Este trabalho de mestrado teve como objetivo o desenvolvimento de uma metodologia analítica focada no preparo de amostra protéica. O objeto de estudo foram os fragmentos solúveis das isoformas da proteína precursora do amilóide (APPs) presentes no plasma rico em plaquetas. As APPs têm sido amplamente estudadas em diversos grupos de pesquisa no Brasil e em outros no mundo como possíveis biomarcadores para a doença de Alzheimer. O preparo de amostra é a etapa fundamental que influencia significativamente nos resultados seguintes, especialmente quando se trata de amostras protéicas que exigem maiores cuidados. Para a avaliação dos melhores preparos de amostra para as APPs, foi utilizado SDS-PAGE e eletrotransferências de proteínas por Western Blotting. A eficiência dos preparos foi avaliada baseando-se nos resultados de revelação com anticorpos específicos para APP e medidas de densitometria de bandas. Após a escolha do melhor preparo de amostra utilizando SDS-PAGE e Western Blotting, as isoformas da APP foram separadas por eletroforese bidimensional (2DE). Durante a etapa de preparo de amostra, os resultados inesperados de massa molecular, o que indicou possível biodegradação das APPs. A identificação da fonte de interferência foi realizada estudando as variáveis dos preparos de amostra. Com isso foi possível determinar a fonte de interferência, mas uma avaliação mais detalhada das isoformas (como utilização de espectrometria de massas) não foi possível. / The goal of this Master\'s work was to develop an analytical methodology focused on protein sample preparation. The analyte studied were soluble amyloid precursor protein isoforms (APPs) which has been studied in many groups in Brazil and around the world as a possible biomarker for Alzheimer\'s disease. Sample preparation is a crucial step that influence significantly on next results, especially about biological samples which require more attention. For the best sample preparation for APPs, was used SDS-PAGE and protein electrotransference by Western Blotting techniques. The efficiency of the sample preparations was evaluated based on specific antibody reactions and densitometry measures of these interactions. After that, the APP isoforms were analyzed by two dimensional electrophoresis (2DE). During the sample preparation, were obtained unexpected molecular mass results, which indicated some APPs biodegradation. For the determination of the interference source, the variants steps of the sample preparation were analyzed. The sample preparation interference source was identified, but a more detailed study of the isoforms (by mass spectrometry) was not possible as well as the analysis of the identity of the possible fragmented isoforms.

Nicotinic acetylcholine receptor (nAChR) activation is neuroprotective and nicotine is a cognitive enhancer. Loss of nAChRs, deposition of tau neurofibrillary tangles, cleavage of amyloid precursor protein (APP) and inflammation are well documented in the pathogenesis of Alzheimer’s disease (AD). Sequential cleavage of APP by β- and γ-secretase enzymes generates soluble Aβ peptides, with oligomeric forms of Aβ implicated in both the control of synaptic excitability and dysregulation of synaptic transmission and induction of neuronal death in AD. Aβ production is inhibited by calcium-dependent recruitment of α-secretase, as exemplified by activation of N-methyl-D-aspartate receptors (NMDAR). All neurodegenerative diseases are associated with inflammation, arising from altered homeostasis of the innate immune system, resulting in heightened activation of immune cells and induction of a pro-inflammatory environment. Stimulation of the α7 subtype of nAChR is anti-inflammatory and also enhances cognition and promotes neuronal survival. This work addressed the hypotheses that stimulation of highly calcium-permeable α7nAChR inhibits Aβ production by promoting α-secretase-mediated processing of APP and also modulates inflammatory cellular behaviour of microglia. Thus, this study assessed the role of α7nAChR at glutamatergic synapses, through probing effects on APP processing and phagocytosis in primary cortical neurons and microglia, respectively. Primary cortical neurons expressed functional α7nAChR and glutamate receptors, and through a number of experimental approaches, including immunoblotting and a cleavage reporter assay, results indicated α7nAChR activation with the α7nAChR-selective agonist PNU-282987 and positive allosteric modulator PNU-120596 had no effect on APP and Tau, in contrast to NMDAR activation that significantly modulated these proteins. Data suggest low expression of α7nAChR, coupled with distinct localisation of presynaptic α7nAChR and postsynaptic APP could explain the lack of effect. In addition, primary microglia were highly responsive to lipopolysaccharide and possessed functional α7nAChR that coupled to ERK phosphorylation. Microglial α7nAChR activation promoted neuroprotective phagocytic behaviour, in agreement with the ‘cholinergic anti-inflammatory pathway’. This study supports the hypothesis that α7nAChR are modulators of anti-inflammatory behaviour, thus α7nAChR-selective ligands are viable candidates for the treatment of AD and promoting cognitive enhancement.

There is a considerable interest in the use of tungsten oxide in the research and development of new materials and devices, such as gas sensors and as photocatalysts. In order to improve the photocatalytic properties of WO3, its combination with metals which allows the preparation of WMxOy materials are believed to be promising photocatalysts under visible light. The present work deals with the synthesis of homo- and hetero-metallic tungsten alkoxide and amide compounds using the single source precursor approach for potential chemical vapour deposition precursors of mixed-metal oxide films.

Dementia, the most common cause of which is Alzheimer's disease (AD), currently affects 850,000 people in the UK, a figure set to rise to over 1 million by 2025. There is currently no disease modifying therapy available to slow or halt this progressive disease. Current understanding of AD implicates the neurotoxic amyloid-β (Aβ) peptide as the primary initiator in a cascade of events leading to the neuronal cell death and brain atrophy associated with the disease. Therefore, inhibiting the production or enhancing the clearance of Aβ within the brain has become a major target for the production of disease modifying therapeutics. Aβ is produced by brain cells through the sequential proteolytic cleavage of a larger transmembrane protein known as the amyloid precursor protein (APP) by β- and γ-secretases. Several aspects of APP physiology can influence its proteolysis, and thus Aβ production, including the isoform of APP which is expressed, its trafficking and subcellular location and its physical interactions with other proteins in the cellular environment. Here we have investigated the influence of subcellular trafficking and location and protein-protein interactions on the differential proteolysis of two APP isoforms, APP695 and APP751 in a neuroblastoma cell line. We have shown that APP751 undergoes less amyloidogenic proteolysis than APP695 and that retention within the early secretory pathway may contribute to this difference. APP751 shows higher co-localisation to the trans-Golgi network than APP695 in immunofluorescence microscopy studies, while addition of a mutation which causes APP proteolysis in the secretory pathway reduces the large difference in amyloidogenic proteolysis of these two isoforms. Targeting APP endocytosis from the cell surface, thought to be a key determinant in Aβ generation, effects APP isoform proteolysis and Aβ production to a similar extent in both the APP isoforms suggesting differences in proteolysis occur before this trafficking event. We also show by immunoblot analysis that the APP isoforms may be differentially cleaved by proteases other than β- and γ-secretase to produce recently identified proteolytic fragments. Using a liquid chromatography - tandem mass spectrometry approach coupled to prior stable isotope labelling of amino acids in cell culture (SILAC), we have identified the interactomes of the two APP isoforms in our model system. Gene ontology analysis identified enrichment of nuclear and mitochondrial proteins specifically in the APP695 interactome. Using siRNA mediated protein knockdown, we have shown interactions with Fe65 and ataxin-10 specifically influence Aβ generation from the APP695 isoform. Fe65 alters proteolysis at the rate limiting β-secretase cleavage step, while ataxin-10 alters proteolysis by γ-secretase. Interaction with growth-associated protein 43 specifically influences Aβ generation from the APP751 isoform, altering proteolysis at the γ-secretase step. Finally we have shown that recently discovered familial AD-linked mutation and protective mutation within the Aβ region of the APP protein have consistent effects on APP proteolysis in both the APP isoforms.

by Betty Shuc Han Wills. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 117-128). / ACKNOWLEDGMENT --- p.i / ABSTRACT --- p.ii / TABLE OF CONTENTS --- p.iv / LIST OF APPENDICES --- p.viii / Chapter CHAPTER 1 --- INTRODUCTION --- p.1 / Chapter CHAPTER 2 --- LITERATURE REVIEW --- p.6 / Parental Responses to the Diagnosis of Acute Lymphocytic Leukaemia (ALL) --- p.7 / Disclosure Of the Child's Diagnosis --- p.10 / Impact of Cancer Treatment on Parents --- p.14 / Sources of Support for Parents --- p.18 / Coping Strategies of Parents of Children With ALL --- p.20 / Coping With The Uncertainty of the Disease --- p.23 / Research Studies Involving Chinese Parents --- p.24 / Summary Of Issues From Literature Review --- p.27 / Chapter CHAPTER 3 --- METHODOLOGY / Research Design --- p.29 / Sampling --- p.31 / Data Collection Method --- p.32 / Data Collection Procedure --- p.34 / Ethical Considerations --- p.38 / Pilot Study --- p.40 / Data Analysis --- p.42 / Issues of Reliability and Validity --- p.45 / Validity --- p.45 / Reliability --- p.48 / Chapter CHAPTER 4 --- RESULTS & DISCUSSION / Introduction --- p.50 / Chapter (I). --- Parents' Profile --- p.51 / Demographic Characteristics Of The Parents / Chapter (II). --- Major categories Corresponding To Interviewing The Mothers --- p.54 / Initial Reactions of the Child's Confirmed Diagnosis --- p.55 / Unpreparedness for the child's Diagnosis / Suddenness of the Diagnosis --- p.56 / Physical and psychological reactions to the child's Diagnosis --- p.58 / Sources of Support for the Mothers --- p.62 / The mothers' main source of support / Other sources of support for the mothers --- p.64 / Disclosure Of Child's Diagnosis --- p.66 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended families --- p.68 / Disclosure of child's diagnosis to non-family members --- p.70 / Uncertainty Brought On By The Illness --- p.71 / Waiting for confirmation of diagnosis / Uncertainty about the success of treatment --- p.73 / Uncertainty about the child's future --- p.74 / Changes In The Family Routine --- p.75 / Needed to normalise family life / Chapter (III). --- Major Categories Corresponding to Interviewing The Fathers Initial reactions to the child's confirmed diagnosis of ALL --- p.78 / Suddenness of diagnosis --- p.79 / Physical and psychological reactions to the diagnosis --- p.80 / Disclosure Of The Child's Diagnosis --- p.82 / Disclosure of the child's diagnosis to the child / Disclosure of the child's diagnosis to members of the immediate and extended family --- p.85 / Disclosure of the child's diagnosis to non-family members --- p.86 / Sources Of Support For The Fathers --- p.87 / Support from immediate and extended families / Support from medical professionals --- p.89 / Support from friends --- p.90 / Changes In The Family Routine --- p.91 / Coping Strategies Utilised By The Fathers --- p.92 / Open communication / Use of religious beliefs and rituals --- p.93 / Chapter (IV). --- Comparison Of Categories Found Between The Mothers And The Fathers --- p.95 / Initial reactions to the child's confirmed diagnosis of ALL / Disclosure of the child's diagnosis --- p.96 / Sources of support for the parents --- p.100 / Changes in the family routine --- p.101 / Summary of findings --- p.103 / Chapter (V). --- Differences between the initial and second interviews --- p.105 / Chapter CHAPTER FIVE --- CONCLUSION / Limitations Of The Study --- p.108 / Implications For Nursing Practice --- p.112 / Recommendations For Future Research --- p.114 / Conclusion --- p.115 / REFERENCES --- p.117 / APPENDIX I - PERSONAL DATA FORM --- p.129 / APPENDIX II - INTERVIEW SCHEDULE --- p.130 / APPENDIX III - CONSENT FORM --- p.134 / APPENDIX IV - SAMPLE SCRIPT OF INTERVIEWS WITH MOTHERS --- p.135 / APPENDIX V - MATRICES ON MOTHERS AND FATHERS --- p.140 / APPENDIX VI - SAMPLE OF FIELD NOTES FOR MOTHERS AND FATHERS --- p.143

The main objective of this thesis is to understand molecular crystallization as a multistep process with or without polymeric additives, including transient liquid-liquid phase separation, nanocrystal nucleation within the dense phase, and subsequent nanocrystal self-assembly or self-organization in sequence. The thesis starts with a quaternary model system, containing DL-Glutamic acid (Glu), polyethyleneimine (PEI), water, and EtOH, for the understanding of multistep precipitation of Glu with PEI as an additive. The experiments were performed by mixing Glu-PEI aqueous solution with a non-solvent EtOH. First, the phase diagram of the quaternary system is determined, obtaining precipitate, coacervates, or homogeneous mixtures by varying Glu/PEI w/w and water/EtOH v/v. Coacervation is observed to occur over a wide range of Glu/PEI with various volumes. The composition of coacervates is conveniently characterized by nuclear magnetic resonance spectroscopy. The observed coacervates are thermodynamically stable phases rich in solute, which is different from metastable polymer-induced liquid precursors. The combination of atomic force microscopy, small angle scattering, and ξ-potential measurements confirms the coexistence of monomers and Glu/PEI complexes and the aggregation of complexes in Glu-PEI-water systems. This suggests that there might be a direct structural transformation between the Glu-PEI complexes in aqueous solution and the metastable liquid precursors in a water-EtOH mixture. The multistep mechanism of Glu precipitation with PEI as an additive is investigated thereafter. The combination of stopped flow and small angle scattering demonstrates that the initially formed liquid precursors pass through an alteration of growth and coalescence. Combined with results from optical microscopy and scanning electron microscopy, the nucleation of nanoplatelets happens within each liquid precursor droplet, and nanoplatelets reorient themselves and self-organize into a radial orientation in the crystalline microspheres. The recipe was then extended to the precipitation of organics in other oppositely charged amino acid-polyelectrolyte systems. After the success in preparing hierarchical microspheres in solution, the similar recipe can be extended to the preparation of patterned thin films on substrate. By dipping a quaternary DL-Lys·HCl (Lys)–polyacrylic acid (PAA)–water–EtOH dispersion on a hydrophilic slide, the fast evaporation process of the volatile solvent EtOH is responsible for the homogeneous nucleation of NPs. Then, the following complete evaporation causes the mesocrystallization of a continuous spherulitic thin film along the receding line of the liquid, which again transforms into a mesocrystalline thin film. Furthermore, annealing is used to optimize the property of mesocrystalline thin films. As evaporation is a non-equilibrium process, it can be used to tune the kinetics of crystallization. Therefore, hierarchical or periodical thin films are obtainable by starting the evaporation from microspheres recrystallization, obtaining mesocrystalline thin films with 4 hierarchy levels. The results reveal that evaporation provides an easy but effective way for the formation of patterned structures via the positioning of NPs after their fast nucleation, resulting in different kinds of patterns by controlling the concentration of NPs, solvent evaporation rate, and other physical forces. Non-classical crystallization is not limited to crystallizations with polymeric additives. We also observed the nucleation and growth of a new molecular layer on the growing DL-Glu·H2O crystals from a supersaturated mother liquor by using an in-situ atomic force microscopy (AFM), where the nucleation and growth of a molecular layer proceed via amorphous nanoparticle (NP) attachment and relaxation process before the observation of the growth of a newly formed molecular layer. NP attachment to the crystal surface is too fast to observe by using in-situ AFM. The height shrinkage of NPs, combined to the structural transformation from 3D amorphous NPs to 2D crystalline layer, is observed during the relaxation process. The nucleation and growth of a newly formed molecular layer from NP relaxation is contradictory to the classical nucleation theory, which hypothesizes that nuclei show the same crystallographic properties as a bulk crystal. The formation of a molecular layer by NP attachment and relaxation rather than attachment of single molecules provides a different picture from the currently held classical nucleation and growth theory regarding the growth of single crystals from solution. / Das Hauptziel dieser Arbeit ist das Verständnis der molekularen Kristallisation, sowohl mit als auch ohne polymere Additive, als einen mehrstufigen Prozess. Dieser beinhaltet eine transiente flüssig-flüssig Phasentrennung, die Nukleation von Nanokristallen in der dichten flüssigen Precursor-Phase so wie eine anschließende nanokristalline Selbstorganisation. Die Arbeit beginnt mit Untersuchungen an einem quaternären Modelsystem bestehend aus DL-Glutamat (Glu), Polyethylenimin (PEI), Wasser und Ethanol. Das Phasendiagramm dieses quaternären Systems wird durch Variation der Glu/PEI w/w und Wasser/EtOH v/v Verhältnisse bestimmt, wobei Präzpitat aus polymerinduzierten flüssigen Precursor, Koazervate oder homogene Mischungen erhalten werden Das thermodynamisch stabile Koazervat kann als Referenz für das Verständnis von flüssigen Precursorn angesehen werden, welche in der Natur metastabil und transient sind. Der mehrstufige Mechanismus der Glu-Präzipitation mit PEI als Additiv wird dann mittels Neutronen Kleinwinkelstreuung untersucht. Dies zeigt, dass die ursprünglich gebildeten flüssigen Precursor noch vor der Nukleation von Nanokristallen einen Wechsel von Wachstum und Koaleszenz durchlaufen. Die Ergebnisse aus optischer- und Eletronenmikroskopie zeigen, dass sowohl die flüssigen Precursor Superstrukturen ausbilden als auch, dass die Nukleation von Nanoplättchen in jedem einzelnen Precursor Tropfen von statten geht. Dies geschieht noch bevor sich die Nanoplättchen selbst in einer radialen Orientierung ausrichten. Diese Studie liefert die Kinetik der Präzipitation von organischen Stoffen in Gegenwart von polymeren Additiven. Eine ähnliche Vorgehensweise wie für die Herstellung von Mikrokügelchen kann für die Darstellung von gemusterten Filmen angewandt werden. Die homogene Nukleation von Nanopartikeln (NPs) findet während der Verdampfung einer quarternären DL-Lys·HCl-Polyacrylsäure-Wasser-Ethanol Dispersion auf einer hydrophilen Oberfläche statt. Die darauffolgende vollständige Verdampfung löst die Mesokristallisation eines kontinuierlichen sphärolithischen dünnen Films aus, welcher sich wiederum in einen mesokristallinen dünnen Film umwandelt. Mesokristalline Filme mit 4 Hierarchiestufen bzw. auch periodische Filme werden durch die Verdampfung der Mikrokügelchen-Dispersion erhalten. Die Ergebnisse zeigen, dass die Verdampfung eine einfache aber effektive Methode zur Herstellung von verschieden gemusterten hierarchischen Filmen darstellt. Nicht-klassische Kristallisation wird auch in der Abwesenheit von polymeren Additiven beobachtet. Wir verfolgen mittels Rasterkraftmikroskop (AFM) die Nukleation und das Wachstum einer neuen molekularen Schicht auf wachsenden DL-Glu·H2O Kristallen aus übersättigter Mutterlauge. Die Bildung einer neuen molekularen Schicht verläuft durch die Anlagerung von amorphen Nanopartikeln. Das Schrumpfen der NPs zusammen mit der strukturellen Änderung von dreidimensionalen NPs zu 2D Schichten wird während dieses Relaxationsprozesses beobachtet. Schließlich kommt es zu der Ausbildung einer neuen molekularen Schicht. Die Bildung einer molekularen Schicht durch die Anlagerung von Nanopartikeln aus der Lösung und die darauffolgende Relaxation liefert ein abweichendes Bild zu der bisher gängigen klassischen Theorie des Kristallwachstums.

Disinfection byproducts (DBPs), such as organic chloramines, THMs, HAAs, and nitrosamines, are formed during mandatory disinfection processes in drinking water treatment. Many of these DBPs have been shown to be potentially carcinogenic. Extensive research has been conducted on the occurrence and formation of these DBPs. However, there has been limited research on their relationships with each other, which may be important for the understanding of their formation mechanisms, and the nature of their precursors is still relatively unknown. Ultimately, this information will be key for the development of possible improvements in treatment technologies. Results of this study improve the understanding of DBP formation in swimming pool water. Some BFAs and PCP additives were identified as potential DBP precursors. Influence of BFAs and PCP additives on DBP formation in swimming pool water was also illustrated. Results provided feasible strategies to minimize DBP formation while maintaining the efficiency of disinfection.

Disinfection byproducts (DBPs), such as organic chloramines, THMs, HAAs, and nitrosamines, are formed during mandatory disinfection processes in drinking water treatment. Many of these DBPs have been shown to be potentially carcinogenic. Extensive research has been conducted on the occurrence and formation of these DBPs. However, there has been limited research on their relationships with each other, which may be important for the understanding of their formation mechanisms, and the nature of their precursors is still relatively unknown. Ultimately, this information will be key for the development of possible improvements in treatment technologies. Results of this study improve the understanding of DBP formation in swimming pool water. Some BFAs and PCP additives were identified as potential DBP precursors. Influence of BFAs and PCP additives on DBP formation in swimming pool water was also illustrated. Results provided feasible strategies to minimize DBP formation while maintaining the efficiency of disinfection.

Following the concept utilize binary selenides as precursors with rapid thermal process (RTP) to fabricate CuInSe2 (CIS) thin film. In order to find the most promise process to get high quality CIS, several precursor stacking sequences have been tested which including SLG/In-Se/Cu-Se/Se, SLG/Cu-Se/In-Se/Se, SLG/0.1In-Se/Cu-Se/0.9/In-Se/Se, and SLG/0.5In-Se/Cu-Se/0.5/In-Se/Se, and the experiment result shows SLG/In-Se/Cu-Se/Se is the most suitable stacking sequence. Subsequently, varying Se flux to obtain several kinds copper selenides (Cu7Se4, Cu3Se2, CuSe, CuSe2) and indium selenides, try to find the suitable pairs through these binary selenides in SLG/In-Se/Cu-Se/Se structure. The suitable combination phase in Cu-Se precursor layer is CuSe blend with CuSe2. Large grain size CIS, about 1£gm, can be prepared in such precursor phase with film thickness between 700nm to 1£gm, strong (112) prefer-orientation vertical with substrate as well as good adhesion. Films were characterized through scanning election microscopy (SEM) to obtain grain size, surface morphology as well as film thickness. The X-ray diffractometer (XRD) was used to identify phase contained in whole film, and the phase constitution near surface layer was examined by Raman spectroscopy. If there are some second phases remaining in the thin film, combining the phase examination result of XRD and Raman spectroscopy, it can be estimate the second phase exist in the surface layers or internal film area.

ABSTRACT Key words: PCDD/FS , aromatic precursor compounds , transition metal catalysts , chlorine donor , surface of fly ash particles In the last 20 years , increasing concern has focused on the environmental chemicals that mimic hormone functions , some of them toxic , which producing cancer , suppression of the immune system , and death from undefined causes . These chemicals are not made intentionally , but are formed as contaminants in combustion sources , including PCDD/FS of dioxin-like compounds that emitted from municipal solid waste incinerators (MWSI) and hazardous waste incinerators (HWI) . This study investigated the role that fly-ash plays in the formation of PCDD/FS using a commercial scale fluidized bed waste incinerators (FBWI) , which rated capacity at 3750000 kcal/hr (LHV) . In this design , a lay of sand is placed on the bottom of the combustion chamber. During combustion, the hot gases are channeled through the sand and crushed solid waste at relatively high velocity . This generated about eight times more mass of fly-ash will be produced from combustion zone than the others, and also makes much greater of PCDD/FS through the air pollutants control devices(APCD) to emission stack . The general reaction in this formation pathway is an interaction between an aromatic precursor compound and chlorine promoted by a transition metal catalyst on a reactive fly-ash surface. Since these reactions involve heterogenous chemistry , the rate of emissions is less depended on reactant concentration than conditions that promote formation such as temperature , retention time , transition metal catalysts (e , g,. Cu , Fe , Pb , Zn , Sn) and availability on catalytic surfaces of fly ash particle . These forming conditions will be proceeding a series of well study and experiment on fly-ashes from 4 zones (F1 , F2 ,F3 , F4) of FBWI . PCDD/FS synthesis from combustion of FBWI can potentially be explained by three principal mechanisms that results can be divided into several major parts as follows¡G 1. The fly-ash from zone F3 generated about 47 times more mass of PCDD/FS than zone F1 . 2. The F3 fly-ash proved to be the most active catalytic (Cu , Zn) medium , despite similarities with respect to specific surface area and average pore diameters . In addition , there are up to 75.9 percent by weight of Zn and 97.6 percent of Cu has been found in F3 of overall ashes. 3. In both fly- ash and transition metal catalysts in formation mechanisms are the dominant controlling factor for rates of PCDD/FS.