Lam Yuk Lan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PUBLICATIONS --- p.vii / TABLE OF CONTENTS --- p.ix / LIST OF FIGURES --- p.xiii / LIST OF TABLES --- p.xiv / LIST OF ABBREVIATIONS --- p.xvi / Chapter Section 1: --- Background --- p.1 / Chapter Chapter 1: --- Cell-free circulating DNA --- p.1 / DNA and Man --- p.2 / Cell-free Circulating DNA in Plasma and Serum --- p.4 / The Discovery and Early Development --- p.4 / Clinical Implications --- p.5 / Cancers --- p.5 / Prenatal diagnosis --- p.11 / Pregnancy abnormalities --- p.14 / Organ transplantation --- p.15 / Trauma and post-traumatic complications --- p.15 / "Origin, Mechanisms and Characteristics" --- p.16 / Methods of Analysis --- p.22 / Chapter Chapter 2: --- Trauma and Organ Failure --- p.25 / Trauma and Society --- p.25 / The Problem of Organ Failure --- p.26 / Definitions --- p.27 / Pathogenesis --- p.228 / Inflammation --- p.29 / Predictions --- p.30 / Trauma Scoring Systems --- p.31 / Abbreviated Injury Scale --- p.32 / Injury Severity Score --- p.32 / Other scoring systems --- p.33 / Definition of Trauma --- p.33 / Chapter Chapter 3: --- Stroke --- p.35 / The Burden of Stroke --- p.35 / What is a Stroke ？ --- p.36 / The Causes --- p.40 / Pathophysiology --- p.41 / Diagnosis and Tests --- p.42 / Assessments and prognosis --- p.44 / Biochemical Markers --- p.47 / Chapter Chapter 4: --- Aims of the study --- p.48 / Chapter Section 2: --- Materials and Methods --- p.50 / Chapter Chapter 5: --- Methods of analysis on cell-free circulating DNA --- p.51 / Materials --- p.51 / DNA Extraction from the Plasma Samples --- p.51 / Real-time Quantitative PCR --- p.52 / Methods --- p.54 / DNA Extraction from the Plasma Samples --- p.54 / Real-time Quantitative PCR --- p.56 / Principle --- p.56 / The β-globin TaqMan Assay --- p.59 / Calibration of the β-globin TaqMan System --- p.62 / Contamination Control --- p.64 / Chapter Section 3: --- Cell-free circulating DNA after trauma --- p.65 / Chapter Chapter 6: --- Cell-free circulating DNA concentration as a prognostic marker in patients after trauma --- p.66 / Introduction --- p.66 / Methods --- p.68 / Results --- p.71 / Discussion --- p.84 / Chapter Chapter 7: --- Temporal changes of cell-free circulating DNA after trauma --- p.89 / Introduction --- p.89 / Methods --- p.90 / Results --- p.92 / Discussion --- p.106 / Chapter Section 4: --- Cell-free circulating DNA concentration after stroke --- p.109 / Chapter Chapter 8: --- Cell-free circulating DNA concentration in patients with stroke --- p.110 / Introduction --- p.110 / Methods --- p.111 / Results --- p.115 / Discussion --- p.129 / Chapter Chapter 9: --- Daily changes of cell-free circulating DNA concentration after stroke --- p.132 / Introduction --- p.132 / Methods --- p.132 / Results --- p.133 / Discussion --- p.137 / Chapter Section 5: --- Conclusion and future perspectives --- p.139 / Chapter Chapter 10: --- Conclusion and Future Perspectives --- p.140 / Conclusion --- p.140 / Future perspectives --- p.145 / BIBLIOGRAPHY --- p.147 / APPENDIX 1: Goriśةmultiple organ failure score --- p.168 / "APPENDIX 2: Definitions and criteria for ARDS, ALI and MODS" --- p.170 / APPENDIX 3: Computed axial tomography and magnetic resonance imaging --- p.172 / APPENDIX 4: Glasgow Coma Scale --- p.173 / APPENDIX 5: Post-Stroke Modified Rankin Scale --- p.174

In conclusion, the studies in this thesis have provided new information on the molecular nature of circulating DNA. Alteration of the size of circulating DNA is demonstrated in different physiological and pathological conditions. The non-bisulfite-based approach described in this thesis has provided a more sensitive and precise way for the detection and quantification of aberrantly methylated DNA sequences in the circulation. This method has tremendous potential for being applied for noninvasive cancer detection and prenatal diagnosis. / In the second chapter of this thesis, the molecular nature of circulating Epstein-Barr virus (EBV) DNA is studied. Circulating EBV DNA has previously been shown to be a valuable marker for the detection, monitoring and prognostication of nasopharyngeal carcinoma and several cancers associated with EBV infection. Using DNase digestion and ultracentrifugation analysis, circulating EBV DNA was shown to be free DNA fragments instead of being associated with viral particles. Furthermore, a quantitative system was developed for measuring the size of these EBV DNA molecules and showed that over 80% of the circulating EBV DNA molecules are shorter than 180 bp. / In the subsequent chapters, this DNA size measurement technique has been applied for analyzing the integrity of plasma genomic DNA in cancer patients and pregnant women. Increased plasma DNA integrity was observed in both of these groups of individuals. Moreover, the size of plasma DNA in cancer patients was shown to normalize after successful treatment and the failure of such normalization was shown to be associated with poor prognosis. In pregnant women, in addition to the overall increase in plasma DNA size, the maternal-derived DNA molecules were further shown to be longer than the fetal-derived ones. This observation opens up the possibility for fetal DNA enrichment through size fractionation of maternal plasma DNA. / The discovery of circulating nucleic acids in plasma and serum has led to the development of numerous promising noninvasive diagnostic tests. To date, circulating nucleic acid analysis has been applied to many different areas, including cancer detection, prenatal diagnosis, monitoring of organ transplant recipients and acute medicine. However, despite the extensive investigations on their clinical applications, the information on the molecular characteristics of the circulating nucleic acids is lacking. / The latter part of the thesis describes the principle of a non-bisulfite-based method for the detection of aberrant DNA methylation in plasma/serum. Using this technique, a universal fetal DNA marker has been developed based on an epigenetic approach. The placentally derived hypermethylated RASSF1A sequence has been developed as a gender and polymorphism-independent marker for fetal DNA in maternal plasma. In pregnant women undergoing noninvasive prenatal rhesus D genotyping, false negative cases were successfully identified through the analysis of this new fetal DNA marker. The quantitative analysis of circulating methylated RASSF1A sequences has further been shown to be useful for the detection and prognostication of hepatocellular carcinoma. / Chan, Kwan Chee. / Adviser: Y.M. Dennis. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3307. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.

In recent years, a number of nuclear transcription factors have been shown to be present in the mitochondria where they have distinct roles in regulating mitochondrial function. Signal Transducer and Activator of Transcription 3 (STAT3), classically activated by the JAK family of receptor associated tyrosine kinases to drive nuclear gene expression, is one such transcription factor with a unique mitochondrial role. There, it has been shown to support oxidative phosphorylation, regulate mitochondrial-encoded transcripts, and be key for the transformation and growth of a number of different cancers. Despite its well-characterized functional importance at the level of the mitochondria, the mechanism through which mitochondrial STAT3 acts and how it is regulated has not been as well studied. Using various cell culture models, we now show that mitochondrial STAT3 is dynamically regulated by oxidative stress and cytokine treatment in the acute setting. Under these conditions we have observed a rapid loss of mitochondrial STAT3 that recovers to baseline conditions with time. During this recovery phase we have noted that mitochondrial STAT3 becomes competent to bind to Cyclophilin D (CypD), the key regulator and activator of the mitochondrial permeability transition pore (MPTP). This is particularly the case with oxidative insults, which we believe may represent an important homeostatic mechanism for the cell. Intriguingly, chronic stimulation with certain stressors seems to increase mitochondrial STAT3 levels suggesting differential regulation in the acute versus chronic setting. The regulation of mitochondrial STAT3 levels by various stimuli points to a novel signaling pathway potentially linking mitochondrial responses with those of the cell. Unification of responses throughout the cell would seem to serve a clear adaptive advantage, particularly in coupling nuclear regulation with metabolic demands as dictated by the mitochondria. Extramitochondrial signaling, also known as the mitochondrial retrograde response, has emerged as an important homeostatic mechanism in lower organisms, but its signaling components have not been well characterized at the mammalian level. Our results point to a role for mitochondrial STAT3 in sensing cellular inputs, whereby its regulation and subsequent association with CypD may have implications in overall mitochondrial quality control. Though the inner workings of this signaling cascade are just beginning to be elucidated, they suggest the existence of a previously unappreciated pathway at the mitochondrial level.

abstract: The fundamental building blocks for constructing complex synthetic gene networks are effective biological parts with wide dynamic range, low crosstalk, and modularity. RNA-based components are promising sources of such parts since they can provide regulation at the level of transcription and translation and their predictable base pairing properties enable large libraries to be generated through in silico design. This dissertation studies two different approaches for initiating interactions between RNA molecules to implement RNA-based components that achieve translational regulation. First, single-stranded domains known as toeholds were employed for detection of the highly prevalent foodborne pathogen norovirus. Toehold switch riboregulators activated by trigger RNAs from the norovirus RNA genome are designed, validated, and coupled with paper-based cell-free transcription-translation systems. Integration of paper-based reactions with synbody enrichment and isothermal RNA amplification enables as few as 160 copies/mL of norovirus from clinical samples to be detected in reactions that do not require sophisticated equipment and can be read directly by eye. Second, a new type of riboregulator that initiates RNA-RNA interactions through the loop portions of RNA stem-loop structures was developed. These loop-initiated RNA activators (LIRAs) provide multiple advantages compared to toehold-based riboregulators, exhibiting ultralow signal leakage in vivo, lacking any trigger RNA sequence constraints, and appending no additional residues to the output protein. Harnessing LIRAs as modular parts, logic gates that exploit loop-mediated control of mRNA folding state to implement AND and OR operations with up to three sequence-independent input RNAs were constructed. LIRA circuits can also be ported to paper-based cell-free reactions to implement portable systems with molecular computing and sensing capabilities. LIRAs can detect RNAs from a variety of different pathogens, such as HIV, Zika, dengue, yellow fever, and norovirus, and after coupling to isothermal amplification reactions, provide visible test results down to concentrations of 20 aM (12 RNA copies/µL). And the logic functionality of LIRA circuits can be used to specifically identify different HIV strains and influenza A subtypes. These findings demonstrate that toehold- and loop-mediated RNA-RNA interactions are both powerful strategies for implementing RNA-based computing systems for intracellular and diagnostic applications. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019

Metabolic engineering has enabled studying microorganisms by the modification of their genetic material and analysis of their metabolism for the isolation of microbial strains capable of producing high yields of high value chemicals and biofuels. In this research, novel tools were developed to improve genetic engineering of microbial cells. In this matter, λ-PCR (lambda-PCR) was developed enabling the construction of plasmid DNA. This technique allows DNA assembly and manipulation (insertion, substitution and/or deletion) at any location of a vector. λ-PCR addresses the need for an easy, highly-efficient, rapid and inexpensive tool for genetic engineering and overcoming limitations encountered with traditional techniques. Then, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system (in vitro) in order to modulate protein expression in biosynthetic pathways. The ability of the sRNAs to regulate mRNA expression with statistical significance was demonstrated. Up to 70% decrease in protein expression level was achieved by targeting specific secondary structures of the mRNA with antisense binding regions of the sRNA. Most importantly, a sRNA was identified capable of protein overexpression by up to 65%. An understanding of its mechanism showed that its mRNA target region(s) likely lead to occlusion of RNase E binding. This mechanism was translated for expression of a diaphorase enzyme, which has relevance to synthetic biology and metabolic engineering in in vitro systems. Results were successful, showing a greater than 75% increase in diaphorase expression in a cell-free protein synthesis reaction. Next, Raman spectroscopy was employed as a near real-time method for microbial phenotyping. Here, Raman spectroscopy was used in combination with chemometric analysis methods through RametrixTM Toolboxes to study the effects of environmental conditions (i.e. illumination, glucose, nitrate deprivation, acetate, sodium chloride and magnesium sulfate) on the phenotypic response of the cyanobacterium Synechocystis sp. PCC6803. The RametrixTM LITE Toolbox for MATLAB® enabled processing of Raman spectra and application of principal component analysis (PCA) and discriminant analysis of principal components (DAPC). Two studies were performed. PCA and DAPC produces distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on the (i) presence of glucose in the growth medium, (ii) illumination, (iii) nitrate limitation, and (iv) throughout a circadian rhythm growth cycle, in the first study. The second study focused on the phenotypic response based on (i) growth in presence of acetate, (ii) presence of high concentrations of sodium chloride and (iii) magnesium sulfate starvation. RametrixTM PRO was applied for the validation of the DAPC models through leave-one-out method that allowed calculation of prediction accuracy, sensitivity and selectivity for an unkown Raman spectrum. Statistical tests (ANOVA and pairwise comparison) were performed on Raman spectra to identify statistically relevant changes in Synechocystis phenotypes. Next, comparison between Raman data and standardized analytical methods (GF-FID, UPLC, spectrometric assays) was established. Overall, good correlation were obtained (R > 0.7). Finally, genomic DNA libraries were enriched to isolate a deoxynivalenol detoxifying enzyme. To do this, library fragments from microorganisms was generated through oligonucleotide primed polymerase chain reaction (DOP-PCR) and transformed in a DON-sensitive yeast strain. Rounds of subculture were performed in the presence of DON and ferulic acid in order to isolate a strain capable of enzymatic degradation of DON. / Doctor of Philosophy / Metabolic engineering is the use of genetic engineering to modify microorganisms in order to produce high yields of valuable commodity chemicals. The goal of this research is to develop new methods to improve genetic modification and selection of microbial cells. The specific objectives were to: (i) develop new tools for DNA assembly and manipulation, (ii) utilize small synthetic RNA to control protein expression level, (iii) use Raman spectroscopy to study phenotypic responses to environmental changes and (iv) enrich for microorganisms that detoxify dangerous toxins. First, a new technique for DNA assembly, named λ-PCR (lambda-PCR), was developed. This method allows the easy manipulation of plasmid DNA with high-efficiency and low-cost compared to traditional techniques. Second, novel synthetic small RNA (sRNA) regulators were designed in a cell-free-system in order to modulate (downregulate or overexpress) fluorescent protein expression. Next, Raman spectroscopy was used to assess phenotypic response of cyanobacterial cells to different environmental modifications (light settings, salts, sugar, etc…). Finally, genomic library was used to discover and characterize enzymes capable of degrading a mycotoxin.

<p>Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. </p><p>In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. </p><p>The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. </p><p>In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.</p>

Advances in genetics, proteomics and chromatography techniques have enabled the successfully generation of a cell-free bacterial translation system composed of highly pure and active components. This system provided an ideal platform for better elucidating the mechanism of each individual step of the prokaryotic protein biosynthesis and the function of the translation factors involved in the process. In doing so, we have discovered that the N-terminal domain or complete deletions of the ribosomal protein L11 reduced the termination efficiency of RF1 on cognate stop codons by four to six folds. The L11 deletions also conferred a two folds decrease in the missense error suggesting the increased nonsense termination accuracy of RF2 by two folds, which would clarified previous in vivo observations. The versatility of the cell-free system has provided the additional possibility to study the effects of class II release factor RF3 mutations in mediating fast dissociation of class I release factors RF1 and RF2 from the post-termination ribosome complexes. The results show a series of mutations within RF3 conferring considerable reduction of the class I release factors recycling rate. These observations together with sequence alignment studies suggest the possible location on RF3 of the class I release factors interaction site. In addition, the utilization of the cell-free system has made it possible to develop a new biotechnological approach for continuous production of polypeptides, based on gel filtration chromatography. The pilot trials have so far resulted in a six fold production increase of the MFTI test peptide compared to the conventional batch method.

[pt] Sistemas multi-input multi-output (MIMO) massivos livres de células são uma variante de sistemas MIMO multi-celulares que consideram a ausência de células. Desta forma, a interferência entre as células é minimizada e a capacidade de cobertura do sistema é melhorada devido à menor distância entre os pontos de acesso (APs) e os usuários. É uma solução de comunicação MIMO massiva multi-usuário que envolve um número estendido de APs que podem ser equipados com tecnologia MIMO para fornecer serviço a usuários simultaneamente. Os APs são controlados por uma unidade central de processamento (CPU) para garantir a coordenação dentro da rede e para processamento e decodificação de informação. Possíveis arranjos para a arquitetura livre de células incluem esquemas centralizados e descentralizados. Para a configuração centralizada, os APs enviam todas as suas estimativas de canal e informações recebidas para a CPU por meio de enlaces de transporte frontais para processamento e detecção de sinais. Além disso, na arquitetura centralizada, os APs atuam como repetidores na rede. Outro nível de cooperação para sistemas MIMO massivos livre de células é o esquema descentralizado. Nesta proposta de tese, a arquitetura dos sistema MIMO massivos livres de células no canal reverso é estudada para as implementações centralizadas e descentralizadas. Em particular, estuda-se o desempenho de técnicas de mitigação de interferência para essas redes supondo-se conhecimento perfeito de canal e usando técnicas de detecção lineares e não lineares, seleção de APs, e esquemas iterativos de detecção e decodificação com códigos LDPC para melhorar o desempenho do sistema e reduzir a carga de sinalização. Para o caso em que há falta de compartilhamento de informações sobre os canais, o uso de pilotos para obter estimativas de canais é considerado e explorado. / [en] Cell-free massive multiple-input multiple-output (CF-mMIMO) is an advanced variant of network multiple-input multiple-output (MIMO) which considers absence of cell boundaries. Thus, the interference between cells in cellular systems is greatly minimised and the system s coverage capacity is improved due to the shorter distances between the access points (APs) and the users. It is a multi-user massive MIMO communications solution that involves an extended number of APs that can either be equipped with MIMO or single antennas to provide service to users simultaneously. The APs are controlled by a central processing unit (CPU) to ensure coordination within the network and for information processing and decoding. Possible arrangements for the CF-mMIMO architecture include, but are not limited to: centralized and decentralized schemes. In this thesis, the uplink of a CF-mMIMO system architecture is studied for the centralized and decentralized implementations. In particular, we study the performance of interference mitigation techniques for CF-mMIMO networks using iterative detection and decoding (IDD) schemes. The performance of the system is studied assuming perfect and imperfect channel state information (CSI). Access point selection based on the effective channel gain to make the network more practical and scalable are devised. The use of low-density parity check (LDPC) codes that adopt message passing has been investigated. Furthermore, log likelihood ratio (LLR) refinement strategies have been proposed to improve decentralized processing for CF-mMIMO networks. Finally, the performance of the considered schemes is analyzed theoretically and simulations are used to assess the performance in terms of BER, number of fronthaul signaling, and computational cost.

La NADPH oxydase du phagocyte est une enzyme impliquée dans la défense immunitaire contre les pathogènes. Après activation du phagocyte, cette enzyme produit des ions superoxyde par réduction du dioxygène par le NADPH. Elle est constituée de quatre sous- unités cytosolubles (p47phox ; p67phox ; p40phox et Rac), et deux membranaires (gp91 ; p22phox). Son activation fait intervenir un processus complexe qui met en jeu des changements d’interaction entre les protéines la constituant et qui permet l’assemblage des six sous- unités. Afin d’obtenir des informations sur les processus d’assemblage et d’activation, j’ai reconstitué le complexe dans un système cell free à l’aide de protéines recombinantes pour pouvoir contrôler tous les paramètres. Dans ce travail nous avons comparé les modes d’activation de p47phox par phosphorylation, par mutation substitutionelle sérine - aspartate en position S303,S304 et S328 pour mimer la phosphorylation et enfin par addition d’acide arachidonique (AA) activateur connu de l’enzyme in vitro mais aussi in vivo. Bien qu’il ai été montré que ces trois méthodes ouvrent la protéine vers une conformation ayant des propriétés similaires, nous avons trouvé que les effets de ces méthodes d’activation sont significativement différents. Ainsi, les changement de conformation observés par dichroisme circulaire, sont dissemblables. Pour p47phox, l’addition de AA déstructure la protéine. La phosphorylation induit un déplacement bathochrome des bandes de CD qualitativement similaire, alors que les mutations S-D de p47phox provoquent un déplacement opposé. Pour le complexe p47phox-p67phox l’addition d’AA destructure le mélange tandis que la mutation induit relativement peu de changement. Nous avons mesuré les constantes de dissociation Kd du complexe p47phox-p67phox. Alors que pour les protéines « sauvages », le Kd est faible (4±2 nM), les mutations de p47phox ainsi que l’addition d’AA augmentent cette valeur jusqu’à environ 50 nM, montrant une diminution de l’affinité entre p47phox-p67phox. De même, sur le complexe entier, l’effet de la phosphorylation de p47phox est différent de la mutation. Nous avons mesuré les valeurs de EC50 relatives à p67phox pour les différentes formes de p47phox. L’activation de p47phox par phosphorylation diminue l’EC₅₀, alors que les doubles ou triple mutations augmentent sa valeur. Nous avons confirmé que la phosphorylation et la mutation sont insuffisantes pour activer l’enzyme. La présence de AA est indispensable pour le fonctionnement du complexe. L’ordre de fixation des sous unités cytosoliques semble indifférent mais il faut que tous les composants soient présents lors de l’ajout de AA. Enfin, la délétion de p47phox dans la partie C-terminale (aa 343 à 390, domaine d’interaction avec p67phox) il n’y a plus de formation du dimère mais l’enzyme fonctionne normalement. Ces résultats apportent des éléments nouveaux sur le rôle de la dimérisation p47 phox-p67 phox, non indispensable à l’activité du système et sur le rôle mineur de la phosphorylation dans l’activation de la NADPH oxydase in vitro. / The NADPH oxidase of phagocytes is an enzyme involved in the innate defense of organisms against pathogens. After phagocyte activation, this enzyme produces superoxide ions by reduction of dioxygen by NADPH. It is constituted of four cytosolic sub-units (p47phox ; p67phox ; p40phox et Rac) and two membrane proteins (gp91 ; p22phox). Its activation takes place through a complex process that involves protein-protein interaction changes leading to assembly and functionning of the catalytic core. In order to obtain information on this process, I have reconstituted the enzyme in a cell free systeme using recombinant proteins, to be able to fully control all the measurement conditions. In this work, we have compared different activation modes of p47phox i) phosphorylation; ii) substitution serine - aspartate by mutations at positions S303, S304 and S328 to mimic phosphorylation; iii) addition of arachidonic acid (AA), a well known activator molecule in vitro. It has been shown that these three activating methods transform p47phox to an open configuration with similar characteristics. However, we have found that the effects of these methods are significantly different. Indeed, the conformational changes observed by circular dichroism are different. For p47phox, the addition of AA destructures the protein. Its phosphorylation induces a bathochromic displacement of the bands, whereas the mutations S-D lead to an opposite displacement. For the dimer p47phox-p67phox , the addition of AA destructures the proteins while mutations induce hardly no changes. We have measured the dissociation constant Kd of the complex p47phox-p67phox. For wild type proteins, Kd value is low (4±2 nM), while mutations of p47phox as well as addition of AA increase its value up to 50 nM, showing a decrease of affinity between p47phox and p67phox. Moreover, on the whole complex, the effect of phosphorylation of p47phox is different from mutations. We have shown that the EC50 values relative to p67phox are sensitive to the various modifications of p47phox. Phosphorylation of p47phox decreases EC₅₀, while double or triple mutations increase its value. We have confirmed that phosphorylation and mutation are not sufficient to activate the enzyme. The presence of AA is a prerequisite for the functionning of the complex, i.e. production of superoxide. The binding order of the cytosolic proteins seems random but it is necessary that all the components be present during the activation by AA. Finally, deletion of the C terminal part of p47phox (aa 343 to 390, interaction domain with p67phox) leads to the absence of dimer formation but does not affect the enzyme activity. These results bring new information on the role of dimerisation of p47-p67 and on that of phosphorylation in the activation of NADPH oxidase in vitro.