Glioblastoma Multiforme (GBM) is an extremely aggressive grade IV brain tumour that is highly infiltrative and can spread to other parts of the brain quickly. It is the most common primary brain tumour where patients have a median survival of 14.6 months. Symptoms vary depending upon the location of the tumour and include seizures, progressive headaches and focal neurological deficit. The poor prognosis is characterised by deregulation of many key signalling pathways involving survival, growth, apoptosis and evasion of immune surveillance. In this study, we investigated whether complement factor H related protein 5 (CFHR5) from primary GBM cells direct from patients exhibited functional activity similar to factor H. The presence of CFHR5 was validated by western blot and ELISA technique from B30, B31 and B33 primary GBM cells. The functional capacity of CFHR5 was examined through the alternative pathway, co-factor, and decay acceleration assay. We demonstrated that CFHR5 was able to inhibit the alternative pathway through the same mechanism as factor H. Emerging evidence had shown that the innate immune protein surfactant protein D (SP-D) and recombinant human SP-D (rhSP-D) were able to induce apoptosis in eosinophilic leukaemic cells. We studied the ability of rhSP-D to induce apoptosis in U87 GBM cells through apoptotic and viability assays. rhSP-D was unable to mediate cell death and instead increased cell viability. This led us to investigate the expression of SP-D in U87 and B30 GBM cells through western blot, ELISA and immuno-fluorescence detection. We demonstrated novel information about the production of SP-D by GBM cells. To extend our study, we investigated the interaction of THP-1 macrophage with rhSP-D bound U87 cells. We carried out live cell imaging, RT-qPCR, and cell viability assays, to study the changes in cytokine expression and viability of cells. THP-1 did not engulf U87 cells; however, it did reduce the number of cells and decrease the expression of pro-tumourigenic cytokines. This study highlights the ability of primary GBM cells to evade innate immune detection by the secretion of functionally active CFHR5. It also demonstrated the ability of U87 to evade destruction by rhSP-D and THP-1 highlighting the extremely aggressive behaviour of the tumour and lack of new treatment to improve prognosis in over a decade.

Activation of innate immune surveillance mechanisms during the development of cancer is well-documented. However, knowledge of how these innate immune proteins, when added exogenously, independent of tumour microenvironment, affect tumour cells is limited. In Chapter 3, the effects of human C1q and its individual globular domains (ghA, ghB and ghC) on an ovarian cancer cell line, SKOV3, have been examined. C1q and globular head modules induced apoptosis in approximately 55% of cells, which involved upregulation of TNF-α and Fas and activation of the caspase cascade. This occurred in parallel to the downregulation of mTOR, RICTOR and RAPTOR survival pathways, which are often over-expressed in the majority of the cancers. Thus, this study provided evidence for another complement-independent role of C1q. The second part of this thesis was to investigate the effect of Human Surfactant Protein-D (SP-D), which is known to modulate secretion of a range of cytokines and chemokines by effector immune cells, such as TNF-a and TGF-β, at mucosal surfaces during infection and inflammation. Our hypothesis was that SP-D can influence these soluble factors as a part of its putative role in the immune surveillance against pancreatic cancer, where the inflammatory tumour microenvironment contributes to the epithelial-to-mesenchymal transition (EMT) invasion and metastases. In this study, a recombinant fragment of human SP-D (rfhSP-D) inhibited TGF-β expression in a range of pancreatic cancer cell lines, thereby reducing their invasive potential by downregulating Smad2/3 expression that may have interrupted signal transduction negatively, which affected the transcription of key mesenchymal genes such as Vimentin, Zeb1 and Snail. Furthermore, prolonged treatment with rfhSP-D induced apoptosis in the pancreatic cancer cell lines via activation of the caspase cascade. Thus, this study added another layer to the well-known protective role of SP-D.

O final do período gestacional é marcado por importantes processos que caracterizam a maturação pulmonar fetal, dentre os quais destacam-se alterações estruturais, como a expansão das áreas de troca gasosa; e funcionais, tais como o aumento na produção de surfactante, cuja principal função é reduzir a tensão superficial na interface ar-líquido alveolar, evitando o colapso dos alvéolos na fase final da expiração. Estudos realizados em diferentes espécies animais indicam a influência de fatores endócrinos, incluindo os glicocorticóides, no desenvolvimento pulmonar fetal e transição para a vida extra-uterina. Até o momento, não há estudos na espécie canina, com o objetivo primordial de avaliar a ação da corticoterapia materna na melhora da função pulmonar. Desta forma, são objetivos deste estudo identificar as alterações morfométricas e funcionais pulmonares de neonatos pré-termos e termos submetidos à corticoterapia materna pré-natal e correlacioná-las à melhora da função pulmonar no período neonatal. Para tanto, 25 neonatos da espécie canina, nascidos por cesariana programada, foram alocados aleatoriamente em 2 grupos: Grupo Controle (CONT) (sem corticoterapia materna; n=15) e Grupo Betametasona (BETA) (corticoterapia materna aos 55 dias de gestação; n=10), por aplicação de betametasona (Celestone Soluspan®) em dose única de 0,5 mg/Kg de peso materno, por via de administração intra muscular (IM). No grupo Controle, os neonatos foram avaliados aos 55, 57 e 63 dias de gestação, enquanto no Grupo Betametasona, aos 57 e 58 dias de gestação. Perfez-se a avaliação clínica por escore Apgar, hemogasometria e radiografia pulmonar. Ainda, as modificações pulmonares estruturais e funcionais foram verificadas por análise morfométrica e imunoistoquímica para detecção do número de pneumócitos tipo II produtores da proteína B do surfactante (SP-B) no parênquima pulmonar. Houve melhor evolução clínica nos neonatos pertencentes ao grupo BETA 57 já aos 60 minutos de vida. Os valores de freqüência cardíaca foram estatisticamente maiores nos grupos tratados e controle termo, em comparação ao grupo CONT 57. O escore de freqüência e padrão respiratórios foi estatisticamente superior nos grupos BETA 57 e CONT 63, seguido pelo grupo BETA 58. Valores estatisticamente semelhantes de irritabilidade reflexa foram observados entre os grupos tratados e termo. Do nascimento aos 60 minutos de vida não houve diferença estatística na avaliação do tônus muscular entre os grupos, com valores significativamente superiores aos 240 minutos de vida nos grupos tratados e controle termo. Os neonatos do grupo CONT 63 apresentaram escore de mucosas aparentes da avaliação Apgar estatisticamente superior em relação aos demais grupos ao nascimento, com valores estatisticamente iguais aos grupos tradados e CONT 57 aos 60 minutos de vida. Ao nascimento e após 2 horas de vida, todos os neonatos apresentaram acidemia, com melhor resposta compensatória ao desequilíbrio ácido-básico no grupo BETA 58. Houve maior septação nos grupos tratados e controle termo, em relação aos demais grupos. Um percentual estatisticamente superior de alveolização foi observado no grupo CONT 63, seguido pelo grupo BETA 58. Um menor percentual de sáculos foi identificado no grupo CONT 63 seguido pelos grupos BETA 57 e CONT 55. Não evidenciou-se diferença estatística quanto ao número de pneumócitos tipo II marcados para a proteína SP-B entre os grupos tratados e CONT 57. A avaliação radiográfica mostrou menor percentual de broncograma aéreo, bem como áreas de atelectasia, no grupo BETA 57, associado à melhor visualização do parênquima pulmonar. Em conclusão, a administração de betametasona materna no período pré-natal induz alterações estruturais do parênquima pulmonar, resultando em melhores valores de escore Apgar. Houve melhor resposta compensatória nos grupos tratados, reflexa ao aumento da capacidade de troca gasosa pulmonar. Não foi possível identificar aumento na síntese de surfactante pulmonar entre os grupos, em resposta à administração pré-natal de betametasona. / The final gestational period is marked by an important processes that characterize the lung fetal maturation, like structural changes such as expansion of the areas of gas exchange, and functional changes, such as increased production of surfactant, whose main function is to reduce the surface tension in the air-liquid interface alveolar, preventing the alveoli from collapsing during late expiration. Studies in different species indicate the influence of endocrine factors, including glucocorticoids in fetal lung development and transition to extrauterine life. Up till now, there is not studies in dogs, with the primary objective to evaluate the action of maternal corticosteroid therapy in improving lung function. Thus, the objectives of this study was to identify morphological changes in lung function in preterm and terms neonates submitted to prenatal maternal corticosteroids and correlate them to the improvement in lung function during the neonatal period. For it 25 canine neonates, born by scheduled cesarean section, were randomly divided into 2 groups: control group (CONT) (no maternal corticosteroid therapy, n = 15) and Group betamethasone (BETA) (maternal corticosteroid therapy at 55 days gestation; n = 10), by application of betamethasone (Celestone Chronodose Injection ®) in a single dose of 0.5 mg / kg of maternal weight, route of administration by intra-muscular (IM). Control group neonates were evaluated at 55, 57 and 63 days of gestation, and the betamethasone group, at 58 and 57 days of gestation. The clinical assessment was made by Apgar score, blood gas and pulmonary radiography. Still, the structural and functional lung changes were verified by morphometric analysis and immunohistochemistry to detect the number of type II pneumocytes producers surfactant protein B (SP-B) in the lung parenchyma. There was better clinical outcome in the groups BETA 57 at 60 minutes of life. The values of heart rate were significantly higher in term treatment and control groups compared to the group CONT 57. The score of respiratory frequency and pattern was statistically higher in groups BETA 57 e 63 followed by the group BETA 58. Statistically similar reflex irritability were observed between the treated groups and term. From birth to 60 minutes of life there was not statistical difference in the assessment of muscle tone between the groups, with significantly higher values at 240 minutes of life in term treatment and control groups. Neonates of CONT 63 has mucous apparent assessment of Apgar statistically superior to other groups at birth, with values statistically equal to tratads and group CONT 57 at 60 minutes of life. At birth and after 2 hours of life, all neonates had acidemia, with better compensatory response to acid-base balance in the group BETA 58. There was an increased septation in treated and control groups comparing other groups. A statistically higher percentage of alveolarization was observed in group CONT 63, followed by the group BETA 58. A lower percentage of saccules was identified in the group CONT 63 followed by groups BETA 57 and CONT 55. There was not statistical differences in the number of type II pneumocytes marked for protein SP-B between the treated groups and CONT 57. The radiographic evaluation showed a lower percentage of air bronchogram, and atelectasis in the group BETA 57, associated with better visualization of the pulmonary parenchyma. In conclusion, maternal administration of betamethasone in prenatally period induced structural changes of the lung parenchyma, resulting in higher values of Apgar score. There was greater compensatory response in the treated groups, the reflex of an increase capacity of pulmonary gas exchange. It was not possible to indentify increases synthesis of surfactant between the groups in response to prenatal administration of betamethasone.

acase@tulane.edu

"March 2001". Bibliography: leaves 193-238. Relates changes in the development of the pulmonary surfactant system in response to birth strategy, lung morphology and phylogeny in order to determine the extent of conservation in this process, by quantifying the total of phsospholipid, disaturated phospholipid and cholesterol in the lung washings of embryonic and hatchling chickens, oviparous bearded dragons and viviparous sleepy lizards, snapping turtles and green sea turtles throughout the final stages of incubation and gestation. Finds that the pattern of development of pulmonary surfactant lipids is consistent with that of mammals.

Includes bibliographical references.

The fetal lung undergoes extensive physiological and biochemical maturation prior to birth in preparation for its postnatal function as an organ for gas exchange. Pulmonary surfactant, a substance that reduces surface tension and prevents alveolar collapse, is produced by type II pneumocytes within the lung. Reduced ability to produce surfactant leads to neonatal respiratory distress syndrome. Synthesis of the phospholipid component of surfactant, phosphatidylcholine (PC), is stimulated by fibroblast-pneumocyte factor (FPF), a protein expressed by fibroblast cells within the fetal lung. Although its function is well known, the identity of this important protein has remained a mystery. Recent research has suggested that FPF may be neuregulin-1, a growth factor found in many tissues during development. Enhanced synthesis of PC (and therefore detection of FPF) is measured using a tissue culture-based method. Primary cultures of lung fibroblasts and type II pneumocytes are prepared, and fibroblast-conditioned medium (FCM) is exposed to the type II cells. Resultant PC synthesis is measured using radioisotope-labeled PC-precursor and a chloroform-based lipid extraction method. Initial results using this method were very inconsistent, so a study was undertaken to determine which parts of the method could be contributing to this inconsistency. Cell density of type II cultures (measured in μg DNA.plate-1) was shown to have a significant effect on results. Treatment of fibroblasts with 100 nM dexamethasone and exposure of type II cultures to the resultant FCM caused a mean 9.17% increase in PC synthesis, but when only type II cultures with a cell density below 25 μg DNA.plate-1 were analyzed, this value increased to 17.56%. Type II cultures with cell density above this threshold value showed a mean increase in synthesis of only 3.39%. The consistent application of [3H]-choline chloride also had a significant effect on results. Experiments utilizing phorbol 12-myristate 13-acetate to stimulate fibroblasts were very inconsistent. The mean activity of the initial [3H]-choline chloride solution prepared for these experiments was found to be 2.04 μCi.mL-1, compared to a mean of 4.79 μCi.mL-1 for all other experiments. Observations from this section of the study led to considerable revision of the method used to measure PC synthesis. Quadrupolar ion trap mass spectrometry (MS) was used to analyze FCM and determine if neuregulin-1 (NRG1) could be FPF. A mass spectrum was obtained for recombinant NRG1, with predominant ions of 1068, 1142 and 1246 m/z. All three of these ions were also detected in both control and dexamethasone-treated FCM. Partial fragmentation of 1068 m/z of NRG1 was achieved using MS2, and generated a base peak of 1047 m/z. This fragmentation was also observed in 1068 m/z from FCM. LC/MS was utilized to quantify NRG1 in FCM, using a standard curve generated using recombinant NRG1. Control FCM had a NRG1 concentration of 19.85 μg.mL-1, while the concentration in dexamethasone treated FCM was 41.59 μg.mL-1. FCM which had given no positive response to dexamethasone when tested using the indirect cultured cell system had a control NRG1 concentration of 20.85 μg.mL-1, and a dexamethasone treated concentration of 22.84 μg.mL-1. These values were not significantly different from the control value for FCM in those fibroblast cultures that had generated a positive response to dexamethasone. Results of this section of the study have provided strong evidence that NRG1 is a major component of FPF, and a review of the NRG1 signaling pathway further supports this conclusion. Insulin-like growth factors (IGFs) are functionally related to neuregulins and are known to be important in fetal development. The effect of IGF-II on synthesis of surfactant PC and its subsequent secretion from type II pneumocytes was studied. In terms of PC synthesis, IGF-II was tested at concentrations of 0.4, 0.6 and 0.8 μM. The mean increase in synthesis was found to be 6.00, 6.15 and 6.91%, respectively. These values were not significantly different from control values. Secretion of PC was tested over the concentration range of 0.1 to 1.6 μM, with no significant effect observed. Possible inhibition by IGF-II was also studied, using the known stimulants of secretion, neuromedin C and isoproterenol. No significant effect on the enhanced level of secretion was observed when IGF-II was added with either secretagogue. Lack of an appropriate receptor and/or the possibility that cultured cells may not exactly mimic the situation in vivo are probably the reasons IGF-II has no effect on either synthesis or secretion.

Adsorption kinetics and adsorption-desorption of atrazine on organoclay composites prepared with the surfactant 6-piperazin-1-yl-N,N'-bis-(1,1,3,3-tetramethyl-butyl)-(1,3,5)triazine-2,4-diamine and Houston Black clay were studied using the indirect batch equilibration procedure. The organoclay composites sorbed significantly more atrazine than the Houston Black clay. Adsorption equilibrium was reached after 72 h for the organoclay composites. Atrazine adsorption isotherms were described by linear partitioning. The Koc values ranged from 605 to 5271 L kg-1 for the organoclay composites compared to a value of 41 L kg-1 for the Houston Black clay. The organoclay composite containing 20% surfactant on a total weight basis provided the most efficient adsorption of atrazine, although organoclay composites containing much lower amounts of surfactant also adsorbed significant amounts of atrazine. An average of 11% of sorbed atrazine was released during desorption. Characterization of desorption products showed only atrazine molecules being released from the organoclay composites.

In recent years, the aim of supramolecular syntheses is not only the creation of particular structures but also the introduction of specific functions in these supramolecules. The present work describes the use of the ionic self-assembly (ISA) route to generate nanostructured materials with integrated functionality. Since the ISA strategy has proved to be a facile method for the production of liquid-crystalline materials, we investigated the phase behaviour, physical properties and function of a variety of ISA materials comprising a perylene derivative as the employed oligoelectrolyte. Functionality was introduced into the materials through the use of functional surfactants. <br><br> In order to meet the requirements to produce functional ISA materials through the use of functional surfactants, we designed and synthesized pyrrole-derived monomers as surfactant building blocks. Owing to the presence of the pyrrole moiety, these surfactants are not only polymerizable but are also potentially conductive when polymerized. We adopted single-tailed and double-tailed N-substituted pyrrole monomers as target molecules. Since routine characterization analysis of the double-tailed pyrrole-containing surfactant indicated very interesting, complex phase behaviour, a comprehensive investigation of its interfacial properties and mesophase behavior was conducted. The synthesized pyrrole-derived surfactants were then employed in the synthesis of ISA complexes. The self-assembled materials were characterized and subsequently polymerized by both chemical and electrochemical methods. The changes in the structure and properties of the materials caused by the in-situ polymerization were addressed.<br><br> In the second part of this work, the motif investigated was a property rather than a function. Since chiral superstructures have obtained much attention during the last few years, we investigated the possibility of chiral ISA materials through the use of chiral surfactants. Thus, the work involved synthesis of novel chiral surfactants and their incorporation in ISA materials with the aim of obtaining ionically self-assembled chiral superstructures. <br><br> The results and insights presented here suggest that the presented synthesis strategy can be easily extended to incorporate any kind of charged tectonic unit with desired optical, electrical, or magnetic properties into supramolecular assemblies for practical applications. / Supramolekulare Chemie zielt auf den Aufbau großer Moleküle und neuer Materialien aus kleineren Einheiten. Durch supramolekulare Wechselwirkungen d.h. nicht-kovalente Bindungen, können definierte Ordnungen von Molekülverbänden in Größen von mehreren 100 Mikrometern hergestellt werden. Diese Wechselwirkungen und die daraus resultierenden Anordnungen von Molekülen bestimmen die für Anwendungen relevanten makroskopischen Materialeigenschaften. <br><br> Es gibt viele verschiedene Wechselwirkungen, die in der Supramolekularen Chemie angewendet werden können. Eine davon ist die ionische Wechselwirkung, die in dieser Arbeit als Triebkraft für die Herstellung supramolekularer Materialien verwendet wird. Diese Strategie wurde vor kurzem ISA (Ionic-Self-Assembly, d.h. Ionische Selbsorganisation) genannt. <br><br> Die vorliegende Arbeit beschäftigt sich mit der Herstellung funktioneller Nanomaterialen durch die Anwendung der ISA-Strategie. Da sich die ISA-Strategie als einfache Methode für die Produktion von Flüssigkristallen herausstellte, untersuchten wir die Eigenschaften vieler ISA-Materialen, die einen Farbstoffbaustein integriert haben. Die Funktion der Materialien wurde hierbei durch die Verwendung funktioneller Tenside geschaffen. Um die Anforderungen für die Produktion funktioneller ISA-Materialen durch die Nutzung funktioneller Tenside sicherzustellen, wurden Pyrrol-Monomere als Tenside hergestellt. Durch die Pyrrol-Einheiten sind die Tenside polymerisierbar und zeigen danach Potential für Leitfähigkeit. <br><br> Es wurden Pyrrol-Tenside sowohl mit Einzel- als auch mit Doppelketten synthetisiert. Da die Standardcharakterisierung des Doppelketten-Tensids ein interessantes Phasenverhalten zeigte, wurden umfassende Untersuchungen der Grenzflächeneigenschaften und des mesophasen Verhaltens durchgeführt. Beide Tenside wurden dann in der Produktion von ISA-Materialien verwendet. Die hergestellten Materialen wurden charakterisiert und konnten durch die Pyrrol-Einheit sowohl chemisch als auch elektrochemisch polymerisiert werden. Die aus dieser Polymerisierung resultierenden Änderungen der Eigenschaften, Struktur und Function der Materialen wurden ebenfalls untersucht. <br><br> Der zweite Teil dieser Arbeit klärt Nutzungsmöglichkeiten chiraler Tenside für die Herstellung chiraler ISA-Strukturen. Obwohl Chiralität keine eigentliche Funktion sondern eine Eigenschaft ist, haben chirale Strukturen in den letzten Jahren viel Aufmerksamkeit bekommen. Deshalb wurden, mit dem Ziel chirale ISA-Strukturen zu erhalten, neue chirale Tenside hergestellt und diese als Bausteine in ISA-Materialien benutzt.<br><br> Die mit dieser Arbeit gewonnenen Ergebnisse und neuen Einsichten zeigen, dass die ISA-Strategie leicht erweitert werden kann, um jede Art von Bausteine zu integrieren. Dadurch können nanostukturierte Materialien mit gewünschten spezifischen optischen, elektrischen oder elektromagnetischen Eigenschaften für praktische Anwendungen geschaffen werden.

Cationic and zwitterionic polymerizable surfactants bearing tri- and tetraethyleneglycol spacer groups between the polymerizable moiety and the surfactant structure were prepared and polymerized. Monomers and polymers were investigated with respect to their aggregation behavior in aqueous systems and compared to analogous monomers and polymers lacking spacer groups. In the case of the monomeric surfactants, the spacer groups depress both the Kraffttemperature and the critical micelle concentration. the area occupied per molecule at the air-water interface is substantially enlarged by the spacers, whereas the depression of surface tension is nearly constant. Although the monomers with and without spacers are true surfactants, all the polymers are water-insoluble, but form monomolecular layers at the air-water interface. In analogy to the monomer behavior, the incorporation of the spacer groups increases the area occupied per repeat unit at the air-water interface substantially, but hardly affects the surface activity.