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Vid låga mängder av DNA, vilket det ofta är vid forensiska prover, är en lyckad DNA-analys beroende av ett effektivt DNA-extraktionsprotokoll. Bland flera kommersiellt tillgängliga extraktionsmetoder för DNA har tidigare studier visat att Chelex 100 resin, som idag används på Nationellt Forensiskt Centrum i Linköping, är en snabb och enkel metod utan toxiska ämnen, långa väntetider eller större kostnader och som även ger ett högt DNA-utbyte. På teknisk mikrobiologi i Lund har olika chelex-baserade protokoll för spårsäkring och extraktion tagits fram för att optimera det aktuella protokollet på Nationellt Forensiskt Centrum. I detta arbete kombinerades spårsäkringsteknik och DNA-extraktionsprotokoll från tidigare studier med olika rör och tops, med syftet att i bästa fall generera ett protokoll som är optimalt för de vanligast förekommande brottsplatsproverna. I resultatet observerades högre DNA-utbyte med reducerade volymer lyseringsbuffert, vilket användning av rör-i-rörsystem tillät. När rör-i-rörsystem sedan kombinerades med extraktionsprotokoll innehållande SDS, observerades en ökning i DNA-utbytet. När dessa två faktorer vidare kombinerades med en tops vars skaft kan klippas rakt av ökade DNA-utbytet ytterligare. Ett byte från mikrofugrör, som används på Nationellt Forensiskt Centrum idag, till ett rör-i-rörsystem reducerar lyseringsvolymen från ca. 600 µl till 200 µl vilket är en avgörande faktor för effektivare extraktioner. / When handling low amounts of DNA, which often is the case with forensic samples, a successful DNA analysis is dependent on an effective DNA extraction protocol. Among many commercially available extraction methods for DNA, previous research has proved Chelex 100 resin, which is used at National Forensic Center in Linkoping, to be a quick and simple method without toxic substances, long waiting time or large costs, which in addition provides a high DNA exchange. At Applied Microbiology, Lund´s University, different chelex-based protocols for sampling and DNA extraction have been developed to optimize the present protocol at the National Forensic Center. In this study, sampling techniques and DNA extraction from previous studies was further combined with different tubes and swabs with the aim to, in best case, generate a protocol that is optimal for the most common crime scene samples. The results showed improved DNA yields with less extraction volume, which the tube in tube system made possible. When tube in tube system was combined with extraction protocols containing SDS, a higher DNA yield was observed. When these two factors were further combined with a cotton swab, whose shaft can be cut straight of, the DNA yield improved even more. A change from microfuge tubes, which is used at the National Forensic Center, to tube in tube system, the lysis volume could be reduced from approximately 600 µl to 200 µl which is a deciding factor for higher DNA yield.

This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.

Predmetom tejto diplomovej práce bolo štúdium vplyvu detergentov na aktivitu, termostabilitu a agregáciu voľnej a imobilizovanej formy komerčného preparátu lipázy izolovanej z mikroskopickej huby Rhizopus arrhizus. Teoretická časť obsahuje ucelenú rešerš popisujúcu štruktúru, mechanizmus účinku a priemyselný význam spomínanej hydrolázy spolu s popisom chemických účinkov detergentov, pričom dôraz bol kladený predovšetkým na skupinu neionogénnych detergentov s názvom tweeny. V experimentálnej časti bol študovaný efekt tweenov na rozpustnej a imobilizovanej forme RA lipázy. Imobilizácia spočívala v priamej adsorpcii enzýmu na neupravený nosič. Ako nosič bol použitý oxidovaný grafén ošetrený tweenom (tween 20, 60, 80). Aktivita enzýmu bola stanovená spektrofotometricky za pomoci substrátu p-nitrofenyl laurátu. Zvýšenie aktivity voľnej lipázy (104 % oproti maximálnej hodnote) bolo zaznamenané pri použití tweenu 20 o koncentrácii vysoko nad hodnotou kritickej micelárnej koncentrácie (10 mmol•dm-3). Na základe štúdie imobilizačných podmienok, boli nastavené ideálne parametre pre dosiahnutie účinnej imobilizácie v spojení s čo najvyššou lipolytickou aktivitou (koncentrácia enzýmu 0,1 mg•ml-1, fosfátový tlmivý roztok pH 7,2, koncentrácia tweenu 10,8 mmol•dm-3, čas imobilizácie 1 hodina). Obe formy lipázy vykazovali maximálnu aktivitu pri 35 °C. Optimálne pH sa u imobilizovanej lipázy posunulo na hodnotu 8, v porovnaní s voľnou formou, ktorej pH optimum bolo stanovené na 9. Tepelná stabilita vykazovala približne rovnaký priebeh u oboch foriem skúmanej hydrolázy. Avšak v prípade štúdia stability enzýmu pri dlhodobej úschove bolo po imobilizácii zistené výrazné zlepšenie tohto parametru.

El presente proyecto se desarrolló con el objetivo de analizar la viabilidad de negocio en crear un detergente ecológico elaborado con insumos naturales, en donde se utiliza como principal materia prima la saqta, una raíz andina utilizada por los incas para el lavado de textiles. De esta manera, buscamos contribuir con la conservación del medio ambiente frente un escenario actual de degradación de los recursos naturales. Este modelo de negocio se dirige principalmente a personas interesadas en el cuidado del ambiente, basándonos en la situación actual del incremento de la cantidad de personas que tienden a adquirir productos eco amigables, que se reflejó en el concierge efectuado durante el desarrollo de la idea de negocio. Actualmente llegamos al público objetivo a través de redes sociales, ferias ecológicas y ventas directas, en donde por medio de anuncios en plataformas digitales, demostraciones en las ferias y flyers publicitarios buscamos que el cliente conozca la saqta y sus propiedades para poder incentivar la venta y crear recordación de marca en nuestros clientes. La conclusión a la que se llegó de este proyecto es que el modelo de negocio planteado tiene aceptación en el público objetivo y que, además, genera rentabilidad para los accionistas que se refleja en los estados de resultados. / The present project was developed with the objective of analyzing the business viability in creating an ecological detergent made with natural inputs, where saqta is used as the main raw material, an Andean root used by the Incas to wash textiles. In this way, we seek to contribute to the conservation of the environment against a current scenario of degradation of natural resources. This business model is mainly aimed at people interested in caring for the environment, based on the current situation of the increase in the number of people who tend to acquire eco-friendly products, which was reflected in the concierge made during the development of the idea of deal.   Currently we reach the target audience through social networks, ecological fairs and direct sales, where through advertisements on digital platforms, demonstrations at fairs and advertising flyers we seek that the client knows the saqta and its properties in order to encourage sales and create brand reminder in our customers. The conclusion that came from this project is that the proposed business model has acceptance in the target audience and generates profitability for shareholders that is reflected in the income statements. / Trabajo de investigación

Orientador: Ronivaldo Rodrigues da Silva / Resumo: Atualmente, a avicultura é um dos setores de grande impacto na economia brasileira. Nos últimos anos, tem sido observado um aumento na produção de frangos de corte, fazendo com que este segmento da indústria gere toneladas de queratina com o descarte de penas. Isso aponta para a necessidade de degradar este material que emerge como um problema ambiental. Neste cenário, as enzimas queratinolíticas têm despertado interesse biotecnológico devido a peculiar capacidade para a degradação de queratina e a possibilidade de aplicar o hidrolisado protéico para suplementação de ração animal e uso como biofertilizantes. Desta forma, neste trabalho, nós propomos prospectar queratinases pela bactéria Citrobacter diversus e o fungo Coriolopsis byrsina e, em seguida, investigar as características bioquímicas destas enzimas, a fim de propor aplicação na degradação de penas de frango. Em nossos resultados, a bactéria C. diversus foi capaz de degradar quase completamente as penas de frango (0,5%) em meio submerso após 36 h de fermentação. O estudo com o extrato enzimático mostrou máxima atividade caseinolítica a pH 9-10,5 e 50-55 ºC, e queratinolítica a pH 8,5-9,5 e 50 ºC. Em destaque, conforme a estabilidade em incubação por 1 h a 50ºC, foi detectado aproximadamente 50% e 100% da atividade queratinolítica e caseinolítica, respectivamente. Sob estabilidade a pH por 48 h a 4ºC, o extrato enzimático manteve maior atividade na faixa de pH 6-8. A atividade caseinolítica foi inibida por EDTA e PMSF,... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Currently, poultry is one of the sectors of great impact on the Brazilian economy. In recent years, there has been an increase in the production of broilers, causing this segment of the industry to generate tons of keratin with the disposal of feathers. This points to the need to degrade this material, which emerges as an environmental problem. In this scenario, keratinolytic enzymes have aroused biotechnological interest due to the peculiar capacity for the degradation of keratin and the possibility of applying protein hydrolyzate to supplement animal feed and use as biofertilizers. Thus, in this work we propose to prospect keratinases for the bacterium Citrobacter diversus and the fungus Coriolopsis byrsina and, next, to investigate the biochemical characteristics of these enzymes, in order to propose application in the degradation of chicken feathers. In our results, the bacterium C. diversus was able to degrade chicken feathers almost completely (0.5%) in submerged medium after 36 h. The study with the enzymatic extract showed maximum caseinolytic activity at pH 9-10.5 and 50-55 ºC, and keratinolytic activity at pH 8.5-9.5 and 50 ºC. Notably, after enzyme pre-incubation for 1 h at 50 ºC, approximately 50% and 100% of keratinolytic and caseinolytic activity were detected, respectively. Under pH stability for 48 h at 4ºC, the enzyme extract maintained greater activity in the pH 6-8 range. Caseinolytic activity was inhibited by EDTA and PMSF, and keratinolytic activity was i... (Complete abstract click electronic access below) / Mestre

Indiana University-Purdue University Indianapolis (IUPUI) / During the late 1960s and early 1970s, the polluted Great Lakes became a central focus of the North American environmental movement. A majority of this pollution stemmed from phosphate-based laundry detergent use, which had become the primary product households used to wash fabrics after World War II. The large volume of phosphorus in these detergents discharged into the lakes caused excess growths of algae to form in waterways, which turned green and smelly. As the algae died off, it reduced the oxygen in the water, making it less habitable for fish and other aquatic life, a process known as eutrophication. As primary consumers of laundry detergents during the time period, women, particularly white, middle-class housewives in the United States and Canada, became involved in state/provincial, national, and international discussions involving ecology, water pollution, and sewage treatment alongside scientists, politicians, and government officials. Their work as volunteers, activists, and lobbyists influencing the debate and ensuing policies on how best to abate this type of pollution, known as eutrophication, has often been ignored. This thesis recognizes the work women completed encouraging the enactment of key water quality regulations and popularizing the basic tenets of environmentally-conscious consumption practices during the environmental movement in the early 1970s.

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Le transporteur d'ADP/ATP est une protéine de la membrane interne mitochondriale qui joue un rôle physiologique majeur en catalysant l'échange d'un ADP cytoplasmique contre un ATP néo-synthétisé dans la matrice mitochondriale. Cette protéine peut être inhibée de manière très spécifique par deux poisons naturels, le carboxyatractyloside (CATR) et l'acide bongkrekique (BA) qui stabilisent la protéine dans deux états conformationnels distincts adoptés au cours du mécanisme de transport. Afin de mieux appréhender cette dynamique fonctionnelle, une étude du transporteur d'ADP/ATP bovin en complexe avec le CATR ou le BA dans le détergent Triton X-100 a été réalisée par échange hydrogène/deutérium couplé à la spectrométrie de masse. Ces travaux se sont déroulés en 4 parties. La première a consisté à adapter cette technique à l'étude des protéines membranaires intégrales. En effet, la présence de détergents, nécessaires au maintien en solution de l'état natif de ces protéines, n'a pas permis jusqu'ici de les étudier par cette approche. Pour pallier cette difficulté, un protocole automatisé de chromatographie liquide permettant l'élimination du Triton X-100 a été mis au point. Les cinétiques de deutération des différents complexes ont alors pu être analysées dans le deuxième volet de cette étude. Les données obtenues ont permis de proposer des modèles conformationnels du transport de nucléotides à travers la membrane interne mitochondriale, dans lesquels le transporteur présenterait une cavité ouverte tour à tour vers l'espace intermembranaire et vers la matrice. Afin d'apporter d'autres éléments de réponse sur ce mécanisme de transport et de s'affranchir de différents problèmes liés à l'utilisation des détergents, des essais de deutération du transporteur d'ADP/ATP bovin dans les mitochondries ont été entrepris et représentent le troisième volet de ces travaux. Cette approche, qui nécessite encore plusieurs améliorations, a permis d'obtenir les premières données de deutération d'une protéine membranaire dans son environnement natif. Le transporteur d'ADP/ATP est aussi impliqué dans des pathologies humaines plus ou moins graves. Dans une dernière partie, l'étude de ces mutations a été abordée en réalisant chez la levure Saccharomyces cerevisiae une étude phénotypique et biochimique de plusieurs mutants du transporteur de l'amibe Dictyostelium discoideum correspondant aux mutations humaines. Cette étude a mis en évidence un problème dans le mécanisme intrinsèque de transport, induit par la mutation V291M, qui pourrait être à l'origine de la pathologie associée.