A disponibilidade de hemoderivados é um parâmetro importante para medir a qualidade da saúde em um país. Dentre os produtos hemoderivados, imunoglobulinas tem alto valor agregado. O Instituto Butantan tem por objetivo o estabelecimento de uma planta industrial para fracionamento de plasma, com um processo produtivo baseado principalmente em cromatografias. Eventos tromboembólicos a partir de infusões de imunoglobulinas por via intravenosa (IgIV) foram relacionados com presença de Fator XI de coagulação (FXI) como contaminantes nas preparações de IgIVs. Com objetivo de detectar o FXI nas frações, o processo cromatográfico foi testado em escala piloto, bancada, e em cromatografia direta e o FXI foi dosado nas frações iniciais e no produto final IgIV. Foram estabelecidos os métodos de dosagem de atividade de FXI por tempo de coagulação e ensaio cromogênico. Concluímos que o FXI acompanha a IgG nas etapas iniciais dos processos cromatográficos e verificamos que ocorre a presença de FXI nos produtos finais. Este trabalho contribui para o desenvolvimento do conjunto de testes de controle de qualidade de biofármacos derivados de plasma humano. / The availability of hemoderivatives is an important parameter to measure a countrys health quality. Among hemoderivatives products, immunoglobulin have high value.. Instituto Butantan, aims the establishment of an industrial plant for plasma fractionation with a process drawn, based mainly in chromatographies. Thromboembolic events from infusions of intravenous immunoglobulins (IVIg) have been related with the presence of coagulation Factor XI (FXI) as contaminant in the IVIG preparations. With an objective of tracking FXI in the chromatographic fractions, the process was tested in pilot and bench scales and in direct chromatography and the FXI was measured in the initial fractions and in the final product IVIg. The methods of measurement of FXI activity thru coagulation time and chromogenic assay were established. We have concluded that FXI accompanies IgG in the early stages of the chromatographic processes and verified that the presence occurs in the final products, This work contributes to the development of the set of quality control tests of biopharmaceuticals derivatives from human plasma.

The ultimate objective of this work was to develop a real-time method for detecting and monitoring β-D-galactosidase as a suitable indicator of the potential presence of total coliform bacteria in water environments. Preliminary comparison of the chromogenic substrate, chlorophenol red β-D-galactopyranoside and the fluorogenic substrate, MuGAL, revealed unreliable results with the fluorogenic technique due to interference from compounds commonly found in environmental water samples. Thus, the chromogenic assay was further explored. Hydrolysis of the chromogenic substrate chlorophenol red β-D-galactopyranoside by β-D-galactosidase to yield chlorophenol red was the basis of this assay. Fundamental studies with chlorophenol red β-Dgalactopyranoside showed that β-D-galactosidase occurs extracellularly and in low concentrations in the polluted water environment. A direct correlation between enzyme activity and an increase in environmental water sample volume, as well as enzyme activity with total coliform colony forming unit counts were observed. Spectrophotometric detection was achieved within a maximum period of 24 h with a limit of detection level of 1 colony forming unit 100 ml[superscript -1]. This enzyme also exhibited physical and kinetic properties different from those of the pure commercially available β-D-galactosidase. Cell permeabilisation was not required for releasing enzymes into the extracellular environment. PEG 20 000 offered the best option for concentrating β-D-galactosidase. The source of β-D-galactosidase in the polluted environmental water samples was confirmed as Escherichia coli through SDS-PAGE, tryptic mapping and MALDI-TOF, thus justifying the further use of this method for detecting and/or monitoring total coliforms. Several compounds and metal ions commonly found in environmental water samples (as well as those used in water treatment processes) did have an effect on β-D-galactosidase. All the divalent cations except Mg [superscript 2+], at the concentrations studied, inhibited the relative activity of β-D-galactosidase in both commercial β-D-galactosidase and environmental samples. Immobilisation of chlorophenol red β-D-galactopyranoside onto a solid support material for the development of a strip bioprobe was unsuccessful, even though the nylon support material yielded some positive results. A monthly (seasonal) variation in β-Dgalactosidase activity from the environmental water samples was observed, with the highest activity coinciding with the highest monthly temperatures. Electro-oxidative detection and/or monitoring of chlorophenol red was possible. Chlorophenol red detection was linear over a wide range of concentrations (0.001-0.01 μg ml[superscript -1]). Interference by chlorophenol red β-D-galactopyranoside in the reduction window affected analysis. A range of phthalocyanine metal complexes were studied in an attempt to reduce fouling and/or increase the sensitivity of the biosensor. The selected phthalocyanine metal complexes were generally sensitive to changes in pH with a reduction in sensitivity from acidic pH to alkaline pH. The tetrasulphonated phthalocyanine metal complex of copper was, however, more stable with a minimum change of sensitivity. The phthalocyanine metal complexes were generally stable to changes in temperature. While only two consecutive scans were possible with the unmodified glassy carbon electrode, 77 consecutive scans were performed successfully with the CuPc-modified glassy carbon electrode. Among the phthalocyanine metal complexes studied, the CuPc-modified glassy carbon electrode therefore provided excellent results for the development of a biosensor. The CuPc modified-glassy carbon electrode detected 1 colony forming unit 100 ml[superscript -1] in 15 minutes, while the plain unmodified glassy carbon electrode required 6 hours to detect the equivalent number of colony forming units. CoPc, ZnPc and CuTSPc required 2, 2.25 and 1.75 h, respectively, to detect the same numbers of colony forming units. The CuPcmodified glassy carbon electrode detected 40 colony forming units 100 ml[superscript -1] instantly. In general, a direct correlation between colony forming units and current generated in the sensor was observed (R2=0.92). A higher correlation coefficient of 0.99 for 0-30 coliform colony forming units 100 ml[superscript -1] was determined. Current was detected in some water samples which did not show any colony forming units on the media, probably due to the phenomenon of viable but non-culturable bacteria, which is the major disadvantage encountered in the use of media for detecting indicator microorganisms. This novel biosensor therefore presents a very robust and sensitive technique for the detection and/or monitoring of coliform bacterial activity in water.

Background: Vancomycin-resistant enterococci (VRE) is a common nosocomial infection. It classifies as Enterococcus faecalis or faecium carrying vanA or vanB gene that alters the bacterial cell wall hence lowering affinity for Vancomycin. Screening for VRE in Swedish hospitals are performed with stool sample pre-grown in selective broth followed by PCR and culture on selective media. Viasures Vancomycin resistance, Real Time PCR Detection Kit indicates that pre-growth in broth is not needed for the analyze. Aim: Comparison between the PCR kit and the subsequent culture on chromogenic agar with or without pre-growth in selective broth. Method: E. faecium with vanA gen (CCUG 36804) and E. faecalis with vanB gen (ATCC 51299) were suspended in different concentrations and added to ESwab transport medium. Thereafter small samples from the ESwab tube were enriched in selective broth. Samples from both selective broth and ESwab medium were analyzed with Viasures PCR kit on BD-MAX system and cultivated on chromogenic agar. Results: With pre-growth in selective broth the genes were found in every sample regardless of pre-concentration in the ESwab medium. Without enrichment the PCR kit always amplified the genes when the concentration was 40 000 cfu/ml for E. faecium (vanA) and ≥ 10 000 cfu/ml for E. faecalis (vanB). Colonies grew on chromogenic agar in every concentration from both ESwab and selective broth. Conclusion: Culture on chromogenic agar is comparable with or without pre-growth in selective broth but Viasure’s PCR kit is not equal for both methods in lower concentrations of the bacteria.

Master of Science / Food Science Institute / Daniel Y.C. Fung / ECA Check® Easygel® Plus (ECA) is a pectin-based gelling system that reacts with calcium ions bound to a pre-treated Petri dish, eliminating autoclaving prior to use. It can chromogenically and/or fluorogenically distinguish three organisms: Escherichia coli, Salmonella spp., and coliforms. This study compared the recovery of these organisms to conventional media using stock culture, inoculated, and non-inoculated ground beef and ground turkey. ECA was compared to Violet Red Bile Agar (VRB), Violet Red Bile Agar with 4-methylumbelliferyl-β-D-glucuronide (VRB-MUG), Xylose Lysine Desoxycholate Agar (XLD), Escherichia coli/Coliform (ECC) Count Plate Petrifilm™, and Tryptic Soy Agar (TSA). The stock culture recovery of Salmonella Typhimurium for ECA, TSA, and XLD were 8.62, 8.69, and 6.82 log CFU/ml, respectively. There was very little difference between the media in the recovery of Escherichia coli and Cronobacter spp., formerly referred to as Enterobacter sakazakii. Mean counts of presumptive E. coli in ground beef were 7.24 and 7.41 logs for ECA and VRB-MUG. Total coliform mean counts were 7.43, 7.63, and 7.37 logs for ECA, Petrifilm™, and VRB. Presumptive Salmonella means were 6.68 and 6.21 logs on ECA and XLD, while total aerobic counts were 7.84 and 6.51 logs on ECA and TSA. At 6.72 logs, ECA recovered considerably more Salmonella than XLD (5.71 logs) from the inoculated ground turkey; ECA recovered 7.62 logs total aerobic count which was significantly more than TSA at 6.89 logs. Total counts for both non-inoculated ground meats resulted in significant differences between TSA recovery and all other media. ECA also recovered significantly more than Petrifilm™ from both non-inoculated foods. The randomly selected organisms recovered from ECA were identified using BBL™ Crystal™ Enteric/Nonfermenter ID or Gram-Positive kits, and correlated precisely to the chromogenic reaction of the colonies. ECA Check® Easygel® was efficient, less labor-intensive, comparable to, and, in some instances, better than conventional media at recovering target organisms.

Energieffektivisering börjar bli ett eftersträvande mål runtom i världen. Detta grundar sig i att energiförbrukningen för byggnader uppgår till ca 40 % globalt, en siffra som man vill få ner. Men att uppnå en energieffektiv byggnad är inte lätt. Detta kan göras på många och olika sätt. Ett av dem är att energieffektivisera fönstren, som är en byggnads svagaste punkt pga dess höga U-värde. Val av fönster är inte lätt, och det finns ett flertal alternativ att välja bland. I kalla klimat som Sverige söker man fönster med lågt U-värde och högt g-värde, samt en hög avskärmningsfaktor. I varmare länder vill man också ha ett lågt U-värde hos fönster men fokusen ligger främst på en låg avskärmningsfaktor. Syftet med uppsatsen var att hitta de mest energieffektiva fönstren, oavsett kostnad, för byggnader som befinner sig i länder med kallare klimat. Det var också av vikt att få veta lönsamheten för fönstren i fråga, därför har även kostnadsfrågor belysts. Metodvalen var informationssökning i olika databaser och litteratur samt att olika företag inom fönsterbranschen kontaktades, vilket ledde till att relevant och önskvärd information erhölls. Därefter fortskred arbetet genom kalkyleringar för energibalansen och lönsamheten. Vacuumfönster, aerogelfönster samt kromogena fönster hör till framtida fönster som kan tillföra positiva inverkan på energibalansen för byggnader. Men dessa fönster är i nuläget inte helt färdigutvecklade, fast har potential att bli världsledande. Vacuumfönster och kromogena fönster är i nuläget bättre lämpade för varmare klimat. Lyckas man komma längre med deras nutida utveckling är det inte omöjligt att anpassa de för kallare klimat. Aerogelfönster ger mest energibesparing vid byte av fönster, men pga vissa optiska egenskaper och komplicerad tillverkning av produkten är den i nuläget inte optimal vid ett fönsterbyte. De framtida fönstren är ej heller ekonomiskt försvarbara, det finns i dagsläget kommersiella energieffektiva fönster som är billigare att införskaffa och ger ett ansenligt bra resultat för en byggnads energibalans. / Energy optimization is starting to be a pursued worldwide main goal. This is based on the global energy consumption that occurs in buildings, which is about 40 percent. There is no doubt that this value needs to be lowered. But to achieve an energy efficient building is not easy. Although, this can be done in many and different ways. One of them is to optimize the windows, which is a buildings weakest point due to its high U-value.The choice of windows can be a harsh decision, there’s plenty of windows to choose among. In heating dominated climates, as the one in Sweden, it is necessary to choose windows with low U-values and high g-values, also a high solar heat gain coefficient/shading coefficient is required. A window with a low U-value is also important in cooling dominated climates but the main focus is instead on a low shading coefficient, which is not the case in this thesis. The purpose is to find the most energy efficient window that lowers the need for active heating in buildings, and also reveal and discuss the cost issues for the chosen windows.By searching in scientific databases and contacting companies active in the window industry the desired information was obtained. Calculations including the energy balance and present value were made, which gave an indication of the profitability for the different windows. Vacuum, aerogel and chromogenic window are examples of future windows which can have a positive impact on the energy balance in buildings. Yet these windows are currently not fully developed, but have potential to be highly valuable types of windows. Vacuum and chromogenic windows are better suited for cooling dominated climates. However if the development succeed where a big progress will be made it will not be impossible to suit them for heating dominated climates too. Aerogel windows have the best impact on the energy savings when replacing windows, but due to some optical attributes and a complicated manufacturing of the product aerogel windows are currently not an optimal choice for window replacement. The future windows isn’t either economically viable. For now, there are other commercially energy efficient windows that are cheaper to purchase. They also show an acceptable good result on the energy balance for a building.

Urinvägsinfektion är en av de mest förekommande infektionerna hos människan. För att visualisera urinvägspatogener kan det kromogena mediet UriSelectTM4 användas för diagnostik. Det primära syftet med studien var att utvärdera om det är möjligt att inkubera det kromogena mediet UriSelectTM4 i 5 % koldioxid istället för aerob miljö utan att totalväxt och morfologi påverkas. Vidare utvärderades totalväxt och antal fria kolonier vid odling på en halv UriSelectTM4-agarplatta med två odlingstekniker för att undersöka om fortsatt diagnostik är möjligt. Urinprover inkuberades i aerob miljö och i 5 % koldioxid och jämfördes visuellt utifrån totalväxt, antal fria kolonier, morfologi samt färgförändring på kolonier och agarn. Resultatet visade att totalväxt och antal fria kolonier endast skiljer i liten grad mellan inkubationsmiljöerna. Däremot förekom skillnader i morfologi och färg. Vidare kunde en halv agarplatta användas vid odling och fortsatt diagnostik. Studien visar därmed att UriSelectTM4 kan inkuberas i 5 % koldioxid utan att totalväxt, fria kolonier och agarn påverkas. / Urinary tract infection is one of the most common infections among humans. For diagnostics, the chromogenic media UriSelectTM4 can be used to visualize the urinary tract pathogens. The primary purpose of the study was to evaluate if the chromogenic media UriSelectTM4 could be incubated in 5% carbon dioxide instead of aerobic environment without impacting total growth and morphology. Furthermore, total growth and number of free colonies was evaluated when cultivating on a half UriSelectTM4 agar media with two streak patterns to examine if further diagnostics is possible. Urine samples were incubated in aerobic environment and in 5% carbon dioxide and visually compared for total growth, number of free colonies, morphology and color change of bacterial colonies and the agar media. The results showed that total growth and free colonies only had slight differences between the incubation environments. On the other hand, morphology and color of the colonies may vary. Further a half agar media could be used for cultivation and further diagnostics. Consequently, the study shows that UriSelectTM4 can be incubated in 5% carbon dioxide without any impact on total growth, free colonies or of the chromogenic media.

Numerous chemical species are important to the health of biological systems. Some species can be beneficial at low doses and harmful at high doses. Other species are highly reactive and trigger serious cell damage. Improved methods to detect the presence and activity of such species are needed. In this work, several biologically important species were studied using appropriate analytical techniques. Fluoride is an important species in human physiology. It strengthens teeth and gives protection against dental caries. However, elevated concentrations of fluoride in the body can lead to health problems such as dental and skeletal fluorosis. Reported fluoride sensors used fluorescence quenching methods in determining fluoride concentration. Our study explored synthesis and characterization of 1,8-bis(phenylthioureido) naphthalene (compound 1) as a fluoride sensing molecule. Compound 1 showed a remarkable 40 fold enhancement in fluorescence with 5 eq of fluoride addition. Compound 1 also showed possibility of visual colorimetric sensing with fluoride. Free radical mediated oxidations of biomolecules are responsible for different pathological conditions in the human body. Superoxide is generated in cells and tissues during oxidative burst. Moderately reactive superoxide is converted to peroxyl, alkoxyl and hydroxyl radicals by various enzymatic, chemical, and biochemical processes. Hydroxyl radical imparts rapid, non specific oxidative damage to biomolecules such as proteins and lipids. Superoxide also reacts with nitric oxide in cells to yield peroxynitrite, which is highly reactive and damages biomolecules. Both hydroxyl radical and peroxynitrite readily react with amino acids containing aromatic side chains. Low density lipoprotein (LDL) carries cholesterol in the human body. Elevated concentration of LDL is a potential risk factor for atherosclerosis. Previous research drew a strong correlation between oxidized low density lipoprotein (ox-LDL) and plaque formation in the arterial wall. More importantly, oxidative damage causes structural changes to the LDL protein (apo B-100) which might facilitate the uptake of LDL by macrophages. In this study LDL was exposed to various concentrations of hydroxyl radical peroxynitrite and hypochlorite. Thereafter oxidized amino acid residues in apo B-100 were mapped by LC-MS/MS methods. We found widely distributed oxidative modifications in the apo B-100 amino acid sequence.

Apixaban och lågmolekylärt heparin (LMH) är antikoagulantia som förhindrar blodproppsbildning genom att hämma faktor Xa. Allt mer patienter använder apixaban och LMH, vilket gör att laboratoriemedicin på länssjukhuset Ryhov är i behov av att utvärdera analysmetoder för apixaban och LMH för att kunna implementera analyserna i klinisk rutin. Syftet med studien var att utvärdera analysmetoden för bestämning av anti-FXa aktivitet i plasma hos patienter behandlade med apixaban eller LMH med hjälp av kromogen substratmetod. Metodutvärderingen bestod av fyra steg: repeterbarhet, mellanliggande precision, överensstämmelse med validerad metod och analys av normalpopulation. Utvärderingen genomfördes med hjälp av Sysmex CS-2100 där det analyserades 20 respektive 40 patientprover för apixaban och LMH samt 10 normalprover. Aktivitet av faktor Xa bestämdes kvantitativt med användning av ljusabsorption vid 405 nm. Repeterbarhet och mellanliggande precision visade låg CV. Patientprover visade överensstämmande resultat med referensvärden från andra laboratorium där r2 för apixaban och LMH var 0,95. Avvikande resultat kan bero på mätfel eller förväxling mellan prover. Analys av normalpopulation visade att värden låg under det lägsta tillförlitliga värdet. Utvärdering av analysmetoden apixaban och LMH på Ryhovs laboratorium visade goda resultat vilket bekräftar att analysmetoden kan användas i klinisk rutin. / Introduction: Apixaban and low molecular weight heparin (LMWH), are anticoagulants that prevent clot formation by inhibiting factor Xa. Increasingly more patients use apixaban and LMWH, for this reason the laboratory medicine at the county hospital Ryhov needs to evaluate methods of analysis for apixaban and LMWH to be able to implement the analyzes in clinical routine. Aim: The purpose of the study was to evaluate the assay method for determining anti-FXa activity in plasma in patients treated with apixaban or LMWH using chromogenic substrate method. Method: The method evaluation consisted of four steps: repeatability, intermediate precision measures, compliance with validated method and analysis of normal population. The evaluation was performed using Sysmex CS-2100 where 20 respective 40 patient samples were analyzed for apixaban and LMWH as well as 10 normal population samples. Factor Xa activity was quantitatively determined using light absorption at 405 nm.Result and discussion: Repeatability and intermediate precision showed low CV. Patient samples showed consistent results with reference values from other laboratories where r2 for apixaban and LMWH were 0.95. Deviant results may be due to measurement errors or confusion between samples. Analysis of normal population showed that values were below the lowest reliable value. Conclusion: Evaluation of the analysis method apixaban and LMWH at Ryhov's laboratory showed good results, which confirms that the assay method can be used in clinical routine.

No description available.

Tesis por compendio / El presente proyecto de investigación está enfocado al desarrollo de sensores químicos fluoro-cromogénicos, para la detección y determinación de especies químicas de interés biológico, industrial y medioambiental de forma selectiva y con alta sensibilidad. En forma general, se busca el diseñar nuevos sistemas sensores basados en compuestos (receptores) formados por dos unidades: una unidad coordinante que interacciona con el anión a determinar y una unidad generadora de señal que alerta del reconocimiento molecular efectuado. Durante este estudio se están preparando diversas moléculas receptoras funcionalizandas con grupos modificadores de estructura para evaluar su influencia sobre las capacidades de detección y selectividad como receptores de especies específicas en diferentes condiciones y medios. Las diferentes aproximaciones en prueba implican a su vez el diseño y síntesis molecular, así como el análisis de las diferentes señales ópticas producidas en el reconocimiento, con el fin de diseñar sistemas de alta eficacia y eficiencia, y con posibilidades reales de aplicación. / Santos Figueroa, LE. (2014). New approaches for the development of chromo-fluorogenic sensors for chemical species of biological, industrial and environmental interest [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/43216 / Premios Extraordinarios de tesis doctorales / Compendio