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Rapid Diagnostic Tests (RDTs) for malaria are restricted to a few biomarkers and antibody-mediated detection. However, the expression of commonly used biomarkers varies geographically and the sensibility of immunodetection can be affected by batch-to-batch differences or limited thermal stability. In this study we aimed to overcome these limitations by identifying a potential biomarker and by developing molecular sensors based on aptamer technology. Using gene expression databases, ribosome profiling analysis, and structural modeling, we find that the High Mobility Group Box 1 protein (HMGB1) of Plasmodium falciparum is highly expressed, structurally stable, and present along all blood-stages of P. falciparum infection. To develop biosensors, we used in vitro evolution techniques to produce DNA aptamers for the recombinantly expressed HMG-box, the conserved domain of HMGB1. An evolutionary approach for evaluating the dynamics of aptamer populations suggested three predominant aptamer motifs. Representatives of the aptamer families were tested for binding parameters to the HMG-box domain using microscale thermophoresis and rapid kinetics. Dissociation constants of the aptamers varied over two orders of magnitude between nano- and micromolar ranges while the aptamer-HMG-box interaction occurred in a few seconds. The specificity of aptamer binding to the HMG-box of P. falciparum compared to its human homolog depended on pH conditions. Altogether, our study proposes HMGB1 as a candidate biomarker and a set of sensing aptamers that can be further developed into rapid diagnostic tests for P. falciparum detection. / Grand Challenges Canada / Revisión por pares

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPSinduced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.

Chez les eucaryotes pluricellulaires, la différenciation des cellules repose en partie sur l’activation oula répression des gènes. Les profils d’expression génique mis en place vont perdurer d’une générationcellulaire à l’autre. Ce phénomène met en jeu des mécanismes épigénétiques qui remodèlentlocalement la structure de la chromatine. Chez Drosophila melanogaster, les protéines des groupesPolycomb (PcG) et Trithorax (TrxG) participent au maintien du profil d’expression des gènes au coursdu développement. Les protéines PcG maintiennent les gènes réprimés tandis que les protéines TrxGmaintiennent les gènes activés. Une troisième classe de protéines nommée Enhancers of Trithoraxand Polycomb (ETP) module l’activité des PcG et TrxG. Dorsal Switch Protein 1 (DSP1) est uneprotéine HMGB (High Mobility Group B) classée comme une ETP. Par tamisage moléculaire, nousavions montré que la protéine DSP1 était présente au sein de complexes de poids moléculaire de 100kDa à 1 MDa. Le travail de thèse présenté ici a pour but d’identifier les partenaires de la protéineDSP1 dans l’embryon et de mieux connaître les propriétés biochimiques de DSP1. Premièrement, j’aimis en place puis effectué l’immunopurification des complexes contenant DSP1 dans des extraitsprotéiques embryonnaires. Cette approche nous a permis d’identifier 23 partenaires putatifs de laprotéine DSP1. Parmi ces protéines, nous avons identifié la protéine Rm62 qui est une ARN hélicaseà boîte DEAD. Les relations biologiques entre DSP1 et Rm62 ont été précisées. Deuxièmement, j’aidéterminé, par une approche biochimique, de nouvelles caractéristiques physico-chimiques de laprotéine DSP1. / In multicellular organism, the identity of cell is determined by several factors playing on genesexpression. Once established, the gene expression pattern is transmitted to daughter cells through aprocess involving epigenetic mechanisms that locally reshape the structure of chromatin. In Drosophilamelanogaster, the Polycomb (PcG) and trithorax (trxG) group genes are involved in the maintenanceof gene expression profile during development. Inside multimeric complexes, PcG proteins maintaingenes in repressed state whereas TrxG maintain genes active. A third class of proteins, calledEnhancers of Trithorax and Polycomb, regulate PcG and TrxG activities. Dorsal Switch Protein 1(DSP1) is a High Mobility Group B protein acting as an ETP. But DSP1 has not yet been identified inPcG or TrxG complexes. On the basis of gel filtration analysis of protein complexes in embryo nuclearextracts, it appears that the majority of DSP1 is present in complex(es) from 100 kDa to 1MDa. Aimsof present work are the identification of DSP1 protein partners in drosophila embryo and thecharacterization of biochemical properties of DSP1. Firstly, I used immunopurification from drosophilaembryonic nuclear extracts. The proteins purified with DSP1 were characterized through sequencingof peptides from individual protein bands by mass spectrometry. Among identified proteins, wefocused on the DEAD Box RNA helicase, Rm62. The role of interaction between DSP1 and Rm62 hasbeen characterized. Secondly, I have identified a new physicochemical aspect of DSP1 protein.

High mobility group box 1 (HMGB-1) protein is a non-histone chromosomal protein. As a DNA binding protein, HMGB-1 is involved in the maintenance of nucleosome structure, regulation of gene transcription and it is active in DNA recombination and repair. It has been known that HMGB-1 is a late mediator of endotoxemia and sepsis. HMGB-1 is released from activated macrophages, induces the release of other proinflammatory mediators, and mediates cell death when overexpressed. We speculated that the course of sepsis maybe different without the involvement of HMGB-1. The aims of this study are to investigate the role of HMGB-1 in mediating sepsis and to observe the effects by using RNAi to affect the production of HMGB-1. Lipopolysaccharide (LPS) was used to simulate sepsis in culture as well as stimulate the release of HMGB-1 from RAW 264.7 cells. Levels of HMGB-1 in the culture medium were subsequently measured by Western blot. Other proinflammatory cytokines (TNF-£\, IL-6 and TGF-£]) were measured by ELISA. HMGB-1 could not be detected in the culture medium in the absence of LPS stimuli, but after 0.5 £gg/ml LPS treatment HMGB-1 release could be detected. HMGB-1 the amount of released from LPS activated RAW 264.7 cells was in a time- and dose-dependent manner. The present study demonstrated that RNAi in the treatment of LPS-stimulated RAW264.7 cells resulted in the blockade of HMGB-1 and decreased LPS-induced inflammatory response. The results demonstrated that HMGB-1 plays a pivotal role in macrophage inflammatory responses by modulating the production of inflammatory mediators.

In the eukaryotic nucleus, an immense length of DNA is compactly packaged to generate an ordered three-dimensional hierarchical structure called chromatin (van Holde, 1988; Wolffe, A.P, 1998). This organization forms a template for various DNA transaction processes like replication, transcription, recombination etc. The different stages of organization of the chromatin finally results in the 10,000-fold compaction observed in the metaphase chromosome. The problem of how the fibres of chromatin are folded has interested biologists and biochemists for decades. It has long been recognized that the Histones play a major part in this folding. However, the distinctly different roles of the Histones H2A, H2B, H3 and H4 on one hand and the lysine rich Histones such as Histone H1 and its cognates on the other, were not understood until after the discovery of the nucleosomes in the early 1970s. Some of the early insights into the structure of chromatin came through the digestion of nuclear chromatin with calcium-dependent endonucleases like micrococcal nuclease. A repeating kinetic intermediate of about 200 bp of DNA with Histones was obtained (Simpson, 1978). Based on repeating pattern of micrococcal nuclease digested chromatin and structural studies, Kornberg (1974) proposed that chromatin is composed of a flexible chain of repeating units of 100 A0 diameter. These units were termed as "nucleosomes" (Oudet et al, 1975). It then became clear that the Histones H2A, H2B, H3 and H4 were constituents of the nucleosome core particle whereas the lysine rich Histone H1 was somehow associated with the linker DNA between core particles. Hence, the formers are called core Histones and the latter as linker Histones. On further digestion of nucleosome, a nucleosome core was obtained in which wrapping of 146 bp of DNA about the Histone octamer to form the core particle provided the first level of folding. Electron microscopy and X-ray diffraction techniques suggested that this particle is a disk, 57 A0 thick and 110 A0 in diameter, and that the DNA is wound around the Histone core (Finch et al, 1977), But this cannot account for the many thousand-fold condensation of the DNA in the eukaryotic nucleus. The "string of beads" structure observed obviously could not satisfy the compaction requirement. It soon became evident that there exists some level of higher order folding of the chromatin fiber. In a classical paper, Finch and Klug (1976), showed that the extended nucleosomal filaments condense into irregular fibers of about 30 nm diameter in the presence of low concentrations of Mg 2+. Based on the data from earlier structural studies, these authors proposed a solenoid model in which nucleosomes were wrapped into a regular helix with a pitch of about 11nm. Later, it was observed that the formation of well defined fibers requires the presence of lysine rich Histones such as Histone H1.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 171-177).

Asthma is a chronic disorder highlighted by intermittent airway inflammation and characterized by paroxysmal dyspnea and airway hyperresponsiveness (AHR). A key feature of severe asthma is the development of airway wall remodeling, which is thought to occur through repeated rounds of inflammation and tissue repair. Remodeling includes structural changes such as increased mass of airway smooth muscle (ASM), and excessive collagen deposition. ASM cells contribute to airway remodeling via the expression and secretion of extracellular matrix (ECM) proteins. This is particularly driven by inflammatory processes, which include mediators such as transforming growth factor (TGF)-β1 and damage associated molecular pattern (DAMP) proteins, such as high mobility group box 1 (HMGB1). HMGB1 is ubiquitously expressed as a non-histone DNA-binding protein that can regulate gene expression, but can also be released in response to stress to underpin inflammation and tissue repair. In this study we tested the hypothesis that extracellular HMGB1 induces signaling pathways that control responses linked to progression of airway inflammation, remodeling and hyperresponsiveness in human ASM cells. We used primary cultured ASM cells as well as hTERT-immortalized human ASM cells. With immunoblotting we demonstrate that exogenous HMGB1 (10 ng/mL) can induce rapid and sustained phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) that is comparable to that induced by a potent mitogen, platelet derived growth factor (PDGF-BB, 10 ng/mL). We also found that TGF-β1 (2.5 ng/mL) promotes the accumulation of secreted HMGB1 in culture medium in a time line concomitant with expression of ECM proteins, collagen and fibronectin, suggesting a role for HMGB1 in pro-fibrotic effects of TGF-β1. By lentiviral delivery, we induced stable expression of short hairpin RNA (shRNA) that silenced expression of endogenous HMGB1 or mammalian diaphanous 1 (mDia1), a cytoplasmic scaffold protein that is required for HMGB1-induced cell responses through one of its receptors, receptor for advanced glycation end products (RAGE). Immunoblot analyses revealed that silencing of mDia1 was associated with markedly decreased induction of p42/p44 MAPK phosphorylation by exogenous HMGB1. In HMGB1-silenced human ASM cells, we observed significantly reduced synthesis and secretion of collagen A1 and fibronectin in response to TGF-β1 (2.5 ng/mL, 0-48 hrs). However, exogenous HMGB1 was not sufficient to rescue ECM synthesis in response to TGF-β1 in HMGB1-silenced cells - this suggests that intracellular, but not necessarily secreted HMGB1, regulates ECM expression and secretion in response to TGF-β1. Consistent with this interpretation, exogenous HMGB1 alone was not sufficient to induce ECM synthesis or secretion in primary cultured ASM cells. In conclusion, we show that though in human ASM cells extracellular HMGB1 alone can activate MAPK signaling, likely via mDia1-dependent pathways involving RAGE. it is not capable of prompting ECM protein expression. Recombinanat exogenous HMGB1 does not appear to directly affect ECM synthesis, rather intracellular (nuclear) HMGB1 likely modulates activity of genes that are affected by TGF-β1. Overall, HMGB1 has potential to regulate tissue repair processes involving ASM through intracellular and extracellular mechanisms, thus our findings support further work to elucidate the role of HMGB1 in pathogenesis of obstructive airway disease.

Objective: The mechanisms by which lipopolysaccharide (LPS) pretreatment induces cardioprotection following ischaemia/reperfusion (I/R) have not been fully elucidated. We hypothesized that activation of phosphoinositide 3-kinase (PI3K)/Akt and high mobility group box 1 (HMGBx1) signaling plays an important role in LPS-induced cardioprotection. Methods: In in vivo experiments, age- and weight-matched male C57BL/10Sc wild type mice were pretreated with LPS before ligation of the left anterior descending coronary followed by reperfusion. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. Akt, phospho-Akt, and HMGBx1 were assessed by immunoblotting with appropriate primary antibodies. In situ cardiac myocyte apoptosis was examined by the TdT-mediated dUTP nick-end labeling (TUNEL) assay. In an in vitro study, rat cardiac myoblasts (H9c2) were subdivided into two groups, and only one was pretreated with LPS. After pretreatment, the cells were transferred into a hypoxic chamber under 0.5% O2. Levels of HMGBx1 were assessed by immunoblot. Results: In the in vivo experiment, pretreatment with LPS reduced the at risk infarct size by 70.6% and the left ventricle infarct size by 64.93% respectively. Pretreatment with LPS also reduced cardiac myocytes apoptosis by 39.1% after ischemia and reperfusion. The mechanisms of LPS induced cardioprotection involved increasing PI3K/Akt activity and decreasing expression of HMGBx1. In the in vitro study, pretreatment with LPS reduced the level of HMGBx1 in H9c2 cell cytoplasm following hypoxia. Conclusion: The results suggest that the cardioprotection following I/R induced by LPS pretreatment involves PI3K/Akt and HMGBx1 pathways.

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