Most theoretical models predict an optimal offspring size that maximises parental fitness. Variation in the quality of the offspring environment can result in multiple offspring size optima and therefore variation of offspring provisioning can evolve. Variation in offspring provisioning is common and found across a variety of taxa. It can be defined as between populations, explained by optimality models, or between and within individuals, neither so easily explained by optimality models. My research focused on the evolution of variable offspring provisioning by testing theoretical models relating to variation in offspring provisioning between and within individuals. Using comparative methods, I found a positive relationship between intraclutch variation in offspring provisioning and variation in the quality of the offspring environment in a suite of pond breeding frogs. This positive relationship provided evidence that patterns of variable offspring provisioning are related to the offspring environment. This study also identified a species (Crinia georgiana) with high variation in offspring provisioning on which to focus further investigations. High variation in offspring provisioning occured between and within individuals of this species independent of female phenotype and a trade-off in offspring size and number existed. In laboratory studies, increased yolk per offspring led to increased fitness per offspring. Parental fitness calculations revealed that in high quality conditions production of small more numerous offspring resulted in higher parental fitness, but in lower quality conditions the production of large offspring resulted in the highest parental fitness. This was confirmed in field experiments under natural conditions using molecular markers to trace offspring to clutches of known provisioning, allowing me to measure exact parental fitness. The strategy of high variation in offspring size within clutches can be of benefit when the future of the offspring environment is not known to the parents: as a form of bet-hedging. Further study of the offspring environment revealed that conditions such as density dependent fitness loss, spatial variation in habitat quality, and non-random offspring dispersal, can combine to create the conditions predicted by theoretical models to maintain a strategy of variable offspring provisioning in the population. My research provides a comprehensive empirical test of the theory of variable offspring provisioning

Human health is increasingly threatened by the emergence of multiply drug resistant malignant organisms. Yet, our understanding of the numerous ways by which such resistance arises is modest. Here, we present evidence of a bet hedging strategy in the budding yeast, Saccharomyces cerevisiae, to counter the effects of cytotoxic drugs through the action of Pdr5, an ATP-binding cassette transporter. We have employed flow cytometry and fluorescent activated cell sorting to probe the expression levels of a GFP-tagged version of PDR5 in individual cells. The results obtained from these experiments demonstrate that each yeast population is variable in the levels of Pdr5 production, and a small subpopulation of cells produces this efflux pump at much higher quantities than the population average. Consequently, cells with high and low levels of Pdr5 grow differentially in presence and absence of cycloheximide, a cytotoxic drug. These properties are highly suggestive of a bet hedging strategy mediated by Pdr5 levels. We further link this bet hedging strategy to the transcriptional regulatory network of PDR5 consisting of two major transcription factors, Pdr1 and Pdr3. Our analysis suggests that a self-activating feedback loop acting on Pdr3 plays an important role in generation of the aforementioned subpopulation. Furthermore, our results point to a large difference in the activity of these two regulators wherein Pdr3 is notably stronger than Pdr1. The disparity in their activity could indicate a mechanism for generation of the observed proportions of subpopulations with regards to the level of Pdr5.

Immune exhaustion can be a major clinical problem for patients who have cancer or chronic inflammation. Persistent antigen stimulation drives T cells to express multiple surface markers called immune checkpoints. When these markers bind to their corresponding ligands that are expressed by antigen (e.g. tumor cells), T cells become metabolically impaired and lose several important functions; some cell signaling pathways are inhibited, while other intracellular mediators are re-modulated. Eventually, both CD4+ and CD8+ T cells behave dysfunctionally in ways that may facilitate cancer progression. Immune checkpoints are a major hallmark of immune exhaustion. In addition, natural killer (NK) cells, a critical immune cell subset in the peripheral immune system, also express immune checkpoint molecules, and are responsible for detecting and destroying circulating tumor cells. Yet, little research has investigated immune checkpoints on NK cells. Here, we explored the role of Bromodomain and ExtraTerminal domain (BET) proteins (BRD2, BRD3, BRD4), which are important transcriptional co-regulators, and critical for proliferation and metastasis in many cancer types, in the regulation of immune checkpoint molecules in several immune cell subsets, including CD4+ and CD8+ T cells, and NK cells. Through binding to acetylated histone tails of nucleosomal chromatin, BET proteins assist in transcription of multiple genes. Deregulated expression of BET proteins promotes cancer development or tumor cell metastasis, and new data show the BET proteins contribute to immune exhaustion. Furthermore, Type 2 diabetes mellitus (T2DM) is another worrisome problem related to cancer. T2DM patients show increased risk of developing cancer. Patients with both T2DM and any type of cancer show higher risks for metastasis. Significantly, T2DM patients also show immune exhaustion, suggesting a hypothesis that BET proteins may couple immune system dysfunction, abnormal metabolism and cancer incidence or progression. Specifically, T2DM has been defined to be a metabolic and a chronic inflammatory disease. The 5' Adenosine Monophosphate-activated Protein Kinase (AMPK) signaling pathway is a key pivot of cell metabolism and as well a significant target of drugs that normalize blood glucose, such as metformin. Based on published data, we considered that it is important to explore the mechanism of how immune checkpoints are regulated through metabolic pathways, focusing on immune exhaustion in T2DM patients. Moreover, considering that the expression of BET proteins promotes cancer development and progression, and metastasis and immune exhaustion are characteristic of many cancers as well, we suspected a potential relationship among BET proteins, the AMPK metabolic signaling pathway and immune exhaustion is worth exploring. Here, we measure expression of the immune checkpoint molecules TIM-3, TIGIT, PD-1, and CTLA-4 on normal T cells and NK cells by flow cytometry. We demonstrate different degrees of regulation of immune checkpoints by BET proteins on stimulated T cells and NK cells. Comparing stimulated-only cells with stimulated-plus AMPK inhibitor cells, we found that inhibition of the AMPK signaling pathway causes divergent expression patterns for TIM-3 and TIGIT, PD-1 and CTLA-4. Simultaneous inhibition of both BET proteins and the AMPK signaling pathway, shows that BET proteins regulate TIM-3 and TIGIT through an AMPK-independent metabolic pathway and regulate PD-1 and CTLA-4 through an AMPK-dependent pathway. Overall, we show TIM-3 and TIGIT, PD-1 and CTLA-4 display different expression patterns under regulation of the AMPK signaling pathway, and we show that BET proteins regulate TIM-3, TIGIT, PD-1 and CTLA-4 through both AMPK-dependent and -independent pathways. These findings are important because they reveal novel mechanisms of immune checkpoint regulation, which may be valuable for targeting in cancer patients who are being treated with checkpoint inhibitors.

The specific surface area, determined by the BET method, is a parameter often used to scale results of mineral studies of surface reactions in terms of rate and capacity to the field scale. Such extrapolations of results from small-scale laboratory experiments to the field-scale are important within many environmental applications. An example of this is for the prediction of radionuclide retention in the bedrock surrounding a deep repository for radioactive waste, following failure of the engineered barriers, where radionuclides may sorb onto minerals constituting the geological environment. As a first step, the approach used in this work is to study the relationship between specific surface area and the particle size (0.075-8 mm) and to approach the field scale via measurements on large, centimetre-sized pieces, for seven natural minerals commonly found in granite: apatite, biotite, chlorite, K-feldspar, hornblende, labradorite and magnetite. The underlying assumption is that sorption of radionuclides can be related to specific surface area of a particular mineral in a continuation of this project.The results show that the phyllosilicates biotite and chlorite have a specific surface area that is about 10 times larger than the other minerals. Over the range of particle size fractions studied, the specific surface area varies between 0.1 and 1.2 m2g-1 for biotite and chlorite. The other studied minerals have specific surface areas varying between 0.01 m2g-1 for the largest fraction and up to 0.06 - 0.12 m2g-1 for the smallest. Results show linear relationships between the specific surface area and the inverse of the particle size for all studied minerals for small particle sizes, as expected. For some minerals, however, the data seemingly can be divided in two linear trends, where a change in internal surface area, surface roughness and/or particle geometry as the particle size decreases may explain this behaviour. Interestingly, for larger particles, there is a deviation from the linearity observed for small particles. Tentatively, this behaviour is attributed to a disturbed zone, created by the mechanical treatment of the material during particle size reduction, extending throughout small particles, but not altering an undisturbed core of the larger particles. In agreement with this, measurements on large pieces show a surface area 5 to 150 times lower than expected from the linear trends observed for the crushed material, implying an overestimation of the surface area and possibly also of the sorption capacities of the rock material from simple extrapolations of experimental results employing finely crushed material to the field situation. / QC 20110929 / Äspö Radionuclide sorption

No description available.

Influenza is one of the most common diseases worldwide, yet the vaccines against influenza are only 35% effective at protecting against infection. Creating a more effective vaccine requires an understanding of the foundation and the factors that contribute to a strong and protective adaptive immune response. T-bet [TBX21] is a transcription factor that plays an instrumental role in the orchestration of the type 1 immune response, which is the specialized response used by the immune system for a cell-mediated response against intracellular pathogens, such as influenza. It has yet to be explored in an influenza setting on the role T-bet in the production of antibodies. The aim of this study is to understand T-bet's role in production of antibody isotypes and identify whether expression of T-bet is more important for antibody production in T cells or B cells. We expected T-bet knockout (KO) mice to have IgG2a and that T-bet expression would be more important in T cells for antibody production. An enzyme-linked immunosorbent assay (ELISA) was used to measure the amount of virus-specific antibody in T-bet KO versus wild type (WT) mice infected with influenza. The results show that the T-bet KO and WT mice have relatively the same amount of IgG and IgG1, but the T-bet KO have a significantly lower level of IgG2a, confirming T-bet's importance for its production. To distinguish the importance of T-bet expression while T-bet expression in T cells was constant, a model was developed to allow us to control expression of T-bet in B cells. The results however were inconclusive, and the experiment will have to be repeated to make a firm conclusion on the roles of lymphocytes in the control of IgG isotypes. Overall, these results indicate that the manipulation of T-bet expression can be used as a vector to control IgG antibody levels, which holds potential for the improvement of vaccines.

L'allergie respiratoire au pollen de bouleau affecte un nombre important de personnes dans le monde. Il est estimé que 100 millions d'individus sont sensibilisés à l'allergène majeur du pollen de bouleau nommé Bet v 1. L’immunothérapie allergénique, basée sur l'administration répétée de l'allergène incriminé, permet la rééducation du système immunitaire du patient d’un profil TH2 vers un profil TH1/Treg et à terme la diminution des symptômes allergiques. Ces travaux de thèse avaient donc pour finalité de produire et de caractériser l'allergène recombinant Bet v 1, à des fins d’immunothérapie allergénique. Dans ce cadre, différentes méthodes analytiques ont été développées et appliquées afin d'optimiser le procédé de production via l'élimination de différentes impuretés liées au produit ou au procédé de production et de documenter la structure de l’allergène. En particulier, l'utilisation de la spectrométrie de masse a permis la détermination de la masse exacte de l'allergène ainsi que la vérification complète de sa séquence en acides aminés. Les travaux en spectrométrie de masse ont également contribué aux détections et identifications de diverses impuretés et produits de dégradations et ont ainsi conduit à plusieurs optimisations du procédé industriel de production de l'allergène recombinant. Les activités immunologiques de certains produits de dégradations ont également été investiguées. La structure tertiaire (spatiale) de l'allergène a été déterminée par diffraction aux rayons X. Enfin, ces travaux ont permis de documenter la qualité de l'allergène recombinant rBet v 1 afin i) de l'établir comme substance de référence pour la Pharmacopée Européenne et ii) de procéder à une étude clinique d’immunothérapie allergénique de phase II auprès de 483 patients allergiques au pollen de bouleau. / Respiratory allergy to birch pollen affects a large number of people in the world. It is estimated that 100 million people are sensitized to the major allergen from birch pollen, namely Bet v 1. Allergen immunotherapy, based on the repeated administration of the allergen of interest, allows the modification of the patient's immune response from a TH2 to a TH1/Treg pattern and thus the reduction of allergic symptoms. This study was therefore aimed to produce and characterize the recombinant Bet v 1 (rBet v 1) allergen, for immunotherapy purpose.In this context, various analytical methods have been developed and applied in order to optimize the production of rBet v 1 via the reduction of process or product-related impurities as well as to document the quality of the purified allergen. In particular, the use of mass spectrometry has allowed the determination of the exact mass of the intact allergen and the complete verification of its amino acid sequence. Mass spectrometry data have also contributed to the detection and identification of impurities and degradation products and have therefore led to several optimizations of the industrial process for the production of the recombinant allergen. Immunological activities of certain degradation products were also investigated and the allergen tertiary structure was determined by X-ray diffraction. Finally, this study was decisive in order i) to establish rBet v 1 as a chemical reference substance for the European Pharmacopoeia as well as ii) to perform a phase II clinical study conducted in 483 patients with birch pollen-induced rhinoconjunctivitis.

No description available.

Nicht-kleinzelliger Lungenkrebs (NSCLC) hat bis heute einen hohen medizinischen Bedarf an effektiveren Therapien. Inhibitoren der Bromodomain and extra-terminal domain (BET) Familie wie JQ1 wirken in verschiedenen Krebsarten, einschließlich Lungenkrebs. Während ihre Aktivität auf die Expression von Onkogenen wie c-Myc in vielen Studien untersucht wurde, bleibt der Effekt von BET-Inhibition auf den Apoptose Signalweg weitgehend unbekannt. In dieser Arbeit wurde die Aktivität von BET-Inhibitoren auf den Zellzyklus und auf Komponenten der Apoptose-Antwort der Zelle untersucht. Genomweite Transkriptionsanalysen haben zusammen mit Chromatin Immunpräzipitation und anschließender Sequenzierung geholfen das MYC Gen und dessen assoziierte Super-enhancer als primäres Ziel des BET-Inhibitors JQ1 zu identifizieren. Mittels einer Gruppe von NSCLC Modellen belegt diese Arbeit, dass Zelllinien die auf die BET-Inhibitoren reagieren in Apoptose gehen und eine Reduktion der S-Phasen Population zusammen mit gleichzeitiger de-regulation der c-Myc Expression aufwiesen. Andererseits konnte die ektopische Überexpression von c-Myc der anti-proliferativen Wirkung entgegenwirken. Die Auswirkung von BET-Inhibition auf die Expression von 370 Genen, die in der Apoptose Regulation involviert sind, wurde in sensitiven und resistenten Zellen verglichen und dabei wurde die starke BET-Abhängigkeit der Expression von zwei Schlüsselgenen der Apoptose FLIP und XIAP festgestellt. Die Kombination von JQ1 mit dem tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) oder dem Chemotherapeutikums Cisplatin die verstärke die Induktion von Apoptose in sowohl BET-Inhibitor sensitiven als auch in resistenten Zellen. Des Weiteren zeigte die Kombination einen verbesserten Antitumor-Effekte in A549 tumortragenden Mäusen. Insgesamt zeigen diese Ergebnisse, dass die Identifizierung von BET-abhängigen Genen unterstützend für die Wahl von therapeutischen Kombinationspartnern in der Krebsbehandlung sein kann. / Non-small cell lung cancer (NSCLC) has a high medical need for more effective therapies. Small molecule inhibitors of the bromodomain and extra terminal domain (BET) family such as JQ1 are active in different cancer types, including lung cancer. While their activity on oncogene expression such as c-Myc has been addressed by many studies, the effects of BET inhibition on the apoptotic pathway remain largely unknown. This work evaluates the activity of BET bromodomain inhibitors on cell cycle distribution and on components of the apoptotic response. Genome-wide transcriptional analyses together with chromatin immunoprecipitation followed by sequencing helped to identify the MYC gene and associated super-enhancers as a primary target of JQ1. Using a panel KRAS-mutated NSCLC models, it was found that cell lines responsive to BET inhibitors underwent apoptosis and reduced their S-phase population, concomitant with down-regulation of c-Myc expression. Conversely, ectopic c-Myc overexpression rescued the anti-proliferative effect of JQ1. The effects of BET inhibition on the expression of 370 genes involved in apoptosis were compared in sensitive and resistant cells and the expression of the two key apoptosis regulators FLIP and XIAP was found to be highly BET-dependent. Consistent with this, combination treatment of JQ1 with the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or the pro-apoptotic chemotherapeutic agent cisplatin enhanced induction of apoptosis in both BET inhibitor sensitive and resistant cells. Furthermore the combination of JQ1 with cisplatin led to significantly improved anti-tumor efficacy in A549 tumor-bearing mice. Altogether these results show that the identification of BET-dependent genes provides guidance for the choice of drug combinations in cancer treatment.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Os nanotubos de carbono apresentam extraordinÃrias propriedades fÃsico-quÃmicas que tÃm sido exploradas para aplicaÃÃes que vÃo desde a ciÃncia de materiais atà a biologia. No entanto, a maioria das aplicaÃÃes requer modificaÃÃes quÃmicas dos nanotubos de modo a explorar ao mÃximo seu potencial. Neste contexto, o desenvolvimento de protocolos de tratamento e de purificaÃÃo à muito importante. O objetivo deste estudo à contribuir para o entendimento de como a mudanÃa de uma variÃvel especÃfica, a temperatura, afeta as propriedades fÃsico-quÃmicas dos nanotubos de carbono de paredes mÃltiplas (MWNTs) durante a purificaÃÃo. Um grama (1g) de MWNTs (Ctube 100, CNT Co. Ltd., Incheon - CorÃia do Sul) foram submetidos a um refluxo de Ãcido nÃtrico 9 mol/L por 12 horas em temperaturas de 25ÂC, 75ÂC, 125ÂC e 175ÂC. ApÃs o refluxo, as amostras foram resfriadas em um banho tÃrmico, filtradas em condiÃÃes de vÃcuo com uma membrana de PTFE de 0, 2 Âm e lavadas com Ãgua deionizada atà que o pH neutro do filtrado foi atingido. Por Ãltimo, os MWNTs oxidados foram secos em vÃcuo durante 48 h. As tÃcnicas utilizadas neste trabalho para a realizaÃÃo das caracterizaÃÃes forneceram resultados que tratam desde caracterÃsticas macro e de superfÃcie atà as associadas ao nÃvel atÃmico. A estabilidade tÃrmica foi analisada por meio de medidas de TGA, uma vez que muitos dos grupos funcionais criados no tratamento sÃo termicamente instÃveis. A oxidaÃÃo resultante do tratamento introduz grupos polares na superfÃcie dos MWNTs e assim cria a estabilidade eletrostÃtica necessÃria para uma dispersÃo estÃvel em meio aquoso. A estabilidade foi avaliada para cada amostra por meio de medidas de potencial zeta. A quantidade e o tipo de resÃduos catalÃticos presentes nas amostras antes e depois do tratamento foram analisados atravÃs de medidas de EDX e UV-VIS. Medidas de espectroscopia Raman Confocal permitiram a anÃlise especÃfica dos defeitos estruturais criados sobre a superfÃcie dos tubos, em conseqÃÃncia do processo de oxidaÃÃo. Constatou-se que os experimentos Raman devem ser realizados sobre muitos pontos para poder acessar as propriedades mÃdias das amostras. A capacidade de adsorÃÃo da superfÃcie de cada amostra apÃs o tratamento foi inspecionada atravÃs de medidas de Ãrea superficial especÃfica (mÃtodo BET). Ao comparar os resultados de diferentes tÃcnicas, foi possÃvel acessar os efeitos da temperatura do tratamento sobre as propriedades fÃsico-quÃmicas relevantes, permitindo assim a obtenÃÃo de amostras bem caracterizadas que serÃo Ãteis para futuros estudos em Ãreas bio-relacionadas. / Carbon nanotubes exhibit remarkable physico-chemical properties which have been exploited for applications ranging from materials science to biology. However, most of the aplications requires the chemical modification of the nanotubes in order to exploit their maximum potential. In this context, the development of protocols for treatment and purification is very important. The purpose of this study is to contribute for the understand on how the change of a particular purification parameter, temperature, affects the physico-chemical properties of multi-walled carbon nanotubes (MWNTs) during the purification. One gram (1g) of MWNTs (Ctube 100, CNT Co. Ltd., Incheon - South Korea) were subjected to a reflux with nitric acid 9 mol/L for 12 hours at temperatures 25ÂC, 75ÂC, 125ÂC and 175ÂC. After refluxed, the samples were cooled down in a heat bath, filtered in vacuum condition using a PTFE membrane of 0, 2 Âm and washed with deionized water until the neutral pH of the filtrate was reached. Afterwards, the oxidized MWCNTs were dried in vacuum for 48 h. The experimental techniques used in this work to perform the samples characterization provide results that allow to get information from macro and surface characteristics to those associated with the atomic level. Thermal stability was analyzed by TGA measurements, since many of the functional groups created during the treatment are thermally unstable. Oxidation resulting from treatment introduces polar groups on the surface of MWNTs thus creating the electrostatic stability required for a stable dispersion in aqueous medium. The stability was evaluated for each sample by zeta potential measurements. The amount and type of catalytic residues present in the samples before and after treatment were analyzed by means of EDX and UV-VIS. Techniques confocal Raman spectroscopy measurements allowed the specific analysis of the structural defects created on the surface of the tubes as a consequence of oxidation process. It was found that Raman experiments should be performed on may points of the sample in order to access their average properties. The adsorption capacity of the surface of each sample after treatment, were inspected through specific surface area measurements (BET method). By comparing the results of different techniques it was possible to access the effects of temperature on the treatment on the relevant physico-chemical properties thus allowing us to have well characterized samples that will be useful for further studies in biorelated areas.