Proefschrift Universiteit van Amsterdam. / Met bibliogr., lit. opg. - Met samenvatting in het Nederlands.

Thesis (Ph. D.)--Michigan State University. Chemistry, Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on July 2, 2009) Includes bibliographical references. Also issued in print.

Thesis (M.S. in environmental engineering)--Washington State University, August 2009. / Title from PDF title page (viewed on Aug. 7, 2009). "Department of Civil and Environmental Engineering." Includes bibliographical references (p. 73-76).

Thesis (Ph. D.)--University of Virginia, 1999. / Includes bibliographical references (p. 136-159). Also available online through Digital Dissertations.

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<P> A great number of cellular processes can lead to local changes in proton concentration and temperature. So, it is desirable to be able to measure pH and temperature with non-invasive and spatially resolved methods. In this thesis, I describe two unique methods to measure pH and temperature using the environmental sensitivity of the proton transfer of fluorescent molecules. The first method is based on the detection of the temperature and pH dependent parameters associated with the blinking of fluorophores by fluorescence correlation spectroscopy (FCS). Employing EGFP as the probe, I used this method to characterize temperature increase at a laser focus due to light absorption in a thin liquid sample. Using pyranine as a probe, I extended the applicability of this method to a range of pH including physiological pH. Also, I investigated in details the effect of buffer composition on the blinking of the fluorophores. Then, I concluded that one limitation of this method is its strong dependence on buffer conditions, which are not well characterized in vivo. The second approach is a pH measurement method based on ratiometric imaging. This method is not as dependent on buffer condition. We improved on current ratiometric imaging techniques by demonstrating the possibility of using two-photon excitation. This method was used to measure pH in pyranine loaded vesicles created during receptor-mediated endocytosis of µ-Opioid receptors expressed in HEK 293 cells. Preliminary results showed that the pH in the endocytic vesicles dropped to a value similar to those measurements in late endosomes roughly~ 10 minutes after triggering the endocytosis, and eventually, the pH reading reached a value similar to that of the pH oflysosomes. </p> / Thesis / Doctor of Philosophy (PhD)

Processos de transferência de H+ de fotoácidos como piranina (POH) em vidros de SiO2, foram atribuídos à presença de água residual no gel. Nestes experimentos, o efeito devido à espécie básica do tampão (HPO4=), todavia não foi investigada. O entendimento de como as reações prototrópicas são mediadas no meio sólido e como a atividade do par ácido - base de Brönsted participa na transferência é o objetivo deste presente estudo. Questões como: o H+ difunde livremente através do gel? A espécie do tampão interage diretamente com o doador de H+, ou água é necessário para intermediar a transferência? Existe um teor limiar de água? Aqui são discutidas. A transferência de H+ da POH foi estudada em monólitos derivados da hidrólise ácida do TEOS contendo diferentes concentrações de tampão fosfato (condições iniciais Si: H2O 1: 16,5). A extensão da transferência de H+ é determinada pelo monitoramento da intensidade de fluorescência devido à base excitada (% PO-*) uma vez que a emissão surge da reação de transferência de H+. Nestes experimentos, géis foram preparados na ausência e presença de tampão fosfato. Até ~ 12 dias, um aumento na transferência de H+ é observada para todas as condições. É também verificado que o aumento na concentração de tampão é paralelo à extensão da reação. Em tempos muito curtos, a atividade da transferência de H+ relativamente baixa é devido à presença de ETOH (hidrólise do TEOS), o qual assim que o sistema envelhece é lixiviado junto com a água do gel. Depois deste envelhecimento inicial, aqueles géis contendo altas concentrações de tampão (50 e 250 mM) mostram uma atividade constante e os demais géis mostram uma queda na transferência de H+. No final do estágio, ~ 50 dias, os géis perderam aproximadamente 80% de sua massa, mas no gel final ainda existe quantia de água residual Si: H2O 1: 7. Com o intuito de explorar a atividade do tampão, misturas secas de KH2PO4 e K2HPO4 na proporção de pHaparente = 6,0 foram trituradas e secas e a fotoacidez do 2 - naftol (NOH) foi investigada. Claramente a baixo teor de água nenhuma reação é observada e somente a emissão devido a NOH* aparece (yem. = 345 nm), adição passo a passo de H2O é seguida pelo aumento na emissão a 413 nm, devido à forma básica (NO-*). Nesta condição, a concentração de água limiar é Xw = 0,44. Por outro lado, para o POH o limite de água para a transferência de H+ é Xw = 0,13. POH e NOH têm pKa no estado excitado (pKa*) de 0,5 e 3,0, respectivamente. Neste estudo, a atividade dos fotoácidos em meio sólido mostra ser dependente do pKa*, da concentração da espécie básica e do teor de água limiar. / H+ transfer processes in SiO2 glasses, from photoacids as piranine (POH), were assigned to the presence of residual water in the gel. In these experiments the effects due to the buffer basic species (HPO4=) were however not investigated. The understanding on how prototropic reactions are mediated in solid media and how the activity of Brönsted acid and base pairs participates in the transfer is the subject of the present study. Questions as: Does the H+ diffuse freely through the gel? Do the buffer species interacts directly with the H+ donors, or water is needed to intermediate the transfer? Is there a threshold in the water content? are here discussed. H+ transfer from POH was studied in monoliths derived from the acid hydrolysis of TEOS having varying concentrations of phosphate buffer (initial conditions Si : H2O 1:16.5). The extent of H+ transfer is determined by monitoring the fluorescence intensity due to the excited base (%PO-*) once this emission arises from the H+ transfer reaction. In these experiments gels were prepared in the absence and presence of phosphate buffer. Up to ~ 12 days an increase in H+ transfer is observed for all conditions. It is also found the increase in buffer concentration parallels the extent of reaction. At very short time the relatively low H+ transfer activity is due to the presence of ETOH (hydrolysis of TEOS), which as the systems age are lixiviated together with water from the gel. After this initial aging, those gels having high buffer concentrations (50 and 250 mM) show a leveled activity, the other gels show a decrease in H+ transfer. At the final stages ~ 50 days the gels lost around 80% of its mass, but in the final gel residual water still amounts to Si:H2O 1:7. In order to further exploit the buffer activity, \"dry\" mixtures of KH2PO4 and K2HPO4 in proportions to pHapp = 6.0 were grinded and dried and the photoacidity of 2-naphtol (NOH) were investigated. Clearly at low water contents no reaction is observed and only the emission due to the NOH* appears (yem = 345 nm), addition step by step of H2O is followed by an increase in the emission at 413 nm, due to the basic form (NO-*). In this situation the threshold water concentration is around Xw = 0.44. By other hand for POH the threshold water for H+ transfer is Xw = 0.13. POH and NOH have excited state pKa\'s (pKa*) of 0.5 and 3.0, respectively. In this study the activity of photoacids in solid media is shown to be dependent on pKa*, on the concentration of basic species and of a threshold water content.

The axilla is a major source of human body odour from which the characteristic musky, urinous or acidic odours emanate, and are predominantly due to bacterial metabolism of the protein-rich fluid secreted by the apocrine and sebaceous glands located in this area (Senol and Fireman, 1999). Clothing has been implicated in contributing to body odour intensity, possibly even increasing the intensity (Dravnieks, et al., 1968; Shelley, et al., 1953) by the transfer of secretions, skin debris and bacteria from the body to the fabric substrate. Despite much anecdotal evidence indicating that some fibres and fabrics are better at limiting odour intensity than others, there appears to be no published research confirming this. The purpose of this study therefore, was to determine whether fabrics varying in fibre content (cotton, wool, polyester) and fabric knit structure (interlock, single jersey, 1x1 rib) differed in the extent to which they retained and emanated axillary odour following wear, and whether the intensity of odour was linked to the number of bacteria transferred to the fabrics. A procedure for collecting odour on fabrics was developed as was a method for evaluating odour through use of a sensory panel. Total aerobic bacteria and aerobic coryneform bacteria extracted from the fabrics were counted to determine if an association between bacterial counts and fabrics existed. Sensory analysis recognises the unique capability of humans as odour-detecting instruments whereas, instrumental analysis has the potential to offer information on the concentration and identification of axillary compounds, which a human assessor cannot. To investigate a new method for detecting axillary odour on apparel fabrics, proton transfer reaction mass spectrometry (PTR-MS) was used to analyse volatiles emitted from fabrics differing in fibre type. After removal of garments from the human body, axillary odour can be detected on fabrics, with the intensity of odour being strongly influenced by the fibre type from which the fabrics had been made. Polyester fabrics emanated odour of high intensity, cotton that of mid-low odour intensity, and wool fabrics were low odour. Fabric structural properties such as thickness, mass per unit area and openness of knit structure also had an effect on odour intensity. However, as the principal factor influencing odour intensity was fibre, only fabrics characterised by a high intensity (i.e. polyester) were influenced by structural properties. Differences in odour intensity among fabrics were not necessarily related to bacterial numbers, and no �inherent antimicrobial� properties were evident for any of the fabrics. Bacterial populations persisted in all fabrics up to 28 days. A decline in numbers was apparent for high-odour polyester fabrics, while numbers in low-odour wool fabrics remained relatively stable. PTR-MS detected compounds likely to be short-chain carboxylic acids which increased in the headspace above the polyester fabrics after 7 days. However, this increase was not evident for either the wool or cotton fabrics. Therefore, bacterial numbers per se cannot be a predictor of the odour intensity emanating from fabrics at least on the basis of these fabrics and fibres. The intensity of axillary odour emanating from fabrics was found inversely related to fibre hygroscopicity. Keywords:fibre content, fabric structure, axillary odour, sensory analysis, bacteria, corynebacteria, instrumental analysis, PTR-MS

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme.