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Dissertations / Theses on the topic 'Fluorimetry'
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1
Aminuddin, Mohammad. "New aromatic dialdehyde labels for analytical fluorimetry." Thesis, Loughborough University, 1987. https://dspace.lboro.ac.uk/2134/27565.
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Fluorigenic aromatic molecules have found wide application in Fluorescence Spectroscopy. Commercially available fluorigenic reagents have been used to detect amines, amino acids, peptides and proteins. Orthophthalaldehyde (OPA) is an aromatic dialdehyde which is specific for a primary amino group. Three polyaromatic dialdehyde molecules similar to this compound have been synthesised and investigated for their analytical applications. They are: (1) naphthalene-2,3- dicarboxaldehyde (NDA), (2) 1-phenylnaphthalene-2, 3- dicarboxaldehyde (0NDA) and (3) anthracene-2,3- dicarboxaldehyde (ADA).
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Tse, Oi Ling. "Development of humidity sensor based on fluorimetric optode membrane." HKBU Institutional Repository, 1999. http://repository.hkbu.edu.hk/etd_ra/191.
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Horne, Andrew James. "Insights into Kv1.2 activation and deactivation using voltage clamp fluorimetry." Thesis, University of British Columbia, 2010. http://hdl.handle.net/2429/27083.
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Voltage-gated potassium (Kv) channels are essential membrane proteins in modulating membrane excitability and related cellular processes. Many details associated with the voltage response are unclear, particularly the complete role of the voltage sensing domain, and not just the densely charged S4 helix. Based on its crystal structure, the Kv1.2 channel represents an ideal model in which to study these questions. This thesis investigates Kv1.2 activation and deactivation gating, using the voltage clamp fluorimetry technique. This technique utilizes an environmentally sensitive fluorophore introduced at locations of interest in order to visualize conformational changes in protein structure. Labelling of Kv1.2 channels at the extracellular end of S4 reports a fast quenching of fluorescence emission upon depolarization that correlates extremely well with gating current measurements, suggesting it is a report of voltage-dependent S4 translocation. In addition, a slow quenching component is observed with a very negative voltage-dependence (V₁/₂ = -73.9 mV ± 1.4 mV), not seen in any other Kv channels studied to date, that involves regions of the voltage sensing domain in S1 and S2. This slow quenching is selectively removed from the fluorescence report with transfer of extracellular S1-S2 or S3-S4 linkers from the homologous Shaker potassium channel, suggesting that it arises from channel-specific interactions between the Kv1.2 linker segments. However, transfer of Kv1.2 linker segments into Shaker fail to recapitulate this quenching component, suggesting that these linker interactions likely underlie further differences in voltage sensor domain gating and/or structure. This slow quenching component correlates with deactivation of ionic current, and is prolonged with co-expression of the N-type inactivation-conferring Kvβ1.2 subunit. In the presence of the beta subunit, this likely reflects unbinding of the inactivation moiety from the pore domain, allowing deactivation and S4 return, but in the α-subunit alone we suggest that this may be a report of a voltage-dependent rearrangement in the voltage sensor domain that stabilizes the S4 in an activated conformation. Such interactions have been reported in other voltage-gated proteins, and provides further evidence that we must consider more than just S4 translocation when it comes to understanding the complete potassium channel voltage response, Kv1.2 or otherwise.
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Drzewianowski, Andrea F. "Temporal Changes in Phytoplankton Variable Fluroescence (FV/FM) and Absorption as a Result of Daily Exposure to High Light." Fogler Library, University of Maine, 2008. http://www.library.umaine.edu/theses/pdf/DrzewianowskiAF2008.pdf.
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Simpson, K. M. "Studies of cosmic ray composition using a hybrid fluorescence detector /." Title page, contents and abstract only, 2001. http://web4.library.adelaide.edu.au/theses/09PH/09phs61261.pdf.
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Afonin, Kirill A. "Design and characterization of novel bio-sensor platform for sequence specific, label-free, fluorescent detection of native RNA molecules." Bowling Green, Ohio : Bowling Green State University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=bgsu1206395144.
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Passmore, James B. "Calibration of a laser induced fluorescence system by raman scattering in hydrogen with application to the detection of hydroxyl radicals." Thesis, Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/27150.
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Primo, Afranio Reis Rodrigues. "Avaliação da influência do reservatório do funil na qualidade da água do rio Paraíba do Sul." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46133/tde-20042007-100003/.
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O rio Paraíba do Sul, após formar o reservatório do Funil, abastece cidades ribeirinhas e a cidade do Rio de Janeiro. A INB, uma empresa da área nuclear, está localizada na margem norte deste reservatório. Neste trabalho, Ag, Al, As, Ba, Cd, Co, Cr, Fe, Mn, Ni e Pb foram determinados por ICP OES em água e sedimentos em pontos a montante e a jusante do Funil e neste. Urânio, usando fluorimetria, também foi determinado em amostras coletadas a montante e a jusante do ponto de descarga de efluentes da INB, no ribeirão da Água Branca. O estudo não mostrou evidências que a INB está provocando impacto ambiental nesse ribeirão. As, Ni e Pb em todos os pontos de amostragem e Al, Cr e Fe na maioria desses pontos apresentaram concentrações em água acima do máximo permitido pela CONAMA 357. A maioria dos elementos apresentou concentrações nas amostras de água a montante do reservatório do Funil superiores às verficadas a jusante, para ambas as estações chuvosa e seca. Os sedimentos estão impactados por As, Cd, Cr e Pb em quase todos os pontos estudados.The Paraiba do Sul River after forming the Funil Reservoir serves as the major source of potable water for downstream cities and for the city of Rio de Janeiro. INB, a company of the nuclear area, is located in the north margin of this reservoir. In the present study, Ag, Al, As, Ba, Cd, Co, Cr, Fe, Mn, Ni and Pb were determined by ICP OES in water and sediments samples at points upstream and downstream from Funil and in this one. Uranium, using fluorimetry, was also determined in samples collected upstream and downstream from the INB effluent discharge point at the Água Branca Creek. The study did not show evidences that INB is provoking environmental impact in this creek. As, Ni and Pb in all the sampling points and Al, C and Fe in most of those points exceeded the CONAMA 357 standards for water. Most of the elements presented concentration in the water samples at the points upstream from Funil reservoir higher than those downstream, for both rainy and dry seasons. Sediments are impacted by As, Cd, Cr and Pb in almost all the studied points.
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Clark, Ian David. "A fluorescence study of the COOH-terminus region of equine platelet tropomyosin." Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26190.
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The use of fluorescent molecules as probes of protein conformation is recognized as a technique which provides very specific information and has been applied, in recent years, to the study of the role of tropomyosin (TM) in the regulation of contractile processes. The isolation and sequencing of TM from horse blood platelets (P-TM) has shown it to be different from muscle TM, especially near the NH₂-and COOH-termini. These differences have been suggested to weaken end-to-end interaction of P-TM molecules. TM's are two chain coiled coils and P-TM has cysteine residues at the penultimate COOH-terminus position of adjacent chains. These can be labelled with
sulfhydryl-specific fluorescent probes that reflect conformational changes in that region of the molecule via changes in their emission characteristics.
The results of experiments on both pyrene (Py) (40) and acrylodan (AD) labelled P-TM show that there is a preferred interaction of the COOH-terminus of P-TM with the NH₂-terminus of cardiac TM over that with the NH₂-terminus of P-TM. This indicates that the altered NH₂-terminus of P-TM, with respect to muscle TM, is responsible for the relative loss of polymerizability of P-TM at low salt concentration. Addition of actin to the Py-P-TM (40) and AD-P-TM species showed changes in emission characteristics indicative of binding to the F-actin filaments, suggesting
that the presence of the probes had not affected the function of the P-TM adversely. However, the presence of pyrenes at the COOH-terminus seemed to reduce further the ability of P-TM to self-polymerize. Thermal denaturation of AD-P-TM, AD-C-TM and AD-labelled truncated P-TM followed by fluorescence polarization suggested that, contrary to the theory of Skolnick and Holtzer on the stability of two chain coiled coils, the region towards the COOH-terminus is among the last to lose its helical character.Science, Faculty of
Chemistry, Department of
Graduate
10
Langford, Stephen Richard. "State-to-state molecular photodissociation dynamics." Thesis, University of Oxford, 1995. http://ora.ox.ac.uk/objects/uuid:771f0638-7d55-4304-b387-7b24de012cc6.
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The water molecule, rotationally state selected in the third and fourth streching overtone (|04>- and |05>-) and stretch-bend combination (|04>|2>-) levels, has been photodissociated at γ ≈ 282 nm via the à state. The OH photofragment rotational distributions, determined by OH(A-X) laser-induced fluoresence (LIF), are found to differ from those reported previously by Andresen and coworkers (H2O|01>- + 193 nm), Grim and coworkers (H2O|04>- + 239.5 nm) and Rosenwaks and coworkers (H2O|01>+ + 193 nm). These variations become more apparent with increasing angular momentum in the parent water molecule and with an increasing number of OH stretching quanta in the intermediate vibrational overtone. The Franck-Condon model of Balint-Kurti is able to qualitatively reproduce the observed trends, provided that dissociation at lower photolysis photon energies and via higher intermediate overtone states is assumed to occur preferentially from extended RH-OH configurations. The calculations suggest that the variation in the photofragment rotational distributions lies in a gradual change in the inertial properties of the bound state water molecule as the H-OH bond is stretched. In a second study, the partially deuterated water molecule, rotationally state selected in the third and fourth OH stretching vibrational overtone levels have been photodissociated via the à state at γ ≈ 288 nm. A branching ratio betweem the H + OD and D + OH dissociation channels is estimated from OD and OH (A-X) LIF measurements to be φ(OD)/φ(OH) > 20; this compares well with the previous measurements of Grim and coworkers, and the theoretical work of Imre and coworkers. The small shift in the centre of mass in the water molecule arising from the substitution of of a deuterium atom for one of the hydrogen atoms is shown to have a marked effect on the rotational distributions of the OD photofragment. Calculations using a modified Franck-Condon model, which includes an approximate exit-torque, are able to reproduce qualitatively the experimental OD rotational distributions at sensible values of RR-OD(~ 1.4 Å). In addition to being sensitive to the dynamics of the parent molecule on the ground state potential, the product OD state distributions are shown to be very sensitive to even the smallest exit channel torque on the excited potential surface.
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Sidhu, Inderjit Kaur. "Expression and mutagenesis of bacteriorhodopsin an integral membrane protein." Thesis, University of Oxford, 1998. http://ora.ox.ac.uk/objects/uuid:fd0d323c-5f02-4cd2-98a9-eb57e2277fa3.
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Although integral membrane proteins represent nearly a quarter of the genes present in both prokaryotes and eukaryotes, progress in this area of research is often hindered due to the nature of their hydrophobic environment. Elucidating the folding pathway of these proteins is essential to understand many membrane mediated biological processes such as signal transduction, ion transport and chemotaxis. The wealth of structural and genetic information on bacteriorhodpsin renders it an ideal model system for the study of membrane proteins. Detailed studies however, necessitate efficient methods for its overexpression and purification. Previous expression systems have reported difficulty in obtaining good yields and simple purification procedures. This thesis investigates a variety of alternative expression and purification systems for the bacterio-opsin gene in Escherichia coli. With sufficient protein, site directed mutagenesis is performed to mutate three proline residues present in the membranous region of bacteriorhodopsin to alanine. The folding kinetics of these mutants is investigated using stopped flow fluorimetry to determine whether proline isomerisation is responsible for a slow step in the folding pathway of bacteriorhodopsin. Comparison of the results with those of the folding kinetics of wild type showed proline isomerisation not to be responsible for the slow step in the pathway. More recent studies have suggested that the slow step may be due to refolding conditions and lateral pressure the lipids impose upon the protein as well as pH. Separate structural studies using mass spectrometry aimed to study the rates of isotopic exchange of amide and side chain protons in bacteriorhodopsin. Low resolution results obtained using matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) prompted the investigation of electrospray ionisation mass spectrometry (ESI-MS). Techniques for sample preparation were optimised by investigating a variety of solvent systems and initial deuteration experiments performed.
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Lawlor, Susan Elizabeth. "Synthesis and metal binding properties of novel Câ†2-symmetric tetraaza ligand systems." Thesis, University of Southampton, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342639.
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Martinez, Carmen Ivette. "Study of photolytic interference on HO measurements by LIF-FAGE." PDXScholar, 1989. https://pdxscholar.library.pdx.edu/open_access_etds/3931.
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For many years there has been a great interest among the scientific community in the study of the hydroxyl radical, HO. This interest stems from the fundamental role played by this molecule in the photochemistry of the atmosphere, mainly as a cleansing agent of environmental pollutants. Knowing the concentration of the radical would enable scientists to corroborate current atmospheric models and to predict future trends in the atmosphere. Even though there is a great interest in the determination of atmospheric concentrations of this molecule, the task has been very difficult. This is mainly due to the lack of a method sensitive enough to detect concentrations around 106 molecules per cubic centimeter. The most accurate method presently available is the method of laser induced fluorescence using the fluorescence assay with gas expansion technique (LIF-FAGE). This method involves low pressure excitation of HO from its ground state to its lowest electronic excited state and observing the consequent fluorescence around 309 nm. The procedure is done at a pressure of 5 Torr to maximize the fluorescence lifetime of the radical and to minimize the interference of photolytic species. Background determination is achieved by chemical modulation using isobutane in a second channel of the same cell which removes the HO signal.
In this study an assessment of the level of ozone interference in LIFF AGE has been done by calculating the relative population distribution of HO among its rotational levels and from this, determining its temperature. When the laser passes through the excitation detection cell it photolyses the ozone present producing in this way the highly reactive 0 1(D). When this molecule reacts with water or with isobutane it produces HO, and this is the source of interference in the actual measurements.
In the determination of the relative population distributions of the different HO species, it was found that the naturally occurring HO has a thermal distribution with a temperature of about 300 K. The HO molecules produced from the reaction of 0 1(D) with isobutane also showed a thermal distribution with a temperature of about 230 K. On the other hand, the HO produced from the reaction of 0 1(D) with water did not show a thermal distribution. Two distinct temperatures were observed for this case: one around 200 K for values of K = 1 to 4, and the second one around 3000 K for values of K = 5 to 6. These values agree with previous experimental results for LIF methods by other authors except for the fact that the deviation from the first temperature determined by other authors starts at K = 6 or 7.
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Wang, Ping. "Diffusion of small organic molecules in fluoroelastomers /." Thesis, Connect to Dissertations & Theses @ Tufts University, 1995.
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Thesis (Ph.D.)--Tufts University, 1995.Adviser: Nak-Ho Sung. Submitted to the Dept. of Chemical Engineering. Includes bibliographical references. Access restricted to members of the Tufts University community. Also available via the World Wide Web;
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Lo, Chung Keung. "Voltammetric and spectroscopic studies of dye-immobilised poly(vinyl chloride) membranes." HKBU Institutional Repository, 2003. http://repository.hkbu.edu.hk/etd_ra/425.
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George, Linda Acha. "Development of a Direct, Low Pressure, Laser-Induced Fluorescence Measurement Technique for NO2., Ambient Measurements and Urban NOx Chemistry." PDXScholar, 1991. https://pdxscholar.library.pdx.edu/open_access_etds/1144.
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Nitrogen oxides control the global formation of ozone in the lower atmosphere and influence the much higher levels of ozone formed in areas subjected to photochemical air pollution. As an important member of the nitrogen oxide family, N02 plays a significant role in serving as the only known source of ozone (through photolysis) in the lower atmosphere and as sink for HO via the formation of nitric acid. Ozone can be destroyed by reaction with another member of the nitrogen oxide family, nitric oxide (NO), to reform N02. This cycle between NO, N02 and 03 is known as the NOx-03 photostationary state (PSS). Imbalances in this cycle have been used to calculate ambient levels of oxidants (such as R02 and H02) responsible for ozone production. Consequently, accurate N02 measurements are critical to making meaningful measurements of the imbalances in the NOx-03 photostationary states (PSS). A low pressure laser-excited fluorescence technique (FAGE) for the direct determination of atmospheric N02 has been developed. This technique has been explored with both a Nd-YAG laser (1.4 W, 532nm, 30Hz) and a Cu-vapor (1.2 W, 511nm, 5.6kHz) laser. The detection limits for these instruments, under laboratory conditions and a signal collection time of 20s (lOs each signal and background), have been determined to be 450 and 350pptv N02, respectively. In these systems, the background was measured by chemically reducing N02 with FeS04°7H20. Ambient measurements of the NOx-03 photostationary state (PSS) were undertaken on a rooftop monitoring site in downtown Portland, Oregon. N02 was monitored with the Cu-vapor system. Nitric oxide and ozone were monitored with standard instruments. Data for three days in 1990 are presented. Overall these data sets clearly show that despite daily changes in concentration of NO, N02 and 0 3 of factors of 4-10 each, the PSS remains relatively constant to within -50%. This is in itself strong confIrmation of the primary importance of the NOx-03 photo stationary state in controlling the concentrations of these species at these levels. In addition, these experiments also serve to demonstrate that the monitoring instruments, including FAGE-N02, are not subject to serious interferences or artifacts at these concentration levels.
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Belshe, Elizabeth F. "Evaluating pulse-amplitude modulated fluorometry for landscape scale assessment of photosynthetic characteristics /." Electronic version (PDF), 2005. http://dl.uncw.edu/etd/2005/belshee/elizabethbelshe.pdf.
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Halverson, Peter Georges. "Detection of high-energy cosmic ray showers by atmospheric fluorescence." Diss., The University of Arizona, 1989. http://hdl.handle.net/10150/184779.
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A novel detector for ultra-high energy cosmic rays, and its prototype are discussed. It detects events with primary energy greater than 100 PeV. (1 PeV = 1000 TeV; 1EeV = 1000 PeV.) The detector operates by sensing the near-ultraviolet scintillation light of ionized nitrogen molecules created by the passage of ionizing particles in extensive air showers. (The concept is loosely based on the highly successful Fly's Eye detector situated at Dugway, Utah.) Typical events should consist of 1 to 100 EeV primary energy showers, with near-vertical cores, passing through the detector's field-of-view at distances of 1 to 20 km. The optical field of view of the hypothetical detector would be 60 degrees wide by several (≈ 3) degrees high and would look in a near-horizontal direction at a distant mountain range or other suitably dark background roughly 20 Ian away. A typical good location would be the rim of a canyon, looking slightly downward at the other side. The field-of-view would be subdivided into 3 or more thinner ''wedges'', 60 degrees wide by, perhaps, 1 degree high. A single detector provides timing and brightness information only. Three widely-separated detectors with overlapping fields-of-view provide sufficient data to determine the core location, the zenith and azinruthal angles of the core axis, and the absolute luminosity of the cascade. Interpretation of the luminosity data would be a challenge, but it should be possible to estimate primary energy from it. The advantage of this new scheme is the enormous effective detector area per relatively low-cost detector module. Each triplet of detectors "sees" 300 square km with a typical core axis acceptance of roughly 1 sr. The construction and testing of a prototype unit has been accomplished. The field-of-view was 41 degrees wide by 2 degrees high. Light was collected by a 4.7 square meter mirror and focused onto a wave-shifter PMT system. 8 events with primary energies in the 0.1 to 1 EeV range were observed in an 8.5 hour period. Representative events are shown and preliminary data analysis is discussed.
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Araújo, Fabiano Tófoli de. "Clonagem, expressão e purificação da proteína ligadora de alcano sulfonatos do sistema de transporte ABC de Xanthomonas axonopodis pv.citri." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-07012009-121801/.
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O genoma de Xanthomonas citri (Xac) possui mais de 20 tipos de transportadores do tipo ABC incluindo o operon ssuABC associado ao transporte de alcano sulfonatos. Deste operon, escolhemos a proteína periplasmática ligadora de alcano sulfonatos SsuA2, para caracterização e análises espectroscópicas e estruturais. A rSsuA2 foi expressa no citoplasma de células de Escherichia coli e utilizada para a preparação de anticorpos em camundongos, que foram capazes de reconhecer a proteína recombinante, mas não a nativa no extrato de células de Xac. A rSssuA2 apresenta estrutura característica de proteínas alfa-beta, maior estabilidade em pH neutro (7.0), como também foi evidenciado pela obtenção de cristais somente nesta faixa, e pouca flexibilidade ao desenovelamento térmico. Os cristais difratam com resolução de 1.8 Å e pertencem ao grupo espacial de simetria P21. Além do o operon ssu (ssu2) altamente conservado, Xac apresenta o operon tau (ssu1) para captação de taurina. O papel do operon para Xac é discutido.Xanthomonas citri (Xac) genome has more than 20 different ABC transporters, including the ssuABC operon. In this work, the alkanosulphonate-periplasmic binding protein SsuA2 was chosen for spectroscopic and structural analysis. The rSsuA2 protein was expressed as a soluble form and purified by immobilized metal affinity chromatography. Antibodies produced from the recombinant protein were able to recognize the rSsuA2, but not the native protein in the Xac extract samples. The protein presents secondary structure defined by alfa helices and beta-sheets, high stability in neutral pH and low flexibility to the thermal denaturation. The determination of the optimal pH range was important to produce crystals of high quality diffracting at 1.8 Å with symmetry of the P21 spatial group. Besides the highly conserved operon ssu (ssu2), Xac has the tau operon (ssu1) for taurine uptake.
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Ribeiro, Jean Francisco Rosa. "Estudo in vitro do metabolismo microssomal hepático de agentes tripanossomicidas." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/75/75133/tde-04042013-145753/.
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Em face das recentes exigências das agências regulatórias quanto à aprovação de novos fármacos, os estudos de biotransformação têm-se tornado uma etapa indispensável para a identificação e otimização de compostos bioativos. O objetivo desses estudos é identificar, já nas fases iniciais da descoberta de fármacos, candidatos que apresentam propriedades indesejáveis como a (i) presença de metabólitos ativos ou tóxicos; (ii) inibição de enzimas metabolizadoras; (iii) depuração metabólica inadequada, entre outras. Neste estudo, foi realizada a caracterização metabólica e a identificação de possíveis inibidores das enzimas do citocromo P450 de oito promissores candidatos a fármacos, identificados através de ensaios virtuais como inibidores da TcGAPDH, Cruzaina e TcDHODH, todas do Trypanosoma cruzi, agente causador da doença de Chagas. Esses compostos foram testados contra as três principais isoformas do citrocromo P450: CYP 3A4, CYP 2D6 e CYP2C9. Os valores de IC50 de 1,4 µM e 1,3 µM contra a CYP2C9 foram encontrados para os compostos Nequimed53 e Nequimed125, enquanto o Nequimed42 inibiu a CYP 3A4 com um valor de IC50 de 7,12 µM. Posteriormente foi conduzida a caracterização metabólica dos compostos Nequimed53 e 125 com foco na identificação dos principais metabólitos, sítios de metabolismo e vias de biotransformação através da técnica de LC-ESI-QqTOF-MS. Para ambos os compostos, a biotransformação por microssomas extraídos de fígado de ratos deu-se preferencialmente por uma única via dependente de NADPH. No caso do Nequimed54, o metabólito formado apresentou uma variação da razão m/z de +16, indicando a ocorrência da hidroxilação do composto parental, enquanto que para o composto Nequimed125, o metabólito formado apresentou uma variação da razão m/z de -28, condizente com a perda de um fragmento etila do composto parental.In the light of recent demands from regulatory agencies for the acceptance of new drugs, the biotransformation studies have become an essential step for the identification and optimization of bioactive compounds. The objective of these studies is to identify compounds that have undesirable properties such as (i) the presence of toxic or active metabolites, (ii) inhibition of metabolizing enzymes, (iii) excessive metabolic clearance, inter alia. In this study we characterized the metabolism and cytochrome P450 inhibition of eight compounds identified by virtual screening as inhibitors of TcGAPDH, Cruzain and TcDHODH which are of interest as targets for intervention in treatment of Chagas Disease. These compounds were tested against cytochrome P450 isoforms 3A4, 2D6 and 2C9. IC50 values of 1.4 µM and 1.3 µM against CYP 2C9 were observed for Nequimed53 and Nequimed125.while Nequimed42 inhibited CYP 3A4 with an IC50 of 7.1 µM. Subsequently, we characterized the in vitro metabolism of Nequimed53 and 125 with a focus on metabolite identification and biotransformation pathways using the LC-ESI-MS-QqTOF technique. For each, the biotransformation by rat liver microsomes occurred by a single NADPH-dependent pathway. For Nequimed54, the observed metabolite [M+16]+ indicated hydroxylation of parent compound. The metabolite [M-28]+ observed for Nequimed125 indicated desethylation of the parent compound.
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Ho, Cheuk Lam. "Conjugated metal-organic phosphorescent materials and polymers containing fluorene and carbazole units." HKBU Institutional Repository, 2007. http://repository.hkbu.edu.hk/etd_ra/808.
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Wang, Zhen Hua. "The application of parallel light detection to plasma deposition processes." Thesis, The University of Sydney, 1993. https://hdl.handle.net/2123/26606.
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This thesis is concerned with the application of parallel light detection to a variety of physical processes, many of which are plasma processes. Parallel light detection is carried out using an optical multichannel analyser (OMA) coupled with a 1024channel photodiode array detector, and a spectrograph if the spectral information is required. The plasma processes investigated include a planar DC sputtering deposition system, a cathodic titanium vacuum arc and a magnetic solenoid filtered vacuum arc deposition system.
An application of the optical multichannel analyser in spectrophotometry is presented. Because of parallel detection of light the OMA-base d spectrophotometer is able to collect optical reflectance and transmittance data at a rate of up to 1024 samples in 17 ms compared with a commercial spectrophotometer which normally has the highest useable rate of 10 samples per second. Using parallel light detection an in—situ monitoring system is set up on a planar DC sputtering deposition system. A number of metal thin films Cu, Ag and Au are produced and their reflectance and transmittance are measured in-situ. It is shown the percolation thickness of silver film produced by sputtering is about 4.7 nm. The optical constants of these films at thicknesses ranging from discrete island form to tens of nanometres are derived from the in-situ measured data using an inverse analysis technique.
The light emission from the cathode spot of a titanium vacuum arc is studied using Fizeau interferometry combined with the OMA. The temperatures of neutrals and ions in the cathode spot are determined from Doppler broadening of the emission lines. Temperatures of -3x 105K and 3.5x 104K are assigned for titanium ions and titanium atoms present in the cathode spot respectively. The light emission from a vacuum arc deposition system which has a curved magnetic solenoid between the vacuum arc chamber and the deposition chamber is also studied. The light is detected in the region near the substrate so the effect of magnetic field, ambient gases and substrate bias to the film deposition can be investigated. It is shown that the curved magnetic solenoid effectively removes the neutral species. The introduction of substrate increases the neutral emission due to resputtering of the coated substrate. The ambient gases increase ion emission as a function of their atomic weight, and the substrate bias also increases the ion emission.
Defects of optical fibres are studied by cathodoluminescence spectroscopy using the OMA and an electron microscope. The cathodoluminescence spectra obtained for various optical fibre preform samples exhibit a number of defect centres GeE', SiE' and drawing induced defects (DID). It is shown the codopant phosphorus decreases the E' defects and DID while codopants boron and fluorine decreases the DID. It is also shown the oxygen-deficient deposition enhances the GeE' centres.
Finally, the optical properties of a Bicron wavelength shifting panel (BC-480) is studied using parallel light detection technique. The quantum efficiency of the panel is found for six incident UV radiation of different wavelengths. This data is crucial in studies into solar neutrinos in high energy particle physics. They range from 43% at 365 nm to 1.5% at 254 nm.
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Braga, Mauro Sergio. "Sistemas optoeletrônicos portáteis para detecção de gases, oxigênio dissolvido e de metais pesados aplicados no controle ambiental." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/3/3140/tde-19122016-100630/.
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O monitoramento ambiental de forma contínua e em tempo real são desafios da realidade atual dos grandes centros urbanos, com a finalidade de prevenir desastres ambientais que possam pôr em perigo a saúde dos seres humanos e a existência de sistemas biológicos. Na presente tese foram propostos e desenvolvidos sistemas optoeletrônicos portáteis aplicados na detecção de O2, OD e íons de metais pesados, visando seu emprego no monitoramento de sistemas hidrológicos como mares, rios, lagos e lençóis freáticos. A definição final da estrutura do sistema portátil foi atingida após o desenvolvimento sistemático de diferentes ensaios experimentais. Primeiramente, foram estudadas e analisadas matrizes hospedeiras em estado sólido que fossem capazes de hospedar sistemas moleculares corantes sensíveis a certas substâncias específicas. A seguir, foi proposta e executada a integração direta dos filmes hospedeiros, dopados com moléculas corantes ativas, diretamente na superfície ativa de dispositivos fotodetectores para detecção de O2 e OD. Os resultados obtidos com estes sistemas que integram o detector e o filme ativo mostraram o mesmo nível de desempenho aos espectrômetros de bancada. Finalmente, de posse destes resultados, foi projetado e desenvolvido um sistema colorimétrico e fluorimétrico portátil e embarcado em uma placa de aquisição (myRIO-1900) da National Instruments, aplicado na detecção e classificação de íons metálicos. Destaque principal é outorgado à aplicação do colorímetro que, juntamente com o processamento de sinais e análises de padrões, utilizando o método de discriminante de Fisher, permitiu obter resultados excelentes na detecção e classificação dos íons de Pb2+, Cd2+, Zn2+, Cu2+, Fe3+ e Ni2+, com os mesmos níveis de desempenho que os obtidos a partir de espectrômetros de bancada de elevada resolução espectral. O sistema portátil desenvolvido sugere sua aplicação no controle ambiental in situ e em tempo real, podendo ser integrado em uma rede de sensores que possam fornecer dados de maneira contínua e receber comandos de centros de controle de monitoramento ambiental. No entanto, seria necessária a formulação de algoritmos eficientes no processo de mineração de dados da rede de sensores.The continuous and in real time environmental monitoring are challenges of the current days of large urban centers, with the aim of preventing environmental disasters that could endanger human health and the existence of biological systems. In this thesis we have proposed and developed portable optoelectronic systems applied to the detection of O2, OD and heavy metal ions, aiming its use in monitoring hydrological systems such as oceans, rivers, lakes and groundwater. The final definition of the portable system structure was achieved after the systematic development of different experimental assays. First, host matrices were studied and analyzed in solid state which were able to host dye molecular systems sensitive to certain substances. Then, we proposed and executed the direct integration of the host film, doped with active dye molecules directly on the active surface of photodetector devices to the detection of O2 and OD. The results obtained with these systems that integrate the detector and the active film showed the same level of performance than those of benchtop spectrometers. Finally, with these results, we designed and developed a colorimetric and fluorimetric portable system with an embedded acquisition board (myRIO-1900) from National Instruments, applied to the detection and classification of metal ions. Main focus is given to the application of the colorimeter which, along with the signal processing and pattern analysis using the Fisher discriminant method, allowed to obtain excellent results in the detection and classification of Pb2+, Cd2+, Zn2+, Cu2+, Fe3+ and Ni2+ ions, with the same level of performance related to those obtained from high spectral resolution benchtop spectrometers. The portable system developed in the present thesis suggests its application in environmental control in situ and in real time, so that it can be integrated in a network of sensors that may provide continuous data and receive commands from environmental monitoring control center; nevertheless, requiring for that the development of efficient algorithms in data mining process of the sensor network.
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Holubová, Zuzana. "Fluorimetrické stanovení skandia." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2008. http://www.nusl.cz/ntk/nusl-216411.
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The submitted thesis deals within the sensitive fluorimetric determination of scandium with a new reagent 8-Hydroxyquinoline-5-sulphonic acid. All important factors such as time, pH, reagent concentration, buffers, surfactants and selected interferents have been studied with respect to the selectivity, sensitivity, precision and detection limit on the determination by using classic fluorescence spectra and their first derivation. This reagent was also used for the determination of scandium in real waters. The new reagent was shortly compared to morin and lumogallion when used for scandium.
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Brown, Peter N. "Biophysical and structural characterisation of protein-peptide interactions." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/3982.
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Proliferating cell nuclear antigen (PCNA) is an essential protein in the cell. It is involved in transcription and many types of DNA repair and replication. Homologues of this protein are found in all orders of life. The high level of conservation and essential nature of PCNA infers that it may be a potential drug target for anti-caner drugs in humans and also a potential anti-parasitic target. X-ray structures of PCNA from Homo sapiens (Hs), Schizosaccharomyces pombe (Sp) and Leishmania major (Lm) are now available and can be used as a template for structure based drug design. In this work PCNA from these three species have been prepared in milligram quantities for biochemical and biophysical studies. The previously unknown structure of LmPCNA has been solved in an uncomplexed form and also complexed with a dodecapeptide to a resolution of 3.0Å. A comparison of PCNA structures and their peptide complexes for the three species identifies structural differences which may be relevant in analysing thermodynamic contributions of binding. All eukaryotic PCNA molecules exist as ring shaped trimers which form around DNA. In this work the oligomeric state of LmPCNA has been determined to be hexameric both in solution and in the crystal. It has also been hypothesised that HsPCNA is hexameric however these would seem to form hexamers in which the trimeric rings associate “back-to-back” while LmPCNA trimers would seem to associate “face-to-face”. The binding affinities for these three PCNAs have been determined with a selection of peptides derived from the Hs p21 protein. This work has shown, using a selection of different techniques including Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC) and Dynamic Scanning Fluorimetry (DSF); that HsPCNA and SpPCNA have similar affinities for a 12mer peptide (Kd of ~1μM) however LmPCNA shows significantly weaker interactions (Kd of ~10μM). This is most likely due to divergence in the sequence and structure of LmPCNA. A systematic investigation by SPR on the effect of peptide linker length on binding has been carried out using a series of synthesised peptides with different lengths of chemical spacer. The series of streptavidin immobilised peptides show that longer spacers are required for the recovery of the PCNA peptide binding affinity. The results presented in this work indicate that a linker length of at least 20Å is required for measurable protein binding activity. This interaction is improved with longer peptide spacers.
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Kleinschmidt, Ross. "An investigation of fluorometry techniques for in-vivo bone mineral determination." Thesis, Queensland University of Technology, 2001.
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Patel, Sandeep A. "Photophysics of fluorescent silver nanoclusters." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/28110.
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Thesis (M. S.)--Chemistry and Biochemistry, Georgia Institute of Technology, 2009.Committee Chair: Dickson, Robert; Committee Member: Brown, Ken; Committee Member: Curtis, Jennifer; Committee Member: Payne, Christine; Committee Member: Perry, Joseph.
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Erlandsson, Lisa-Marie. "Understanding the Involvement of Leukocyte Cell-derived Chemotaxin 2 (LECT2) in Amyloidosis." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-162631.
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Leukocyte cell-derived chemotaxin 2 (LECT2) is a zinc-binding multi-functional protein comprising three disulfide bonds, that is involved in multiple disorders of worldwide concern. Recently LECT2 was found to be involved in amyloidosis (ALECT2) and is believed to be the third most common form of systemic amyloidosis. The disease progression of ALECT2 is relatively slow, and the aggregation assembly is foremostly associated with the kidneys and the liver, but also other organs in the later onset of the disease. This study involved developing a protocol for producing His6-TEV-LECT2 including expression in E.coli BL21(DE3), refolding, and purification. The protocol resulted in a sufficient yield for initial measurements for characterization and biophysical analysis with the following methods: mass spectrometry (MS), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD), and fluorimetry. The produced protein was characterized as LECT2 predominantly in its oxidized form. A brief biophysical analysis was made where LECT2 started to unfold already at physiological temperature with a midpoint at 50°C. Additionally, under chemical denaturation LECT2 unfolded with a midpoint of 3 M urea in a cooperative transition without any intermediates. Further on, wavelengths for monitoring the unfolding and the aggregation simultaneously were identified. The unfolding process occurred under 20 sec in 6 M urea and correlates with a double-exponential model. The LECT2 aggregates resemble protofibril-like structures and aggregates species from monomer up to hexamer were found, suggesting simple monomeric addition towards a growing fibril as the aggregation mechanism. The content of aggregates in the sample was notably decreased upon disulfide bond reduction highlighting the importance of further investigating the role of the disulfide bonds in the destabilization and aggregate formation of LECT2.
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Guglielmino, Maud. "Développement d'une nouvelle méthode analytique du formaldéhyde dans l'air basée sur un dispositif microfluidique." Thesis, Strasbourg, 2014. http://www.theses.fr/2014STRAF048.
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Le formaldéhyde (HCHO) est un polluant majeur de l’air intérieur. L’objectif de cette thèse est de réaliser les avancées scientifiques et technologiques nécessaires à l’obtention d’une méthode analytique basée sur un dispositif microfluidique de mesure du formaldéhyde dans l’air associant précision, sélectivité, rapidité d’analyse avec pour objectif majeur une autonomie suffisante sur de longues durées, typiquement un mois. Le principe de la méthode reposait initialement sur trois étapes clés, à savoir le piégeage du formaldéhyde gazeux en solution, la réaction du formaldéhyde avec un agent dérivatif, puis la détection du produit de dérivation par colorimétrie ou fluorimétrie. La méthode a finalement évolué vers seulement deux étapes distinctes grâce à l’utilisation d’un dispositif microfluidique innovant dans lequel le piégeage et la réaction ont lieu simultanément. L’étude des performances analytiques du dispositif a permis de valider la méthode développée pendant cette thèseFormaldehyde (HCHO) is a major pollutant in indoor air. The objective of this work is to realize the scientific and technological advances required to obtain an analytical method based on a microfluidic device to measure air formaldehyde combining precision, selectivity, analysis speed with for major objective a sufficient autonomy on a long time, typically one month. The principle of the method was initially based on three key steps, the gaseous formaldehyde uptake in solution, the formaldehyde derivatization reaction, then the detection of reaction product by colorimetry or fluorimetry. The method has finally advanced toward only two definite steps thanks to the use of an innovative microfluidic device in which uptake and reaction take place simultaneously. The study of analytical performances of the device allows to validate the method developedduring this work
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Dong, Jian. "Photochemical and Photophysical Studies of Synthetic Derivatives of the Green Fluorescent Protein Chromophore." Diss., Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/24655.
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We have synthesized dimethyl derivatives of the GFP chromophore (p-HOBDI) and several of its derivatives, and their photochemistry and photophysics were investigated using various steady-state and time-resolved techniques as follows.
We first consider the effect of the £]-barrel on the optical properties of the GFP chromophore (p-HOBDI) experimentally by selective variation of the protonation state of chromophores and different solvents. Each of these forms shows a complex solvatochromic behavior and is governed by both polar and acid/base properties of the solvents. In contrast to their solution behavior, some O-alkyl GFP chromophore (alkoxy-BDI) derivatives exhibit large fluorescent enhancement in the solid state. The color of the crystalline BDI is tuned by substituent-mediated crystal packing, showing the potential applications in optoelectronic devices.
Using femtosecond polarization-sensitive infrared (IR) spectrosceopy of the C=O stretching mode of the HOBDI, we have then discovered a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation. Cis/trans isomerization induced by the rotation around the bridge is thought to be responsible for the behavior of blinking in fluorescent protein; however, the mechanism of the thermal reverse isomerization is more problematic. Thus we synthesized BDI derivatives with decreasing para-donating ability, HO, CH3O, CH3, H, and Cl, and used a Hammett plot for the rate study. With a positive â value, we conceived, for the first time, a novel nucleophilic addition/elimination mechanism.
Finally, the GFP chromophore falls into the general category of hydroxyarene photoacids, which exhibit high excited-state acidities but neutral ground states. A hydroxyl substituent at the meta position shows enhanced charge transfer and greater acidity in the excited state. As a result, we have demonstrated that the fast quenching of the excited state by internal conversion to the ground state is much slower in meta- than in para-HOBDI derivatives. This allows studies of this ultrafast intermolecular ESPT that competes with isomerization. The photoinduced dynamics of the meta isomer of GFP chromophore was further investigated using femtosecond transient absorption and fluorescence upconversion spectroscopies.
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Merchant, David Frank. "Optical fibre fluorimeter for online measurement." Thesis, Liverpool John Moores University, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.313161.
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Barnett, Alexandre. "Régulation de l'activité photosynthétique du microphytobenthos et conséquence sur la dynamique temporelle de la production primaire dans les vasières intertidales de la côte atlantique de l'Europe de l'Ouest." Thesis, La Rochelle, 2013. http://www.theses.fr/2013LAROS412/document.
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Le microphytobenthos (MPB) des latitudes tempérées est dominé par les diatomées. Deux grands groupes se distinguent, les épipéliques (mobiles) des sédiments vaseux, et les épipsammiques (fixées) des sédiments sablo-vaseux. Afin de mieux comprendre la production des vasières, le MPB a été étudié par des approches du niveau physiologique au niveau écologique. Dans un premier temps, l’étude s’est focalisée sur des expérimentations en laboratoire. La réponse des différents groupes à la lumière a montré que la forme de vie et la mobilité sont en lien étroit avec la capacité de photoprotection physiologique. Ainsi, les diatomées non-mobiles présentent une meilleure photoprotection physiologique que les diatomées mobiles qui peuvent fuir les excès de lumière. Dans une deuxième partie, le travail s’est effectué sur des échantillons ramenés en laboratoire. Des profils de migrations ont été réalisés par mesure continue de la fluorescence. Il a été établi que le MPB présente un rythme de migration interne pouvant être modulé par la lumière. De plus la qualité de la lumière modifie les profils de migration. Il est communément admis que les phases de division cellulaire se dérouleraient en profondeur. La cytométrie en flux permet de vérifier cette hypothèse. Finalement les mesures effectuées en laboratoire ont été comparées à des mesures effectuées directement sur le terrain à l’échelle de la communauté. Il a ainsi pu être vérifié que la photoprotection sous lumière fluctuante est fonction de la population. Pour les populations épipéliques, la photoprotection physiologique ne varie pas au cours des fluctuations lumineuses, laissant supposer que la migration module ces fluctuations. Les populations épipsammiques, quant à elles, modifient leur réponse physiologique en fonction des fluctuations lumineusesMicrophytobentos (MPB) from temperate latitude is mainly composed of diatoms. Those microorganisms can be separated in two groups: the epipelic one from muddy sediments (composed of mobile diatoms) and the epipsammic one from sandy-muddy sediments (composed of diatoms living attached to their substrate). In order to investigate mudflats’ primary production, the MPB compartment was studied through diverse approaches from the physiological level to the ecological one. In the first place, laboratory experiments (in vitro experiments), focusing on light reaction of epipelic and epipsammic diatoms, showed that their life form and their mobility were strongly connected to their physiological photoprotection ability. Thereby, the motionless diatoms were characterized by higher physiological photoprotection abilities than the mobile ones, which could avoid excess of light. In the second place, the fluorescence of collected samples (in vivo experiments) was measured to acquire diatoms’ migration profiles. The results pointed out an internal and light-regulated migration pattern of the MPB and furthermore highlighted the effect of light quality on migration profiles. Besides, the commonly accepted hypothesis of deep cell division phases was tested and confirmed through flow cytometry experiments. Eventually, laboratory measurements were compared to in situ ones realized at the scale of the whole community. These comparisons revealed that diatoms photoprotection in fluctuating light depended on the targeted populations. Epipelic organisms were indeed characterized by an unvarying photoprotection, diatoms migration regulating alone the effect of light fluctuations. On the contrary, motionless epipsammic populations required a light-regulated photoprotection
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Means, John A. "Fluorescence and NMR Characterization of a T Box Antiterminator-tRNA Complex." View abstract, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3289332.
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Whiteside, Ian Robert Crosby. "Fluorimetric determination of amines in non-aqueous media." Thesis, University of Hull, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.278393.
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Pokora, Zdeněk. "Využití fluorimetrie pro detekci stopovačů proudění podzemních vod." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2008. http://www.nusl.cz/ntk/nusl-216445.
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The thesis studies detection of fluorescein for coloration experiments in surface and underground water. The first part of the work deals with the adsorption of fluorescein on active charcoal from water and desorption by means of different desorption solutions. The results of measurements are used for practice of coloration experiments in karst research. In the second part of thesis it is researched the option of automated record of fluorescence concentration and its detection by means of laser induced fluorescence with confocal microscope.
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Ali, Rashid Majid Yousif. "Fragment-screening by X-ray crystallography of human vaccinia related kinase 1." Thesis, Linköpings universitet, Institutionen för fysik, kemi och biologi, 2020. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-166811.
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Fragment-screening by X-ray crystallography (XFS) is an expensive and low throughput fragment drug discovery screening method, and it requires a lot of optimization for each protein target. The advantages with this screening method are that it is very sensitive, it directly gives the three-dimensional structure of the protein-fragment complexes, and false positives are rarely obtained. The aim of this project was to help Sprint Bioscience assess if the advantages with XFS outweigh the disadvantages, and if this method should be used as a complement to their differential scanning fluorimetry (DSF) screening method. An XFS campaign was run using the oncoprotein vaccinia related kinase 1 (VRK1) as a target protein to evaluate this screening method. During the development of the XFS campaign, a diverse fragment library was created which consisted of 298 fragments that were all soluble in DMSO at 1 M concentration. The crystallization of the protein VRK1 was also optimized in this project to get a robust, high throughput crystallization set up which generated crystals that diffracted at higher resolution than 2.0 Å when they were not soaked with fragments. The soaking protocol was also optimized in order to reduce both the steps during the screening procedure and mechanical stress caused to the crystals during handling. Lastly, the created fragment library was used in screening VRK1 at 87.5 mM concentration with XFS. 23 fragment hits could be obtained from the X-ray crystallography screening campaign, and the mean resolution of the crystal structures of the protein-fragment complexes was 1.87Å. 11 of the 23 fragment hits were not identified as hits when they were screened against VRK1 using DSF. XFS was deemed as a suitable and efficient screening method to complement DSF since the hit rate was high and fragments hits could be obtained with this method that could not be obtained with DSF. However, in order to use this screening method a lot of time needs to be spent in optimizing the crystal system so it becomes suitable for fragment screening. Sprint Bioscience would therefore need to evaluate the cost/benefit ratio of using this screening method for each new project.
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Chang, Q. "Colorimetric and fluorimetric plastic film sensors for carbon dioxide." Thesis, Swansea University, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.636226.
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Medical diagnosis and treatment of critically ill patients require frequent monitoring of carbon dioxide partial pressure in the human body. Along with the traditional methods (i.e. IR capnography and Severinghaus electrode), colorimetric and fluorimetric CO2 sensors are playing an increasingly important role in detection of correct intubation, intensive care and blood CO2 analysis, due to its advantages of cheapness in cost, miniature in size, and mechanically robust. In Chapter One an outline of the recent development of such sensor systems is given, and their applications background in the biomedical area is discussed. Chapter Two focuses on the experimental techniques used in these studies. In Chapters Three and Four the equilibrium responses of three new colorimetric and one fluorescence plastic film sensors for CO2 as a function of % CO2 and temperature are described, respectively. The results fit a model in which there is a 1:1 equilibrium reaction between the deprotonated form of the dye (present in the film as an ion pair) and CO2. The basic theory behind conventional colorimetric and fluorimetric optical sensors for CO2 is proposed and examined in Chapter Five. Special attention is given to the effect on sensor response of the key parameters of initial base concentration and dye acid dissociation constant, KD. In Chapter Six the diffusion-controlled response and recovery behaviour of a naked optical film sensor with a hyperbolic-type reponse changes in analyte concentration in a system under test is approximated using a numerical model, followed by a second part in which a systematic, experimental investigation on the kinetics, responses and recovery behaviour of the colorimetric film sensors is described. Finally, in Chapter Seven, a plasticised and unplasticised polymer colorimetric film sensor for gaseous CO2 is tested as a sensor for dissolved CO2. The plasticised form of the film sensor develops a measurable degree of opacity when exposed to aqueous solution, while an unplasticised polymer remains largely clear upon exposure to aqueous solution and it is shown that it also functions as a quantitative sensor for dissolved CO2 over the range 0-4% CO2.
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McConnell, Brendan Neil. "Fragment-based approaches to targeting EthR from mycobacterium tuberculosis." Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/290255.
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Tuberculosis affects millions of people worldwide every year. The current treatment for TB is divided into a regimen of both first- and second-line drugs, where first-line treatments are more tolerated and require shorter treatment lengths. With rising levels of resistance, alternative treatment regimes are urgently needed to fight this disease. Ethionamide, a second-line drug is administered as a prodrug which is activated in vivo by the enzyme EthA, which is in turn regulated by EthR. The disruption of the action of EthR could lead to novel therapeutics which could enhance the efficacy of ethionamide, and raise it to a first-line treatment. The work reported in this thesis examines the elaboration of three chemical scaffolds using fragment-based approaches to develop novel inhibitors capable of disrupting the EthR-DNA interaction. The first scaffold, 5-(furan-2-yl)isoxazole was investigated by fragment-merging approaches and produced compounds with the best of these having a KD of 7.4 uM. The second scaffold, an aryl sulfone was elaborated using fragment-merging strategies. This led to several modifications of the fragment, leading to several variants with KDs around 20 uM. With both of these series the affinity could not be improved below 10 uM and due to the synthetic complexity a further scaffold was prioritised. The third scaffold was explored was a 4-(4-(trifluoromethyl)phenyl)piperazine using fragmentgrowing from the NH of the piperazine to probe deeper into the EthR binding pocket. In addition to this, SAR around the 4-(trifluoromethyl)phenyl group was assessed to explore the interactions with EthR. These modifications led to compounds with nanomolar IC50s. A range of compounds were then screened by REMAssay to determine the boosting effect on ethionamide, and this identified compounds with up to 30 times boosting in the ethionamide MIC. The final chapter examines a concept where compounds were designed to exploit the dimeric nature of EthR by linking two chemical warheads with a flexible linker. These compounds are examined using mass spectrometry to investigate the stoichiometry of the interaction to provide insight into the binding of these extended compounds and exploring an alternative strategy to inhibit EthR. The work in this thesis demonstrated the successful use of fragment-based approaches for development of novel EthR inhibitors which showed significant ethionamide boosting effects.
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Silva, Carlos Eduardo Tanajura da. "Classificação tipo/titulação de óleos almentícios por fluorimetria e redes neurais." Escola Politécnica /Instituto de Matemática, 2014. http://repositorio.ufba.br/ri/handle/ri/22924.
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O Brasil destaca-se entre os maiores exportadores de grãos do mundo, sendo assim, cada vez mais surgem novos produtos e derivados destas Commodity. Os métodos para classificação desses produtos muitas vezes são custosos e demorados, quase sempre se valendo de técnicas de química analítica e métodos matemáticos como PCA (Principal Component Analysis), PCR (Principal Components Regression) ou PLS (Properties of Partial Least Squares) e RNA (Redes Neurais Artificiais) para aumentar sua eficiência. Devido à grande variedade de produtos são necessários métodos mais eficientes para qualificar, caracterizar e classificar estas substâncias, uma vez que o preço final deve refletir a excelência do produto que chega ao consumidor. Este trabalho propõe uma solução para classificação de óleos vegetais: Canola, Girassol, Milho e Soja colocados no mercado por diferentes marcas e fabricantes. O método de análise empregado é a fluorescência induzida por LED de amostras de óleo diluídas em heptano, com diferentes concentrações, sendo que a classificação dos espectros de fluorescência foi feita por RNA. Foram produzidas e caracterizadas 640 amostras, sendo 480 para treinamento da rede neural e 160 para sua validação. Para a classificação das amostras de fluorescência, os dados foram organizados em dois estudos, o primeiro com referência ao tipo das amostras, o segundo a titulação, este por final contento três arranjos dos dados e RNAs distintas. Na classificação do tipo das amostras, a rede conseguiu identificar 115 amostras, tendo acertado aproximadamente 72% destas amostras de validação. A classificação por titulação, utilizou a metade das amostras de fluorescência, o universo de treinamento passou a ter 240 amostras, as de validação 80. Para esse segundo estudo houve 3 arranjos desses dados, o resultado do primeiro arranjo teve 33 amostras classificadas com sucesso de 80, o segundo 49 e o terceiro 31.
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El, Kirat Sofiane. "Développement d’outils cellulaires et moléculaires pour l’étude des interactions Candida - phagocytes ; Application à la caractérisation du gène OLE2 codant une désaturase chez C. lusitaniae." Thesis, Bordeaux 2, 2010. http://www.theses.fr/2010BOR21774/document.
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Les levures Candida sont des pathogènes opportunistes responsables d’infections graves chez les patients immunodéprimés. Au cours de ce travail, nous avons développé un modèle cellulaire in vitro pour la caractérisation multiparamétrique des phénotypes d’interaction entre les levures Candida et les macrophages et les neutrophiles, principaux effecteurs de la défense anti-Candida. Il repose sur l’utilisation de marqueurs fluorescents pour le suivi quantitatif de l’interaction en cytométrie en flux et en fluorimétrie. Ce modèle a été validé par la comparaison de l’interaction de trois espèces de levures, C. albicans, C. glabrata et C. lusitaniae, avec des macrophages murins et des neutrophiles humains. Deux stratégies principales de survie des levures à la phagocytose ont été mises en évidence : par la résistance à la phagolyse et la multiplication des levures à l’intérieur des phagocytes jusqu’à leur éclatement, ou par l’évitement de la phagocytose et la multiplication des levures à l’extérieur des phagocytes. L’interprétation des données quantitatives a été confirmée par microscopie à fluorescence et vidéo-microscopie. Afin de mieux comprendre les interactions Candida-phagocytes, nous avons mis au point des outils pour l’analyse fonctionnelle de gènes chez C. lusitaniae. Une stratégie de PCR chevauchante a été développée pour l’obtention de mutants nuls de C. lusitaniae, sans étape de clonage. C’est ainsi que le gène OLE2, codant une Δ9 désaturase d’acides gras potentiellement impliquée dans la biosynthèse de la prostaglandine PGE2, a été invalidé. Le mutant ole2Δ présentait de très nets défauts de filamentation et de reproduction sexuée. Par rapport à une souche sauvage, le mutant ole2∆ était massivement phagocyté par les macrophages, et la survie des phagocytes était plus importante, ce qui suggère un rôle important des lipides insaturés et des oxylipides dans la signalisation cellulaire au cours de l’interaction Candida-phagocytes. Dans la dernière partie de notre travail, nous avons construit une banque de 10 000 mutants de C. lusitaniae par l’intégration aléatoire d’un marqueur dans le génome. Le criblage de cette banque à travers notre modèle cellulaire d’interaction permettra d’identifier de nouveaux gènes impliqués dans l’interaction avec les phagocytes afin de mieux comprendre la physiopathologie des candidoses et de trouver de nouvelles pistes thérapeutiquesCandida species are opportunistic pathogens causing severe infectious diseases in immunocompromised patients. In this work, we developed a tool for a multi-parameter characterization of the cell interactions between the yeasts Candida and both macrophages and neutrophils, which constitute the main defense against candidiasis. It relies on the labelling of each population with specific fluorescent markers, and on the use of fluorimetry and flow cytometry to assess interactions. The tool has been validated by comparing the interactions of three yeast species C. albicans, C. glabrata and C. lusitaniae, with murine macrophages and human neutrophils. We found that yeasts use two main ways for escaping phagocytosis, which has been confirmed using video-microscopy: either (1) by surviving to phagolysis and dividing into the phagosome until phagocytes burst, or (2) by avoiding phagocytosis and dividing outside phagocytes. In order to better understand the cellular and molecular mechanisms involved in Candida-phagocytes interactions, we developed new molecular tools for the functional analysis of genes in C. lusitaniae, notably a two-step cloning-free PCR-based method for the deletion of genes. This method was successfully used for the deletion of OLE2, a gene encoding a Δ9-desaturase of fatty acids, possibly implicated in prostaglandin PGE2 biosynthesis. The ole2Δ mutant exhibited strong defects in both pseudofilamention and sexual mating. During macrophages infection, ole2Δ yeast cells were massively internalized and triggered less phagocytes cell death than the wild type strain, suggesting that unsaturated fatty acids and/or oxylipids could play a role during interaction with phagocytes. Lastly, a bank of 10,000 mutants was constructed in C. lusitaniae by the random integration of a genetic marker in the genome. The screening of this bank through our tool to analyse cellular interactions will be undertaken to gain insights into understanding of the early stages of the infectious process
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Lee, Lok Yan. "Study of the photodegradation and photostability of anti-cancer drugs in different media towards the development of both new actinometers and liquid formulations." Thesis, De Montfort University, 2016. http://hdl.handle.net/2086/12188.
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This study aims at tackling some of the problems often encountered in photostability testing and liquid formulation development. Three anti-cancer drugs will be employed as models; Dacarbazine (DBZ) has well established photostability issues, Axitinib (AXI) and Sunitinib (SUT) are two new drugs only commercially available in solid dosage forms. In ethanol, the photokinetics of these drugs were well described by the newly proposed Φ-order kinetic mathematical model. This has confirmed the photoreversible character of AXI and SUT’s and unimolecular photoreaction of DBZ’s photodegradations. Also, the Φ-order kinetics is proven to describe them better than the usually used classic thermal reaction orders. In aqueous solution, the drugs were found to undergo thermal and photochemical complex degradations, involving at least 3 photoproducts. A new photokinetic approach has been proposed in this work to solving and unravelling the attributes of such complex mechanisms. For the first time, the quantum yields (QY) of the three drugs were determined and found to increase with irradiation wavelength. SUT’s QY were comparable in ethanol and water (QY460 = 0.02), DBZ was found to be more photoefficient in water (QY330 = 0.04 and 0.1, respectively) and AXI in water (QY330 = 0.06 and 0.03). Φ-order kinetics’ potential for the development of reliable actinometers of the three drugs, without prior knowledge of unknown reaction parameters, has also been established. A general equation to describe the isotherm of a (Gn:Hm) guest-host multicomponent complex was proposed in this work to palliate the lack of a strategy for characterising nanosponge-drug complexes. It provides information on both stiochiometry and association constant of the complex. The results indicate that hydrophobic AXI forms a 1:0.8 complex, indicating the possibility of multiple association sites and/or different types of binding. The newly developed AXI/nanosponge liquid formulation has significantly increased solubility (5000-fold) and thermal stability. Furthermore, the photostability of DBZ and SUT were considerably improved by using a strategy based on light-absorption competitors. Their initial velocities reduced from 10 and 3 s-1 (respectively) to 1 and 0.13 s-1. The successful application of these methods to the model anti-cancer drugs has set out new approaches that might be found useful for future treatments of photodegradation data, development of drug-actinometers and liquid formulations of drugs.
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Williams, Mark R. "pH and calcium regulation in the lens epithelial cells : a fluorimetric dye study." Thesis, University of East Anglia, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.334394.
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Duschl, Josef. "Messung des Anisotropieabklingverhaltens einer Porphyrinprobe in Mizellen mit Hilfe eines selbstgebauten Frequenz-Domäne-Fluorimeters." kostenfrei, 2004. http://www.opus-bayern.de/uni-regensburg/volltexte/2004/244/.
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Milich, Kacy. "A ratiometric fluorometer for reduced sensitivity against solvent artifacts." Diss., Columbia, Mo. : University of Missouri-Columbia, 2005. http://hdl.handle.net/10355/5837.
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Thesis (M.S.)--University of Missouri-Columbia, 2005.The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 24, 2007) Includes bibliographical references.
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Reis, Rodrigo Alexandre [UNESP]. "Desenvolvimento de equipamento multifuncional portátil de baixo custo para determinações fotométricas, turbidimétricas, nefelométricas e fluorimétricas." Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/110845.
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000775320.pdf: 1787970 bytes, checksum: f55fdff75708b286b90a8cbdd7ba34d9 (MD5)Foi desenvolvido um equipamento de baixo custo, portátil e multifuncional que integra num único dispositivo quatro técnicas analíticas: fotometria, turbidimetria, nefelometria e fluorimetria. Os principais componentes do equipamento proposto são: diodo emissor de luz (LED) como fontes de radiação; resistor dependente de luz (LDR) como fotodetector; multímetro digital como dispositivo de leitura; um bloco cilíndrico de Nylon preto. Para avaliar o desempenho do equipamento, fez-se a determinação de fósforo (fotometria) e de enxofre (turbidimetria) em fertilizante, de enxofre em material vegetal (nefelometria) e de quinina em água tônica (fluorimetria). As determinações também foram feitas em um equipamento comercial para fins comparativos. Para a determinação de fósforo pelo método do fosfovanadomolibdato, a faixa de trabalho foi de 0,07 a 6,52 mg L-1 (r= 0,9944) e o limite de detecção foi de 0,06 mg L-1. Para a determinação turbidimétrica de sulfato pelo método da precipitação de sulfato de bário, a calibração foi de 2,5 a 100 mg L-1 (r= 0,9982), sendo o limite de detecção 0,23 mg L-1. Na determinação nefelométrica de sulfato, a faixa linear foi de 0,2 a 1,8 mg L-1 (r= 0,9948) e o limite de detecção de 0,10 mg L-1. Na determinação fluorimétrica de quinina, a faixa linear foi de 0,16 a 8,0 mg L-1 (r= 0,9982) e limite de detecção de 0,011 mg L-1. Os resultados obtidos foram comparados com aqueles fornecidos por equipamentos comerciais e resultados concordantes foram observados ao nível de 95% de confiança. Materiais de referência foram analisados pelo equipamento proposto e os resultados para fósforo e enxofre foram concordantes com os respectivos valores certificados ao nível de 95% de confiança. Além do potencial analítico, o equipamento proposto apresenta utilidade como ferramenta didática. O aprendizado do conteúdo de métodos ópticos de análise pode ser estimulado quando há melhor...
A new portable, low cost and multifunctional equipment comprising four analytical techniques (photometry, turbidimetry, nephelometry and fluorimetry) in one single device was developed The main components of the developed device are light emitting diode (LED) as radiation source, light-dependent resistor (LDR) as photodetector, digital multimeter as reading device, and a black plastic container. The performance of the device was checked by determining phosphorus in fertilizer (photometry), sulfur in fertilizer (turbidimetry), sulfur in vegetable samples (nephelometry) and quinine in tonic water (fluorimetry). All determinations were also made in commercial equipments for comparison purposes. The phosphovanadomolibdate method was employed for phosphorus determination within the 0.07 – 6.52 mg L-1 concentration range ((r= 0.9944), and the detection limit was 0.06 mg L-1. The precipitation of barium sulfate was employed for turbidimetric and nephelometric determination of sulfate. For the turbidimetric technique, the calibration range was in the 2.5 - 100 mg L-1 (r= 0.9982), and the detection limit was 0.23 mg L-1. For nephelometry, calibration within 0.2 – 1.8 mg L-1 (r= 0.9948) resulted in detection limit of 0.10 mg L-1. The fluorimetric determination of quinine in the 0.16 – 8.0 mg L-1 linear working range (r= 0.9982) resulted in 0.011 mg L-1 detection limit. Results found by the proposed device were in agreement with those obtained by commercial equipments at 95% confidence level. Standard reference materials were also analyzed, and results for P and S were in agreement with certified values. Besides analytical capabilities, the proposed device may be useful for teaching. The learning goals of some optical techniques may be easier using easy-to-make instruments.
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Yamazaki, Naho. "Hyperosmotic sensitivity of the Na'+ x H'+ exchanger, NHE1, in bovine articular chondrocytes." Thesis, University of Oxford, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365787.
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Pereira, Eduardo Ferreira. "Desenvolvimento de sistema automatizado para o monitoramento da degradação de resíduos de brometo de etídio." reponame:Repositório Institucional da UnB, 2015. http://repositorio.unb.br/handle/10482/20496.
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Tese (doutorado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2015.Submitted by Fernanda Percia França (fernandafranca@bce.unb.br) on 2016-03-03T12:48:10Z No. of bitstreams: 1 2015_EduardoFerreiraPereira.pdf: 3187364 bytes, checksum: 50b7a71b45f3b71d8af9c22fc9653768 (MD5)
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O Brometo de Etídio (BE) é um composto fluorescente que tem seu rendimento quântico muito aumentado quando intercalado à moléculas de DNA e RNA e, por isto, é usado para detecção destas espécies em procedimentos de eletroferese em gel. Devido a estas características, esta substância é considerada perigosa, pois pode causar danos ao material genético humano sendo classificado como um agente mutagênico /tóxico. Para realização do seu descarte é necessário um processo de inertização, sendo um dos mais aplicados o proposto por Lunn e Sansone em que são utilizados o ácido hipofosfórico e o nitríto de sódio para oxidação da substância. Porém, esta é uma reação de longa duração e a sua finalização deve ser atestada com o uso da detecção por fluorescência. Considerando este aspecto, o principal objetivo do trabalho foi construir e avaliar um sistema automatizado de baixo custo com detecção fluorimétrica, empregando-se um LED (máximo de emissão em 535 nm) para monitorar a degradação do BE. Este instrumento é controlado por microcontroladores (PIC 16F877A e PIC 16F628) que são responsáveis pelos controles dos processos de fluxo das amostras e do fluido carreador e por adquirir e processar o sinal do sistema de detecção, composto de dois fotodiodos e como filtros para, emissão do BE, foram utilizados plásticos de coloração alaranjada. O Fluorímetro proposto foi avaliado tanto em batelada quanto em um sistema de fluxo e apresentou limites de detecção de 0,33 mg L-1 no sistema de fluxo com abordagem stoped-flow e 0,04 mg L-1 em batelada (utilizando cubeta). As medidas realizadas com o instrumento foram comparadas ao de um fluorímetro comercial de bancada e em todas as comparações não foram observadas diferenças significativas ao nível de 95% de confiança. Foram realizados testes para verificar se o sistema de decisão do instrumento proposto era confiável para indicar automaticamente o final da degradação sendo observada uma taxa de 100% de acerto para as amostras testadas com concentrações acima do limite máximo permitido e abaixo deste limite (5 mg L-1). Alternativamente, avaliou-se a possibilidade de uso de um sistema de detecção de BE utilizando um LED como fonte de excitação e medidas das componentes RGB de imagens a partir de um aplicativo e da câmera de um aparelho celular. Para esta abordagem foi encontrado um limite de detecção de 0,91 mg L-1 utilizando apenas componente R. Comparando-se os resultados obtidos para amostras fortificadas com BE realizadas com o fluorímetro proposto e com a detecção via celular com os resultados obtidos com um espectrofluorímetro comercial, não foram observadas diferenças significativas ao nível de 95 % de confiança. O Fluorímetro de LED proposto se mostrou eficiente e teve um rendimento satisfatório a um baixo custo (cerca de US$ 200) podendo ser aplicado para a detecção de BE no procedimento de descarte ou na detecção destes em amostras residuais. ______________________________________________________________________________ ABSTRACT
Ethidium Bromide (BE) is a fluorescent compound which has a significant enhancement of quantum yield when intercalated with RNA and DNA molecules and, due to such effect, it is applied for detection of these species in gel electrophoresis. For that reason, BE is considered dangerous, producing damages to human genetic material and classified as a mutagenic/toxic agent. In order to discard BE residues, is necessary a prior process to turn the substance inert and one of the most applied protocols is the proposed by Lunn and Sansone, where hipofosforic acid and sodium nitrite are used to oxidize the BE. However, this reaction requires a long time period and its finalization must be attested by fluorescence measurements. Considering this, the main objective of this work was to fabricate and evaluate a low cost automated system with fluorometric detection, using a LED (maximum emission in 535 nm) to to monitor BE degradation. The proposed instrument is controlled by microcontrollers (PIC 16F877A and PIC 16F628) that controls the sample and carrier flows and performs the acquisition and processing of the analytical signal from the detector, comprising two photodiodes covered by two pieces of transparent orange plastic. The proposed fluorometer was tested with the use of batch or flow based strategies. Using a stopped-flow system, the limit of detection of 0.33 mg L-1 was achieved and for the batch determination the detection limit was 0.04 mg L-1. The determinations performed with the proposed LED fluorometer were compared to the determinations performed with a commercial spectrofluorometer with no significant differences at 95 % confidence level. Tests were also conducted to verify if the decision system of the proposed instrument was reliable to automatically indicate the end of degradation, being checked 100 % of accuracy for the tested samples with BE concentrations beyond and below the limit of concentration of 5 mg L-1. Alternatively, the detection of BE using a green LED as excitation source and measurements of RGB pattern from photographic images acquired by a cell phone was evaluated. For this approach, the detection limit of 0.91 mg L-1 was stimated using only the Red component of RGB. By comparing the results acquired with the proposed fluorometer/cell phone detection with the data obtained with a commercial spectrofluorometer for BE fortified samples, no significant differences were observed at the 95 % confidence level. The proposed LED Fluorometer was efficient and had a satisfactory performance at a low cost (US$ 200) and can be applied to the detection of BE in the disposal procedure or detecting in residual samples.
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Reis, Rodrigo Alexandre. "Desenvolvimento de equipamento multifuncional portátil de baixo custo para determinações fotométricas, turbidimétricas, nefelométricas e fluorimétricas /." Araraquara, 2014. http://hdl.handle.net/11449/110845.
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Orientador: José Anchieta Gomes NetoBanca: Marisa Spirandeli Crespi
Banca: Jorge Luiz Raposo Júnior
Resumo: Foi desenvolvido um equipamento de baixo custo, portátil e multifuncional que integra num único dispositivo quatro técnicas analíticas: fotometria, turbidimetria, nefelometria e fluorimetria. Os principais componentes do equipamento proposto são: diodo emissor de luz (LED) como fontes de radiação; resistor dependente de luz (LDR) como fotodetector; multímetro digital como dispositivo de leitura; um bloco cilíndrico de Nylon preto. Para avaliar o desempenho do equipamento, fez-se a determinação de fósforo (fotometria) e de enxofre (turbidimetria) em fertilizante, de enxofre em material vegetal (nefelometria) e de quinina em água tônica (fluorimetria). As determinações também foram feitas em um equipamento comercial para fins comparativos. Para a determinação de fósforo pelo método do fosfovanadomolibdato, a faixa de trabalho foi de 0,07 a 6,52 mg L-1 (r= 0,9944) e o limite de detecção foi de 0,06 mg L-1. Para a determinação turbidimétrica de sulfato pelo método da precipitação de sulfato de bário, a calibração foi de 2,5 a 100 mg L-1 (r= 0,9982), sendo o limite de detecção 0,23 mg L-1. Na determinação nefelométrica de sulfato, a faixa linear foi de 0,2 a 1,8 mg L-1 (r= 0,9948) e o limite de detecção de 0,10 mg L-1. Na determinação fluorimétrica de quinina, a faixa linear foi de 0,16 a 8,0 mg L-1 (r= 0,9982) e limite de detecção de 0,011 mg L-1. Os resultados obtidos foram comparados com aqueles fornecidos por equipamentos comerciais e resultados concordantes foram observados ao nível de 95% de confiança. Materiais de referência foram analisados pelo equipamento proposto e os resultados para fósforo e enxofre foram concordantes com os respectivos valores certificados ao nível de 95% de confiança. Além do potencial analítico, o equipamento proposto apresenta utilidade como ferramenta didática. O aprendizado do conteúdo de métodos ópticos de análise pode ser estimulado quando há melhor...
Abstract: A new portable, low cost and multifunctional equipment comprising four analytical techniques (photometry, turbidimetry, nephelometry and fluorimetry) in one single device was developed The main components of the developed device are light emitting diode (LED) as radiation source, light-dependent resistor (LDR) as photodetector, digital multimeter as reading device, and a black plastic container. The performance of the device was checked by determining phosphorus in fertilizer (photometry), sulfur in fertilizer (turbidimetry), sulfur in vegetable samples (nephelometry) and quinine in tonic water (fluorimetry). All determinations were also made in commercial equipments for comparison purposes. The phosphovanadomolibdate method was employed for phosphorus determination within the 0.07 - 6.52 mg L-1 concentration range ((r= 0.9944), and the detection limit was 0.06 mg L-1. The precipitation of barium sulfate was employed for turbidimetric and nephelometric determination of sulfate. For the turbidimetric technique, the calibration range was in the 2.5 - 100 mg L-1 (r= 0.9982), and the detection limit was 0.23 mg L-1. For nephelometry, calibration within 0.2 - 1.8 mg L-1 (r= 0.9948) resulted in detection limit of 0.10 mg L-1. The fluorimetric determination of quinine in the 0.16 - 8.0 mg L-1 linear working range (r= 0.9982) resulted in 0.011 mg L-1 detection limit. Results found by the proposed device were in agreement with those obtained by commercial equipments at 95% confidence level. Standard reference materials were also analyzed, and results for P and S were in agreement with certified values. Besides analytical capabilities, the proposed device may be useful for teaching. The learning goals of some optical techniques may be easier using easy-to-make instruments.
Mestre
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Surribas, i. Casalprim Anna. "Millora en el procés de producció d'una lipasa de Rhizopus oryzae en Pichia pastoris mitjançant tècniques de monitoratge i estratègies de cultiu alternatives." Doctoral thesis, Universitat Autònoma de Barcelona, 2009. http://hdl.handle.net/10803/5328.
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En aquest treball s'exposen dues línies de recerca centrades en el procés de producció d'una lipasa de Rhizopus oryzae (ROL) en Pichia pastoris: la utilització de diferents tècniques de monitoratge i l'aplicació d'estratègies de cultiu que permetin millorar la producció de la ROL en una soca Mut+ d'aquest sistema d'expressió. En els cultius discontinus alimentats amb P. pastoris cal disposar d'una mesura en línia i en temps real de la concentració de substrat, el metanol. Per això es va desenvolupar un analitzador d'injecció seqüencial. Es va comprovar que aquest té una freqüència d'anàlisi òptima per a cultius amb la soca Muts però baixa per a la soca Mut+. Amb aquesta última soca, es van utilitzar i comparar dos mesuradors comercials en fase gas.
Es va avaluar també la fluorimetria com a tècnica de monitoratge per a la determinació de tres variables clau: la biomassa, el substrat (glicerol/metanol) i la proteïna recombinant.
Inicialment es va fer un seguiment de l'evolució de la biomassa a partir del senyal de fluorescència off-line del triptòfan. Es pot predir la biomassa correctament però es van evidenciar diferents desavantatges: la necessitat de dilució de les mostres i d'un sistema de presa de mostra específic. Per això es va decidir aplicar i avaluar la fluorimetria multivariable in situ.
Amb la utilització d'una sonda fluorimètrica in situ, combinada amb mètodes quimiomètrics de tractament de dades multivariables, es va aconseguir predir la biomassa i el substrat a partir de la determinació del senyal de diversos fluoròfors. No es va aconseguir una bona predicció de la producció de ROL.
Per millorar el seguiment de la proteïna, es va fusionar a la GFP. Es van detectar dos inconvenients: els nivells de producció disminuïen respecte a la producció de ROL únicament i la riboflavina, que Pichia excreta al medi, interferia amb el senyal de fluorescència del mutant de GFP escollit. Tant amb la filtració de la mostra abans de la determinació del senyal de GFP com si s'hagués escollit un mutant de GFP amb emissió més allunyada de la riboflavina es podria haver evitat aquest inconvenient i fer un seguiment de la ROL excretada. Es pot seguir la ROL intracel·lular.
Quant a la producció de la ROL, es va estudiar l'efecte del nivell de metanol residual en cultius discontinus alimentats amb la soca Mut+. Existeix una concentració òptima entorn els 2.5 g·l-1 de metanol al medi. A concentracions superiors es va apreciar inhibició per substrat. Es va observar un fenomen de limitació per transferència d'oxigen al final del cultiu i una important disminució de la viabilitat cel·lular.
Per això, es van avaluar estratègies de cultiu alternatives. Primer es va aplicar una estratègia de metanol limitant (MLFB) per evitar la limitació d'oxigen al final d'un cultiu a 2.5 g·l-1 de metanol residual (MNLFB). Es va millorar la productivitat un 40%. En segon lloc, es va aplicar una estratègia de temperatura limitant per avaluar el seu efecte sobre la producció. No es van millorar els resultats. Finalment, es va aplicar una estratègia MNLFB a 2.5 g·l-1 de metanol però amb un medi amb una menor osmolaritat i una fase final amb limitació de temperatura per evitar la limitació d'oxigen. Es va aconseguir reduir la mortalitat cel·lular però la productivitat disminuïa respecte el cultiu on s'aplica una fase de MLFB al final de la inducció. Tot i això, es va obtenir un producte final un 30% més pur en quant a activitat lipolítica respecte la proteïna total.
Posteriorment es va procedir a escalar la producció en planta pilot. Es va observar limitació en la transferència d'oxigen en fases inicials de la inducció. Això va originar l'aparició d'un subproducte, associat a una reducció en la producció de ROL extracel·lular. En millorar la transferència d'oxigen es va minimitzar aquesta limitació i va millorar la producció. No es van aconseguir els mateixos nivells de productivitat que a escala laboratori però es continua treballant en la millora de la transferència de matèria per assolir-los.
In this work two research lines, applied to the production of a Rhizopus oryzae lipase (ROL) in Pichia pastoris, are shown: the application of different monitoring and cultivation techniques to improve ROL production in a P. pastoris Mut+ strain.
During P. pastoris fed-batch cultures, methanol needs to be on line measured and monitored in real time. For this purpose, a sequential injection analyzer was developed. Although it presented a suitable analysis frequency for a Muts strain it was too low for a Mut+ strain. When the later was used, two different methanol commercial sensors in the outlet gas steams were utilized and compared.
Fluorometry was also evaluated as a monitoring technique for three key variables: biomass, substrate (glycerol/methanol) and recombinant protein.
Initially, biomass was followed by the off-line determination of tryptophan's fluorescence. Biomass was correctly predicted with this system but different disadvantages appeared: the need of a sample dilution procedure and a specific sampling device. Therefore, multivariable in situ fluorometry was subsequently evaluated.
By means of an in situ multivariable fluorimetric probe, combined with chemometric methods to data processing, biomass and substrate prediction was properly achieved. ROL production could not be satisfactorily estimated.
To improve ROL monitoring, it was fusioned to the green fluorescent protein (GFP). Two main disadvantages were found: production levels were lower when compared to solely ROL expression and riboflavin, naturally excreted by the yeast, interfered to GFP's signal. This problem could be solved with sample filtration prior to GFP's measurement and also if a different GFP mutant had been chosen with emission signal further to riboflavin's.
With respect to ROL production, the effect of methanol concentration was studied in Mut+ fed-batch cultures. There is an optimal methanol concentration about 2.5 g·l-1. Substrate inhibition was observed at higher methanol levels. Oxygen transfer limitation at the end of the induction phase and an important cell viability decrease were also found.
Therefore, alternative culture techniques were evaluated. First a methanol limited fed-batch phase (MLFB) was applied when oxygen limitations appeared at the end of a 2.5 g·l-1 methanol fed-batch phase (MNLFB). Productivity increased up to 40% with this strategy. Secondly, a temperature limited fed-batch was applied. No better results were obtained compared to the reference MNLFB culture. Finally, a 2.5 g·l-1 methanol fed-batch was applied with a lower salt content medium and a final temperature limited phase when oxygen limitation appeared. Cell death was reduced but productivity decreased with respect to the reference MNLFB culture. However, a 30% purer lipase was obtained in terms of lipase activity to total protein.
Thereafter, the scaling of the production process in a pilot plant was evaluated. Oxygen limitation was found in the early induction phase. This caused a byproduct secretion associated to a ROL production decrease. When oxygen transfer was enhanced, byproduct secretion was reduced and ROL production improved. Similar laboratory scale productivities were not achieved but oxygen mass transfer is being further enhanced to reach the objective levels.
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Sapelli, Evandro. "Parâmetros físico-químicos das reações de Zn2+ e Cd2+ com 8-hidroxiquinolina acompanhado por espectrofluorimetria em meio micelar." Florianópolis, SC, 2006. http://repositorio.ufsc.br/xmlui/handle/123456789/89305.
Full textAbstract:
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-Graduação em QuímicaMade available in DSpace on 2012-10-22T19:16:13Z (GMT). No. of bitstreams: 1 233926.pdf: 2391303 bytes, checksum: 6f228aab91d9ad1d4906ec949bb6bdc1 (MD5)
O intenso uso dos metais pesados aliados as suas toxicidades, fazem com que novos métodos de análise sejam importantes. No presente trabalho estuda-se um método para determinação de Zn2+ e Cd2+ com diferentes surfactantes (CTABr, SDS, TRITON X-100, Lauril e Palmitil sulfobetaína) por espectrofluorimetria. O método proposto emprega a sonda fluorescente 8-hidroxiquinolina (8-HQ). O realce da fluorescência da 8-HQ pelos íons metálicos Zn2+ e Cd2+, mostra que é possível analisar metais em soluções de composição conhecida. Com base nos resultados verificou-se que a técnica de espectroscopia de fluorescência em meio micelar mostra-se eficiente e de fácil aplicabilidade. A utilização do meio micelar é importante, pois permite a solubilização do complexo formado entre 8-HQ com Zn2+ e Cd2+, o qual logo precipita na ausência de surfactante. Nas condições experimentais de análise ([8-HQ] = 2,0 x 10-4 mol.L-1 e [Me2+] na faixa de 0 até 6,0 x 10-5 mol.L-1), os resultados indicam que o surfactante CTABr é o que atua de forma mais favorável na solubilização do complexo, tanto no caso do Zn2+ quanto Cd2+, e nenhuma precipitação foi observada até 24 horas depois. Os resultados dos testes de viscosidade mostram que adição de Zn2+ afeta a viscosidade macroscópica das soluções de CTABr, fato que pode estar relacionado com a estabilidade do complexo em solução. No caso do CTABr pode-se calcular que o LD é equivalente a 2,39 nano moles de Zn2+ e no caso do Cd2+ 2,74 nano moles. Em ambos casos, em sistemas cromatográficos onde as amostras são injetadas em micro litros, e/ou pré-tratadas em coluna de pré-concentração, o método pode atingir pico moles de metais.