Title: Spectroscopic Study of Singlet Oxygen in Cells and Model Systems Author: Marek Scholz Department: Department of Chemical Physics and Optics Supervisor: doc. RNDr. Roman Dědic, Ph.D., KChFO Abstract: Singlet oxygen (1O2), the first excited state of molecular oxygen, plays many important roles in nature and technology. The work is aimed at development of novel methods for monitoring of 1O2 in cells and other biological samples. Two main ap- proaches were employed: direct detection of the very weak near-infrared phospho- rescence of 1O2, and detection of Singlet Oxygen-Feedback Delayed Fluorescence (SOFDF), which is the emission from the photosensitizer induced by energy transfer from 1O2. The first part of the thesis introduces the basic concepts of photophysics and photochemistry of 1O2: its generation, deactivation, applications, and overview of detection methods. The second part presents the experimental results. Wide-field mi- crospectroscopic detection of 1O2 phosphorescence enabled us to acquire 1O2-based images and near-infrared spectra from single cells incubated with photosensitizers. However, the direct detection suffers from the inherently very low phosphorescence quantum yield. It is shown that SOFDF may overcome this problem and become a promising alternative tool for studies of 1O2 and...

Orientadores: José Euclides Stipp Paterniani, Cristiano de Mello Gallep / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agrícola / Made available in DSpace on 2018-08-25T21:52:58Z (GMT). No. of bitstreams: 1 Santos_SamuelRicardodos_D.pdf: 51294263 bytes, checksum: 55a8bb93513a62aa29e85fe9467412c6 (MD5) Previous issue date: 2014 / Resumo: Giardia spp. e Cryptosporidium spp. são organismos desafiadores em monitoramento ambiental, podendo afetar os seres humanos e os animais com grandes impactos na saúde pública. Métodos para detectar esses organismos são descritos na literatura ¿ p.ex.: o método EPA 1623.1. No entanto, muitos não são capazes de detectar a viabilidade destes parasitos. Este trabalho avaliou o uso de marcadores fluorescentes combinados com a técnica de detecção de fluorescência retardada na detecção de viabilidade de Giardia intestinalis e Cryptosporidium parvum. Testes de incubação com 6-carboxifluorceina-succinimidil-diacetato-éster (CFDA-SE), C12-resazurina e SYTOX Green foram desenvolvidos com cistos de G. intestinalis e oocistos de C. parvum. Medidas de fluorescência retardada em câmara escura projetada e em dispositivo comercial foram aplicados em amostras purificadas e concentradas de G. intestinalis após incubação com CFDA-SE. Grupos contendo cistos vivos e infecciosos, mortos a 100° C, estressados com luz UV-C e envelhecidos foram analisados via fluorescência retardada e microscopia de epifluorescência em oito séries experimentais. Os resultados demonstram que (oo)cistos vivos e infecciosos não são marcados com os marcadores fluorescentes. Dupla marcação em (oo)cistos mortos é observada após 30 minutos de incubação com C12-resazurina 5,0 ?M e SYTOX Green 100 nM. (Oo)cistos mortos apresentam marcação verde após incubação de CFDA-SE 5,0 ?M. O envelhecimento da amostra foi acompanhado pelo aumento da taxa de marcação celular com cistos apresentando ~50% de marcação aos 30 dias de idade e ~100% aos 50 dias de idade. Testes com fluorescência retardada demonstram que cistos vivos e com idade inferior a 20 dias apresentam intensidades superiores aos cistos mortos e estressados após excitação com 365 nm. A excitação com 365 nm apresentou correlação R2 > 95% após análise de cinética de decaimento com modelo exponencial de segunda ordem. Os dados indicam que o decaimento da fluorescência retardada é acompanhado por duas componentes k1 e k2 onde k2 = 5?k1, estando estas conectadas com as condições fisiológicas da Giardia. O procedimento pode ser efetuado em 10 passos laboratoriais em aproximadamente 60 minutos de análise. A fluorescência retardada apresenta futuro promissor na análise de viabilidade de parasitos em amostras purificadas / Abstract: Giardia spp. and Cryptosporidium spp. are challenging and important organisms in modern environmental monitoring. These protozoa can affect humans and animals seriously, as reflection of sanitation problems in water quality control, with huge impact over economics and public health. Methods to detect such organisms are well described in literature - i.e. the EPA Method 1623.1 and AWWA 2012. But those ones are not able to detect infectivity. For that, the usual procedures include infection of animal model leading to at least one week for confirming infectivity. Some research with dye probes are being developed in order to provide useful, reliable and low cost procedures for detection of protozoa viability, i.e. enabling to distinguish dead samples cells from living ones. This work describes the screening tests for viability detection of protozoa samples - Giardia intestinalis and Cryptosporidium parvum - using carboxifluorcein-succinimidyl-diacetate-ester (CFDA-SE), C12-resazurin and SYTOX Green. Living, heat-killed and UV-C stressed (oo)cysts were analyzed using these chemical probes. G. intestinalis in concentrated samples and stained with CFDA-SE were analysed by fluorescence imaging as well as by delayed fluorescence (DF) after UV-A and white-light excitation. The weak DF profiles were detected in photon-counting setups, in 8 series of tests for intact, for heat-killed and for UV-C-stressed samples are shown. Results show that fresh, i.e. living and viable (oo)cysts cannot be stained by the mentioned neither with CFDA-SE nor C12-resazurin and SYTOX Green dyes. Double-marked (oo)cysts are observed when C12-resazurin and SYTOX Green are applied to old cysts as well to dead ones. Aged samples show increasing number of stained organisms: 30-day-old with ~50% while samples older than 50 days with almost 100% marked. Intact samples present stronger fluorescence and DF than the stressed ones, with good replication after UV-A excitation. After excitation @365nm samples present DF better fitted by double exponential decay kinetics, with the decay constant k2 five times higher than the k1 constant. The procedure can be easily reproduced in 10 steps, taking around 1h of laboratorial work with purified samples / Doutorado / Agua e Solo / Doutor em Engenharia Agrícola

As humans we have a remarkable capacity to sacrifice for the future, and an equally remarkable capacity to shortchange it, leaving our future selves to pay the price. The research in this dissertation suggests that sacrifice for the future (patience) may be governed by the same rules that govern sacrifice for others (altruism). Studies found that (1) patience was positively correlated with altruism, and (2) patience was positively correlated with empathic accuracy, a measure that predicted altruism. A third set of studies found that (3) a pervasive influence on altruism--group membership--also influenced patience. Participants who expected to switch groups in the future felt less connected to their future selves and left more than twice as much unpleasant work to the future. This research provides converging support for the hypothesis that common factors promote altruism and patience, suggesting that we may be able to draw on the decades of research studying kindness toward others to promote a little more kindness toward our future selves. / Psychology

The purpose of this study was to estimate disparities in late human immunodeficiency virus (HIV) diagnosis and all-cause mortality among varying populations of HIV-positive Latinos, and to identify neighborhood-level predictors. Florida HIV surveillance data for years 2000–2011 were merged with 2007–2011 American Community Survey (ACS) data. Multilevel logistic regressions were used to estimate adjusted odds ratios (aOR) for late HIV diagnosis (acquired immunodeficiency syndrome within 3 months of HIV diagnosis). Multilevel weighted Cox regressions were used to estimate adjusted hazard ratios (aHR) for mortality. Of 5522 Latinos diagnosed 2007–2011, males were at increased odds of late diagnosis compared with females (aOR 1.37, 95% confidence interval [CI] 1.13-1.67). Associated factors included residing in the lowest quartile of neighborhood education for females, and in the 3 highest quartiles of unemployment for males. Foreign-born compared with United States (US)-born Latinos were also at risk (aOR 1.24, 95% CI 1.08-1.42). Among foreign-born, residing in areas with

Chinese society has greatly emphasized filial piety and other related societal norms. This thesis explores how Mainland Chinese international students, especially those who are from one-child households, maintain their familial responsibilities while studying in Canada away from home. The research is framed by the family migration strategy literature and empirically adopts a qualitative approach. Semi-structured face-to-face interviews with Mainland Chinese international students are undertaken. The findings show that as with many other types of migration, student migration, in this case, is a familial decision. To a certain extent, obtaining a foreign degree is the foundation for fulfilling familial responsibilities in the future. In addition, a new concept, delay remittances, is introduced as a way of conceptualizing the concrete meaning of familial responsibility for Mainland Chinese international students. The findings also show that the most significant tension for Mainland Chinese international is related to the uncertainty of how and where they will be able to fulfill their familial obligations to their parents.

Ces travaux visaient à développer de nouveaux implants biodégradables se formant in situ pour le traitement des parodontites, les infections les plus fréquentes au monde. Ces implants permettront de délivrer localement le principe actif et de contrôler sa libération. L’un des pré-requis pour ces nouveaux systèmes est de présenter une bonne bioadhésion et des propriétés mécaniques permettant d’éviter une expulsion prématurée hors de la poche parodontale.Tout d’abord, de nouveaux implants se formant in situ avec un potentiel prometteur pour surmonter l'un des inconvénients majeurs liés au traitement local de la parodontite: l’adhérence limitée aux tissus environnants ont été développés. L'addition de diverses concentrations de différents types de plastifiants (l’acetyltributyl citrate, ATBC et le dibutyl sebacate, DBS) et de polymères adhésifs (l'hydroxypropyl méthylcellulose, HPMC) ont permis d’obtenir une augmentation significative de l’adhésion des implants à base de l’acide poly(lactique-co-glycolique) (PLGA). Ces systèmes sont formés in situ à partir des formulations liquides de N-méthyl-2-pyrrolidone (NMP). Dans le même temps, une bonne aptitude à la déformation plastique des implants a été obtenue et les cinétiques de libération du principe actif souhaitées ont pu être affinées à l'aide de plusieurs outils de formulation. L'activité antimicrobienne de ce nouveau type d'implants se formant in situ, chargés à l’hyclate de doxycycline, a été démontrée en utilisant la méthode de diffusion en gélose sur plusieurs souches de Streptococcus isolées à partir de la microflore buccale des patients souffrant de parodontite.Ensuite, une meilleure compréhension des mécanismes de formation in situ des implants a été suivi en utilisant de différentes techniques tels que: la résonance paramagnétique électronique (EPR), la résonance magnétique nucléaire (1H NMR), le suivi de l’évolution de la masse et la cinétique de libération du principe actif dans différentes conditions, la microscopie optique, la chromatographie d'exclusion stérique (SEC). Des implants se formant in situ à base de PLGA, d’ATBC, de chlorhydrate de minocycline, de NMP et d’HPMC, ont été préparés et caractérisés en détail in vitro. Ces résultats ont révélé une vision approfondie sur les phénomènes physico-chimiques impliqués dans la formation de l'implant et sur le contrôle de la libération du principe actif. Par exemple, les effets de l'ajout d’HPMC dans la formulation, qui améliore l'adhérence de l'implant et réduit le gonflement, ont pu être expliqués. De manière importante, les implants se formant in situ ont efficacement empêché la croissance bactérienne dans les poches parodontales des patients. Enfin, l’impact de la composition des implants sur la performance des systèmes a été étudié. Afin d’élucider ces relations, des techniques de caractérisation de pointe, telles que l'analyse EPR ont été utilisées. Il est intéressant de noter que l’ajout d’HPMC et de PLGA de plus faible poids moléculaire a légèrement diminué la libération du principe actif, alors que dans le cas de PLGA de poids moléculaire plus élevé, la vitesse de libération a substantiellement augmenté. Ces tendances peuvent être expliquées en se basant sur la cinétique du transport de masse au cours de la formation de l'implant et des structures internes des systèmes. En outre, l'activité antimicrobienne des implants contre les micro-organismes présents dans les poches parodontales de patients atteints de parodontite a été évaluée. Ces systèmes gênent plus efficacement la croissance des bactéries pathogènes que celle des micro-organismes physiologiques. Ainsi, une recolonisation de la flore saine dans les poches des patients peut être envisagée in vivo. / This work aimed to develop new biodegradable in situ forming implants for the treatment of periodontitis, the most common infections in the world. These implants would locally deliver the active ingredient and control its release. One of the prerequisites for these new systems is to provide a good bioadhesion and mechanical properties to prevent premature expulsion from the periodontal pocket. Firstly, new in situ forming implants with promising potential to overcome one of the major drawbacks for the local treatment of periodontitis: limited adhesion to the surrounding tissue were developed. The addition of various concentrations of different types of plasticizers (acetyltributyl citrate, ATBC and dibutyl sebacate, DBS) and adhesive polymers (hydroxypropyl methylcellulose, HPMC) resulted in a significant increase in the adhesion of poly(lactic-co-glycolic acid) (PLGA)-based implants. The systems are formed in situ from N-methyl pyrrolidone (NMP)-based liquid formulations. Importantly, at the same time, good plastic deformability of the implants could be provided and desired drug release patterns could be fine-tuned using several formulation tools. The antimicrobial activity of this new type of in situ forming implants, loaded with doxycycline hyclate, was demonstrated using the agar well diffusion method and multiple Streptococcus strains isolated from the oral microflora of patients suffering from periodontitis.Secondly, a better understanding of the mechanisms of the in situ implant formation was followed using different techniques such as electron paramagnetic resonance (EPR), nuclear magnetic resonance (1H NMR), mass change and drug release measurements under different conditions, optical microscopy, size exclusion chromatography (SEC). The in situ forming implants containing PLGA, ATBC, minocycline hydrochloride, HPMC and NMP were prepared and characterized in detail in vitro. Based on these results, deeper insight into the physico-chemical phenomena involved in implant formation and the control of drug release could be gained. For instance, the effects of adding HPMC to the formulations, resulting in improved implant adherence and reduced swelling, could be explained. Importantly, the in situ formed implants effectively hindered the growth of bacteria present in the patients’ periodontal pockets.Finally, the impact of the composition of the implants on system performance was investigated using advanced characterization techniques, such as EPR analysis. Interestingly, HPMC addition to shorter chain PLGA slightly decreased drug release, whereas in the case of longer chain PLGA the release rate substantially increased. These tendencies could be explained based on the mass transport kinetics during implant formation and the systems’ inner structures. Furthermore, the implants’ antimicrobial activity against microorganisms present in the periodontal pockets of patients suffering from periodontitis was evaluated. Interestingly, these systems more effectively hinder the growth of pathogenic bacteria than of physiological microorganisms. Thus, a re-colonization of the patients’ pockets with healthy flora can be expected to be favored in vivo.

Purpose: To refute the alleged practice of "pseudoscience" by P. Finn, A. K. Bothe, and R. E. Bramlett (2005) and to illustrate their experimental and systematic bias when evaluating the SpeechEasy, an altered auditory feedback device used in the management of stuttering. Method: We challenged the experimental design that led to the seemingly predetermined outcome of pseudoscience rather than science: Limited preselected literature was submitted to a purposely sampled panel of judges (i.e., their own students). Each criterion deemed pseudoscientific was contested with published peer-reviewed data illustrating the importance of good rhetoric, testability, and logical outcomes from decades of scientific research. Conclusions: Stuttering is an involuntary disorder that is highly resistant to therapy. Altered auditory feedback is a derivation of choral speech (nature's most powerful stuttering "inhibitor") that can be synergistically combined with other methods for optimal stuttering inhibition. This approach is logical considering that in stuttering no single treatment is universally helpful. Also, caution is suggested when attempting to differentiate science from pseudoscience in stuttering treatments using the criteria employed by Finn et al. For example, evaluating behavioral therapy outcomes implements a post hoc or untestable system. Speech outcome (i.e., stuttered or fluent speech) determines success or failure of technique use, placing responsibility for failure on those who stutter.

Bakgrund Träningsvärk är ett vanligt förekommande fenomen som uppstår efter ovan, excentrisk eller högintensiv träning. Svullnad, muskelömhet, samt nedsatt muskelfunktion och ledrörlighet är symptom som är förknippade med träningsvärk. Det finns ett antal olika behandlingsmetoder för att lindra dessa symptom. För närvarande finns dock en diskrepans i litteraturen angående de olika behandlingarnas effekt.   Syfte Syftet med studien var att undersöka hur ett kylbandage applicerat över m. quadriceps femoris direkt efter utförd träning påverkar muskelstyrka, knäledens rörlighet och upplevd träningsvärk 48 timmar efter träning.   Metod Nitton personer rekryterades till studien. Vid första tillfället utvärderades låromfång, upplevd träningsvärk och m. quadriceps femoris flexibilitet med Ely’s test. I en isokinetisk träningsmaskin mättes maximalt och genomsnittligt vridmoment vid 60°/s och 180°/s. Försökspersonerna genomgick ett styrketräningsprotokoll innehållande koncentriska och excentriska moment. Vänster m. quadriceps kylbehandlades sedan med kylbandage i 60 min. Fyrtioåtta timmar senare upprepades samtliga mätningar. Muskelflexibilitet, låromfång, muskelstyrka samt smärta användes som utvärderingsmått.   Resultat Resultatet visade signifikant mindre upplevd träningsvärk i behandlat ben än i obehandlat vid aktiv knäextension. Det fanns en signifikant ökning av genomsnittligt vridmoment för obehandlat ben vid 180°/s. Ingen signifikant skillnad erhölls gällande knäledens rörlighet, upplevd träningsvärk vid Ely’s test, eller skillnad i maximalt vridmoment och totalt utfört arbete.   Konklusion Kylbandage som appliceras över m. quadriceps femoris direkt efter högintensiv träning kan eventuellt lindra upplevd träningsvärk, men minskar ej de relaterade funktionsnedsättningarna. Mot tidigare litteratur och diskrepansen i denna, är vidare forskning nödvändig för att fastställa kylbehandlingens effekter på träningsvärk. / Background Delayed onset muscle soreness (DOMS) is common after unaccustomed, eccentric or high-intensity exercise. Swelling, soreness, impaired muscle function and joint mobility are associated with DOMS. There are several types of treatment strategies to relieve these symptoms. However, there is a discrepancy in the literature regarding the effect of these treatments.   Objective The purpose was to investigate how a cold bandage applied on quadriceps femoris immediate after performed high-intensity exercise, affects strength, muscle flexibility and perceived DOMS 48 hours post-exercise.   Methods Nineteen subjects were recruited. At the first occasion thigh circumference, Ely’s test and DOMS were evaluated. Peak torque and average peak torque were measured with a training device at 60°/s and 180°/s. The participants underwent a strengthening exercise protocol consisting of concentric/eccentric components. The left quadriceps femoris was then treated with a cold bandage for 60 minutes. All tests were repeated 48 hours post-exercise. Muscle flexibility, thigh circumference, strength and perceived DOMS were evaluated.   Results There was significantly less self-estimated DOMS in the treated leg compared to the untreated, during an active extension of the knee. There was a significant increase of the average peak torque in the untreated leg at 180°/s. No significant difference was obtained regarding muscle flexibility, self-estimated DOMS during Ely’s test, peak torque or total work.   Conclusion A cold bandage applied on quadriceps femoris after high-intensity exercise may be effective as treatment of self-estimated DOMS, but has no beneficial effect on the loss of function. Further research is required to establish the effect of the treatment strategy.

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Ship-mounted cranes are used to transfer cargo from large container ships to smaller lighters when deep-water ports are not available. The wave-induced motion of the crane ship produces large pendulations of hoisted cargo and causes operations to be suspended. In this work, we show that in boom type ship-mounted cranes, it is possible to reduce these pendulations significantly by controlling the slew and luff angles of the boom. Such a control can be achieved with the heavy equipment that is already part of the crane so that retrofitting existing cranes would require a small effort. Moreover, the control is superimposed on the commands of the operator transparently. The successful control strategy is based on delayed-position feedback of the cargo motion in-plane and out-of-plane of the boom and crane tower. Its effectiveness is demonstrated with a fully nonlinear three-dimensional computer simulation and with an experiment on a 1/24 scale model of a T-ACS (The Auxiliary Crane Ship) crane mounted on a platform moving with three degrees of freedom to simulate the ship roll, pitch, and heave motions of the crane ship. The results demonstrate that the pendulations can be significantly reduced, and therefore the range of sea conditions in which cargo-transfer operations could take place can be greatly expanded. Furthermore, the control strategy is applied experimentally to a scaled model of a tower crane. The effectiveness of the controller is demonstrated for both rotary and gantry modes of operation of the crane. This work was supported by the Office of Naval Research under Contract #N00014-96-1-1123. / Ph. D.