Introduction: In South Africa, breast cancer has the highest prevalence with a life time risk of 1 in every 9 women being diagnosed annually. There are four sub-types of breast cancer and according to the stage of the cancer, various treatment regimens are prescribed. A major obstacle is that majority of cancers have developed multi-drug resistance and new treatment regimens need to be developed in order to obtain therapeutic efficacy. Cancer cells use aerobic glycolytic metabolism for energy generation and inhibition of this pathway increases sensitivity of the cells to anti-neoplasic treatments. 2-Deoxyglucose (2-DG) competes with and inhibits glucose uptake inhibiting the glycolytic pathway which can result in depolarisation of the mitochondrial membrane potential releasing cytochrome c. Two 2-Methoxyestradiol (2-ME) derivatives, ESE- 15-ol and ESE-16 have shown to be promising anti-cancer agents and combination therapy could allow the use of these compounds with a decreased side effect profile. The combination of these compounds with 2-DG was therefore investigated. Aim: To investigate combinations of two oestrone analogues and the glycolysis inhibitor 2- deoxyglucose for potential synergistic effects using a cell enumeration assay, mitochondrial membrane potential and cell cycle analysis, on breast cancer cells in an in vitro setting. Cell apoptosis, necrosis and autophagy pathways were assessed to indicate the mechanism of cytotoxicity. Methods: The breast cancer MCF-7 and non-tumorigenic MCF-12A cell line were used. Cells were exposed to ESE-15-ol, ESE-16 and 2-DG alone and in combination. Mechanistic studies were performed using the various research methodologies including the sulforhodamine B assay for cell enumeration, Annexin-V FITC and propidium iodide labeling for apoptosis/necrosis studies, PlasDIC and light microscopy for morphological analysis, propidium iodide staining for cell cycle progression, JC-1 for mitochondrial membrane potential studies, transmission electron microscopy and western blotting for the analysis of autophagy. Results: A GI50 of 34.1 nM was reported for MCF-7 cells after treatment with ESE-15-ol, 141 nM for ESE-16 and 1.3 mM 2-DG. The GI50 of ESE-15-ol treated MCF-12A cells was 141 nM, 140.1 nM for ESE-16 treated cells and 1.7 mM for 2-DG. ESE-16 had the greatest effect on cell viability in MCF-7 cells and a shift from an inhibitory effect to the initiation of cell death was evident after treatment of 100 nM of ESE-15-ol and ESE-16. 2-DG had a lower cytotoxic effect than the oestrone analogues. The MCF-12A cell line was less susceptible to the experimental compounds. The combination of the oestrone analogues with 2-DG elicited a greater effect on cell enumeration than each of the compounds alone with a less pronounced effect on the MCF- 12A cell line in comparison to the MCF-7 cells. The experimental compounds initiated apoptosis with ESE-16 eliciting a greater effect than ESE-15-ol. The combination of the oestrone analogues with 2-DG resulted in increased apoptosis in contrast to the compounds alone. ESE-16 alone and in combination with 2-DG lead to the most prominent morphological changes, with ESE-15-ol decreasing cell density slightly. The combination of ESE-15-ol with 2-DG decreased cell density with membrane blebbing apparent. The MCF-12A cell line was less susceptible to morphological changes after treatment of ESE-15-ol with 2-DG however ESE-16 and the combination with 2- DG resulted in similar attributes seen in MCF-7 treated cells. ESE-15-ol resulted in accumulation of cells in the G2 cell cycle phase which was further amplified after the combination of 2-DG. A sub-G1 accumulation was observed after treatment with ESE-16 with a shift to a G2 accumulation after the combined treatment of ESE-16 with 2-DG. After 48 hours, ESE-15-ol alone and in combination with 2-DG on MCF-7 cells resulted in depolarisation of the mitochondrial membrane. A slight decrease in the membrane potential was observed after treatment with ESE-16 and this was further increased after the combined treatment of ESE-16 with 2-DG. The MCF-12A were less susceptible after 24 hour treatment than 48 hour exposure of the experimental compounds. The presence of autophagic-like vacuoles were apparent in all treatment groups as well as the increased expression of LC3-II. Conclusion: The combined treatment of synthetic oestrone analogues with 2-DG displayed greater therapeutic efficacy than each of the compounds alone. As a result, the apoptotic and autophagic pathways were induced and a shift in cell cycle progression was observed. Mitochondrial involvement was apparent and the compounds significantly affected cell viability. This suggests that the combinations between the antimitotic oestrone analogues and glycolysis inhibitor 2-DG act synergistically to induce apoptosis and autophagy in MCF-7 breast cancer cells. / Dissertation (MSc)--University of Pretoria, 2017. / Pharmacology / MSc / Unrestricted

Urban emissions of antibiotics into the environment have the potential to adversely affect terrestrial and aquatic organisms. Developed standardized test methods allow the quantification of the resulting ecotoxicological risk, which strongly relies on a comprehensive situation analysis by predicting or measuring a representative antibiotic concentration of interest. Predicting the input loads of antibiotics to wastewater treatment plants using secondary input data (e.g. prescriptions) is a reasonable method if no analytical data is available. The absence of such data poses the question of an aquired reasonable sample quantity to capture local seasonal differences in prescriptions as well as flow conditions within the catchment area. Both, the theoretical and measurement based determination of environmental concentrations have been scarcely verified in practice. Hence, high resolution prescription data in combination with an extensive monitoring campaign at the wastewater treatment plant Dresden-Kaditz (WWTP) were used as a basis to evaluate the reliability of predicting and measuring urban antibiotic emissions. As expected, the recovery of antibiotic input loads strongly varies among substances. The group of macrolides as well as sulfamethoxazole and trimethoprim were almost fully recovered whereas nearly all substances of the beta-lactam family exhibit high elimination rates during the wastewater transport in the sewer system. Yet other antibiotics (e.g. fluoroquinolones) show distinct fluctuations through the year, which was not obvious from relatively constant prescriptions. The latter substances are an example that available data are not per se sufficient to predict the actual release into the environment which, in certain cases, emphasizes the necessity of adequate measuring campaings. The extensive data pool of this study was hence used to calculate the necessary number of samples to determine a representative annual mean load to the WWTP. Based on the applied approach, a minimum number of 20 to 40 samples per year is proposed to reasonably estimate a representative annual input load of antibiotics and other micropollutants. Regarding the WWTP, the mass flow analysis revealed that macrolides, clindamycin/ clindamycin-sulfoxide and trimethoprim were mainly released with the effluent, while penicillins, cephalosporins as well as sulfamethoxazole were partly degraded in the studied WWTP. Levofloxacin and ciprofloxacin are the only antibiotics under investigation with a significant mass fraction bound to primary, excess and digested sludge. In this context, the sludge concentrations are considered to be highly inconsistent which leads to questionable results. It remains unclear whether the inconsistencies are due to insufficiencies in sampling and/or analytical determination or if the fluctuations can be considered reasonable for digesters. Subsequently, verified antibiotic loads were evaluated regarding their ecotoxicological effects in the aquatic environment. Two approaches were applied (1) to address the ecological impact on individual trophic levels algae, daphnia and fish, and (2) to assess the possible synergistic potential of antibiotic combinations. Ciprofloxacin, levofloxacin and the group of cephalosporins showed to significantly affect the aquatic environment. They either have the highest impact on (one of) the lowest trophic level(s) or disproportionately increase the ecotoxicological risk due to their synergistic characteristics. In this regard, the deficiencies regarding the input prediction of these antibiotics is of particular concern. The underestimation of such critical mass flow conditions weakens the approach of assessing environmental risks on the basis of secondary data like prescriptions. Hence, efforts must be made to further develop the projection model by improving the quality of secondary data, identifying additional emitters and understanding possible retention and degradation dynamics of antibiotics within the sewer system. / In der Humanmedizin eingesetzte Antibiotika werden im menschlichen Körper nicht vollständig metabolisiert und gelangen über die Ausscheidungen in das kommunale Abwasser. In der Kläranlage erfolgt nur eine unvollständige Elimination dieser Stoffe, so dass der Kläranlagenablauf einen Hot Spot für Antibiotikaemissionen in die Umwelt darstellt. Das induzierte ökotoxikologische Risiko kann anhand standardisierter Testverfahren und allgemein anerkannter Bewertungsansätze für Einzelsubstanzen abgeschätzt werden. Erfolgt jedoch die Betrachtung von Antibiotikagemischen, wie es für den gereinigten Ablauf einer Kläranlage sinnvoll ist, sind aufgrund zumeist unspezifischer Wirkmechanismen und dem Mangel an repräsentativen Daten eine Reihe von Vereinfachungen und Annahmen zu treffen. Es besteht in der Folge die Gefahr einer Unterschätzung des durch Substanzgemische hervorgerufenen ökotoxikologischen Risikos. Eine vielversprechende Möglichkeit den Entscheidungsprozess über mögliche Vermeidungs- und Eliminationsmaßnahmen zu unterstützen besteht in der Priorisierung von Antibiotika entsprechend ihres Effektpotentials. Hierbei sind Substanzen zu identifizieren, die den größten Einfluss auf die Nahrungskette im Gewässer bzw. das höchste (negative) Synergiepotential mit anderen Substanzen aufweisen. Die Verringerung dieser Substanzen führt zu einer hohen ökologischen Effektivität und Effizienz der eingesetzten Mittel. Wie im Fall des klassischen Bewertungsansatzes, ist auch für den Priorisierungsansatz eine umfängliche und zuverlässige Situationsanalyse die Grundvoraussetzung für verwertbare Ergebnisse. Die Situationsanalyse beruht auf der analytischen Bestimmung bzw. der Abschätzung von emittierten Antibiotikafrachten zur Berechnung von repräsentativen Umweltkonzentrationen. Analytisch ermittelte Umweltkonzentrationen vieler Antibiotika weisen aufgrund saisonaler Verschreibungsmuster eine hohe zeitliche und räumliche Variabilität auf. Die für eine adäquate Erfassung der Situation notwendigen Messkampagnen sind kostenintensiv, wobei die tatsächlich notwendige Häufigkeit der Probenahme von zumeist nicht hinreichend bekannten substanzspezifischen Informationen, wie der chemischen Stabilität im Rohabwasser und der saisonal beeinflussten Applikation, abhängt. Alternativ können Antibiotikaeinträge in die Kanalisation anhand von Verschreibungsdaten abgeschätzt und mit Hilfe von Stoffflussanalysen (SFA) zur ökotoxikologischen Bewertung herangezogen werden. Eine vom Umfang befriedigende, direkte Gegenüberstellung von prognostizierten und analytisch ermittelten Frachten ist bisher jedoch nicht erfolgt, so dass die Verifizierung dieses Ansatzes noch aussteht. Für den Fall einer bestehenden Verschreibungspflicht für Antibiotika besitzen Verschreibungsdaten eine vergleichsweise hohe zeitliche und räumliche Informationsgüte. In Verbindung mit einer an diese Datenqualität angepassten Messkampagne, ergibt sich die Möglichkeit einer detaillierten SFA mit substanzspezifischer Bewertung der Eignung des Prognoseansatzes. Die am Beispiel der Stadt Dresden durchgeführte Bewertung des Prognoseansatzes fußt auf einer 15-monatigen Messkampagne und den für das Einzugsgebiet der Zentralkläranlage Dresden-Kaditz verfügbaren Verschreibungsdaten der AOK PLUS. Erwartungsgemäß ergibt der Abgleich von erwarteten und analytisch ermittelten Frachten eine starke Variation der für den Zulauf der Kläranlage ermittelten Wiederfindungsdaten verschiedener Substanzen. Die analytisch ermittelten Frachten von Sulfamethoxazol, Trimethoprim sowie der Gruppe der Makrolid-Antibiotika entsprechen nahezu den prognostizierten Mengen. Die Beta-Laktam-Antibiotika unterliegen bereits während des Abwassertransports einer umfänglichen, zumeist biologisch bedingten, Elimination, was zu hohen Unterbefunden im Zulauf der Kläranlage führt. Andere Substanzen hingegen (z.B. Fluorchinolone) weisen messtechnisch eine signifikante Jahresdynamik auf, die aufgrund der weitgehend konstanten Verschreibung in dieser Ausprägung nicht zu erwarten ist. Die Auswertung zuletzt genannter Substanzen zeigt deutlich, dass die Nutzung von Verschreibungsdaten nicht per se ausreicht, um die Emission von Antibiotika (und anderer Pharmazeutika) sowie die sich daraus ergebenden Umweltkonzentrationen mit ausreichender Sicherheit prognostizieren zu können. Für eine nachgelagerte ökotoxikologische Bewertung ist in diesen Fällen die Durchführung von Messungen unumgänglich. Zur effizienten Planung derartiger Kampagnen wurde der umfassende Datenpool dieser Studie hinsichtlich der erforderlichen Probenanzahl zur Bestimmung einer repräsentativen mittleren Jahresfracht ausgewertet. Es ergibt sich ein Minimum von 20 bis 40 homogen über das Jahr verteilten Proben, um die jährlich in die Kläranlage eingetragene Fracht an Antibiotika bzw. anderer Mikroschadstoffe mit ausreichender Sicherheit abschätzen zu können. Im Rahmen der SFA in der Kläranlage Dresden-Kaditz wird deutlich, dass Makrolide, Clindamycin und dessen Humanmetabolit Clindamycin-Sulfoxid sowie Trimethoprim in der nahezu keiner Elimination unterliegen, wohingegen Penizilline, Cefalosporine und auch Sulfamethoxazol teilweise bis vollständig abgebaut werden. Mit Levofloxacin und Ciprofloxacin handelt es sich um die einzigen untersuchten Antibiotika, welche zu einem signifikanten Massenanteil an Primär-, Überschuss- und Faulschlamm gebunden vorgefunden werden. Aufgrund der hohen Relevanz dieses Eliminationspfades für die zuvor genannten Antibiotika bedarf die Beobachtung von z. T. widersprüchlichen Schwankungen einer kritischen Betrachtung der Ergebnisse. Es ist nicht abschließend geklärt, ob die beobachteten Fluktuationen auf eine unzureichende Qualität der Probenahme und/oder der Analytik zurückzuführen sind oder sich die Schwankungen in einem für Faulbehälter tolerierbaren Bereich befinden. Im Anschluss an die verifizierten Antibiotikaemissionen erfolgte die Priorisierung der betrachteten Antibiotika nach ihrem ökotoxikologischen Effektpotential. Zum einen wurde der ökologische Einfluss auf verschiedene, die Nahrungskette bildende trophische Ebenen (Alge, Daphnie, Fisch) untersucht. In Anlehnung an die humanmedizinische Kombinationstherapie erfolgte im zweiten Ansatz die Beurteilung der Antibiotika hinsichtlich ihres möglichen Potentials zur Verstärkung von negativen Effekten durch das gleichzeitige Auftreten mit anderen Substanzen. Für Ciprofloxacin, Levofloxacin und die Gruppen der Makrolide und Cefalosporine konnten signifikante Beeinträchtigungen der aquatischen Umwelt nachgewiesen werden. Diese Stoffe und Stoffgruppen führten im Rahmen der untersuchten Substanzen entweder zur höchsten Schadwirkung gegenüber der niedrigsten trophischen Ebene oder besitzen das höchste Synergiepotential in Kombination mit anderen Substanzen. Die Auswertung der SFA bestätigt die grundsätzliche Eignung der Verschreibungsdaten sowie des entwickelten Prognosemodells zur Vorhersage von Antibiotikaemissionen im urbanen Raum. Die Stoffflussanalyse stellt somit ein strategisches, im Vergleich zur Messung kostengünstiges Instrument zur Identifikation von Hot Spots der Antibiotikaemission dar und erleichtert die Entscheidungsfindung für monetär aufwendige Reduktionsmaßnahmen am Ort der Entstehung oder in der Kläranlage (z.B. 4. Reinigungsstufe). Die Vorgehensweise zur Priorisierung von Substanzen hinsichtlich ihres ökotoxikologischen Effektpotentials eignet sich sehr gut, Antibiotika mit dem höchsten Schadpotential zu identifizieren. Die Verschneidung der Kenntnis dieser Substanzen mit den Ergebnissen der SFA macht deutlich, dass mit Ausnahme der Makrolide, alle ökotoxikologisch priorisierten Antibiotika eine mangelhafte Prognosefähigkeit aufweisen. Die unvollständige Abbildung kritischer Stoffströme, wie z.B. Frachtspitzen, führt insbesondere im Fall der ökotoxikologisch priorisierten Substanzen zu einer Minderung der Aussagekraft des auf Verschreibungsdaten beruhenden Prognoseansatzes. An diesem Punkt ist in zukünftigen Betrachtungen anzusetzen, um die Qualität von Verschreibungsdaten zu verbessern, potentiell nicht erfasste Emittenten in die Betrachtungen einzubeziehen, sowie die Dynamik der Rückhalte- und Eliminationsprozesse in der Kanalisation adäquat beschreiben zu können. Die ergänzende Betrachtung weiterer Anlagentechnologien (z.B. Festbettreaktoren) kann zur Bestätigung der am Beispiel der Kläranlage Dresden-Kaditz gewonnenen Ergebnisse beitragen bzw. Unterschiede bei der Elimination von Antibiotika das Potential, die Problematik der Antibiotika und anderer Mikroschadstoffe bereits während der Planung von Abwasseranlagen berücksichtigen zu können.

As florestas tropicais contêm mais da metade de todas as espécies terrestres existentes, mas sofrem com a crescente influência das atividades humanas. A destruição e a degradação de habitats são, atualmente, as principais ameaças à biodiversidade. Embora exista uma extensa literatura sobre extinção de espécies em paisagens antropizadas, muitos aspectos ainda foram pouco estudados. Desta forma, buscamos contribuir para o entendimento sobre: i) o atraso e as taxas de extinções locais de espécies após a perda e a fragmentação do habitat florestal; e ii) como as interações entre variáveis de fragmentação e de degradação do habitat (perda de qualidade do habitat) podem agravar a taxa de extinção local de espécies. Para responder a estas questões, amostramos grupos de aves em uma paisagem intensamente fragmentada ao norte do Estado do Mato Grosso, no Arco do Desmatamento Amazônico. Para quantificar o débito de extinção, inventariamos as espécies de papa-formigas em dez pontos de escuta, durante três dias, em 29 localidades amostrais, durante dois períodos separados por quase uma década. Avaliamos o legado do histórico de fragmentação da paisagem na extinção local de espécies por meio de um modelo que considera o tamanho do fragmento e o tempo desde o seu isolamento. Para investigar o papel das interações entre a degradação e a fragmentação de habitats na extinção de espécies, consideramos a assembleia de aves de sub-bosque, inventariadas por redes-de-neblina durante 14.400 horas em 30 localidades amostrais. Utilizamos a seleção de modelos para determinar quais interações e variáveis de degradação e de fragmentação são melhores preditoras do número de espécies, abundância e a composição da avifauna nos fragmentos. Nossos resultados revelaram que há duas etapas para a extinção de espécies: a extinção imediata e a extinção com atraso. Mesmo considerando a extinção com atraso, mais da metade das espécies desaparece em menos de duas décadas nos fragmentos, independentemente do tamanho do fragmento. Encontramos que a grande maioria das espécies nos fragmentos pequenos (<150 ha) foi extinta localmente logo após o isolamento, enquanto que nos fragmentos grandes, a perda de espécies ocorre com o tempo e as taxas de extinção local são mais elevadas. Ademais, os efeitos das interações entre as variáveis de degradação e fragmentação de habitat contribuem para a extinção local de espécies na paisagem estudada. Identificamos que o sinergismo entre o tamanho do remanescente florestal e a incidência de fogo é a principal causa da extinção de espécies nos fragmentos. No entanto, a abundância e a composição de espécies na comunidade foram, principalmente, influenciadas pelas interações aditivas entre o tamanho do fragmento e a presença de gado. Observamos também que as alterações causadas pela presença do gado nos fragmentos resultam na substituição de espécies especialistas de habitat florestal e típicas de sub-bosque por espécies que habitam o dossel, bordas e áreas perturbadas. As implicações de nossos resultados para a conservação são: o intervalo de oportunidade para mitigação dos efeitos da fragmentação e perda de habitat, sobre os papa-formigas, devido ao débito de extinção, mesmo que ainda presente, é muito curto para a implementação de ações eficazes para conservação, especialmente para os fragmentos pequenos, onde a perda da maioria das espécies ocorre imediatamente após o isolamento do fragmento. Portanto, ações de conservação deveriam otimizar seus esforços em fragmentos grandes (>150 ha), onde há maior chance de resguardar as espécies já comprometidas com a extinção. Além disso, as espécies nos fragmentos pequenos são negativamente e de forma mais intensa influenciadas pelos efeitos interativos com a incidência de fogo e penetração do gado. Concluímos que a preservação apenas dos remanescentes florestais não é suficiente para conservação da biodiversidade. Assim, as políticas públicas devem ser direcionadas à coibição de novos desmatamentos e queimadas, além do incentivo para a utilização de cercas ao redor dos remanescentes florestais em propriedades privadas / Tropical forests contain over half of all terrestrial species on Earth, but are succumbing to the growing impact of human activities. Habitat destruction and degradation are the main current threats to biodiversity. While there is an extensive literature on species extinction in human-modified landscapes, many aspects are yet to be explored. This study aims to contribute to our understanding of (i) the rates of time-lagged local extinctions of species in the aftermath of habitat loss and fragmentation; and (ii) the combined effects of habitat fragmentation and degradation (i.e. reduction in habitat quality) in aggravating rates of local extinctions. To develop this research, we sampled different functional groups of forest birds within an intensively fragmented landscape representative of the Arc of Deforestation of southern Brazilian Amazonia. To quantify the magnitude of extinction debt we inventoried antbirds at 29 forest sites during two periods, separated by nearly a decade. At each site, we carried out ten standardized point-counts over three days, which were validated with simultaneous digital recordings. We examined species extinction rates induced by historical landscape fragmentation using a model that considers forest fragment size and time since their isolation. In relation to interactions between habitat degradation and fragmentation, we considered all understorey birds inventoried by mist-nets during 14,400 hours at 30 sampling locations. We used model selection to determine which metrics and interactions of forest degradation (intensity of fires, selective logging and cattle presence within the forest remnants) and fragmentation (fragment size, amount of surrounding forest cover) best predicted the number, abundance and composition of species in the fragments. Our results revealed that there are two main stages for species extinctions: immediately after and time-lagged forest isolation. Even where there is a delay in species extinctions, over half of all species disappeared within less than two decades post-isolation, regardless of forest fragment size. We also found that the majority of species in small fragments (<150 ha) disappear immediately after isolation, whereas species losses in large fragments occur over time and they present higher local extinction rates. Moreover, interactions between habitat degradation and habitat fragmentation contributed to the local species extinctions in the studied landscape. Forest patch size operated synergistically with fire incidence as the main cause of local extinctions in fragments. However, the composition and abundance of species was influenced by the additive interactions between fragment size and cattle intrusion, which resulted in the replacement of understorey forest specialists with generalist species typically found in disturbed areas. The conservation implications of our results include: there is a narrow window of opportunity for mitigating the effects of habitat loss and fragmentation on antbirds, especially for small fragments, where most species were lost immediately after isolation. Conservation actions should be focused on large fragments (> 150 ha) where there is greater potential for retaining species committed to extinction. Also, species in small fragments were more affected by the detrimental effects of fire incidence and cattle intrusion. We therefore conclude that the preservation of remaining forest fragments in itself is not enough for forest biodiversity conservation; public policy should be directed to fire suppression and fencing to deter cattle access to remaining forest fragments within private proprieties

Introdução: Várias são as propostas descritas para tratar farmacologicamente a lesão traumática da medula espinal. A metilprednisolona já foi padronizada para uso clínico. O fator estimulador de colônia de granulócitos (G-CSF) tem sido promissor em estudos experimentais e clínicos. Não há pesquisas quanto ao efeito da associação dos dois fármacos. Objetivo: Avaliar o efeito do tratamento com o fator estimulador de colônia de granulócitos associado a metilprednisolona na lesão medular aguda experimental em ratos. Material e métodos: Foram avaliados 40 ratos Wistar submetidos a lesão medular moderada com o NYU-Impactor. Os animais foram divididos em quatro grupos de 10 ratos. O Grupo Controle não recebeu tratamento; o Grupo G-CSF, foi tratado com G-CSF no momento da lesão e diariamente ao longo dos cinco dias subsequentes; o Grupo Metilprednisolona, com metilprednisolona durante 24 horas; e o Grupo G-CSF/Metilprednisolona, com metilprednisolona durante 24 horas e G-CSF no momento da lesão e ao longo de cinco dias. Os animais foram mantidos vivos durante 42 dias; a avaliação funcional foi realizada com a aplicação da escala funcional de Basso, Beattie e Bresnahan (BBB) nos dias 2, 7, 14, 21, 28, 35 e 42 subsequentes à lesão. A avaliação dos potenciais evocados motores foi realizada no dia 42 e a avaliação histológica da lesão da região da medula espinal lesada, realizada logo após a eutanásia ocorrida no dia 42. Resultados e conclusões: A associação de metilprednisolona e G-CSF no tratamento do traumatismo medular contuso experimental em ratos promoveu melhora neurológica avaliada pela escala BBB superior à melhora promovida pela metilprednisolona e G-CSF quando utilizadas isoladamente. A associação teve também efeito sinérgico que resultou em melhora nos parâmetros histológicos no local da lesão. Não houve diferença entre os grupos quanto à avaliação neurofisiológica / Introduction: There are several proposals to pharmacologically treat traumatic spinal cord injury. Methylprednisolone has already been standardized for clinical use. Granulocyte colony stimulating factor (G-CSF) has been promising in experimental and clinical studies. There is no research on the effect of the association of the two drugs. Objective: to evaluate the effect of combined treatment of the granulocyte colony-stimulating factor (G-CSF) associated with methylprednisolone in experimental acute spinal cord injury in rats. Material and methods: Forty male Wistar rats were submitted to a moderate spinal cord injury with the NYU-Impactor. The animals were divided into four groups of ten rats each. The Control Group was not treated; the G-CSF Group was treated with G-CSF at the time of injury and daily over the next five days; the Methylprednisolone Group was treated with methylprednisolone for 24 hours; the G-CSF/methylprednisolone Group, was treated with methylprednisolone for 24 hours and G-CSF at the time of injury and daily over the next five days. The animals were kept alive for 42 days; Functional evaluation was performed using the Basso, Beattie and Bresnahan (BBB) score on days 2, 7, 14, 21, 28, 35 and 42 following the spinal cord injury. Evaluation of motor evoked potentials was held and histological examination of the lesion of the spinal cord was done immediately after euthanasia on day 42. Results and conclusions: The combination of methylprednisolone and G-CSF in the treatment of experimental spinal cord injury in rats promoted neurological improvement as assessed by BBB scale with greater improvement than with methylprednisolone or G-CSF when used alone. The combination of treatment had also a synergistic effect resulting in improvement in histological parameters at the injury site. There was no difference between groups regarding neurophysiological evaluation

Nos últimos anos a incidência de infecções causadas por bactérias Gram-negativas multirresistentes aumentou expressivamente. Esse fenômeno não foi acompanhado pelo desenvolvimento de novos antimicrobianos, resultando em infecções cuja opção de tratamento é a combinação de antimicrobianos. Acinetobacter baumannii, Pseudomonas aeruginosa e Serratia marcescens são importantes agentes de infecções relacionadas à assistência à saúde (IRAS), e capazes de adquirir resistência a várias classes de antimicrobianos, como os carbapenêmicos e polimixinas. Os testes que avaliam sinergismo in vitropodem ser uma ferramenta importante na escolha do tratamento antibiótico adequado para infecções causadas por microrganismos multirresistentes. O objetivo deste estudo foi avaliar métodos de sinergismo in vitroque possam ser utilizados na rotina de laboratórios de microbiologia clínica como alternativa ao método considerado padrão ouro (time-kill). Foram selecionados para o estudo 48 isolados Gram-negativos não fermentadores (20 A. baumannii e 28 P. aeruginosa) e 14 fermentadores (S. marcescens) do banco de cepas do LIM-54 com diferentes mecanismos de resistência identificados por PCR e sequenciamento total do genoma. Foram realizados, concentração inibitória mínima dos antimicrobianos e avaliação do sinergismo pelos métodos time-kill (TK), disco aproximação (DA) e CIM:CIM razão.A concordância dos métodos foi avaliada por teste de kappa e os resultados discordantes foram classificados em tipos de erros baseado no FDA.Os isolados apresentaram alta proporção de resistência a meropenem e 7 isolados de A. baumanniiapresentaram resistência a colistina. A combinação de colistina com fosfomicina ou meropenem apresentou elevado efeito sinérgico para os isolados de A. baumanniiresistentes a colistina pelo DA e TK. Por outro lado, a combinação de fosfomicina-meropenem apresentou concordância boa pelo teste de kappa e baixo número de erros entre os métodos TK e DA para todos os isolados de A. baumannii. Para os isolados de P. aeruginosa não foram detectados efeitos sinérgicos pelos métodos DA e CIM:CIM razão.O método CIM:CIM razão apresentou alta concordância com o TK entre os isolados de A. baumannii resistentes a colistina. A combinação ceftazidima-avibactam com meropenem apresentou elevado efeito sinérgico para os isolados de S. marcescensportadores do gene blaKPC-2 pelo DA e TK. O método de DA apresentou uma boa correlação com o TK para a combinação de fosfomicina-meropenem e ceftazidima-avibactam-meropenem, com baixa porcentagem de erros menores, podendo ser utilizado para avaliar sinergismo in vitro para essas combinações. Não foi identificado no estudo efeito antagônico, portanto, foram encontrados somente erros menores / In recent years, the incidence of infections caused by multiresistant Gram-negative bacteria has increased. This event has not been accompanied by the development of new antimicrobials, resulting in infections which treatment option is the combination of antimicrobials. Acinetobacter baumannii, Pseudomonas aeruginosa and Serratia marcescens are important causesof Hospital Acquired Infection (HAI), and able to acquire resistance to multiple classes of antimicrobials, such ascarbapenems and polymyxins. Synergism testing may be an important tool in choose the appropriate antibiotic treatment. The aim of this study was to evaluate in vitro synergism methods that can be used in a clinical microbiology laboratory as an alternative to the method considered the gold standard (time-kill).For the study were selected 48 non-fermenting (20 A. baumannii and 28 P. aeruginosa)and 14 fermenting(S. marcescens) Gram-negativeisolates from LIM-54strains bench with different resistance mechanisms identified by PCR and total genome sequencing.Minimum antimicrobial inhibitory concentration and synergism evaluation by time-kill (TK), disk approximation (DA) and MIC: MIC ratio were performed. Agreement of the methods was evaluated by kappa test and the discordant results were classified in types of errors based on the FDA. The isolates showed high proportion of resistant to meropenem and 7A. baumannii isolates showed resistance to colistin. The combination of colistin with fosfomycin or meropenem showed high synergistic effect for colistin-resistant A. baumannii isolates by DA and TK.On the other hand, the combination of fosfomycin-meropenem showed good concordance by kappa test and low number of errors between TK and DA for all A. baumannii isolates.For P. aeruginosa isolates no synergistic effects were detected by DA and MIC: MIC ratio.The method MIC: MIC ratio showed high concordance with the TK among the colistin-resistant A. baumannii isolates.The combination ceftazidime-avibactam with meropenem showed high synergistic effect for the isolates of S. marcescenscarryingblaKPC-2 gene by DA and TK. The DA showed a good agreement with TK for the combination of fosfomycin-meropenem and ceftazidime-avibactam-meropenem, with a low percentage of minor errors, and could be used to evaluate synergism in vitro for these combinations

The model of basal ganglia function proposed by Albin, Young and Penney (1989) describes two anatomically independent motor pathways, the direct and indirect. However, under normal conditions striatal dopamine (DA) is required for the expression of motor behavior, and DAergic control of the two pathways (via D1 and D2 receptors, respectively) is dependent on co-activation. We tested for a possible breakdown of D1/D2 synergism using transgenic R6/1 mice bearing the human huntingtin allele (Htt). Motor stereotypy, observed prior to the onset of HD-related symptoms, was rated on a 5-point scale following activation of: A) D1 receptors alone, B) D2 receptors alone, and C) stimulation of both D1 and D2 receptors. Results revealed that mice with the HD allele, like their WT litter mates, depend on the co-activation of the indirect and direct motor pathways to facilitate deliberate behavior.

<p>The hydrolysis kinetics of bacterial cellulose and its derivatives by <i>Trichoderma reesei</i> cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. </p><p>A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. </p><p>The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.</p>

Adsorption on solid surfaces from solution is a fundamental property of a surfactant. It might even be the most important aspect of surfactant behavior, since it influences many applications, such as cleaning, detergency, dispersion, separation, flotation, and lubrication. Consequently, fundamental investigations of surfactant adsorption are relevant to many areas. The main aim of this thesis has been to elucidate the adsorption properties, primarily on the solid/water interface, of a particular class of polyhydroxyl based surfactants: the alkyl glucosides. By the use of ellipsometry, the equilibrium and kinetic aspects of adsorption on titanium dioxide with respect to structural effects has been studied. Furthermore, the effects of small amounts of cationic surfactant additives on the adsorption on silica have been investigated. The results have been compared with similar studies for other nonionic surfactants. We have found that the surfactant structure has a strong effect on the adsorption properties. An increase in the surfactant chain length increases the cooperativity of the system. An increase in the head group polymerization decreases the cooperativity and the plateau adsorbed amount at equilibrium. The effect of surfactant structure on the adsorption kinetics depends on the concentration relative to the cmc, while the there is a decrease in the rate of desorption with increasing hydrophobic chain length independent of the concentration. The adsorption/desorption process is concluded to be diffusion driven, as suggested by the model used. When comparing these results with studies on ethylene oxide based surfactants, we conclude that the two types of surfactants exhibit similar trends on surfaces onto which they adsorb. Adsorption from binary surfactant solutions is even more interesting than adsorption from single surfactant solutions, since it brings us one step closer to the systems used in applications. In addition, adsorption from a mixture can be very different from adsorption from any of the single surfactants in the mixture. Alkyl glucosides alone do not adsorb on silica, but addition of small amounts of a cationic surfactant to the alkyl glucoside solution allows for adsorption on silica. A comparison between the adsorption and bulk properties has shown that mixed micellization explains most, but not all, effects of the coadsorption properties. Changing the pH in the mixed systems reveals that a surfactant with a pH-dependent charge and the ability to adapt its charge to the environment, e.g. a surface, enhances the adsorbed amount over a wider range of pH values than a purely cationic surfactant. It is well known that alkyl glucosides and ethylene oxides adsorb differently on different types of hydrophilic surfaces. As a consequence, replacing ethylene oxides with alkyl glucosides might not be all straight-forward; however, we have shown that the effect of the surface can be eliminated by the use of a cosurfactant. / <p>QC 20101018</p>

The hydrolysis kinetics of bacterial cellulose and its derivatives by Trichoderma reesei cellulases was studied. The cellulose surface erosion model was introduced to explain the gradual and strong retardation of the rate of enzymatic hydrolysis of cellulose. This model identifies the decrease in apparent processivity of cellobiohydrolases during the hydrolysis as a major contributor to the decreased rates. Both enzyme-related (non-productive binding) and substrate-related (erosion of cellulose surface) processes contribute to the decrease in apparent processivity. Furthermore, the surface erosion model allows, in addition to conventional endo-exo synergism, the possibility for different modes of synergistic action between cellulases. The second mode of synergism operates in parallel with the conventional one and was found to be predominant in the hydrolysis of more crystalline celluloses and also in the synergistic action of two cellobiohydrolases. A mechanism of substrate inhibition in synergistic hydrolysis of bacterial cellulose was proposed whereby the inhibition is a result of surface dilution of reaction components (bound cellobiohydrolase and cellulose chain ends) at lower enzyme-to-substrate ratios. The inhibition of cellulases by the hydrolysis product, cellobiose, was found to be strongly dependent on the nature of the substrate. The hydrolysis of a low molecular weight model substrate, such as para-nitrophenyl cellobioside, by cellobiohydrolase I is strongly inhibited by cellobiose with a competitive inhibition constant around 20 μM, whereas the hydrolysis of cellulose is more resistant to inhibition with an apparent inhibition constant around 1.5 mM for cellobiose.

Background: The aim of the present article is to explore interaction and correlation effects between familial depression liability and selected adverse (separation and traumatic) events in predicting the first onset of a major depressive episode (MDE) in a 10-year prospective longitudinal community survey. Methods: Analyses are based on 1982 subjects (14 to 24 years at baseline) without baseline MDE who participated during the whole study period and for whom diagnostic information about psychopathology in both parents was available. The offspring’s familial depression liability was determined by aggregating information on parental depressive symptoms obtained from family history data and direct interviews with parents. Data were assessed with the Munich-Composite International Diagnostic Interview according to its DSM-IV algorithms. Results: Adverse events predicted a substantially increased incidence of MDE among respondents with familial liability but not in those without familial liability. There was a significant interaction between familial liability and traumatic events with the strongest effect for the number of severe traumatic events (risk difference = 11.3%; 95% confidence interval = 3.55–19.15). Associations with familial liability were most pronounced for separation events. Conclusions: Adverse events are particularly pathogenic in individuals with familial liability. The involvement of interactions and correlations between familial liability and adversity might depend on type, severity, and number of events. Both processes are suggested to be concomitant rather than exclusive.