolar constituída pelos 3 padrões. Analisámos 55 amostras de urina de indivíduos saudáveis, pelos métodos de Soulsby, Elder e HPLC como forma de comparar os diferentes métodos e inferir sobre a qualidade de resultados. O método de Soulsby falhou na extracção e quantificação de uro I, mas foi bem-sucedido para a copro I e III. O método de Elder permitiu quantificar os padrões de copro e uro, individualmente e sob a forma de mistura de padrões. Os métodos de Soulsby e HPLC apresentaram uma relação linear, ainda que fraca (R2=0,268), para a determinação de copro total na urina. Quando comparados, os métodos de Elder e HPLC para a análise de porfirinas totais, estes também apresentaram uma fraca relação linear entre si (R2= 0,086). Na última parte do trabalho, foram voluntariamente cedidas amostras de urina de 63 indivíduos de uma amostra de população portuguesa, divididos em vários grupos de acordo com o nível e forma de exposição a metais pesados: pessoas que vivem numa cidade de uma ilha portuguesa (Grupo I), pessoas que vivem numa grande área urbana (Grupo L), mineiros (Grupo M), pessoas que vivem numa área rural (Grupo A), técnicos hospitalares de Raios-X (Grupo R), pessoas que vivem numa área urbana não-industrializada (Grupo S), pessoas que vivem numa área urbana industrializada (Grupo V) e fumadores (Grupo F). Determinámos o perfil urinário de porfirinas por HPLC e os níveis de ALA na urina por um método colorimétrico. Estes valores foram combinados usando análise discriminante para criar um modelo capaz de identificar se um individuo tem um contexto de exposição igual a uma área/grupo específico. Estes biomarcadores foram posteriormente integrados em procedimentos preditivos recorrendo a ferramentas estatísticas. Aplicaram-se 3 procedimentos preditivos: o primeiro combinando todos os grupos com excepção do grupo I; o segundo, combinando apenas os grupos expostos ambientalmente (Grupo L, A, S e V), e o terceiro considerando apenas os grupos expostos activamente (Grupo M, R e S). Pela combinação dos biomarcadores determinados, o primeiro procedimento, apenas conseguiu classificar correctamente 76,8% dos individuos. No segundo procedimento, já foi possível classificar correctamente 96,8% dos indivíduos expostos ambientalmente nos diferentes contextos, em áreas urbanas industrializadas e não-industrializadas, numa área rural e numa grande área urbana, de Portugal. No terceiro procedimento, a discriminação de indivíduos expostos activamente a metais pesados de forma ocupacional (mineiros ou técnicos de Raios-X) ou estilo de vida (fumadores), foi alcançada com uma classificação de 100%. Do nosso estudo podemos concluir que para garantir a conservação de todas as porfirinas, especialmente as copro e uro, é necessário adicionar um conservante. Enquanto o Na2CO3, aumenta o pH do meio e conserva os analitos sob a forma de porfirinogénios, o HCl, acidifica a urina e promove a conversão de porfirinogénios a porfirinas. Sugerimos ainda que as amostras sejam armazenadas em alíquotas para garantir uma maior estabilidade das porfirinas urinárias. Este trabalho veio confirmar que a uro não é solúvel em éter, e por isso os métodos espectrofotométricos que o utilizem na extração não o conseguem quantificar. No entanto, estes métodos já são eficazes para análises de copro. As porfirinas totais conseguem ser detectadas em urina acidificada com HCl por espectrofotometria UV-Vis, e estimadas quantitativamente. O método de preferência deve ser HPLC, uma vez que permite a separação de cada porfirina e a sua quantificação individual, mas os métodos espectrofotométricos podem ser utilizados para uma análise rápida e preliminar. A combinação dos perfis de porfirinas com os níveis de ALA na urina pode ser utilizada como biomarcador de exposição e efeito de metais pesados, na amostra de população estudada, uma vez que os resultados dos procedimentos preditivos foram satisfatórios. A aplicação destes biomarcadores pode ser uma ferramenta útil para prever o tipo de exposição e a magnitude dos efeitos tóxicos provocados pelos metais em populações expostas, sendo por isso necessário continuar a investir no estudo das porfirinas e nas suas aplicações em Toxicologia Preditiva.
Throughout our lives we are simultaneously exposed to single or multiple sources of metal mixtures in environmental or occupational contexts. Lead (Pb), Arsenic (As) and Manganese (Mn) are classified as human carcinogens and may also cause neurotoxic effects. These metals induce porphyrins and delta-aminolevulinic acid (ALA) accumulation, that results from the interference at specific points of the heme synthetic pathway. For this, porphyrins and ALA can be used as biomarkers of exposure and effect, contributing to prevent the risk of neurotoxicity in human populations. The aim of this work was to determine the best preservation conditions of urinary porphyrins and the most accurate method of extraction and quantification of specific and total porphyrins. We also intended to provide biomarkers for heavy metals, through the characterization of urinary porphyrins profiles and urinary levels of ALA, in a Portuguese sub-population sample. We experienced various storage conditions (aliquots or pools subjected to freeze-thaw cycles), different temperatures (4ºC, -20 ºC and -80 ºC), and two different preservatives, over time. The addition of a preservative, like HCl or Na2CO3, to urine aliquots preserved at -80ºC succeeded to stabilize uroporphyrin (uro) and coproporphyrin (copro) for at least 90 days. Through the analysis of copro (I and III) and uro (I) standards we conclude that spectrophotometric methods, that use ether to extract the porphyrins from urine, are not effective for uro determination, while copro, uro and total porphyrin can be detected in acidified urine by spectrophotometry, without extraction. However, HPLC should be the method of choice, once the various porphyrins are able to be separated and individually quantified through this method. We combined the urinary porphyrin profiles and ALA levels of 63 subjects, using discriminant analysis, to create a model aiming to identify whether an individual is in an exposure context according with a specific area/group. It was possible to correctly classify 96,8% of individuals environmentally exposed to metals in an urban, rural and industrial context, and 100% of the individuals actively exposed to metals (workers or smokers). The investigation of porphyrins and their precursors seems to provide tools for the development of Occupational Toxicology, highlighting predictive biomarkers of effect, and consequently the improvement of public health.

Algae “super blooms” are a commonly encountered environmental issue in fresh and brackish water that occurs due to the buildup of cyanobacteria. Many of the commonly encountered cyanobacteria such as Mycrocystis aeruginosa (M. aeruginosa) produce potent cyanotoxins (microcystins) that pose serious health threats and even death to local wild life and humans. Microcystin contaminated fresh-water that empties into the ocean has been shown to lethally affect marine life in the area of contamination. Human consumption of tainted sea life and other routes of mycrocystin exposure can lead to serious liver damage and even death. Thus, a method was developed for forensic postmortem analysis of microcystins RR, LR and YR by Two-Dimensional (2D) Liquid Chromatography (LC) - tandem Mass Spectrometry (MS/MS). A final 2D LC-MS/MS method was selected from 6x6 automated method development experiments. Each microcystin were subjected to a total of 36 methods, which were completed over an 18hr period. The extraction process was performed using a reverse-phase sorbent (Oasis HLB, Waters Corporation, Milford, MA) with a 3cc solid phase extraction (SPE) barrel using sequential elution. From acetonitrile (ACN) and methanol (MeOH) stock solutions, 10 μL of the internal standard (IS) Nodularin was added to the final extract. The concept of sequential micro extraction was designed to capture the retention behaviour of the target analyte in response to various extraction parameters (sorbent strength, elution polarity, and solubility). Therefore, optimized conditions were selected to excise the region of interest during extraction. The elution solvents chosen for the microcystins were acetonitrile, methanol and acetone with 10 % sequential increments. Since microcystins exhibit a zwitterionic structure, three sets of elution solutions were created to evaluate their elution profile (pH 3, pH 7, pH 10). When the elution profile for low pH and high pH are compared, microcystin RR was eluted over the 40% to 70% methanol fractions under low pH conditions with a slight shift towards higher organic % (50%-70% fractions) under high pH. This elution behaviour suggests that the basic moieties of the structure demonstrate a stronger retention for the stationary phase. Microcystin LR and YR however, eluted at a higher organic solvent percentage under low pH conditions and at a lower organic solvent percentage under high pH conditions, indicates that the acidic moieties of the structures have stronger retention. The urine sample gave recovery values for all three microcystins in the 80-90% range, as to be expected with type of complexity associated with biological samples. The sequential extraction protocol produced an extraction method that delivered a clean extract after a 30 min workflow using a single and optimized 2D LC-MS/MS method. The total analytical run time was set at 10 minutes.

A 2000 mm long saturated laboratory soil column was used to simulate soil aquifer treatment under saturated conditions to assess the removal of chemical and biochemical oxygen demand (COD and BOD), dissolved organic carbon (DOC), nitrogen and phosphate, using high strength artificial wastewater. The removal rates were determined under a combination of constant hydraulic loading rates (HLR) and variable COD concentrations as well as variable HLR under a constant COD. Within the range of COD concentrations considered (42 mg L(-)(1)-135 mg L(-)(1)) it was found that at fixed hydraulic loading rate, a decrease in the influent concentrations of dissolved organic carbon (DOC), biochemical oxygen demand (BOD), total nitrogen and phosphate improved their removal efficiencies. At the high COD concentrations applied residence times influenced the redox conditions in the soil column. Long residence times were detrimental to the removal process for COD, BOD and DOC as anoxic processes and sulphate reduction played an important role as electron acceptors. It was found that total COD mass loading within the range of 911 mg d(-)(1)-1780 mg d(-)(1) applied as low COD wastewater infiltrated coupled with short residence times would provide better effluent quality than the same mass applied as a COD with higher concentration at long residence times. The opposite was true for organic nitrogen where relatively high concentrations coupled with long residence time gave better removal efficiency.

Shing, Ka Fai.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (leaves 158-188).
Abstracts in English and Chinese.
ABSTRACT --- p.i
摘要 --- p.iv
ACKNOWLEDGEMENTS --- p.vi
TABLE OF CONTENTS --- p.vii
LIST OF TABLES --- p.x
LIST OF FIGURES --- p.xii
Chapter SECTION I: --- BACKGROUND --- p.1
Chapter CHAPTER 1: --- CELL-FREE NUCLEIC ACIDS IN HUMAN BODILY FLUIDS --- p.2
Chapter 1.1 --- Early studies on the presence of cell-free nucleic acids in human bodily fluids --- p.2
Chapter 1.2 --- Circulating nucleic acids in plasma and serum --- p.2
Chapter 1.2.1 --- Cancer Detection --- p.3
Chapter 1.2.1.1 --- Circulating tumor-derived DNA --- p.3
Chapter 1.2.1.2 --- Circulating tumor-derived RNA --- p.5
Chapter 1.2.2 --- Prenatal diagnosis --- p.7
Chapter 1.2.2.1 --- Circulating fetal DNA --- p.7
Chapter 1.2.2.2 --- Circulating fetal messenger RNA --- p.11
Chapter 1.2.2.3 --- Circulating placental microRNA --- p.13
Chapter 1.3 --- Cell-free nucleic acids in urine --- p.14
Chapter 1.3.1 --- Transrenal DNA (Tr-DNA) --- p.15
Chapter 1.3.1.1 --- Biology of Tr-DNA --- p.15
Chapter 1.3.1.2 --- Detection of fetal-derived Tr-DNA --- p.15
Chapter 1.3.1.3 --- Potential problems associated with the detection of Tr-DNA --- p.16
Chapter 1.3.2 --- Cell-free DNA in urine as released from the urinary tract --- p.17
Chapter 1.4 --- Other bodily fluids with cell-free nucleic acids --- p.18
Chapter 1.4.1 --- Amniotic fluid --- p.19
Chapter 1.4.2 --- Cerebrospinal fluid (CSF) --- p.20
Chapter 1.4.3 --- Peritoneal fluid --- p.20
Chapter CHAPTER 2: --- MICRORNA IN HUMANS --- p.21
Chapter 2.1 --- Introduction --- p.21
Chapter 2.2 --- Biogenesis --- p.21
Chapter 2.2.1 --- Transcription of microRNA genes --- p.21
Chapter 2.2.2 --- Processing and maturation of microRNA precursors --- p.23
Chapter 2.3 --- Mechanisms of gene regulation --- p.24
Chapter 2.3.1 --- Cleavage of target mRNA --- p.24
Chapter 2.3.2 --- Translational repression of mRNA --- p.25
Chapter 2.4 --- Functional roles of microRNAs --- p.25
Chapter 2.4.1 --- Oncogenesis --- p.25
Chapter 2.4.2 --- Programmed cell death --- p.26
Chapter 2.4.3 --- Cellular differentiation and development --- p.27
Chapter 2.4.4 --- Regulation of physiological and cellular processes --- p.28
Chapter 2.5 --- Aim of this thesis --- p.28
Chapter SECTION II: --- MATERIALS AND METHODS --- p.30
Chapter CHAPTER 3: --- QUANTITATIVE ANALYSIS OF CIRCULATING AND URINARY NUCLEIC ACIDS --- p.31
Chapter 3.1 --- Preparation of samples --- p.31
Chapter 3.1.1 --- Preparation of plasma --- p.31
Chapter 3.1.2 --- Preparation of blood cells --- p.32
Chapter 3.1.3 --- Preparation of placental tissue --- p.32
Chapter 3.1.4 --- Preparation of urine and urine cell pellet --- p.32
Chapter 3.2 --- Nucleic acid extraction --- p.33
Chapter 3.2.1 --- "Extraction of small RNA-containing total RNA from plasma, blood cells and placental tissue" --- p.33
Chapter 3.2.2 --- Extraction of DNA from urine --- p.37
Chapter 3.3 --- Quantitative measurements of nucleic acids --- p.38
Chapter 3.3.1 --- Principle of real-time quantitative PCR --- p.38
Chapter 3.3.2 --- One-step QRT-PCR assays for mRNA quantification --- p.40
Chapter 3.3.2.1 --- Principle --- p.40
Chapter 3.3.2.2 --- Quantification of human placental lactogen （hPL) mRNA --- p.40
Chapter 3.3.3 --- Two-step QRT-PCR assays for microRNA quantification --- p.45
Chapter 3.3.3.1 --- Principle --- p.45
Chapter 3.3.3.2 --- Advantages --- p.46
Chapter 3.3.3.3 --- TaqMan® MicroRNA Assays --- p.47
Chapter 3.3.4 --- QPCR assays for DNA quantification --- p.53
Chapter 3.3.4.1 --- Principle --- p.53
Chapter 3.3.4.2 --- Quantification of the leptin gene and the sex-determining region on Ychromosome gene --- p.53
Chapter 3.4 --- Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) --- p.57
Chapter 3.4.1 --- Principle --- p.57
Chapter 3.4.2 --- Zinc finger protein gene assay for determining the fractional concentration of male DNA --- p.58
Chapter 3.5 --- Statistical analyses --- p.65
Chapter SECTION III: --- CIRCULATING PLACENTAL MICRORNAS IN MATERNAL PLASMA AS MARKERS FOR PRENATAL DIAGNOSIS　 --- p.66
Chapter CHAPTER 4: --- THE EXISTENCE AND QUANTITATIVE DETECTION OF CELL-FREE MICRORNAS IN PLASMA --- p.67
Chapter 4.1 --- Introduction --- p.67
Chapter 4.2 --- Materials and methods --- p.69
Chapter 4.2.1 --- Sample collection --- p.69
Chapter 4.2.2 --- Experimental design --- p.69
Chapter 4.2.3 --- RNA extraction and quantification --- p.72
Chapter 4.3 --- Results --- p.75
Chapter 4.3.1 --- Validation of two-step QRT-PCR system for miRNA quantification --- p.75
Chapter 4.3.2 --- Detection of cell-free miRNA in maternal plasma --- p.82
Chapter 4.4 --- Discussion --- p.82
Chapter CHAPTER 5: --- SYSTEMATIC IDENTIFICATION AND CHARACTERIZATION OF PLACENTAL MICRORNAS IN MATERNAL PLASMA --- p.86
Chapter 5.1 --- Introduction --- p.86
Chapter 5.2 --- Materials and methods --- p.88
Chapter 5.2.1 --- Sample collection --- p.88
Chapter 5.2.2 --- Experimental design --- p.88
Chapter 5.2.3 --- RNA extraction and miRNA quantification --- p.91
Chapter 5.3 --- Results --- p.93
Chapter 5.3.1 --- A systematic search for placental miRNAs in maternal plasma using two-step QRT-PCR assays --- p.93
Chapter 5.3.2 --- Detection rate and clearance kinetics of placental miRNAs in maternal plasma --- p.97
Chapter 5.3.3 --- Effects of filtering maternal plasma on the concentration of placental miRNA and mRNA --- p.99
Chapter 5.3.5 --- Temporal profile of placental miRNA concentrations in maternal plasma across different trimesters of pregnancies --- p.103
Chapter 5.4 --- Discussion --- p.115
Chapter SECTION IV: --- DETECTION OF CELL-FREE DNA IN URINE --- p.119
Chapter CHAPTER 6: --- HEMATOPOIETIC STEM CELL TRANSPLANTATION RECIPIENTS AS A MODEL TO STUDY CELL-FREE DNA IN URINE --- p.120
Chapter 6.1 --- Introduction --- p.120
Chapter 6.2 --- Materials and methods --- p.123
Chapter 6.2.1 --- Sample collection --- p.123
Chapter 6.2.2 --- Experimental design --- p.124
Chapter 6.2.3 --- DNA extraction and quantification --- p.125
Chapter 6.3 --- Results --- p.128
Chapter 6.3.1 --- Validation of the zinc finger protein gene assay --- p.128
Chapter 6.3.2 --- Fractional concentration of male DNA in blood cells and plasma of sex-mismatched HSCT patients --- p.129
Chapter 6.3.3 --- Fractional concentration of male DNA in the urine and the urine cell pellets of sex-mismatched HSCT patients --- p.131
Chapter 6.3.4 --- Size distribution of cell-free DNA in peripheral blood and urine samples of sex-mismatched HSCT patients --- p.132
Amplicon size --- p.138
Chapter 6.4 --- Discussion --- p.143
Chapter SECTION V: --- CONCLUDING REMARKS --- p.147
Chapter CHAPTER 7: --- CONCLUSION AND FUTURE PERSPECTIVES --- p.148
Chapter 7.1 --- Circulating miRNA is a valuable resource for molecular analysis --- p.148
Chapter 7.2 --- The presence of donor-derived DNA in the urine of HSCT recipients --- p.150
Chapter 7.3 --- Prospects for future work --- p.152
APPENDIX 1 --- p.154
REFERENCES --- p.158

The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility. Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples. Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use. The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating. To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use. ‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up. Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.

A simple, sensitive and reliable high-performance liquid chromatographic procedure has been developed for the determination of erythromycin in human serum and urine using amperometric detection. A solid-phase extraction procedure was used followed by chromatography on a reverse-phase column. The mean recovery of erythromycin from serum and urine was 80%. This method allows both erythromycin and its principle degradation product, anhydroeythromycin, to be determined during a period of sample storage at 4 degree C and minus 15 degree C. The method is sufficiently sensitive and precise and is thus highly suited for use in both pharmacokinetic and stability studies.

碩士
國立成功大學
環境醫學研究所
93
Biological fluids, such as serum, urine, cerebrospinal fluid (CSF), and lavage, are characteristics of different organ systems and body cavities and can be used for pathology monitoring. Especially, the proteins in body fluids are not only used for diagnosis but also contain important information related to pathology pathways. Proteomics refers to the systematic investigation of the proteins in a cell culture or a tissue, that is, the proteome, by separation, quantification, and identification of the complicated protein mixture. Today, the most popular analytical method for proteomic study is the combination of two-dimensional gel electrophoresis (2D-GE) and mass spectrometry (MS) protein identification (ID). However, 2D-GE process is time-consuming and highly labor-intensive. Alternative approaches that eliminating 2D-GE steps in proteomic study are desired and under intensive investigation. Recently, liquid chromatography tandem mass spectrometry (LC-MS/MS) has been used for proteome identification without tedious 2D-GE. We foresee that LC-MS/MS will be very useful to analyze biological fluids for the search of biomarkers related to various diseases. This study design and setup an LC-MS/MS-based system for proteomic analysis of biological fluids. This system can be used for the identification and quantification of proteomes in various biological fluids for biomarker discovery. The system should shorten the analysis time and reduce experimentation variations by automation. This analytical system has been used to study the changes in the proteome of the bronchoalveolar lavage fluid (BALF) of rats exposed directly to a fuming oil-releasing environment in a metal processing factory. The results revealed that 29 proteins exhibited significant changes after exposure. These proteins included surfactant-associated proteins (SP-A and SP-D), inflammatory proteins (complement component 3, immunoglobulins, lysozyme, etc.), growth factors (e.g. transforming growth factor alpha), calcium-binding proteins (calcyclin, calgranulin A, calreticulin, and calvasculin), and other proteins (e.g., cathepsin D, saposin, and intestinal trefoil factor). A large decrease in protein levels of SP-A and SP-D (0.24- and 0.38-fold, respectively) following exposure was observed. In contrast, protein levels of transforming growth factor alpha and calcium-binding proteins were significantly increased (4.46- and 1.4-1.8-fold, respectively). Due to the diverse functions of these proteins, the results might contribute to understand the mechanisms involved in lung disorders induced by oil mist exposure.