<p> A fluorometric method using lumogallion has been shown to be adequate to measure the degree and rate of complexation of aluminum at low concentrations, (1.85 xl0^-6 M), by other ligands such as fluoride and citrate, at a pH of 5.2.</p> <p> The species H2PO4 -, H4SiO4 and SO4 2- do not compete with lumogallion for aluminum at this pH for concentration ratios of less than 100, (competing ion: lumogallion). </p> <p> A rate law for the destruction of the lumogallion complex by fluoride ion has been determined as -r = (-dC/dt)A = ((4.3 x 10^3)[F^-1]^1.8 CA)/(1 - (1.0 x 10^3)[F^-1]^-0.7 CA) where CA is the corrected concentration of the fluorescent complex at any time.</p> <p> This method is proposed to measure aluminum complexes in natural waters.</p> / Thesis / Bachelor of Science (BSc)

Magister Scientiae - MSc (Bioinformatics) / Biobanks are collections of biological samples and associated data for future use. The day to day activities in a biobank laboratory is underpinned by a laboratory information management system (LIMS). For example, the LIMS manages the execution of tests on biospecimens and track their movement and processing through the laboratory. There are a range of commercially available Biobank LIMS systems on the market but their costs are prohibitive in a resource limited setting. The cost of Commercial off-the-shelf software includes the initial cost of acquiring the system, as well as the cost of maintenance and support throughout the software's life cycle. The Bika LIMS system on the other hand is Free and open source software (FOSS) with decreased license cost, used routinely in non-medical laboratories. Ideally, if Bika LIMS could be customised to handle human biospecimens, then both biobanks and genetics laboratories could benefit. Central to any biobank functionality in Bika LIMS is the ability to import information from routine biomedical equipment. We identified two instruments that are key to human biobanking and are lacking in Bika LIMS namely BioDrop ?LITE and the Qubit Fluorometric instrument. Import interfaces for importing DNA/RNA concentration analyses from these instruments and management of the results with associated sample information would add value to the LIMS. The aim of the thesis was to customise Bika LIMS for utility in a biomedical laboratory. In collaboration with colleagues at Tygerberg medical school, the Bika LIMS software was customised to accommodate the DNA and RNA concentration analyses results for a pathology laboratory and the LIMS workflows customised for use at Tygerberg medical school. In this process the manual operations of Tygerberg medical school laboratory would migrate to the use of Bika LIMS. The analytical module in Bika LIMS was implemented using PYTHON, by using logic that allows importing of specific analyses. A template was created for the BioDrop ?LITE and Qubit Fluorometric instruments used for developing the interface for an analysis import form. The instruments generate results in CSV file format. A parser was created to read and parse the files uploaded from the import form, by splitting them into parts, extracting the data, and populating key-value pairs. The controller manages the submission of the form by initialising the parser that imports the specific file into the LIMS where it is managed by the configured Bika LIMS workflow.

<p>ABSTRACT</p><p>Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent.</p>

"A number of fluorometric techniques have been applied to characterize contamination associated with oil discharges and spills in the environment. While these techniques provide quick and lower cost alternatives to the many of the advanced techniques for characterizing oil-related constituents, their applicability still isn’t fully understood. The objectives of this research were to understand the characteristics of organic transport in a linked surface-water/ground-water system, and develop some practical approaches using fluorometry to characterize the pathways of organic transport. The approach included modeling, field sampling and comparisons of laboratory analyses to assess basic field fluorometry techniques for characterizing sources and distributions of polycyclic aromatic hydrocarbons (PAHs) associated with oil discharges. The primary field site included a canal and nearby river, which resulted in generally uniform hydraulic gradient, such that petroleum and PAH contamination at the site could be characterized. Historical data provided general information on the distribution of contamination. Modeling using the Modflow groundwater flow package provided basic information on groundwater flow pathways and rates. Samples were collected from the canal, groundwater, the river and a treatment facility. Additional samples were collected from Bayou Corne sinkhole in Lousiana and the Deepwater Horizon crude oil spill in the Gulf of Mexico. The samples were analyzed for fluorometric absorbance using a 10AU field fluorometer, a Shimadzu absorbance spectrometer and a LS5 luminescence spectrometer (which provided fluorescence over a spectrum of frequencies). Additional analyses were completed using a gas chromatograph with a flame ionization detector (GC-FID) to provide a more complete qualitative description of the oil composition. Analysis of the results from the 10-AU field fluorometer confirmed the capability of the field fluorometer to detect organic contamination resulting from crude and refined oil spills. Absorbance spectrometer results demonstrated possibility of using the PAH absorbance spectra to distinguish between the different types of oil, although more detailed analyses using various types of oil is recommended. The results using the luminescence spectrometer were consistent with GC FID results, and provided useful comparisons indicating the characteristics of fresh and weathered oil. The comparisons provide insight into the applicability of fluorometric approaches for characterizing transport pathways and concentrations of organic constituents associated with discharges of oil and other PAHs."

ABSTRACT Chemotherapy is a common method used for cancer treatment. Especially when it concerns cancers that have grown invasively it seems to be the only efficient treatment due to the substances ability to reach and affect almost the entire body. One major obstacle regarding chemotherapy is that the patients often develop resistance to the cytotoxic substances used. Fluorometric microculture cytotoxicity assay (FMCA) is a method developed to measure sensitivity of tumor cells to different cytotoxic substances in vitro. The assay is based on hydrolysis of fluorescein diacetate to fluorescein by cells with intact cell membranes after incubation with drugs for 72 hours. This study investigated the impact of two methodological factors that may cause errors in the achieved results; namely the possible occurrence of drug decay during incubation and the use of an inappropriate amount of cells. These factors were tested by exposing the cytotoxic drugs to pre-incubation in absence of tumor cells for different times and to use suspensions with different concentrations of cells. The results indicated occurrence of drug decay in 3 of the 18 substances tested and that the amount of cells affected the results for most of the drugs tested but to different extent.

Electroporation (EP) is a physical non-viral technique used to deliver therapeutic molecules across the cell membrane. During electroporation an external electric field is applied across a cell membrane and it causes pores to form. These pores then allow the surrounding media containing the therapeutics to diffuse across the membrane. This technique has been specifically studied as a promising gene and drug delivery system. Colloidal particles have also proven to be promising for a variety of biological applications including molecular delivery, imaging, and tumor ablation, due to their large surface area and tunable properties. In more recent years researchers have explored the use of both electroporation and particles simultaneously. In this research, the main objective was to investigate and determine the role of sub-micron particles in the electroporation process. Presented in this dissertation are results from the synthesis and characterization of colloidal particles of various sizes and different compositions. The use of these dielectric and metallic particles during in vitro electroporation were investigated along with various other electrical parameters associated with EP such as pulse length, number of pulses, and field strength. Computationally, aspects such as particle composition and particle concentration were explored in an attempt to predict experimental outcomes.

Incluir como ag?ncias financiadoras: Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) e Funda??o de Amparo ? Pesquisa do Estado de Minas Gerais (FAPEMIG). / Submitted by Jos? Henrique Henrique (jose.neves@ufvjm.edu.br) on 2018-02-07T21:39:03Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) / Approved for entry into archive by Rodrigo Martins Cruz (rodrigo.cruz@ufvjm.edu.br) on 2018-03-09T19:17:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) / Made available in DSpace on 2018-03-09T19:17:22Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) marcelo_henrique_fernandes_ottoni.pdf: 3524322 bytes, checksum: c7675d522b34cfb2455e18dbca1699da (MD5) Previous issue date: 2017 / Conselho Nacional de Pesquisas (CNPq) / A presen?a de fluoresc?ncia interferente em uma dada amostra ? considerada como um importante problema em m?todos fluorim?tricos, devido ? poss?vel sobreposi??o espectral entre ela e a emiss?o fluorescente de sondas. Por isso, ? importante conhecer se h? fluoresc?ncia interferente em uma dada amostra e subst?ncias de interesse nas condi??es de trabalho pretendidas. No presente estudo, foi avaliada a presen?a de fluoresc?ncia interferente em linf?citos humanos ap?s o tratamento com extratos de tr?s diferentes plantas medicinais, sendo estas, objeto de estudo do nosso grupo de pesquisas. Os extratos s?o: o extrato etan?lico das partes a?reas de Ageratum fastigiatum, extrato etan?lico das partes a?reas de Eriosema campestre e o extrato etan?lico do caule de Pseudobrickellia brasiliensis. Foi coletado o sangue de tr?s volunt?rios para a separa??o das c?lulas mononucleares de sangue perif?rico, para a confec??o de culturas in vitro em meio RPMI devidamente suplementado. Foram feitas uma cultura controle n?o-tratada (CON), culturas tratadas com cada extrato em tr?s concentra??es diferentes, al?m de uma cultura de c?lulas tratadas com dimetilsulf?xido (DMSO), solvente usado na solubiliza??o dos extratos vegetais. As culturas celulares foram incubadas por 24 horas a 37 ?C e 5% de CO2. Ap?s esse per?odo, as c?lulas foram lavadas e avaliadas por citometria de fluxo ou por microscopia confocal. A presen?a de fluoresc?ncia interferente foi determinada com base em histogramas de intensidade de fluoresc?ncia feitos para oito intervalos de comprimento de onda distintos, tendo sido feitas an?lises quali e quantitativas dos dados. A fluoresc?ncia dos extratos vegetais e DMSO foram avaliadas isoladamente por fluorimetria, usando-se as mesmas concentra??es utilizadas para as culturas celulares. Atrav?s da citometria de fluxo, foi identificado que o tratamento de linf?citos com qualquer um dos tr?s extratos de plantas levou ao aparecimento de fluoresc?ncia interferente detect?vel em v?rias faixas de comprimento de onda. Culturas tratadas com DMSO n?o apresentaram fluoresc?ncia interferente. Pela fluorimetria foi visto que os extratos n?o emitem fluoresc?ncia, o que sugere que a fluoresc?ncia interferente foi induzida nas c?lulas ap?s intera??es entre elas e os extratos. Este estudo levanta precau??es que visam avaliar a poss?vel presen?a de fluoresc?ncia interferente em condi??es de trabalho, possibilitando evitar esse vi?s e aumentar a confiabilidade dos resultados. / Disserta??o (Mestrado) ? Programa de P?s-gradua??o em Ci?ncias Farmac?uticas, Universidade Federal dos Vales do Jequitinhonha e Mucuri, 2017. / The presence of interfering fluorescence in a given sample is considered as an important problem on fluorometric methods due to the possible spectral overlap between it and the fluorescent emission of probes. Therefore, it is important to know if there is interfering fluorescence in a given sample, as well as, in substances of interest, in the desired working conditions. In the present study, it was evaluated the presence of interfering fluorescence in human lymphocytes after the treatment with extracts from three different medicinal plants, being such plants the object of study of our research group. The extracts are: the ethanolic extract from the aerial parts of Ageratum fastigiatum, ethanolic extract from the aerial parts of Eriosema campestre and the ethanolic extract from the stem of Pseudobrickellia brasiliensis. Blood was collected from three volunteers to get the peripheral blood mononuclear cells, for in vitro culture preparation in properly supplemented RPMI medium. It was made an untreated control culture (CON), cultures treated with each extract at three different concentrations, and a culture of cells treated with dimethylsulfoxide (DMSO), the solvent used in the plant extracts solubilization. Cell cultures were incubated for 24 hours at 37?C and 5% CO2. After that, cells were washed and evaluated by flow cytometry or by confocal microscopy. The presence of interfering fluorescence was determined based on making fluorescence intensity histograms at eight wavelength ranges for each cell culture. Qualitative and quantitative data analyzes were performed with those graphs. Plant extracts and DMSO fluorescences were evaluated by fluorometry, using the same concentrations used for the cell cultures. In flow cytometry results, it was identified that the treatment of lymphocytes with any of the three plant extracts led to the appearance of interfering fluorescence detectable in several wavelength ranges. Cultures treated with DMSO showed no interfering fluorescence. By fluorometry it was seen that the extracts are not fluorescent, which suggests that the interfering fluorescence was induced in the cells by interactions between them and the extracts. This study raises precautions to evaluate the possible presence of interfering fluorescence in working conditions, making possible to avoid this bias and increase the results reliability.

Modern techniques like automated microscopy and spectroscopy now make it possible to study quantitatively, across multiple phenotypic and molecular parameters, how cell populations are affected by different treatments and/or environmental disturbances. As the technology development at the instrument level often is ahead of the data analytical tools and the scientific questions, there is a large and growing need for computational algorithms enabling desired data analysis. These algorithms must have capacity to extract and process quantitative dynamic information about how the cell population is affected by different stimuli with the final goal to transform this information into development of new powerful therapeutic strategies. In particular, there is a great need for automated systems that can facilitate the analysis of massive data streams for label-free methods such as phase contrast microscopy (PCM) imaging and spectroscopy (NMR). Therefore, in this thesis, algorithms for quantitative high-throughput phenotypic and systemic profiling of in vitro growing cell populations via label-free microscopy and spectroscopy are developed and evaluated. First a two-dimensional filter approach for high-throughput screening for drugs inducing autophagy and apoptosis from phase contrast time-lapse microscopy images is studied. Then new methods and applications are presented for label-free extraction and comparison of time-evolving morphological features in phase-contrast time-lapse microscopy images recorded from in vitro growing cell populations. Finally, the use of dynamic morphology and NMR/MS spectra for implementation of a reference database of drug induced changes, analogous to the outstanding mRNA gene expression based Connectivity Map database, is explored. In conclusion, relatively simple computational methods are useful for extraction of very valuable biological and pharmacological information from time-lapse microscopy images and NMR spectroscopy data offering great potential for biomedical applications in general and cancer pharmacology in particular.

Background: Cancer is a common disease, and the choice of treatment becomes more difficult over time due to chemotherapy resistant in cancer cells. To improve the in vitroassay and the individual cancer treatment, a luminescence-based endpoint assay, CellTiter Glo 2.0 was compared with the currently in use fluorescence endpoint assay, fluorometric microculture cytotoxic assay.  Aim: The aim of this study was to validate and compare the CellTiter Glo 2.0 assay with awell-established method (FMCA) and MTT [3-(4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2Htetrazolium bromide] assay. Moreover, investigate whether the generated data can be used as a reference database for validation of patient samples in the future.   Materials and methods: The validation was performed on peripheral blood mononuclear cells from different healthy donors and two cell lines (HCT116-wt and HT-29) of colorectal cancer carcinoma were ordered frozen from American Type Culture Collection. Analysis was also done in solid samples (ovarian and kidney cancer cells). To get as correct evaluation as possible all materials were analyzed in parallel between the two methods.  Results and conclusion: A clear trend was observed when using CellTiter Glo 2.0 assay,post FMCA directly on tumor cells. This setup, makes it possible to collect reference data in the future. In addition, a high spread of the survival index data was noted between the two methods. The reason is still unknown but could be due to the low number of tested tumor cells, therefore more tumor cells need to be tested in future studies

Gastric cancer (GC) is one of leading causes of death in the world, and peritoneal metastases (PM) are a major site of recurrence. PM from GC implies a poor prognosis, with median overall survival (mOS) approximately 3 months and no survival at five years. The aims of this thesis were to explore the incidence and evaluate prognostic factors for mOS of PM from GC in a defined population; to investigate the outcome of a new multimodal treatment; to analyse the treatment costs, and to investigate differences in drug sensitivity between individual patient samples and between various tumours. The incidence of loco-regional advanced GC was 3.8 per 100,000 person-years. Synchronous loco-regional GC in combination with synchronous distant metastasis was a negative prognostic factor while chemotherapy and good performance status, and radiotherapy plus chemotherapy were positive prognostic factors . There were no significant differences in mOS for the group of patients included during the period 2000-2004 versus 2005-2009, and this lack of improvement in mOS during the past decade justifies new treatment approaches. In a Phase II study of patients treated with neoadjuvant systemic chemotherapy followed by cytoreductive surgery + hyperthermic intraperitoneal chemotherapy, mOS was 14.3 months and for patients with macroscopically radical surgery mOS was 19.1 months. The mean overall cost of the loco-regional treatment was $145,700 compared to $59,300 with systemic chemotherapy treatment. In an ex vivo chemo-sensitivity test, it was determined that GC samples were equivalent to colorectal cancer in chemo-sensitivity to standard drugs and targeted drugs, whereas ovarian cancer samples were more sensitive. The individual GC samples varied considerably in sensitivity to increasing concentrations of the drugs, arguing for individualized drug selection. The incidence of loco-regional advanced GC was more common than previously reported and there were no improvements in mOS over the past decade. The mOS for patients with neoadjuvant systemic chemotherapy followed by macroscopically radical cytoreductive surgery + hyperthermic intraperitoneal chemotherapy was better than in recent reports on treatment with systemic chemotherapy. Treatment of advanced GC patients is costly irrespective of treatment modality. The GC samples varied considerably between individuals in terms of sensitivity to increasing concentrations of the drugs and were comparable to colorectal cancer in chemo-sensitivity.