Дисертації з теми "Humour Vitrous"

The vitreous humour is the substance that fills the posterior chamber. The vitreous body ensures the adherence of the retina to the underlying layers and acts as a barrier for heat and drug transport between the anterior and the posterior segments of the eye. In the medical literature many authors have postulated the existence of a connection between the fluid dynamics within the vitreous chamber during eye movements (especially when the substance filling the chamber is a liquid) and the occurrence of the retinal detachment. In the present PhD Thesis the vitreous humour dynamics is studied by means of both an experimental and a numerical point of view. The experiments consist in the measurement of the velocity field on the equatorial plane of a magnified model of the vitreous chamber using PIV techniques. The vitreous chamber has been modeled by means of a cavity carved in a rigid Perspex container. Two different geometries were used: a spherical geometry and a deformed geometry, which closely resembles that of the real eye. The model is filled with Glycerol, a highly viscous Newtonian fluid, and it is mounted on the shaft of a computer controlled motor, which rotates according to a generic time law. Visualizations of the fully three-dimensional flow show that the primary flow occurs on planes perpendicular to the axis of rotation. Secondary flows can be detected, the magnitude of which is however three or four orders smaller than the primary flow. In the case of spherical geometry, theoretical results, based on a simplified solution, are shown to be in very good agreement with the experimental findings. The maximum value of the shear stress at the wall does not significantly depend on the amplitude of saccadic movements but is strongly influenced by the vitreous viscosity. Velocity fields are found to be strongly influenced by the deformed geometry of the domain. When using the deformed model the formation of a vortex in the vicinity of the lens is invariably observed. The path described by this vortex during a period of oscillation is found to depend on the Womersley number of the flow. The second part of the Thesis is devoted to the formulation of a numerical model which provides a direct numerical simulation of the flow of a viscous fluid inside an oscillating sphere. The problem is formulated in the primitive velocity-pressure form. Replacing the continuity equation by the Poisson equation for the pressure supported by an independent condition of integral character an uncoupled formulation of the problem is derived. The equations are discretized in time by means of a non-fractional-step scheme. A spectral technique is used to transform the original problem in a sequence of ordinary differential problems for the coefficients of a spherical harmonics expansion of the variables. Spectral techniques have been tested on a simplified problem providing encouraging results.
L’umor vitreo `e la sostanza che riempie la camera posteriore dell’occhio. Esso assicura l’aderenza della retina agli strati sottostanti e costituisce una barriera per il trasporto di calore o sostanze tra la camera anteriore e la camera posteriore dell’occhio. Molti autori nella letteratura medica hanno ipotizzato una connessione tra la dinamica del vitreo durante i movimenti oculari (specialmente nei casi in cui la sostanza che riempie la camera `e un liquido) e l’insorgenza del distacco retinico. Nella presente Tesi di Dottorato lo studio della dinamica del vitreo oculare `e stato affrontato secondo un approccio sperimentale e numerico. Gli esperimenti hanno riguardato la misura dei campi di moto che si realizzano sul piano equatoriale di un modello in scala amplificata della camera vitrale attraverso l’utilizzo della tecnica PIV. La camera vitreale `e stata modellata per mezzo di una cavit`a ricavata in un contenitore rigido di perspex. I contenitori usati hanno due diverse geometrie: una sferica e una sferica deformata che riproduce verosimilmente la reale conformazione della camera. Il modello `e stato riempito con glicerina, un fluido newtoniano ad elevata viscosit`a, e montato sull’albero di un motore che `e stato fatto ruotare secondo una generica legge di tipo. Attraverso visualizzazioni del moto tridimensionale si `e osservato come il moto principale si sviluppi su piani ortogonali all’asse di rotazione. Sono stati anche osservati moti secondari la cui intensit`a risulta essere di tre-quattro ordini di grandezza inferiori al moto principale. Nel caso di modello sferico i risultati sperimentali sono stati confrontati con una teoria semplificata mostrando un buon accordo con quest’ultima. Il massimo valore della tensione tangenziale`e risultato non dipendere in modo significativo dall’ampiezza del movimento ma di essere influenzato dalla viscosit`a del fluido interno alla camera. I campi di moto sono fortemente influenzati dalla geometria del dominio. Usando il contenitore deformato in tutti gli esperimenti `e stata osservata la formazione di un vortice. Il percorso descritto dal vortice durante un periodo di oscillazione `e risultato dipendere dal numero di Womersley del moto. La seconda parte della Tesi `e dedicata alla formulazione di un modello numerico consistente in una simulazione numerica diretta del moto di un fluido viscoso all’interno di una sfera oscillante. Il problema `e formulato nelle variabili primitive pressione-velocit`a. Sostituendo l’equazione di continuit`a con l’equazione di Poisson per la pressione, associata ad una condizione al contorno indipendente di tipo integrale, si `e ricavata una formulazione disaccoppiata dl problema. Le equazioni sono state discretizzate nel tempo per mezzo di uno schema non frazionario. Il problema originale `e stato trasformato attraverso una tecnica spettrale in una sequenza di problemi alle derivate ordinarie per i coefficienti di espansione in armoniche sferiche delle variabili del problema. Le tecniche spettrali sono state testate per la soluzione di un problema semplificato fornendo risultati confortanti.

Ce travail avait pour objectif d'étudier l'intérêt et les limites de l'analyse de deux matrices alternatives que sont la bile et l'humeur vitrée (HV), en toxicologie médicolégale. Pour chacune des deux matrices, une revue de la littérature visait à investiguer les connaissances utiles à leur application en toxicologie médicolégale. Une place importante de ces revues est réservée à l'anatomie et la physiologie de l'HV et du système biliaire ainsi qu'aux mécanismes de distribution des xénobiotiques dans ces matrices. La partie expérimentale décrit trois études: deux menées sur populations autopsiques et une associant expérimentations animales et études de populations autopsiques. Les deux premières ont permis de proposer des outils statistiques d'interprétation des concentrations de méprobamate mesurées dans ces matrices. Ils peuvent être utilisés dans diverses situations à la place ou en complément de l'interprétation des concentrations sanguines: cadavre exsangue, putréfaction avancée du corps, redistribution post mortem des xénobiotiques… La troisième étude concernait six molécules (diazépam, citalopram, cyamémazine, morphine, caféine et méprobamate). Les molécules détectées dans le sang l'étaient systématiquement dans l'HV et la bile aussi bien dans les prélèvements des populations autopsiques que ceux issus des expérimentations animales. Les concentrations vitréennes chez l'animal et chez l'homme étaient systématiquement corrélées aux concentrations sanguines, exceptées celles de cyamémazine et de citalopram chez l'homme. Pour la bile, une corrélation significative était observée pour le méprobamate et la caféine chez l'homme et l'animal. Il ressort de ces résultats, que l'analyse de l'HV et de la bile permettent de disposer d'informations relatives à la nature des molécules absorbées et à leur rôle dans la survenue du décès
The present study sought to assess the interest and limitations of analyzing two alternative matrices, bile and vitreous humor (VH), in forensic toxicology. For each matrix, a literature review established the state of knowledge relating to their forensic application. The review placed special focus on the anatomy and physiology of VH and the biliary system and the mechanisms of xenobiotic distribution within the specific matrix. The experimental sections describe three studies: two performed on autopsy populations, and one associating autopsy populations to an animal model. The first two studies resulted in statistical tools for interpreting meprobamate concentrations in these matrices, which can be used as alternatives or complements to blood concentrations in various situations: exsanguination, advanced putrefaction, postmortem xenobiotic redistribution, etc. The third study focused on 6 molecules: diazepam, citalopram, cyamemazine, morphine, caffeine and meprobamate. Molecules detected in blood were also systematically detected in VH and bile samples from both the autopsy and animal populations. Animal VH and blood levels showed systematic correlation. In autopsy samples, cyamemazine and citalopram showed no such correlation. In bile, significant correlations with blood concentrations were found for meprobamate and caffeine in both the autopsy and animal populations. This study confirmed the interest of postmortem analysis of bile and VH. Results show that analyzing bile and VH sheds light on drugs intake and on their implication in cause of death

In this work, the dynamics of vitreous humour (VH) and of a range of pharmacological fluids used to replace VH in eye surgery were investigated, both experimentally and numerically. Vitreous humour is a gel-like viscoelastic material that fills the majority of the ocular globe. Age-related changes occur in the VH, and as a consequence VH becomes progressively liquefied with age, which has an impact on the corresponding rheological properties, and leads to the appearance of vitreoretinal pathologies. The most effective treatment for such diseases is the injection of a VH substitute in the vitreous cavity. Most of the vitreous substitutes commercially available are silicone oils (SiO) and perfluorocarbon liquids (PFLC), but so far none of them proven to be used as a permanent substitute. The results of the rheological characterisation of the VH and pharmacological fluids under shear and extensional flow conditions are presented in the first part of this thesis. Vitreous humour samples were obtained from New Zealand specimen rabbit eyes, whereas the pharmacological fluids were purchased from two different pharmaceutical companies. Two distinct VH phases were analysed: a liquid and a gel phase. The average surface tension measured for the VH liquid phase was 47.8 mN/m, at T = 21 ± 2 °C. Steady shear experiments demonstrated that VH liquid phase has a pronounced shear-thinning behaviour. Small amplitude oscillatory shear (SAOS) experiments showed that both gel and liquid VH phases present a solid-like behaviour, which is more dominant in the gel phase, and these results were corroborated by creep experiments. Experiments with the VH liquid phase under extensional flow yielded an average relaxation time of λ = 9.7 ms, at T ≈ 37 °C. None of the pharmacological fluids tested showed a rheological behaviour close to the VH. The PFLCs and SiO-based fluids exhibit a Newtonian behaviour, with the exception of Siluron 2000 fluid, which under extensional flow presents a relaxation time of λ = 6.8 ms, at T ≈ 20 °C. The PFLCs have viscosities close to the viscosity of water, whereas the SiO-based fluids present viscosities between 0.73 and 4.57 Pa s, at T = 37 °C. Surface tension experiments showed that the PFLCs present lower values than the SiO-based fluids, and that the surface tension of the VH liquid phase is more than double of that measured for each of the pharmacological fluids tested. In contact with VH liquid phase from rabbit eyes, all the pharmacological fluids tested yielded interfacial tensions around 30 mN/m. The dynamic response of VH during saccadic movements was studied numerically using the opensource software OpenFOAM®. A comparative study between the VH and pharmacological fluids was performed for a saccadic movement with an amplitude of 40°. The numerical results showed that the VH liquid and gel phases present distinct behaviour. The shear stresses at the walls for the VH gel phase are higher than those produced by the VH liquid phase. This shows that the phase balance in VH significantly affects the flow dynamics of the biofluid. The numerical results also showed that velocity and wall shear stress (WSS) profiles are significantly different for VH and pharmacological fluids, revealing that the latter are unable to mimic accurately the VH flow behaviour in conditions such as those experienced during saccadic eye movements. Finally, different degrees of saccadic movements were studied and the numerical results showed that, for all the fluids investigated, the momentum diffusion across the vitreous cavity and the WSS increase with the increase of the saccadic movement amplitude. The new experimental results provided in this thesis, complemented by the dynamic behaviour computed numerically for saccadic eye movements, offer new insights for the improvement and development of new VH substitutes to be used in eye surgery, with particular focus on their mechanical functionality.

O peixe dourado é extensivamente utilizado em pesquisas sobre visão devido a diversos fatores: é um animal pecilotermo, o que possibilita manter sua retina ex-vivo viável para manipulação experimental por mais tempo; apresenta uma organização retiniana semelhante à de mamíferos; possui neurônios grandes, facilitando estudos eletrofisiológicos e morfológicos e, diferentemente de mamíferos, é dotado de neurogênese constante de alguns tipos celulares retinianos. A produção, composição e escoamento dos meios oculares nesta espécie, fundamentais para a formação de imagens sobre a retina, são, entretanto, pouco conhecidos. A variedade padrão desta espécie apresenta desenvolvimento ocular normal, enquanto a variedade demekin é extremamente míope por possuir crescimento ocular exagerado (buftalmia), condição característica de alguns tipos de glaucoma em mamíferos. O presente trabalho investigou aspectos morfofisiológicos do sistema de produção e drenagem do humor aquoso e a composição do humor vítreo nestas duas variedades de peixe dourado, com o objetivo de melhor compreender as causas subjacentes ao crescimento ocular do demekin. Para estudar a drenagem do humor aquoso, foram realizadas injeções de corante na câmara anterior de animais das duas variedades, com processamento histológico posterior em intervalos de tempo pré-determinados (5, 15, 30, 60, 120 minutos e 1, 2, 3 4 e 7 dias, n <= 3 olhos de cada variedade por grupo experimental). Não foram observadas diferenças na drenagem de humor aquoso entre as duas variedades. Em ambas, o humor aquoso é drenado principalmente pela malha trabecular contida na porção ventral do ângulo de drenagem, entre a íris e o ligamento anular. Duas vias secundárias de drenagem foram encontradas nas duas variedades de peixe dourado estudadas: (i) a via irídica e (ii) a drenagem através de vasos epirretinianos. Estas vias são aparentemente mais lentas do que a via trabecular, uma vez que foi encontrado corante nessas regiões até quatro dias após as injeções de corante na câmara anterior. Foi realizado também estudo ultraestrutural da retina e epitélio ciliar em ambas as variedades através de microscopia eletrônica de transmissão. O epitélio pigmentado da retina do demekin apresentou grânulos de melanina mais escassos, e observamos certa desorganização da camada de fotorreceptores, bem como redução da sua densidade. Através de aparato invasivo construído por Joselevitch e Smith (1997), foi mensurada a pressão intraocular (PIO) de 14 olhos de cada variedade. O demekin apresentou valores de PIO ligeiramente aumentados em relação ao animal padrão (3,6 ± 0,9 mmH2O para o demekin e 1,9 ± 0,6 mmH2O para o peixe dourado padrão). A composição proteica do corpo vítreo foi estudada através de eletroforese em gel de poliacrilamida utilizando um pool de humor vítreo de 4 animais da variedade padrão e 2 da variedade demekin. As proteínas de baixa massa molecular (entre 15 a 25 kDa) estavam expressas em ambas - embora com maior expressão na variedade padrão - e outras, de maior massa molecular, expressas apenas no humor vítreo do demekin (50-75 kDa e 150 kDa). Os resultados obtidos sugerem, portanto, que há diferenças importantes na ultraestrutura retiniana, na composição proteica do humor vítreo e na pressão intraocular do demekin. Para melhor compreensão do crescimento ocular desta variedade faz-se necessária a realização de um estudo proteômico, identificando quais são as proteínas do humor vítreo evidenciadas através da eletroforese, bem como estudos morfológicos em animais de diferentes tamanhos visando a quantificação dos danos retinianos decorrentes do seu crescimento ocular
The goldfish is extensively used in vision research due to several factors: it is a poikilothermic animal, which allows ex-vivo experiments using its retina for longer periods of time; its retinal organization is very similar to that of mammals; it has large neurons, which facilitates electrophysiological and morphological studies and, unlike mammals, it is endowed with constant neurogenesis of some retinal cell types. However, the production, composition and outflow of ocular media in this species, which are fundamental for image formation onto the retina, are poorly understood. The standard goldfish has normal eye development, while the Black Moor goldfish is extremely myopic due to excessive eye growth (buphthalmos), a condition that is present in certain types of mammalian glaucoma. The present study investigated morphophysiological aspects of the aqueous humor production and drainage system and vitreous humor composition in these two varieties of goldfish to better understand the causes underlying the eye growth of the Black Moor. To study the drainage of the aqueous humor, anterior chamber dye injections and histological processing were performed in animals of both varieties after predetermined time intervals (5, 15, 30, 60, 120 minutes and 1, 2, 3, 4, and 7 days, n <= 3 eyes of each variety for each experimental group). No differences were observed in the drainage of aqueous humor between the two varieties. In both, the aqueous humor is drained mainly by the trabecular meshwork contained in the ventral portion of the drainage angle, between the iris and the annular ligament. Two secondary drainage routes were found in the two goldfish varieties studied: (i) the iridic route and (ii) drainage through epiretinal vessels. These pathways are apparently slower than the trabecular route, since the dye was found in these regions 4 days after the injections. An ultrastructural study of the retina and ciliary epithelium was also carried out in both varieties through transmission electron microscopy. The retinal pigment epithelium of the Black Moor presented more scarce melanin granules and we observed some disorganization of the photoreceptor layer, as well as a reduction in its density. Through an invasive device constructed by Joselevitch and Smith (1997), the intraocular pressure (IOP) of 14 eyes of each variety was measured. The Black Moor presented slightly increased IOP values compared to the standard animal (3.6 ± 0.9 mmH2O for demekin and 1.9 ± 0.6 mmH2O for the standard goldfish). The protein composition of the vitreous body was studied by polyacrylamide gel electrophoresis using a vitreous humor pool of 4 animals of the standard variety and 2 of the Black Moor variety. Low molecular weight proteins (15 to 25 kDa) were expressed in both - although with greater expression in the standard variety - and others of higher molecular mass, expressed only in the vitreous humor of the Black Moor (50-75 kDa and 150 kDa). These results suggest, therefore, that there are important differences in the retinal ultrastructure, the protein composition of the vitreous humor and the intraocular pressure of the Black Moor. To better understand the ocular growth in this variety, it is necessary to carry out a proteomic study, identifying which are the vitreous humor proteins evidenced through electrophoresis, as well as morphological studies in animals of different sizes aiming the quantification of the retinal damages caused by its ocular growth

Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2004.
"A dissertation in pharmaceutical sciences and chemistry." Advisor: Ashim K. Mitra. Typescript. Vita. Description based on contents viewed Feb. 22, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 156-188). Online version of the print edition.

O humor vítreo (HV) é um gel aquoso, transparente e incolor, situado entre o cristalino e a retina. Ele pode ser uma ferramenta para determinação de drogas de abuso, sobretudo quando é impossível fazer a determinação em sangue devido à sua ausência ou sua deterioração, por exemplo em casos de exsanguinação, embalsamento e carbonização. As drogas e seus metabólitos passam para o HV por difusão passiva e, de modo geral, a concentração dos analitos no HV é similar às concentrações obtidas no sangue. A cocaína e a benzoilecgonina são facilmente detectadas na matriz. Por outro lado, a difusão da morfina é bem limitada. O maior interesse do estudo de opioides é a análise de 6-acetilmorfina, que pode diferenciar o uso de heroína e morfina. O delta-9-tetrahidrocanabinol é uma molécula muito polar e fortemente ligada a proteínas plasmáticas, o que limita a sua difusão para o HV. Entre as anfetaminas, a metilenodioximetanfetamina e a metanfetamina são as moléculas mais estudadas no HV. Foi desenvolvido e validado um método utilizando GC-MS para análise simultânea de cocaína, anfetaminas, opioides, canabinoides e respectivos metabólitos em HV. Os analitos de interesse foram extraídos do HV utilizando extração em fase sólida e analisadas por GC-MS, utilizando o modo de aquisição SIM. A faixa de linearidade foi de 10 a 1000 ng/mL para todos os analitos, com exceção do éster de metilanidroecgonina (10 a 750 ng/mL). A exatidão variou de 95,6 a 104,0%, a precisão inter-ensaio variou de 1,2 a 10,0% e a precisão intra-ensaio foi menor que 10,4% para todos os analitos. O limite de quantificação para todas as drogas foi de 10 ng/mL e a recuperação variou de 70,4 a 100,1% para compostos básicos e neutros, entretanto os compostos ácidos apresentaram baixa recuperação - menor que 40%. A dosagem de etanol foi realizada por GC-FID e extração por headspace. Os métodos validados foram aplicados em 250 amostras de HV coletadas de vítimas de morte violentas nos anos de 2011 e 2012 que foram necropsiadas no Departamento Médico Legal de Vitória - ES. A maioria das vítimas era do sexo masculino (85,4%) e a causa mais comum de morte foi homicídio (46,2%), destes, 89,5% foram mortos por disparo de arma de fogo. Os acidentes de trânsito corresponderam a 44,1%; suicídio, 2,4%; e outras mortes totalizaram 7,2% das amostras. Substâncias psicoativas (álcool e drogas) foram positivas em 60,4% dos casos. Em 23,2% das amostras foi quantificada cocaína e/ou seu metabólito, e em um terço destes foi identificado o uso de crack. O álcool estava presente em 19,2% dos casos e a associação entre cocaína e álcool em 12,8% dos casos. Outras drogas incluíram anfetaminas (13 casos) e codeína (1 caso). Quando comparadas as concentrações das drogas pesquisadas no sangue e HV, a anfetamina e metanfetamina mostraram boa correlação entre as duas matrizes. A 6-acetilmorfina encontrada no HV foi utilizada para demonstrar o uso de heroína, uma vez que as concentrações foram mais altas do que no sangue. Entretanto, o HV não pode ser utilizado como amostra alternativa para detecção de canabinoides.
Vitreous humor is the aqueous gel located between the lens and retina. Vitreous humor is a useful alternative postmortem matrix for the detection of drugs, particularly in death investigations where postmortem blood is not available or is of limited quality or quantity (e.g. after hemorrhagic shock, burns, embalming or decomposition processes). Drugs and their metabolites enter the vitreous humor by passive diffusion from blood across the blood-vitreous barrier. Vitreous humor concentrations are often similar to the drug concentrations in the circulation blood. Cocaine and benzoylecgonine are easily detected in this matrix. On the order hand, morphine diffusion is limited. The interest in opiates is due to 6-acetylmorphine, which is stable in vitreous humor and can confirm heroin abuse. Drugs that are highly protein-bound, such as THC, achieve lower vitreous humor concentrations as only the free fraction can cross the blood-vitreous barrier. Methamphetamine and methylenodioxymetamphetamine are also detected in vitreous humor. A GC-MS method for simultaneous analysis of cocaine, amphetamines, opiates, cannabinoids and its metabolites in vitreous humor was developed and fully validated. Vitreous humor samples were extracted using solid phase extraction and analyzed by GC-MS in SIM mode. For all analytes the linearity ranged from 10 to 1000 ng/mL, excepted for anydroecgonine methylester which ranged from 10 to 750 ng/mL. Inter-assay imprecision ranged from 1.2 to 10.0% and intra-assay imprecision was less than 10.4% for all analytes at all QC concentrations. Accuracy ranged from 95.6 to 104.0% and recoveries ranging from 70.4 - 100.1% for basic and neutral compounds, the acids compounds had poor recovery (less than 40%). The limits of detection were up to 1.0 ng/mL. Ethanol was quantified by headspace extraction and GC-FID. The validated methodology was applied to 250 vitreous humor samples collected from violent death victims between 2011 and 2012 in the Departamento Médico Legal de Vitória - ES. Most of the victims were male (85.4%) and the most common cause of death was homicide (46.2%), in which 89.5 occurred by firearm shot. Traffic accident represented 44.1%, suicide 2.4% and other deaths 7.2%. Psychoactive substances (alcohol and?or drugs of abuse) were positive in 60.4% of the cases. Cocaine was quantified in 23.2% of the samples and one third was positive for crack cocaine. Ethanol was present in 19.2% of the cases and the association between cocaine and alcohol in 12.8%. Other drugs included amphetamines (13 cases) and codeine (1 case). When comparing the drug concentrations in blood and vitreous humor, amphetamine and methamphetamine showed a good correlation. 6-acetylmorphine in vitreous humor can demonstrate heroine abuse and its concentration is higher in vitreous humor than in blood. However, vitreous humor is not a good matrix for the detection of cannabinoids.

Orientadores: Nelci Fenalti Höehr, Marcos Nogueira Eberlin
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
Made available in DSpace on 2018-08-26T10:40:41Z (GMT). No. of bitstreams: 1 SantosJunior_JulioCesar_M.pdf: 4092105 bytes, checksum: 8809a688f7411f1a87fd956c6674fe41 (MD5) Previous issue date: 2014
Resumo: Embora as anfetaminas estejam proibidas no Brasil, elas continuam sendo adquiridas ilegalmente assim como os canabinóides e cocaína, que representam um dos principais problemas de saúde pública a serem enfrentados no nosso país. Um dos grandes desafios desta área é a dificuldade de obtenção de material para exames periciais. O humor vítreo por encontrar-se isolado em um compartimento relativamente protegido de contaminação externa, invasão de microorganismos e traumatismos em geral, bem como por sua simplicidade/estabilidade analítica e esterilidade durante um longo período após a morte constitui-se como uma excelente amostra para a determinação de xenobióticos em corpos politraumatizados, carbonizados ou em decomposição, auxiliando na delegação da causa mortis. O uso da espectrometria de massas e o advento de novas metodologias de ionização são ferramentas essenciais à toxicologia forense, a V-EASI (venture easy ambient sonic-spray), é uma fonte de ionização de fácil aplicação e instalação, que não requer fluxo de eluente e os demais fatores utilizados nas fontes comerciais. Além disso, o uso da espectrometria de massas de ressonância ciclotrônica de íons por transformada de Fourier (FT-ICR-MS) de ultra-alta resolução e exatidão (valores de m/z exatos) leva a exata composição molecular, alcançando erros abaixo de 1 ppm (partes-por-milhão). Perante isso a avaliação da fórmula molecular normalmente é inequívoca. Quando acoplada a ionização por eletrospray (ESI) espécies moleculares suaves são formadas reduzindo a complexidade do espectro e produzindo informação composicional livre de fragmentos em misturas complexas facilitando sua compreensão. Portanto este trabalho visa o desenvolvimento de metodologias analíticas para análise de drogas de abuso presentes em humor vítreo, empregando o uso de técnicas modernas de espectrometria de massas (FT-ICR-MS e V-EASI-MS)
Abstract: Although amphetamines are banned in Brazil, they remain illegally acquired as cannabinoids and cocaine, which account for a public health task to be faced in this country. The major challenge is to obtain material for investigation exams. The vitreous humor constitutes a good alternative for these exams, since it occurs isolated in a protected space, free of external contamination and of microorganisms and traumatisms, and also due to its analytical stability and sterility preserving it for a long period after death. Moreover, the vitreous humor constitutes an excellent sample for the determination of xenobiotics even in polytraumatized bodies, carbonized or in decomposition, involved in the causa mortis. The use of mass spectrometry and the advent of new ionization methods are essential tools for forensic toxicology, the V-EASI (venture easy ambient sonic-spray), is a source of ionization easy to use and install, not requiring nitrogen flow, eluent flow and other factors used in commercial sources. Furthermore, the use of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) provides ultra high resolution and accuracy in mass analysis and its accurate m/z values lead to the exact molecular composition, reaching errors below 1 ppm (parts-per-million) on normal operational conditions, the assigned molecular formula are normally unequivocal. When electrospray ionization (ESI) is used, soft molecular species are formed reducing spectra complexity and providing fragment-free compositional information about complex mixtures facilitating comprehension. Therefore, this work aims at the development of analytical methodologies for the analysis of drugs of abuse present in the vitreous humor, employing the use of modern techniques of mass spectrometry (FT-ICR-MS and V-EASI-MS
Mestrado
Ciencias Biomedicas
Mestre em Ciências Médicas

Os metilenodioxi derivados estão entre as drogas sintéticas de uso ilícito mais consumidas no ocidente, são substâncias que causam alterações motoras e psíquicas e tem causado grande ameaça a segurança no trânsito, pois estes efeitos afetam o desempenho dos condutores, podendo causar acidentes de trânsito. O humor vítreo é uma matriz biológica disponível apenas nas análises postmortem, e é de grande relevância para toxicologia forense. Constitui uma matriz simples, de fácil coleta e manuseio, boa estabilidade, muito indicada principalmente nos casos de corpos politraumatizados, parcialmente carbonizados ou em estado de decomposição. Isto porque o humor vítreo localiza-se em uma área anatomicamente isolada, relativamente protegida contra a contaminação externa e invasão de microorganismos. O objetivo do presente trabalho foi desenvolver e validar uma metodologia analítica para detectar e quantificar metilenodioxi derivados em amostras de humor vítreo em vítimas fatais de acidentes de trânsito. Para desenvolvimento do método foi utilizado 1 mL de humor vítreo, extração líquido-líquido com solvente acetato de etila, derivatização com anidrido heptafluorobutírico (HFBA) e detecção por cromatografia gasosa acoplada a espectrometria de massas (CG/MS). A metologia foi validada de acordo com os parâmetros preconizados pela ANVISA e demonstrou linearidade de 10 a 400 ng/mL. Os limites de detecção variaram entre 1 a 2,5 ng/mL, a exatidão variou entre 97,1% -103,7%, na precisão intra-ensaio foram obtidos valores entre 4,54%-9,14% e na precisão inter- ensaio, valores entre 6,92%-10,59%. A recuperação encontrada foi superior a 57,87%. A metologia validada foi aplicada em humor vítreo de vítimas fatais de acidentes de trânsito coletadas no Centro de Medicina Legal de Ribeirão Preto (CEMEL).
The methylenedioxy derivatives are among synthetic drugs of illicit use most consumed in the occident, these are substances cause mental and motor disorders and has caused major threat to traffic safety, these effects affect the performance of drivers and may lead to traffic accidents. The vitreous humor is a biological matrix available in postmortem analysis, and has great relevance to forensic toxicology. It is a simple matrix, clean, easy collection and handling, good stability, highly recommended especially in cases of body injuries, partially carbonization or in a state of decomposition. This is because the vitreous humor is located in an anatomically isolated area, relatively protected from external contamination and invasion of microorganisms. The purpose of this study was to develop and validate an analytical methodology to detect and quantify methylenedioxy derivatives in samples of vitreous humor in fatal traffic accidents. To develop the method we used 1 mL of vitreous humor, liquid-liquid extraction with ethyl acetate solvent, derivatization heptafluorobutyric anhydride (HFBA) and detection by gas chromatography-mass spectrometry (GC/MS). The methodology was validated according to the parameters established by ANVISA and demonstrated linearity from 10 to 400 ng /mL. The limits of detection were between 1,0 ng/mL and 2,5 ng / mL. The accuracy had values between 97.1% -103.7%, the intra-assay precision was 4.54% -9.14% and inter assay precision between 6.92% -10.59%. The recovery was higher than 57.87%. The methodology validated was applied in vitreous humor of victims of fatal traffic accidents collected in the Centre of Forensic Medicine of Ribeirão Preto (CEMEL)

O uso abusivo de substâncias psicoativas cresce a cada dia em diferentes segmentos da sociedade mundialmente. Aumentos significativos no número de ocorrências de óbito com envolvimento de tais substâncias têm sido reportados nas últimas décadas. A classe dos opiáceos está figurada entre as substâncias de maior prevalência nesse contexto. Em toxicologia forense, análises toxicológicas conduzidas em amostras postmortem podem auxiliar se substâncias químicas tiveram influência no óbito. A realização dessas análises e interpretação dos resultados obtidos nesses casos é bastante complexa devido à deterioração sofrida pelos cadáveres, e também pela ocorrência de um fenômeno denominado redistribuição postmortem, responsável pela transferência de substâncias após a morte a favor de gradiente de concentração. Em geral, as substâncias são transferidas de órgãos como fígado, coração, pulmões, e trato gastrointestinal, para locais de menor concentração, afetando principalmente o sangue da região central e órgãos adjacentes. O humor vítreo, apesar de considerado um espécime não-convencional, pode ser bastante útil, principalmente em casos onde não há amostras sanguíneas disponíveis para coleta. Esse espécime se apresenta como uma matriz menos propensa à decomposição bacteriana, além de ser menos afetado pela redistribuição postmortem por sua localização mais afastada dos sítios centrais. Desta forma, um método para quantificação de opiáceos (morfina livre e total, codeína e 6-acetilmorfina) em sangue (cardíaco e periférico) e em humor vítreo humanos coletados postmortem foi desenvolvido e validado. O método mostrou ser preciso, eficiente e sensível, com limite de quantificação de 10 ng/ml. Amostras de 7 casos postmortem com envolvimento de opiáceos foram analisadas com o intuito de verificar correlação nas concentrações entre os sítios, e possível ocorrência do fenômeno de redistribuição.
The abuse of psychoactive substances grows every day worldwide in different segments of the society. Significant increases in the number of drug-related deaths have been reported in recent decades. The class of opiates is one of the most prevalent substances in this context. In forensic toxicology, toxicological analyses are performed in postmortem samples to evaluate whether chemical substances were involved in the death. The completion of this analysis and interpretation of results are very complex due to the deterioration suffered by the corpses, and also by the occurrence of the phenomenon called postmortem redistribution, responsible for the transfer of substances along a concentration gradient after death. In general, the transference of the substances occurred from organs such as liver, heart, lungs and gastrointestinal tract to sites of low concentrations, mainly affecting the blood from central sites and adjacent organs. By this reason, the collection of blood from two different sites of the body for comparison is highly recommendable. Despite being considered a non-conventional specimen, vitreous humor can be quite useful, especially in cases where no blood samples are available for collection. This specimen is usually less prone to bacterial decomposition, and it\'s also less affected by postmortem redistribution due to its location further away from central sites. Thus, a method to quantify opiates (free and total morphine, codeine and 6- monoacetylmorphine) in postmortem blood (cardiac and peripheral) and in vitreous humor samples was developed and validated. The method showed good accuracy, efficiency and sensibility, with limit of quantification of the 10 ng/ml. Seven samples of postmortem cases with opiates involvement were analyzed to verify the correlation in the concentrations of the sites, and possible occurrence of the redistribution phenomenon.

Post-mortem blood drug concentrations vary greatly and as a consequence of post-mortem change and redistribution may not reflect the concentration at the time of death. Tissues that are located away from central drug reservoirs and that lack esterase activity, e.g. muscle and vitreous humour (VH), have the potential to provide more reliable post-mortem toxicological specimens. In the absence of a blood sample the toxicologist may have to rely on such tissues yet few studies have been undertaken to examine the relationship between drugs in blood and less conventional tissues at the time of death. The purpose of this study was to investigate the distribution of opiates (heroin specific compounds) and cocaine and their respective metabolites in VH and muscle with a view to elucidating the interpretive value of these tissues. Analytical methods were developed and validated to measure drug concentrations in blood, VH and muscle, including throughout the rectus femoris thigh muscle, in cases of drug related death. To assist with interpretation of drug concentrations measured in post-mortem tissues the in vitro stability of cocaine and 6-acetylmorphine (6AM) was examined during the putrefactive process and under different storage conditions. Relationships between blood and tissue drug concentrations were assessed in relation to case circumstances with particular focus on the approximation of survival time. In contrast to a report previously published in the literature, this study found the concentration of cocaine, and its metabolites, benzoylecgonine (BZE) and cocaethylene (COET), to be uniformly distributed throughout the thigh muscle (n = 7). Concentrations of cocaine in muscle were markedly higher than in blood and correlated well with the blood. The stability of cocaine in muscle tissue was found to greatly exceed that in blood and VH. These preliminary results also indicated that the cocaine to BZE ratio measured in both muscle and VH may be of value in the assessment of survival time. These findings promote the use of muscle as a toxicological specimen for cocaine determinations. Further work is required to validate these findings and to examine the distribution of opiates in muscle, which could not be assessed in this study. The relationship between femoral blood and vitreous humour morphine concentration (n = 70) was found to be dependent on survival time and possibly influenced by accumulation of morphine in the VH. These findings demonstrate that the concentration of morphine in blood cannot be inferred from that measured in the VH. The VH provided a useful adjunct to interpretation owing to the prolonged detection of 6AM in this matrix. The addition of 1.5% sodium fluoride to VH was found to be essential for 6AM stability during storage. The utility of rapidly metabolised heroin specific compounds in blood as indicators of survival period following heroin intake and the role of concomitant drug consumption in heroin fatalities was also discussed in this thesis.

Os antidepressivos pertencem a uma importante classe de medicamentos investigados na toxicologia forense. Em casos de amostras provenientes de cadáveres, o intervalo entre o óbito e a obtenção da espécie biológica pode proporcionar a redistribuição postmortem destes fármacos. Com o objetivo de elucidar esse fenômeno, métodos analíticos foram desenvolvidos e aplicados utilizando sangue total (ST), humor vítreo (HV) e fígado. Para as amostras de ST e HV, o método de extração escolhido e validado foi a microextração em fase líquida (LPME) trifásica. Fibras ocas constituídas de polipropileno, com a extensão de 8 cm cada, foram tratadas com o solvente orgânico dodecano (fase orgânica), resultando em um membrana com permeabilidade seletiva. No lúmen destas fibras, adicionou-se ácido fórmico 0,1 mol/L (fase aceptora). Em frasco de fundo chato com 5 mL de capacidade, pipetou-se 3,5 mL de NaOH 0,1 mol/L (fase doadora) e 0,5 mL de ST ou HV. Ao término da extração, as amostras foram introduzidas no GC-MS, sem a necessidade de reações de derivatização. O estudo com ST contemplou os antidepressivos amitriptilina (AMI), nortriptilina (NTR), imipramina (IMI), desipramine (DES), clomipramina (CLO), desmetilclomipramina (DMC), fluoxetina (FLU) e norfluoxetina (NFL). Os limites de quantificação para estas substâncias ficaram inferiores aos níveis terapêuticos (20 ng/mL). As médias dos coeficientes de variação intradia e interdia foram, respectivamente, de 9,7 e 9,8%. As curvas de calibração apresentaram linearidade entre as concentrações de 20 até 1200 ng/mL. A validação do parâmetro integridade da diluição assegurou a mensuração de quantidades superiores ao limite apresentado na curva de calibração. O método foi aplicado em sete amostras reais postmortem e em apenas um caso foi observada uma diferença significativa (300%) entre os valores quantificados no ST periférico e central. Os antidepressivos tricíclicos AMI, NTR, IMI e DES foram avaliados no HV e o efeito matriz foi detectado para os dois últimos analitos. O método foi otimizado e validado utilizando solução salina adicionada de AMI e NTR. O limite de detecção igual a 5 ng/mL, foi obtido com a redução da voltagem da fonte de íons do espectrômetro de massa para 50 eV. Coeficientes de variação foram inferiores a 15%. Os procedimentos validados foram aplicados em seis amostras reais de HV. A relação encontrada entre os valores obtidos no ST periférico e HV foi de aproximadamente 0,1. A extração acelerada por solvente (ASE) e, posteriormente, a extração em fase sólida (SPE) foram as técnicas de separação dos analitos da matriz fígado. Ao término das citadas extrações, os antidepressivos foram analisados no GC-MS. Para esta matriz sólida, são necessários mais estudos, pois os valores encontrados nos ensaios analíticos estão em desacordo com as diretrizes utilizadas na validação dos métodos.
Antidepressants belong to an important class of drugs investigated in forensic toxicology. In cases of samples from corpses, the interval between death and obtaining the biological specimens can provide the postmortem redistribution of these drugs. Aiming to elucidate this phenomenon, analytical methods were developed and applied using whole blood (WB), vitreous humor (VH) and liver. For samples of WB and HV, the extraction method chosen and validated was the three-phase liquid phase microextraction (LPME). Hollow fibers consist of polypropylene, with a length of 8 cm each were treated with dodecane organic solvent (organic phase) resulting in a membrane with selective permeability. Into the lumen of these fibers was added formic acid 0.1 mol/ L (acceptor phase). In the vial containing 3.5 mL of NaOH 0.1 mol / L (donor phase) was spiked 0.5 ml of biological fluids (WB or VH). Subsequently, the samples were injected in GC-MS without derivatization reactions. The study of the ST included antidepressants amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI), desipramine (DES), clomipramine (CLO), desmethylclomipramine (DMC), fluoxetine (FLU) and norfluoxetine (NFL). The quantification limits for these substances were below the therapeutic levels (20 ng / ml). The mean coefficients of variation and separate intradays were respectively 9.7 and 9.8%. The calibration curves showed linearity between concentrations of 20 to 1200 ng / mL. The validation of the integrity of the dilution parameter assured measurement higher than the limit shown in the calibration curve quantities. The method was applied to seven real postmortem samples and in one case a significant difference (300%) between the measured values in the peripheral and central ST was observed. The tricyclic antidepressants AMI, NTR, IMI and DES were evaluated in VH and the matrix effect was detected in the last two analytes. The method was optimized and validated using saline spiked AMI and NTR. The limit of detection (5 ng/ml) was obtained by reducing the voltage of the ion source of the mass spectrometer 50 eV. Coefficients of variation were below 15%. The procedures were validated in six real samples of HV. The relationship found between the values obtained in the peripheral ST and HV was approximately 0.1. Accelerated solvent extraction (ASE) and subsequently the solid phase extraction (SPE) were the techniques of separation of analytes liver matrix. At the end of the cited extractions, antidepressants were analyzed in GC-MS. To this solid tissue, further studies are needed, because the values found in the analytical tests were not in accordance with the guidelines used in the validation of the methods.

Glaucoma is a progressive optic neuropathy that leads to irreversible blindness. From a molecular point of view it is possible to define glaucoma as a syndrome that develops pro-apoptotic signals towards the optic nerve head. In the pathogenesis of glaucoma a central role is played by the trabecular meshwork (TM) that, together with the juxtacanalicular connective tissue and the inner wall of the Schlemm’s canal, forms the conventional way of outflow of the aqueous humor. Although, the etiology of the disease is partly still unknown, the main features are an elevated intraocular pressure (IOP) related to an alteration of the trabecular outflow resistance, ocular vascular alteration, extracellular matrix (ECM) changes. There are many other conditions that contribute to the development of glaucomatous disease, such as oxidative stress and cellular responses to damage that lead to autophagy or senescence [1–5]. Up-to date the IOP is the only target of therapy, without counteracting blindness. Unfortunately, actually there is not reliable experimental model for analyzing and identifying crucial factors for prevention and therapy towards this multifactorial disease [6–8]. Taking account of those issues, it is crucial to develop in vitro human-based model. During my PhD research activity of these three years, I set up three different in vitro TM models, to check which of those could better mimic the glaucoma onset: 2D conventional and 3D static and innovative biodynamic models. Since in glaucoma TM seems to be the main ocular tissue that is affected by oxidative stress, as first approach, all TM models were subjected to prolonged oxidative stress and at each selected check point I analyzed some markers related to cellular responses to damage. Afterwards, has been added a pressure modulator to 3D dynamic chamber, to examine the role of increased pressure, to better mimic glaucoma onset. Lastly, I evaluated the performance of our dynamic 3D-HTMC model as platform to test the effects of therapeutic compounds for glaucoma disease. I analyzed the effect of iTRAB ®, a concentrate mixture of polyphenols ≥ 2.5%. Preliminary experiments were performed by the analysis of collagens, ECM glycoproteins and regulators, as well as several cytokines by qPCR. The results are reported as two independent sets: 1) response to OS plus iTRAB ®, and 2) response to increased pressure flow plus iTRAB®. The selected genes were found to be differentially expressed in response to the OS and increased pressure flow. However, under both experimental conditions, the administering of the PM down-regulated the expression of crucial genes involved in TM dysfunction, confirming the potential usefulness of our model References: 1. Saccà SC, Gandolfi S, Bagnis A, Manni G, Damonte G, Traverso CE, et al. From DNA damage to functional changes of the trabecular meshwork in aging and glaucoma. Ageing Res Rev. 2016;29: 26–41. doi:10.1016/j.arr.2016.05.012 2. Izzotti A, La Maestra S, Micale RT, Longobardi MG, Saccà SC. Genomic and post-genomic effects of anti-glaucoma drugs preservatives in trabecular meshwork. Mutat Res. 2015;772: 1–9. doi:10.1016/j.mrfmmm.2014.11.006 3. Quigley HA. Number of people with glaucoma worldwide. Br J Ophthalmol. 1996;80: 389–393. doi:10.1136/bjo.80.5.389 4. Acott TS, Kelley MJ. Extracellular matrix in the trabecular meshwork. Exp Eye Res. 2008;86: 543–561. doi:10.1016/j.exer.2008.01.013 5. Awai-Kasaoka N, Inoue T, Kameda T, Fujimoto T, Inoue-Mochita M, Tanihara H. Oxidative stress response signaling pathways in trabecular meshwork cells and their effects on cell viability. Mol Vis. 2013;19: 1332–1340. 6. A. Bouhenni R, Dunmire J, Sewell A, Edward DP. Animal Models of Glaucoma. J Biomed Biotechnol. 2012;2012. doi:10.1155/2012/692609 7. Johnson TV, Tomarev SI. Rodent models of glaucoma. Brain Res Bull. 2010;81: 349–358. doi:10.1016/j.brainresbull.2009.04.004 8. Aires ID, Ambrósio AF, Santiago AR. Modeling Human Glaucoma: Lessons from the in vitro Models. Ophthalmic Res. 2017;57: 77–86. doi:10.1159/000448480

Over the past few years, there has been an intensification in onset of ocular diseases among worldwide population. The incidence of eye pathologies upsurges with advancing age, leading to a decline of normal eye function and even to blindness. Ocular diseases are frequently triggered by chronic disorders, such as diabetes or hypertension, or by alterations in retinal positioning, which occurs in retinal detachment (RD). Recent studies indicates that human vitreous humor (VH) suffers proteome alterations according to the actual physiological and pathological state of the retina. However, there are few published articles regarding RD proteome. Hence, this study is focused in the proteomic analysis of VH samples in RD and posterior identification of the present proteins in these samples, as well as to understand in what way they are connected and how they act. In this way, we can get access to the VH proteome in RD patients, which is our main goal. To achieve these goals, several strategies were combined, including high abundant proteins depletion, protein fractionation and analysis by mass spectrometry (MS), in order to maximize the number of identified proteins. The final optimized strategy combined protein fractionation using liquid chromatography (LC), SDS-PAGE, and protein identification by MALDI-TOF/TOF. Using this methodology, we found 236 proteins with a 95% confidence, using ProteinPilot, where 46 proteins share common biological associations, with a minimum score of 0.900 according to STRING10. The majority of these 46 proteins are involved in regulation processes and share binding functions. Simultaneously, the same VH group sample was analyzed by LC and MALDI-TOF/TOF, in order to understand the importance of the implementation of SDS-PAGE in the process. Thus, we verified that with LC-MALDI only 110 proteins were identified. In conclusion, the developed strategy allowed to find proteins which were not identified using other proteomic strategies.
Nos últimos anos, tem-se verificado um grande incremento de doenças oculares na população mundial. A incidência destas patologias surge com a progressão da idade, levando a um declínio da função ocular normal e, em alguns casos, podendo levar à cegueira. As patologias oculares são frequentemente desencadeadas por doenças crónicas, tais como diabetes ou hipertensão, ou por alterações no posicionamento da retina, o que ocorre no descolamento de retina (DR). Estudos recentes indicam que o humor vítreo (HV) humano sofre alterações proteómicas, de acordo com o estado fisiológico e patológico da retina. No entanto, existem poucos estudos publicados sobre o proteoma do HV no DR. Assim, este estudo centra-se essencialmente na análise proteómica de amostras de HV em DR, e posterior identificação das proteínas presentes nestas amostras, de que forma estão interligadas e como atuam. Desta forma, pode ter-se acesso ao proteoma do HV em doentes com descolamento de retina, que é o principal objetivo deste trabalho. Para atingir estes objetivos, diversas estratégias foram combinadas, incluindo a depleção de proteínas abundantes, fracionamento e análise de proteínas por espectrometria de massa (MS), a fim de maximizar o número de proteínas identificadas. A estratégia final otimizada combinou o fracionamento de proteínas por cromatografia líquida (LC) e SDS-PAGE e a identificação das proteínas por MALDI-TOF/TOF. Usando esta metodologia, foram identificadas 236 proteínas com uma confiança de 95%, usando o ProteinPilot, onde 46 proteínas partilham associações biológicas comuns, com um score mínimo de 0.900 de acordo com o STRING10. A maioria dessas 46 proteínas estão envolvidas em processos de regulação e têm funções de ligação. Em simultâneo, analisou-se o mesmo grupo de amostras através de LC e MALDI-TOF/TOF, de forma a compreender a importância da implementação de SDS-PAGE no processo. Verificou-se que através de LC-MALDI, apenas 110 proteínas foram identificadas. Em conclusão, a estratégia desenvolvida (LC-SDS-MALDI) permitiu encontrar proteínas que não tinham sido anteriormente identificadas usando outras estratégias proteómicas.

Treatment of vision impairing diseases involves drug transport through the vitreous humor. Diffusion cells were used to measure macro-scale (mutual) diffusivity (Dm) to understand how solute size affects diffusion through the vitreous humor of rabbit and porcine eyes. Solutes examined included timolol maleate, dexamethasone sodium phosphate (DMSP), sodium fluorescein, and FITC-dextrans (4, 40, and 150kDa). Diffusivity was inversely dependent on solute size. The Dm's of small solutes in the vitreous were 30 – 65% of that in PBS, while the Dm's of large solutes were 40 – 60% of that in PBS. Extrapolations to the human eye produced similar results using diffusivities based on either species. We used Diffusion Ordered NMR Spectroscopy to measure micro-scale (self) diffusivity (Ds) of DMSP through vitreous humor. The Ds and Dm were significantly different in PBS, but similar in vitreous. A method for obtaining in vivo imagery and data of vitreous fluorophore distribution is also presented.

My PhD research involves multi-disciplinary areas of study such as measuring perfusion of blood vessels in hamster dorsal skin using laser speckle imaging technique. In this study the changes were measured in blood flow velocity and diameters of micro vasculatures after the influence of glycerol application. The second study identifies the changes in morphology and optical properties of eye tissue after applying hyper-osmotic agent such as 100% anhydrous glycerol. Further investigation on the reversal process was performed without any application of 0.9% saline. The third study identified the variation in fluorescence in hamster dorsal skin tissue and enucleated porcine eyes with temperature. This study investigated the variation in fluorescence intensity with temperatures starting at 14°C and compared in vivo and in vitro results for consistency. The fourth study investigated an implantable drug delivery package that was fabricated using PMMA and implanted between the sub-conjunctival and super-scleral space and release the content of the device by either mechanical pressure or light-activated ophthalmic Nd:YAG laser after optically clearing the eye tissue by topical application of a hyper-osmotic agent, 100% anhydrous glycerol. A hyper-osmotic agent creates a transport region in the conjunctiva and sclera to get visual access of the compartments in the drug delivery package. This new technology would provide the option to the patient of one time implantation of the carrier system containing the drug. Each time the patient requires medication a ND-YAG or other laser beam will propagate through the cleared eye tissue to release the drug in measurable doses at the discretion of the doctor from the package directly in to the vitreous humor. In this study we have measured half-life of the dye in the vitreous humor or posterior chamber and biocompatibility. The last study had drawn distinction between the fluorescence signals based on the location (anterior or posterior chamber) of the 10% Na fluorescence dye in the in vivo rabbit and ex vivo pig eyes.
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Dissertação de Mestrado em Química Forense apresentada à Faculdade de Ciências e Tecnologia da Universidade de Coimbra.
O etanol (CH3CH2OH) é uma substância consumida desde tempos remotos, que pode ser obtido pela fermentação de açúcar existente em produtos de origem vegetal tais como frutos, caules e raízes. A sua vasta utilização em bebidas alcoólicas, na indústria farmacêutica, na produção de perfumes e/ou como solvente químico, torna-o uma das substâncias atualmente mais utilizadas. Embora considerada uma substância psicotrópica pela sua atuação ao nível do sistema nervoso central (SNC), a sua comercialização é permitida e o seu consumo quase que incentivado. Uma elevada taxa de álcool no sangue, quando associada a atividades como a condução, aumenta significativamente o risco de ocorrência de acidentes graves. Por essa razão, a sua deteção e quantificação no sangue é muito importante em âmbito forense e corresponde, por esse motivo, a uma percentagem muito significativa do total das análises requisitadas ao Serviço de Química e Toxicologia Forenses (SQTF) do Instituto Nacional de Medicina Legal e Ciências Forenses, I.P. (INMLCF). A tolerância, do ponto de vista social, relativamente ao seu consumo, resulta também numa maior contestação por parte dos indivíduos envolvidos em situações de condução sob o efeito do álcool. Apesar de ser preferível que a deteção e quantificação do etanol seja feita em sangue, existem casos em que não se dispõe deste tipo de amostra para análise (e.g. cadáver em elevado estado de decomposição). O humor vítreo surge como uma matriz alternativa, devido às suas características e localização anatómica. O objetivo deste trabalho foi o de validar um método analítico para deteção e quantificação do etanol em amostras de humor vítreo. Estes ensaios são efectuados no SQTF com recurso a HS-GC/FID, usando uma solução aquosa de n-propanol (100 mg/L) como padrão interno. Este método, amplamente utilizado em toxicologia forense, permite cumprir os requisitos e critérios definidos para este tipo de análises. A separação cromatográfica foi realizada utilizando duas colunas capilares com fases estacionárias de diferentes polaridades, a fim de garantir o cumprimento dos critérios de identificação recomendados para este tipo de análise. O método traduziu-se numa boa separação cromatográfica em ambos os sistemas GC/FID utilizados neste trabalho com o etanol e o padrão interno a eluírem entre os 5 e os 6 min, respetivamente. O processo de validação consistiu na avaliação objetiva de cada um dos parâmetros estabelecidos no procedimento de validação de métodos quantitativos em vigor no SQTF da Delegação do Centro do INMLCF. Após a validação, este método foi aplicado a amostras reais de humor vítreo que foram selecionadas entre os processos enviados ao SQTF para a realização de análises de rotina. Embora os dois equipamentos utilizados possuam diferentes modos de injeção, os resultados obtidos foram concordantes, o que permitiu também obter ilações positivas sobre a robustez do ensaio.
Ethanol (CH3CH2OH) is a substance that is consumed since ancient times. It is obtained by fermentation of sugar existing in vegetable products such as fruits, stems and roots. Its widespread use in alcoholic beverages, pharmaceutical industries, in the production of perfumes and / or as chemical solvents, explains why it is one of the substances most used by mankind. Although considered a psychotropic substance acting mainly in the central nervous system, their marketing is permitted and consumption almost encouraged. The high level of alcohol in the blood, when associated to activities such as driving, triggers serious problems. Therefore, their detection and quantification in blood is very important in forensics and corresponds not only to the most significant proportion of the analyzes required to the Service of Chemistry and Forensic Toxicology of the National Institute of Legal Medicine and Forensic Sciences, but also with greater appeal by individuals involved. Although being preferable that the detection and quantitation of ethanol is performed in blood, there are cases where this type of sample is not available for analysis (eg high decomposition of the body), so the vitreous humor appears as an alternative due to their characteristics and anatomical location. The aim of this study was the validation of an analytical method for detection and quantification of ethanol in samples of vitreous humor. The determination of ethanol in samples of vitreous humor is made in the Service of Chemistry and Forensic Toxicology using HS-GC/FID, as gas chromatography is a widely used technique in the field of chemistry and forensic toxicology, responding to almost all the class of toxicologically relevant compounds. Prior to chromatographic analysis, all samples, including the calibrators, were diluted. For this purpose, 100 μL of vitreous humor were diluted in 1 ml of n-propanol aqueous solution (100 mg/L), used as internal standard. The chromatographic separation was performed using two capillary columns with different polarities in order to ensure compliance with the identification criteria recommended for this type of analysis. The method provides good separation on both chromatographic equipments. The tested substance and the internal standard were eluted in a time interval of 5 min for the GCFI001 and 6 min for the GCFI002. The validation process included the evaluation of each of the parameters established for the validation procedure of quantitative methods adopted in the Service of Chemistry and Forensic Toxicology of the National Institute of Legal Medicine and Forensic Sciences – Centre Branch. After validation of the method, it was applied to real samples of vitreous humor selected among the routine analyses from the Service of Chemistry and Forensic Toxicology. Although the two devices have different modes of injection, the results were consistent, which proves that the method provides reliable results and that the requirements for the specific intended use or application have been met.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Na toxicologia forense postmortem o etanol é a substância mais frequentemente determinada. A sua interpretação é particularmente difícil quando é colhido sangue de cadáveres em putrefação. Nesta situação é de extrema importância a análise de outras amostras biológicas, como a urina e o humor vítreo, que em circunstâncias normais são positivas para etanol apenas quando ocorre ingestão antemortem. A relevância acentua-se quando a concentração de etanol no sangue é menor que 0.5 g/L. Foi realizado um estudo, por um período de 3 anos, tendo os dados sido retirados da informação presente nas requisições recebidas pelo Serviço de Química e Toxicologia Forense do Instituto Nacional de Medicina Legal e Ciências Forenses de Portugal. Dos casos de putrefação, 66 apresentavam etanol no sangue. Destes, 66.7% tinham uma concentração de etanol entre 0.1 g/L e 0.5 g/L, o que é relevante dado que poderá ter ocorrido produção endógena, tornando a interpretação destes valores desafiante. Esta interpretação pode beneficiar da integração da informação disponível do caso, especialmente da pesquisa de etanol em outras substâncias biológicas. Os dados recolhidos permitiram determinar o número de casos que possuíam uma, duas ou três amostras biológicas e foram escolhidos três casos forenses que representavam cada uma destas categorias, exemplificando situações passíveis de acontecerem no estudo da concentração de etanol em cadáveres putrefactos. Este estudo realça, através da revisão da literatura e dos casos apresentados, que quando a concentração de etanol é abaixo de 0.5 g/L em cadáveres putrefactos, a análise das três amostras biológicas (sangue, urina, humor vítreo) permite apoiar a presumida origem do etanol.
Alcohol is the most determined substance in postmortem toxicology and its interpretation is especially complicated when the blood alcohol concentration (BAC) is determined from corpses in putrefaction. Therefore, it is of high relevance the analysis of other biological samples, as urine and vitreous humor, that in normal situations are only positive for alcohol when antemortem ingestion has occurred. This is important mostly when the BAC is lower than 0.5 g/L. This study was made within a period of 3 years, with data material obtained from the Forensic Chemistry and Toxicology Service of the National Institute of Legal Medicine and Forensic Sciences of Portugal. There were 66 cases of putrefaction with a positive result for alcohol. Of these, 66.7% had a BAC between 0.1 g/L and 0.5 g/L, relevant as endogenous production might have happened, making the interpretation of these values challenging. However, this interpretation can benefit from the integration of the available case information, especially the search for alcohol in other biological samples. Using this data, it was determined how many cases presented one, two and three biological samples and three forensic cases were selected, representing each of these categories, exemplifying situations that can easily occur when studying the BAC of putrefied corpses. This study highlights, through the reviewed literature and the presented cases, that in decomposing bodies, when the BAC is lower than 0.5 g/L, the analysis of all three types of biological samples (blood, urine, vitreous humor) is helpful to reach a conclusion regarding the origin of the alcohol.

Vascular Endothelium Growth Factors (VEGFs) are important regulators of endothelium cell proliferation, migration, and permeability not only during embryonic vasculogenesis but also in physiological and pathological angiogenesis. The VEGF family comprises seven members, including VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and Placental Growth Factor (PIGF); each one has a unique expression, specificity, and function. These members with different physical and biological properties act through three specific receptor tyrosine kinases, VEGFR-1, VEGFR-2, VEGFR-3, and two semaphorins receptors neuropilin-1 (NRP-1) and NRP-2. Almost 80% of the volume of the eye is made up of a clear gel-like substance called vitreous humor that fills the center of the eye and is in continuous contact with the retina. It consists of water (~99%), hyalocytes, hyaluronic acid, ascorbic acid, inorganic salts, glucose, and a network of collagen fibrils. In the present study, three cytokines (VEGF-A, VEGF-B and PIGF) were investigated in vitreous humor and serum of patients with ocular pathology, measured by Enzyme-linked immunosorbent assay (ELISA). To compare and characterize the quantitative results obtained by ELISA, the patients were categorized into two groups: 1. Patients with neovascular ocular diseases were categorized into the following subgroups: age-related macular degeneration (AMD), retinal vein occlusion (RVO), and diabetic retinopathy (DR) patients and 2. Patients with non-neovascular ocular diseases: vitreomacular traction syndrome patients of idiopathic etiology or rhegmatogenous retinal detachment patients. Additionally, the growth factors were compared or correlated between: 1. The studied groups of patients; 2. The different ocular pathologies; 3. With patient baseline clinical characteristics such as naïve vs. non-naive patients, previous treatments, the presence of glaucoma, and stage of diabetic retinopathy (non-proliferative diabetic retinopathy vs. proliferative diabetic retinopathy). For further investigation, serum and vitreous concentration levels of VEGF-A, VEGF-B, and PIGF were correlated between each other and between changes assessed by optical coherence tomography (OCT) and visual acuity (VA) measurements. The major conclusions of this study were as follows: 1. VEGF-A, VEGF-B, and PIGF vitreous levels are overexpressed in patients with neovascular ocular diseases in comparison with patients with non-neovascular ocular diseases; 2. In diabetic patients the vitreous vascular endothelial growth factors levels increase with the progression of the disease, being lower in non-proliferative diabetic retinopathy patients and higher in proliferative diabetic retinopathy patients. However, serum levels of VEGF-A, VEGF-B and PIGF were higher in non-proliferative diabetic retinopathy patients in comparison with proliferative diabetic retinopathy patients; 3. There were no statistical differences between the concentration values of serum VEGF-A, VEGF-B or PIGF between neovascular and non-neovascular diseases; 4. There was a positive and strong correlation between vitreous and serum VEGF-A and VEGF-B, suggesting a simultaneous pathological increase in those cytokines; 5. There was no correlation between serum levels and vitreous levels of all growth factors: VEGF-A, VEGF-B and PIGF; 6. There was a correlation between VEGF A or VEGF-B and macular volume; 7. Through the descriptive analysis of previous treatments as well as the comparative analysis between naïve and non-naïve patients, the existence of patients with insufficient response to the current therapies was confirmed, and therefore, there is an unmet need for the research of new drugs to treat neovascular ocular diseases. Taken altogether, these results suggest an overexpression of vitreous levels for the three studied cytokines. The correlations between VEGF-A and VEGF-B growth factors confirmed they may be simultaneously increased in neovascular eye pathologies. The correlations with diabetic retinopathy stage as well with structural and functional characteristics of studied neovascular disorders demonstrated the action of these cytokines in the progression of the disease. Targeting VEGF-A or VEGF-B or PIGF inhibition may have beneficial and impactful implications in the treatment of neovascular diseases, not only in terms of better outcomes but also in the regression of disease (in the case of diabetes). The identification of serum markers or other disease characteristics is easily measurable. Their correlation with vitreous levels will also lead to early intervention in disease prevention through the detention of ocular neovascularization before the appearance of clinical symptoms, subsequently leading to the prevention of blindness.
Os fatores de crescimento endotelial vascular (VEGF) são importantes reguladores da proliferação, da migração e da permeabilidade das células endoteliais, não só durante a fase embrionária e vasculogénese, mas também na angiogénese fisiológica e patológica. A família VEGF é constituída por sete membros, os quais incluem o VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F e o factor de crescimento placentário (PlGF), cada um com uma expressão única, especificidade e função. Estes membros com diferentes propriedades físicas e biológicas específicas, atuam através de três receptores tirosina quinase, VEGFR-1, VEGFR-2 e VEGFR-3, e dois receptores de sinalização neuropilina-1 (NRP-1) e NRP-2. Quase 80% do globo ocular é constituído por uma substância clara, semelhante a um gel, designada por humor vítreo, que preenche o centro do olho e está em contacto contínuo com a retina. O humor vítreo é constituído por água (~ 99%), hialócitos, ácido hialurónico, ácido ascórbico, sais inorgânicos, glucose e uma rede de fibrilhas de colagénio. No presente estudo, foram investigadas três citoquinas (VEGF-A, VEGF-B e PIGF) no humor vítreo e soro de doentes com patologia ocular através da quantificação por ELISA (“Enzyme-Linked Immunosorbent Assay”). Para comparar e caracterizar os achados quantitativos destes fatores de crescimento, os doentes foram divididos em 2 grupos: 1. Doentes com doenças oculares neovasculares classificados posteriormente nos seguintes subgrupos de doentes: degenerescência macular da idade (DMI), oclusão da veia da retina (OVR) e retinopatia diabética (RD); 2. Doentes com doenças oculares não-neovasculares, ou seja, doentes com síndroma de tracção vitreomacular de etiologia idiopática ou doentes com descolamento retiniano regmatogénio. Além disso, os fatores de crescimento foram comparados ou correlacionados entre: 1. Os grupos de doentes estudados (neovascular vs. não neovascular); 2. As diferentes patologias oculares; 3. As características clínicas de “baseline” do doente, como por exemplo, doentes naïve versus doentes não-naïve, tratamentos efetuados antes de serem submetidos a vitrectomia, presença de glaucoma e estadio de retinopatia diabética (retinopatia diabética não proliferativa vs. retinopatia diabética proliferativa). Para uma investigação mais aprofundada, os valores médios das concentrações séricas e vítreas de VEGF-A, VEGF-B e PIGF foram correlacionadas entre si e entre as alterações estruturais ocorridas nestes doentes, avaliadas por tomografia de coerência óptica (OCT) e medições da acuidade visual (AV). As principais conclusões deste estudo foram as seguintes: 1. Os níveis vítreos de VEGF-A, VEGF-B e PIGF encontram-se sobre-expressos em doentes com doença ocular neovascular em comparação com os doentes sem doença ocular neovascular; 2. Nos doentes diabéticos, os níveis de fatores de crescimento endotelial vascular no humor vítreo aumentaram com a progressão da doença, sendo as concentrações obtidas de VEGF-A, VEGF-B e PIGF, menores em doentes com retinopatia diabética não proliferativa e maiores em doentes com retinopatia diabética proliferativa. Contudo, os níveis séricos de VEGF-A, VEGF-B e PIGF apresentaram valores maiores em doentes com retinopatia diabética não proliferativa em comparação com os doentes com retinopatia diabética proliferativa; 3. Não se observaram diferenças estatisticamente significativas entre valores de concentração de VEGF-A, VEGF-B ou PIGF sérico entre doenças neovasculares e não neovasculares; 4. Observou-se uma correlação positiva e forte entre VEGF-A e VEGF-B, tanto no vítreo como no soro analisado, sugerindo um aumento patológico em simultâneo dessas citoquinas; 5. Não foram reportadas correlações entre os níveis séricos e os níveis vítreos para todos os factores de crescimento endotelial vascular: VEGF-A, VEGF-B e PIGF; 6. Foi encontrada uma correlação positiva entre VEGF-A ou VEGF-B e volume macular; 7. Através da análise descritiva de terapêuticas prévias administradas aos doentes com doença ocular neovascular, e das análises comparativas entre doentes naïve e não-naïve, confirmou-se a existência provável de doentes com resposta insuficiente às terapêuticas actualmente aprovadas, reconhecendo-se assim a necessidade de investigação de novos fármacos para o tratamento de doenças neovasculares. Em resumo, todos os resultados sugeriram uma sobre-expressão dos níveis vítreos para as três citoquinas estudadas; as correlações entre fatores de crescimento de VEGF-A e VEGF-B, confirmaram o seu aumento em simultâneo em patologias neovasculares oculares; e as correlações com o estadio da retinopatia diabética e características funcionais e estruturais das doenças neovasculares, demonstraram a ação destas citoquinas na progressão da doença. O enfoque na inibição de VEGF-A ou VEGF-B ou PIGF pode ter implicações benéficas e um grande impacto no tratamento das doenças neovasculares, não apenas em termos de melhor eficácia, mas também em termos de regressão da doença (no caso da diabetes). A identificação de biomarcadores ou outras características da doença quantificáveis e a sua correlação com os níveis de VEGF-A, VEGF-B e PIGF no vítreo poderá conduzir a uma intervenção precoce na prevenção destas doenças, através da detenção da neovascularização ocular antes do aparecimento de sintomas clínicos, conduzindo assim à prevenção da cegueira.

This dissertation introduces a new approach to measure the mechanical properties of soft tissues. A laser-induced microbubble, created by focusing a single nanosecond laser pulse with a custom-made objective lens, was created at desired locations inside a tissue sample. An acoustic radiation force was generated by a low frequency transducer to displace the microbubble. A custom-built high pulse repetition frequency (PRF) ultrasound system, consisting of two 25 MHz single element transducers, was used to track the dynamics of the microbubble. Reconstruction of the mechanical properties at the specific location in a tissue sample was performed using a theoretical model, which calculated the dynamics of a microbubble under an externally applied force in a viscoelastic medium. The theoretical model and the high PRF ultrasound system were successfully validated in both gelatin phantoms and ex vivo bovine crystalline lenses. Age-related sclerosis of the crystalline lenses from bovine was clearly detected, which might be linked to changes in the crystalline. Location-dependent variation explained that the outer cortex and the inner nucleus had different mechanical properties. In the old and young porcine vitreous humors, age-related changes were not found. However, local variations of the mechanical properties were discovered, which may coincide with the different distributions of the molecular compositions. The laser-induced microbubble approach shows potential for future research into the origin of physiological phenomena and the development of inherent disorders in the eye. I hope that further studies – in the development of a more suitable theoretical model for the microbubble dynamics, in extension to in vivo applications, and in defining the relationship of the mechanical properties to molecular components in the eye – may provide a plan for the therapeutic treatment of eye-related diseases.
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