La leucémie Myéloïde Chronique est un néoplasme myéloprolifératif causé par l'expression de la kinase oncogène BCR-ABL. Les Inhibiteurs de Tyrosine Kinase (ITK) spécifiques de BCR-ABL ont révolutionné la prise en charge de la maladie. Les ITK ne sont cependant pas curatifs ; en effet, certaines cellules souches leucémiques (CSL) sont résistantes aux ITK, et persistent dans la moelle osseuse des patients même en rémission prolongée. Ces CSL sont probablement responsables de la rechute chez 60% de ces patients après arrêt des ITK. 30% des patients développent une résistance aux ITK via des mécanismes inconnus. Dans un contexte sain, les Bone Morphogenetic Proteins (BMP) régulent différentes propriétés des cellules souches hématopoïétiques. Nous avons mis en évidence que les patients atteints de LMC présentent une altération de la voie BMP avant leurs mises sous traitement, avec une hausse de l'expression du récepteur dans les cellules leucémiques immatures, amplifiée par de forts taux de BMP2/4 produits par le microenvironnement des CSL, la niche. Ici, nous démontrons que ces altérations sont maintenues chez les patients sous traitement, et sont activement impliquées dans la résistance aux ITK. Les patients résistants présentent une surexpression de BMPR1b dans les CSL et un maintien de forts taux de BMP produits à la fois par les cellules leucémiques mais aussi par les cellules stromales. Les BMP permettent la survie des CSL via l'expression du récepteur BMPR1b et induisent l'expression de TWIST-1, un facteur de transcription précédemment identifié par l'équipe comme induisant la résistance / Chronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm caused by the expression of the oncogenic protein kinase, BCR-ABL. The Tyrosine Kinase Inhibitors (TKI) specifics of BCR-ABL kinase dramatically changed the outcome of CML, turning a life-threatening disease into a chronic illness. However, TKI are not yet curative since most CML patients still retain progenitors and leukemic stem cells (LSC) in bone marrow permanently. Thus, approximately 60% of patients that achieve Complete Molecular Remission =2 years relapse following TKI withdraw. Moreover, some patients develop true resistance to TKI, with ~30% due to unknown mechanisms. In chronic phase CML (CP-CML), LSC survive, sustain interactions with their niche where resistance mechanisms can occur, responsible for disease persistence and relapse following treatment cessation. In normal bone marrow, Bone Morphogenetic Proteins (BMP) pathway regulate the fate and proliferation of normal hematopoietic stem cells, as well as interactions with their niche. The deregulations of this pathway drive early steps of CML development. In newly diagnosed CP-CML patients, high concentration of BMP2/4 in the leukemic niche allows LSC maintenance and sustains a permanent pool of leukemic progenitors expressing elevated levels of BMPR1b receptor. Here, we report that alterations of the BMP pathway persist in TKI-CML resistant patients. As compared to patients in Complete Cytogenetic Remission (CCyR), cells isolated from TKI-resistant patients display a high level of BMPR1b expression in immature cells and high levels of BMP2/4 in bone marrow, provided by the niche and by the leukemic immature cells themselves. BMP allow leukemic stem cells resistance to treatments through binding to BMPR1b. Interestingly, BMP2/4-treated cells overexpressed TWIST-1, a transcription factor that we previously identified as a predictive factor of CML resistance

Le médicament prophylactique qui a drastiquement réduit l’impact et la sévérité du tétanos sur les populations humaines est le vaccin antitétanique. Son principe actif appelé anatoxine tétanique résulte de l’inactivation au formaldéhyde de la toxine tétanique. Cette détoxification chimique est une étape critique qui détermine la sécurité, l’antigénicité et l’immunogénicité du vaccin. Pour une meilleure compréhension de ce processus chimique, à l’échelle moléculaire, nous avons dans un premier temps caractérisé l’anatoxine tétanique par chromatographie liquide couplée à la spectrométrie de masse en tandem haute résolution (LC-MS/MS) afin d’identifier et de localiser exhaustivement l’ensemble des modifications induites par le formaldéhyde sur la structure tridimensionnelle de la protéine vaccinale. Dans un second lieu, pour un meilleur suivi qualité du procédé industriel de fabrication de l’anatoxine tétanique, nous avons développé des méthodes uniques d’expertise in vitro par LC-MS/MS pour réaliser la quantification relative et/ou absolue de la toxine tétanique, de l’anatoxine tétanique, ainsi que pour effectuer la quantification relative des fragments de toxine chimiquement modifiés par le formaldéhyde. Ces outils de caractérisation sont complémentaires aux méthodes de contrôles qualités existantes et contribuent actuellement à un meilleur suivi de la reproductibilité des lots de vaccins antitétaniques / The prophylactic drug, which has drastically reduced the impact and severity of tetanus on human populations, is the tetanus vaccine. Its active ingredient called tetanus toxoid results from the inactivation of tetanus toxin with formaldehyde. This chemical detoxification is a critical step, which determines the safety, antigenicity and immunogenicity of the vaccine. For a better understanding of this chemical process, at the molecular level, we first characterized tetanus toxoid by liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-MS/MS) in order to fully identify and map all the modifications induced by formaldehyde on the three-dimensional structure of the vaccine protein. In a second step, for a better quality control of the industrial process of manufacturing tetanus toxoid, we developed in vitro expertise methods by LC-MS/MS to perform the relative and/or absolute quantification of tetanus toxin, tetanus toxoid, and to carry out the relative quantification of the toxin fragments chemically modified with formaldehyde. These characterization tools are complementary to existing quality control methods and currently contribute to better monitoring the reproducibility of tetanus vaccine batches

<p>The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr⁵⁰ in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites.</p><p>Cys⁴⁸ and Tyr⁵⁰ were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr⁵⁰ with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The K_M^GSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases k_cat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys⁴⁸ restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.</p>

Nanoparticles were synthesized and modified for target drug delivery. The research involved the aqueous synthesis of near infrared (NIR) sensitive Au-Au₂S nanoparticles. An anti-cancer drug (<i>cis-platin</i>) was subsequently adsorbed onto the Au-Au₂S nanoparticle surface via the 11-mercaptoundecanoic acid layers. The results showed that the degree of adsorption of cis-platin onto Au-Au₂S nanoparticles was controlled by the pH value of solution, and the rate of drug release was sensitive to NIR irradiation. The results of the synthesis, drug-release properties and nanoparticle-cell interactions will be discussed. / Singapore-MIT Alliance (SMA)

The glutathione transferases (GSTs) represent a superfamily of dimeric proteins involved in cellular detoxication by catalyzing the nucleophilic addition of the reduced glutathione (GSH) to the hydrophobic electrophiles. The present work focuses on the functional role of the conserved structures of GSTP1-1. The lock-and-key motif is a highly conserved hydrophobic interaction in the subunit interface of Pi, Mu, and Alpha class GSTs. The key residue (Tyr50 in hGSTP1-1) of one subunit is wedged into a hydrophobic pocket of the neighboring subunit. The heterodimer GSTP1/Y50A was constructed from the fully active wild-type GSTP1-1 and the nearly inactive Y50A in order to study how an essentially inactive subunit influences the activity of the neighboring subunit. The results illuminate the vital role of the lock-and-key motif in modulating the GSH binding and the rate of catalysis. Additionally, the two active sites of the dimeric enzyme work synergistically. An observed water network, in hGSTP1-1 structures, connects the two active sites, thereby offering a mechanism for communication between the two active sites. Cys48 and Tyr50 were targeted by mutations and chemical modifications for understanding how the α2 loop residues modulate GSH binding and catalysis. The replacement of Tyr50 with different unnatural amino acids showed that the nature of the key residue side-chain influences the interaction with the lock structure and, consequently, the catalytic activity. The KMGSH, GSH affinity and protein stability can be modulated by fitting key residue into the lock cavity of the neighbor subunit and, consequently, restriction of the flexibility of the α2 loop. Optimization of the interaction between the key residue and the lock-cavity increases kcat. Also, the crystal structure of the Cys-free variant was determined. The result indicated that Cys48 restricts the flexibility of the α2 loop by interacting with surrounding residues and, consequently, contributes to GSH binding and protein stability.

Developing radiation delivery systems for optimisation of absorbed dose to the target without normal tissue toxicity requires advanced calculations for transport of radiation. In this thesis absorbed dose and fluence in different radiotherapy applications were calculated by using Monte Carlo (MC) simulations. In paper I-III external neutron activation of gadolinium (Gd) for intravascular brachytherapy (GdNCB) and tumour therapy (GdNCT) was investigated. MC codes MCNP and GEANT4 were compared. MCNP was chosen for neutron capture reaction calculations. Gd neutron capture reaction includes both very short range (Auger electrons) and long range (IC electrons and gamma) products. In GdNCB the high-energetic gamma gives an almost flat absorbed dose delivery pattern, up to 4 mm around the stent. Dose distribution at the edges and inside the stent may prevent stent edge and in-stent restenosis. For GdNCT the absorbed dose from prompt gamma will dominate over the dose from IC and Auger electrons in an in vivo situation. The absorbed dose from IC electrons will enhance the total absorbed dose in the tumours and contribute to the cell killing. In paper IV a model for calculation of inter-cluster cross-fire radiation dose from β-emitting radionuclides in a breast cancer model was developed. GEANT4 was used for obtaining absorbed dose. The dose internally in cells binding the isotope (self-dose) increased with decreasing β-energy except for the radionuclides with substantial amounts of conversion electrons and Auger electrons. An effective therapy approach may be a combination of radionuclides where the high self-dose from nuclides with low β-energy should be combined with the inter-cell cluster cross-fire dose from high energy β-particles. In paper V MC simulations using correlated sampling together with importance sampling were used to calculate spectra perturbations in detector volumes caused by the detector silicon chip and its encapsulation. Penelope and EGSnrc were used and yielded similar results. The low energy part of the electron spectrum increased but to a less extent if the silicon detector was encapsulated in low z-materials.

Protein-protein interactions (PPIs) are attractive targets because of their therapeutic potential. One approach to design small molecules that can disrupt the PPIs is to use structural information of proteins. With this approach, triazole-based peptidomimetics that mimic beta-turn hot-spot regions in neurotrophins were synthesized. The monovalent mimics were assembled into bivalent mimics via a combinatorial method. Three different bivalent mimics were prepared for different studies. Bivalent mimics with long-linkers bound to TrkA or TrkC receptor and showed partial antagonism for the receptors. Other mimics were conjugated with cytotoxic compounds and they were used for TrkC targeted drug delivery. The last group of bivalent mimics previously showed targeted delivery effects for pancreatic cancer cells. In this study, we synthesized Eu-chelated bivalent mimics to perform a competitive binding assay for pancreatic cancer cells. Previous research in our group focused on design of secondary structures' mimics on rigid scaffolds as "minimalist mimics." We sought to establish structural design criteria for the minimalist mimics, and we wanted to propose that sets of such compounds could mimic local pairs of amino acids in any secondary structures as "universal peptidomimetics." Thus, we designed five compounds, such as oxazoline-, pyrrole-, dyine- "kinked" and "linear" bistrizole-based peptidomimetics, and performed molecular modelings, DFT calculations, and QMD for them to validate our hypothesis. On the concepts of "minimalist mimics" and "universal peptidomimetics," we developed the C alpha ? C beta vector matching program to evaluate preferred orientations of C alpha - C beta coordinates for secondary structures. We applied the program to omegatides and pyrrolinone-pyrrolidine oligomers. The compounds matched better with strands than for helices. We expanded the C alpha ? C beta vector matching idea to a method that ranks preferred conformations of small molecules on any combination of three interface side-chains in all structurally characterized PPIs. We developed a PDB mining program (explores key orientation, EKO) to do this, and EKO applied to pyrrolinone-pyrrolidine oligomers to find targets. EKO found several interesting targets, such as AICAR Tfase, GAPDH, and HIV-1 protease. HIV-1 dimerization inhibition and Zhang-Poorman kinetic assays were performed to validate our hypothesis, and the results showed that pyrrolinone-pyrrolidine derivatives inhibited HIV-1 dimerization.

Severe Traumatic Brain Injury (TBI) and aneurysmal Subarachnoid Hemorrhage (SAH) are severe cerebral emergencies. They are common reasons for extensive morbidity and mortality in young people and adults in the western world. This thesis, based on five clinical studies in patients with severe TBI (I-IV) and SAH (V), is concentrated on examination of pathophysiological developments and of evaluation of therapeutic approaches in order to improve outcome after cerebral emergency. The treatment for severe TBI patients at Umeå University Hospital, Sweden is an intracranial pressure (ICP)-targeted therapy according to “the Lund-concept”. This therapy is based on physiological principles for cerebral volume regulation, in order to preserve a normal cerebral microcirculation and a normal ICP. The main goal is to avoid development of secondary brain injuries, thus avoiding brain oedema and worsened microcirculation. Study I is evaluating retrospectively 41 children with severe TBI, from 1993 to 2002. The boundaries of the ICP-targeted protocol were obtained in 90%. Survival rate was 93%, and favourable outcome (Glasgow Outcome Scale, score 4+5) was 80%. Study II is retrospectively analysing fluid administration and fluid balance in 93 adult patients with severe TBI, from 1998 to 2001.The ICP-targeted therapy used, have defined fluid strategies. The total fluid balance was positive day one to three, and negative day four to ten. Colloids constituted 40-60% of total fluids given/day. Severe organ failure was evident for respiratory insufficiency and observed in 29%. Mortality within 28 days was 11%. Study III is a prospective, randomised, double-blind, placebo-controlled clinical trial in 48 patients with severe TBI. In order to improve microcirculation and prevent oedema formation, prostacyclin treatment was added to the ICP-targeted therapy. Prostacyclin is endogenously produced, by the vascular endothelium, and has the ability to decrease capillary permeability and vasodilate cerebral capillaries. Prostacyclin is an inhibitor of leukocyte adhesion and platelet aggregation. There was no significant difference between prostacyclin or placebo groups in clinical outcome or in cerebral microdialysis markers such as lactatepyruvate ratio and brain glucose levels. Study IV is part of the third trial and focus on the systemic release of pro-inflammatory mediators that are rapidly activated by trauma. The systemically released pro-inflammatory mediators, interleukin-6 and CRP were significantly decreased in the prostacyclin group versus the placebo group. Study V is a prospective pilot study which analyses asymmetric dimethylarginine (ADMA) concentrations in serum from SAH patients. Acute SAH patients have cerebral vascular, systemic circulatory and inflammatory complications. ADMA is a marker in vascular diseases which is correlated to endothelial dysfunction. ADMA concentrations in serum were significantly elevated seven days after the SAH compared to admission and were still elevated at the three months follow-up. Our results show overall low mortality and high favourable outcome compared to international reports on outcome in severe TBI patients. Prostacyclin administration does not improve cerebral metabolism or outcome but significantly decreases the levels of pro-inflammatory mediators. SAH seems to induce long-lasting elevations of ADMA in serum, which indicates persistent endothelial dysfunction. Endothelial dysfunction may influence outcome after severe cerebral emergencies.

A multi-tiered system of supports offers a comprehensive model for the prevention of academic and behavior problems in schools. To date, research has emphasized the impact of universal and intensive interventions. However, the need for research on secondary or targeted group interventions (Tier 2) for those students who do not respond to the universal level of support is growing. This study evaluated CICO, a Tier 2 intervention, in improving student behavior when it is used with three elementary students from a high-need population and in conjunction with student accountability tracking, designed to promote parental involvement. Functional assessments indicated that all three students had attention-maintained problem behavior during instruction sessions. The study employed a concurrent multiple baseline design across students to assess the effects of CICO and CICO with accountability tracking on academic engagement and problem behavior. Results indicated that the team members were able to implement CICO with fidelity and their implementation of the intervention was effective in increasing academic engagement and reducing problem behavior. The CICO with accountability tracking implementation with one student contributed to further improvement of his target behaviors. These effects were shown to be maintained moderately well for two students who underwent fading. Results are discussed in terms of the study limitations and implications for practice and future research.

An educational framework known as School Wide Positive Behavior Support being implemented in school systems across the country provides the schools with three tiers of support to address both academic and behavior challenges. The purpose of this study was to evaluate the use of peer tutors when applied to a Tier 2 intervention known as Check-In Check-Out (CICO). Peer tutors performed the morning check-in with the tutees by setting the expectations for the day and giving the tutees their Daily Progress Report (DPR) form. Throughout the day, the tutees took the DPR form to each class where they received a score from the teacher. At the end of the class period, the peer tutors provided the tutees with feedback on the scores received on the DPR form. Once the school day finished, the peer tutees checked-out with the tutors and received a reward if they met their percentage goal. The results of this study showed that CICO implemented by peers improved classroom behavior for all three participants.