Direct Interrogation of Viral Peptides Presented by the Class I HLA of HIV-Infected T Cells

Identification of CD8+ cytotoxic T lymphocyte (CTL) epitopes has traditionally relied upon testing of overlapping peptide libraries for their reactivity with T cells in vitro. Here, we pursued deep ligand sequencing (DLS) as an alternative method of directly identifying those ligands that are epitopes presented to CTLs by the class I human leukocyte antigens (HLA) of infected cells. Soluble class I HLA-A*11:01 (sHLA) was gathered from HIV-1 NL4-3-infected human CD4+ SUP-T1 cells. HLA-A*11:01 harvested from infected cells was immunoaffinity purified and acid boiled to release heavy and light chains from peptide ligands that were then recovered by size-exclusion filtration. The ligands were first fractionated by high-pH high-pressure liquid chromatography and then subjected to separation by nano-liquid chromatography (nano-LC)���mass spectrometry (MS) at low pH. Approximately 10 million ions were selected for sequencing by tandem mass spectrometry (MS/MS). HLA-A*11:01 ligand sequences were determined with PEAKS software and confirmed by comparison to spectra generated from synthetic peptides. DLS identified 42 viral ligands presented by HLA-A*11:01, and 37 of these were previously undetected. These data demonstrate that (i) HIV-1 Gag and Nef are extensively sampled, (ii) ligand length variants are prevalent, particularly within Gag and Nef hot spots where ligand sequences overlap, (iii) noncanonical ligands are T cell reactive, and (iv) HIV-1 ligands are derived from de novo synthesis rather than endocytic sampling. Next-generation immunotherapies must factor these nascent HIV-1 ligand length variants and the finding that CTL-reactive epitopes may be absent during infection of CD4+ T cells into strategies designed to enhance T cell immunity.

Comparison of three analytical methods for the determination of quinolones in pig muscle samples

This work presents a comparison between three analytical methods developed for the simultaneous determination of eight quinolones regulated by the European Union (marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin, difloxacin, sarafloxacin, oxolinic acid and flumequine) in pig muscle, using liquid chromatography with fluorescence detection (LC-FD), liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The procedures involve an extraction of the quinolones from the tissues, a step for clean-up and preconcentration of the analytes by solid-phase extraction and a subsequent liquid chromatographic analysis. The limits of detection of the methods ranged from 0.1 to 2.1 ng g���1 using LC-FD, from 0.3 to 1.8 using LC-MS and from 0.2 to 0.3 using LC-MS/MS, while inter- and intra-day variability was under 15 % in all cases. Most of those data are notably lower than the maximum residue limits established by the European Union for quinolones in pig tissues. The methods have been applied for the determination of quinolones in six different commercial pig muscle samples purchased in different supermarkets located in the city of Granada (south-east Spain).

O estado da arte da cromatografia associada �� espectrometria de massas acoplada �� espectrometria de massas na an��lise de compostos t��xicos em alimentos

Chromatography combined with several different detection systems is one of the more used and better performing analytical tools. Chromatography with tandem mass spectrometric detection gives highly selective and sensitive analyses and permits obtaining structural information about the analites and about their molar masses. Due to these characteristics, this analytical technique is very efficient when used to detect substances at trace levels in complex matrices. In this paper we review instrumental and technical aspects of chromatography-tandem mass spectrometry and the state of the art of the technique as it is applied to analysis of toxic substances in food.

Valida����o de m��todo multirres��duo para determina����o de pesticidas em alimentos empregando QuEChERS E UPLC-MS/MS

This paper presents a practical and rapid method which was validated for simultaneous quantification and confirmation of 29 pesticides in fruits and vegetables using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The samples were extracted following the method known as QuEChERS. Using the developed chromatographic conditions, the pesticides can be separated in less than 9 min. Two multiple reaction monitoring (MRM) assays were used for each pesticide. Four representative matrices (lettuce, tomato, apple and grapes) were selected to investigate the effect in recoveries and precision. Typical recoveries ranged from 70-120%, with relative standard deviation (RSDs) lower than 20%.

MONITORING OF ORGANOPHOSPHOROUS PESTICIDE RESDUES IN SAMPLES OF BANANA, PAPAYA, AND BELL PEPPER

The objective of this study was to monitor 11 organophosphorus pesticides in samples of papaya, bell pepper, and banana, commercialized in the metropolitan area of Vitória (ES, Brazil). The pesticides were determined by an optimized and validated method using high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). All three samples exhibited a matrix effect for most of the pesticides, mainly with signal suppression, and therefore the calibration curves were produced in matrices. Linearity revealed coefficients of determination (r2) greater than 0.9895 for all pesticides and recovery results ranged from between 76% and 118% with standard deviation no greater than 16%. Precision showed relative standard deviation values lower than 19% and HorRat values lower than 0.7, considering all pesticides. Limits of quantification were less than 0.01 mg/kg for all pesticides. Regarding analysis of the samples (50 of each), none of the pesticides exceeded the maximum residue limit determined by Brazilian legislation.

COMPARA����O DE M��TODOS POR CROMATOGRAFIA L��QUIDA NA DETERMINA����O DE MULTIRRES��DUOS DE AGROT��XICOS EM MORANGOS

Liquid chromatography is often used for the determination of pesticide multiresidues in foods. In Brazil, the strawberry crop is an example of a food with high levels of irregularities because of the application of pesticides. This is a major concern from the perspective of food safety, environmental protection, and certification for food export. The purpose of this study is to evaluate and compare chromatographic separation and detection methods in relation to a newly developed and validated method using ultra high performance liquid chromatography (LC) coupled with tandem mass spectrometry (MS/MS) for the analytical determination of pesticides in strawberries. The comparisons were based on evaluations of the analysis time, consumption of the solvent in the mobile phase, injection volume, detectability, matrix effect, and recovery. The results showed that the LC–MS/MS and UHPLC–MS/MS techniques were both extremely efficient at analyzing pesticide residues with different physico-chemical parameters that were present at low concentrations in a complex matrix. The UHPLC separation method provided better chromatographic performance and productivity, which contributed favorably to routine analytical determinations. Detection by MS/MS had better detectability and selectivity compared with the diode array detector.

Experimental poisoning by Baccharis megapotamica var. weirii in buffalo

Five male 6-8 month-old Murrah buffalo calves were orally dosed with the fresh aerial parts of Baccharis megapotamica var. weirii at doses of 1, 3, 4, 5 and 10g/kg body weight (bw) (~1-10mg macrocyclic trichothecenes/kg/bw). The B. megapotamica used for the experiment was harvested on a farm where a recent spontaneous outbreak of poisoning caused by such plant had occurred. Clinical signs appeared 4-20 hours and 4 buffaloes died 18-49 hours after the ingestion of the plant. Clinical signs were apathy, anorexia, and watery diarrhea, fever, colic, drooling, muscle tremors, restlessness, laborious breathing and ruminal atony, and dehydration. The most consistent gross findings were restricted to the gastrointestinal (GI) tract consisted of varying degrees of edema and reddening of the mucosa of the forestomach. Histopathological findings consisted of varying degrees of necrosis of the epithelial lining of the forestomach and of lymphocytes within lymphoid organs and aggregates. Fibrin thrombi were consistently found in sub-mucosal vessels of the forestomach and in the lumen of hepatic sinusoids. It is suggested that dehydration, septicemia and disseminated intravascular coagulation participate in the pathogenesis of the intoxication and play a role as a cause of death. A subsample of B. megapotamica var. weirii was frozen-dried and ground and analyzed using UHPLC (Ultra High Performance Liquid Chromatography) with high resolution Time of Flight mass spectrometry and tandem mass spectrometry, it was shown that the plant material contained at least 51 different macrocyclic trichothecenes at a total level of 1.1-1.2mg/g. About 15-20% of the total trichothecenes contents was found to be monosaccharide conjugates, with two thirds of these being glucose conjugates and one third constituted by six aldopentose conjugates (probably xylose), which has never been reported in the literature.

Extraction and Simultaneous Determination of Glyphosate, AMPA and Compounds of the Shikimic Acid Pathway in Plants

This study has aimed to develop a method for simultaneous extraction and determination by liquid chromatography and mass spectrometry (LC-MS/MS) of glyphosate, aminomethylphosphonic acid (AMPA), shikimic acid, quinic acid, phenylalanine, tyrosine and tryptophan. For the joint analysis of these compounds the best conditions of ionization in mass spectrometry and for chromatographic separation of the compounds were selected. Calibration curves and linearity ranges were also determined for each compound. Different extraction systems of the compounds were tested from plant tissues collected from sugarcane (Saccharum officinarum) and eucalyptus (Eucalyptus urophylla platiphylla) plants two days after the glyphosate application at the dose of 720 g a.e. ha-1. The plant material was dried in a forced air circulation drying oven and in a lyophilizer, and subsequently the extractions with acidified water (pH 2.5), acetonitrile-water (50:50) [v/v] and methanol-water (50:50) [v/v] were tested. To verify the recovery of the compounds in the plant matrix with acidified water as an extracting solution, the samples were fortified with a solution containing the mixture of the different analytical standards present so that this one presented the same levels of 50 and 100 μg L-1 of each compound. All experiments were conducted with three replicates. The analytical method developed was efficient for compounds quantifications. The extraction from the samples dried in an oven and using acidified water allowed better extraction levels for all compounds. The recovery levels of the compounds in the fortified samples with known amounts of each compound for both plants samples were rather satisfactory.

Understanding the bioactivity of plant tannins: developments in analysis methods and structure-activity studies

Tannins, typically segregated into two major groups, the hydrolyzable tannins (HTs) and the proanthocyanidins (PAs), are plant polyphenolic secondary metabolites found throughout the plant kingdom. On one hand, tannins may cause harmful nutritional effects on herbivores, for example insects, and hence they work as plants��� defense against plant-eating animals. On the other hand, they may affect positively some herbivores, such as mammals, for example by their antioxidant, antimicrobial, anti-inflammatory or anticarcinogenic activities. This thesis focuses on understanding the bioactivity of plant tannins, their anthelmintic properties and the tools used for the qualitative and quantitative analysis of this endless source of structural diversity. The first part of the experimental work focused on the development of ultra-high performance liquid chromatography���tandem mass spectrometry (UHPLC-MS/MS) based methods for the rapid fingerprint analysis of bioactive polyphenols, especially tannins. In the second part of the experimental work the in vitro activity of isolated and purified HTs and their hydrolysis product, gallic acid, was tested against egg hatching and larval motility of two larval developmental stages, L1 and L2, of a common ruminant gastrointestinal parasite, Haemonchus contortus. The results indicated clear relationships between the HT structure and the anthelmintic activity. The activity of the studied compounds depended on many structural features, including size, functional groups present in the structure, and the structural rigidness. To further understand tannin bioactivity on a molecular level, the interaction between bovine serum albumin (BSA), and seven HTs and epigallocatechin gallate was examined. The objective was to define the effect of pH on the formation on tannin���protein complexes and to evaluate the stability of the formed complexes by gel electrophoresis and MALDI-TOF-MS. The results indicated that more basic pH values had a stabilizing effect on the tannin���protein complexes and that the tannin oxidative activity was directly linked with their tendency to form covalently stabilized complexes with BSA at increased pH.

An exploratory study for the discovery of non-invasive hepatocellular carcinoma biomarkers among high-risk hepatitis C virus infected patients

Hepatocellular Carcinoma (HCC) is a major healthcare problem, representing the third most common cause of cancer-related mortality worldwide. Chronic infections with Hepatitis B virus (HBV) and/or Hepatitis C virus (HCV) are the major risk factors for the development of HCC. The incidence of HBV -associated HCC is in decline as a result of an effective HBV vaccine; however, since an equally effective HCV vaccine has not yet been developed, there are 130 million HCV infected patients worldwide who are at a high-risk for developing HCC. Because reliable parameters and/or tools for the early detection of HCC among high-risk individuals are severely lacking, HCC patients are always diagnosed at a late stage where surgical solutions or effective treatment are not possible. Using urine as a non-invasive sample source, two different approaches (proteomic-based and genomic-based approaches) were pursued with the common goal of discovering potential biomarker candidates for the early detection of HCC among high-risk chronic HCV infected patients. Urine was collected from 106 HCV infected Egyptian patients, 32 of whom had already developed HCC and 74 patients who were diagnosed as HCC-free at the time of initial sample collection. In addition to these patients, urine samples were also collected from 12 healthy control individuals. Total urinary proteins, Trans-renal nucleic acid (Tr-NA) and microRNA (miRNA) were isolated from urine using novel methodologies and silicon carbide-loaded spin columns. In the first, "proteomic-based", approach, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to identify potential candidates from pooled urine samples. This was followed by validating relative expression levels of proteins present in urine among all the patients using quantitative real time-PCR (qRT-PCR). This approach revealed that significant over-expression of three proteins: DJ-1, Chromatin Assembly Factor-1 (CAF-1) and 11 Moemen Abdalla HCC Biomarkers Heat Shock Protein 60 (HSP60), were characteristic events among HCC-post HCV infected patients. As a single-based HCC biomarker, CAF-1 over-expression identified HCC among HCV infected patients with a specificity of 90%, sensitivity of 66% and with an overall diagnostic accuracy of 78%. Moreover, the CAF-lIHSP60 tandem identified HCC among HCV infected patients with a specificity of 92%, sensitivity of 61 % and with an overall diagnostic accuracy of 77%. In the second genomic-based approach, two different approaches were processed. The first approach was the miRNA-based approach. The expression levels of miRNAs isolated from urine were studied using the Illumina MicroRNA Expression Profiling Assay. This was followed by qRT-PCR-based validation of deregulated expression of identified miRNA candidates among all the patients. This approach shed the light on the deregulated expression of a number of miRNAs, which may have a role in either the development of HCC among HCV infected patients (i.e. miR-640, miR-765, miR-200a, miR-521 and miR-520) or may allow for a better understanding of the viral-host interaction (miR-152, miR-486, miR-219, miR452, miR-425, miR-154 and miR-31). Moreover, the deregulated expression of both miR-618 and miR-650 appeared to be a common event among HCC-post HCV infected patients. The results of the search for putative targets of these two miRNA suggested that miR-618 may be a potent oncogene, as it targets the tumor-suppressor gene Low density lipoprotein-related protein 12 (LPR12), while miR-650 may be a potent tumor-suppressor gene, as it is supposed to downregulate the TNF receptor-associated factor-4 (TRAF4) oncogene. The specificity of miR-618 and miR-650 deregulated expression patterns for the early detection of HCC among HCV infected patients was 68% and 58%, respectively, whereas the sensitivity was 64% and 72%, respectively. When the deregulated expression of both miRNAs was combined as a tandem biomarker, the specificity and the sensitivity were 75% and 58% respectively. 111 Moemen Abdalla HCC Biomarkers In the second, "Trans-renal nucleic acid-based", approach, the urinary apoptotic nucleic acid (uaNA) levels of 70ng/mL or more were found to be a good predictor of HCC among chronic HCV infected patients. The specificity and the sensitivity of this diagnostic approach were 76% and 86%, respectively, with an overall diagnostic value of 81 %. The uaNA levels positively correlated to HCC disease progression as monitored by epigenetic changes of a panel of eight tumor-suppressor genes (TSGs) using methylation-sensitive PCR. Moreover, the pairing of high uaNA levels (:::: 70 ng/mL) and CAF-1 over-expreSSIOn produced a highly specific (l 00%) multiple-based HCC biomarker with an acceptable sensitivity of 64%, and with a diagnostic accuracy of 82%. In comparison to the previous pairing, the uaNA levels (:::: 70 ng/mL) in tandem with HSP60 over-expression was less specific (89%) but highly sensitive (72%), resulting in a diagnostic accuracy of 64%. The specificities of miR-650 deregulated expression in combination with either high uaNA content or HSP 60 over-expression were 82% and 79%, respectively, whereas, the sensitivities of these combinations were 64% and 58%, respectively. The potential biomarkers identified in this study compare favorably with the diagnostic accuracy of the a-fetoprotein levels test, which has a specificity of 75%, sensitivity of 68% and an overall diagnostic accuracy of 70%. Here we present an intriguing study which shows the significance of using urine as a noninvasive sample source for the identification of promising HCC biomarkers. We have also introduced new techniques for the isolation of different urinary macromolecules, especially miRNA, from urine. Furthermore, we strongly recommend the potential biomarkers indentified in this study as focal points of any future research on HCC diagnosis. A larger testing pool will determine if their use is practical for mass population screening. This explorative study identified potential targets that merit further investigation for the development of diagnostically accurate biomarkers isolated from 1-2 mL urine samples that were acquired in a non-invasive manner.

D��veloppement de techniques analytiques pour la d��termination des agents anti-infectieux dans les eaux environnementales

Les agents anti-infectieux sont utilis��s pour traiter ou pr��venir les infections chez les humains, les animaux, les insectes et les plantes. L���apparition de traces de ces substances dans les eaux us��es, les eaux naturelles et m��me l���eau potable dans plusieurs pays du monde soul��ve l���inqui��tude de la communaut�� scientifique surtout �� cause de leur activit�� biologique. Le but de ces travaux de recherche a ��t�� d�����tudier la pr��sence d���anti-infectieux dans les eaux environnementales contamin��es (c.-��-d. eaux us��es, eaux naturelles et eau potable) ainsi que de d��velopper de nouvelles m��thodes analytiques capables de quantifier et confirmer leur pr��sence dans ces matrices. Une m��ta-analyse sur l���occurrence des anti-infectieux dans les eaux environnementales contamin��es a d��montr�� qu���au moins 68 compos��s et 10 de leurs produits de transformation ont ��t�� quantifi��s �� ce jour. Les concentrations environnementales varient entre 0.1 ng/L et 1 mg/L, selon le compos��, la matrice et la source de contamination. D���apr��s cette ��tude, les effets nuisibles des anti-infectieux sur le biote aquatique sont possibles et ces substances peuvent aussi avoir un effet indirect sur la sant�� humaine �� cause de sa possible contribution �� la diss��mination de la r��sistance aux anti-infecteiux chez les bact��ries. Les premiers tests pr��liminaires de d��veloppement d���une m��thode de d��termination des anti-infectieux dans les eaux us��es ont montr�� les difficult��s �� surmonter lors de l���extraction sur phase solide (SPE) ainsi que l���importance de la s��lectivit�� du d��tecteur. On a d��crit une nouvelle m��thode de quantification des anti-infectieux utilisant la SPE en tandem dans le mode manuel et la chromatographie liquide coupl��e �� la spectrom��trie de masse en tandem (LC-MS/MS). Les six anti-infectieux cibl��s (sulfam��thoxazole, trim��thoprime, ciprofloxacin, levofloxacin, clarithromycin et azithromycin) ont ��t�� quantifi��s �� des concentrations entre 39 et 276 ng/L dans les ��chantillons d���affluent et d���effluent provenant d���une station d�����puration appliquant un traitement primaire et physico- chimique. Les concentrations retrouv��es dans les effluents indiquent que la masse moyenne totale de ces substances, d��vers��es hebdomadairement dans le fleuve St. Laurent, ��tait de ~ 2 kg. En vue de r��duire le temps total d���analyse et simplifier les manipulations, on a travaill�� sur une nouvelle m��thode de SPE coupl��e-LC-MS/MS. Cette m��thode a utilis�� une technique de permutation de colonnes pour pr��concentrer 1.00 mL d�����chantillon dans une colonne de SPE coupl��e. La performance analytique de la m��thode a permis la quantification des six anti-infectieux dans les eaux us��es municipales et les limites de d��tection ��taient du m��me ordre de grandeur (13-60 ng/L) que les m��thodes bas��es sur la SPE manuelle. Ensuite, l���application des colonnes de SPE coupl��e de chromatographie �� d��bit turbulent pour la pr��concentration de six anti-infectieux dans les eaux us��es a ��t�� explor��e pour diminuer les effets de matrice. Les r��sultats obtenus ont indiqu�� que ces colonnes sont une solution de r��change int��ressante aux colonnes de SPE coupl��e traditionnelles. Finalement, en vue de permettre l���analyse des anti-infectieux dans les eaux de surface et l���eau potable, une m��thode SPE coupl��e-LC-MS/MS utilisant des injections de grand volume (10 mL) a ��t�� d��velopp��e. Le volume de fuite de plusieurs colonnes de SPE coupl��e a ��t�� estim�� et la colonne ayant la meilleure r��tention a ��t�� choisie. Les limites de d��tection et de confirmation de la m��thode ont ��t�� entre 1 �� 6 ng/L. L���analyse des ��chantillons r��els a d��montr�� que la concentration des trois anti-infectieux cibl��s (sulfam��thoxazole, trim��thoprime et clarithromycine) ��tait au dessous de la limite de d��tection de la m��thode. La mesure des masses exactes par spectrom��trie de masse �� temps d���envol et les spectres des ions produits utilisant une pente d�����nergie de collision inverse dans un spectrom��tre de masse �� triple quadrip��le ont ��t�� explor��s comme des m��thodes de confirmation possibles.

Identification de biomarqueurs prot��iques pr��dictifs et diagnostiques des maladies coronariennes pour une population de souche canadienne-fran��aise

Les biomarqueurs plasmatiques constituent des outils essentiels, mais rares, utilis��s pour diagnostiquer les maladies, comme les maladies cardiovasculaires (MCV), et stratifier le niveau de risque associ��. L���identification de nouveaux biomarqueurs plasmatiques susceptibles d���am��liorer le d��pistage et le suivi des MCV repr��sente ainsi un enjeu majeur en termes d�����conomie et de sant�� publique. Le projet vise �� identifier de nouveaux biomarqueurs plasmatiques pr��dictifs ou diagnostiques des MCV, �� d��terminer le profil prot��omique plasmatique de patients atteints de MCV et �� d��velopper des m��thodes innovantes d���analyse d�����chantillon plasmatique. L�����tude a ��t�� effectu��e sur une large banque de plasma provenant de 1006 individus de souche Canadienne-Fran��aise recrut��s �� diff��rents stades de la MCV et qui ont ��t�� suivis sur une p��riode de 5 ans. Des s��ries de d��pl��tions ont ��t�� r��alis��es afin de d��pl��ter les 14 prot��ines majoritaires (colonne IgY14TM) de l�����chantillon avant son analyse par trois approches effectu��es en parall��le: 1) Une chromatographie liquide (LC) en 2 dimensions qui fractionne les prot��ines selon le point iso��lectrique puis selon le degr�� d���hydrophobicit��, via le syst��me PF2D, suivie par une chromatographie liquide coupl��e avec une spectrom��trie de masse en tandem (LC-MS/MS). 2) Une s��paration classique sur gel 1D-SDS-PAGE suivie d���une LC-MS/MS; 3) Par une d��pl��tion plus pouss��e du plasma avec l���utilisation en tandem avec la colonne IgY14TM d���une colonne SupermixTM permettant de d��pl��ter ��galement les prot��ines de moyenne abondance, suivie d���une s��paration sur gel 1D-SDS-PAGE et d���une analyse LC-MS/MS de la portion d��pl��t��e (3a) et de la portion li��e �� la SupermixTM (3b). Les r��sultats montrent que le syst��me PF2D permet d���identifier plusieurs profils prot��iques sp��cifiques au groupe MCV. Sur un total de 1156 fractions (��quivalent �� 1172 pics prot��iques pour le groupe contr��le et 926 pics pour le groupe MCV) recueillies, 15 fractions (23 pics prot��iques) pr��sentaient des diff��rences quantitativement significatives (p<0,05) entre les 2 groupes. De plus, 6 fractions (9 pics) sont uniquement pr��sentes dans un groupe, repr��sentant d���autres signatures prot��omiques et biomarqueurs potentiellement int��ressants. Les m��thodes 2, 3a et 3b ont permis l���identification de 108, 125 et 91 prot��ines respectivement avec des chevauchements partiels (31% entre la m��thode 2 et 3a, 61% entre 2 et 3b et 19% entre 3a et 3b). Les m��thodes 2 et 3 ont permis l���identification de 12 prot��ines qui pr��sentaient des diff��rences quantitatives significatives entre les 2 groupes. L���utilisation de plusieurs approches prot��omiques compl��mentaires nous ont d���ores et d��j�� permis d���identifier des candidats biomarqueurs des angines instables avec r��cidive d���infarctus du myocarde (IM).

D��veloppement de m��thodes de s��paration des chitooligosaccharides obtenus par d��ac��tylation enzymatique

M��moire num��ris�� par la Division de la gestion de documents et des archives de l'Universit�� de Montr��al

Detection of methotrexate using surface plasmon resonance biosensors for chemotherapy monitoring

Le m��thotrexate (MTX), un agent anti-canc��reux fr��quemment utilis�� en chimioth��rapie, requiert g��n��ralement un suivi th��rapeutique de la m��dication (Therapeutic Drug Monitoring, TDM) pour surveiller son niveau sanguin chez le patient afin de maximiser son efficacit�� tout en limitant ses effets secondaires. Malgr�� la fen��tre th��rapeutique ��troite entre l���efficacit�� et la toxicit��, le MTX reste, �� ce jour, un des agents anti-canc��reux les plus utilis��s au monde. Les techniques analytiques existantes pour le TDM du MTX sont co��teuses, requi��rent temps et efforts, sans n��cessairement fournir promptement les r��sultats dans le d��lai requis. Afin d���acc��l��rer le processus de dosage du MTX en TDM, une strat��gie a ��t�� propos��e bas��e sur un essai comp��titif caract��ris�� principalement par le couplage plasmonique d���une surface m��tallique et de nanoparticules d���or. Plus pr��cis��ment, l���essai quantitatif exploite la r��action de comp��tition entre le MTX et une nanoparticule d���or fonctionnalis��e avec l���acide folique (FA-AuNP) ayant une affinit�� pour un r��cepteur mol��culaire, la r��ductase humaine de dihydrofolate (hDHFR), une enzyme associ��e aux maladies prolif��ratives. Le MTX libre mix�� avec les FA-AuNP, entre en comp��tition pour les sites de liaison de hDHFR immobilis��s sur une surface active en SPR ou libres en solution. Par la suite, les FA-AuNP li��es au hDHFR fournissent une amplification du signal qui est inversement proportionnelle �� la concentration de MTX. La r��sonance des plasmons de surface (SPR) est g��n��ralement utilis��e comme une technique spectroscopique pour l���interrogation des interactions biomol��culaires. Les instruments SPR commerciaux sont g��n��ralement retrouv��s dans les grands laboratoires d���analyse. Ils sont ��galement encombrants, co��teux et manquent de s��lectivit�� dans les analyses en matrice complexe. De plus, ceux-ci n���ont pas encore d��montr�� de l���adaptabilit�� en milieu clinique. Par ailleurs, les analyses SPR des petites mol��cules comme les m��dicaments n���ont pas ��t�� explor��s de mani��re intensive d�� au d��fi pos�� par le manque de la sensibilit�� de la technique pour cette classe de mol��cules. Les d��veloppements r��cents en science des mat��riaux et chimie de surfaces exploitant l���int��gration des nanoparticules d���or pour l���amplification de la r��ponse SPR et la chimie de surface peptidique ont d��montr�� le potentiel de franchir les limites pos��es par le manque de sensibilit�� et l���adsorption non-sp��cifique pour les analyses directes dans les milieux biologiques. Ces nouveaux concepts de la technologie SPR seront incorpor��s �� un syst��me SPR miniaturis�� et compact pour ex��cuter des analyses rapides, fiables et sensibles pour le suivi du niveau du MTX dans le s��rum de patients durant les traitements de chimioth��rapie. L���objectif de cette th��se est d���explorer diff��rentes strat��gies pour am��liorer l���analyse des m��dicaments dans les milieux complexes par les biocapteurs SPR et de mettre en perspective le potentiel des biocapteurs SPR comme un outil utile pour le TDM dans le laboratoire clinique ou au chevet du patient. Pour atteindre ces objectifs, un essai comp��titif colorim��trique bas�� sur la r��sonance des plasmons de surface localis��e (LSPR) pour le MTX fut ��tabli avec des nanoparticules d���or marqu��es avec du FA. Ensuite, cet essai comp��titif colorim��trique en solution fut adapt�� �� une plateforme SPR. Pour les deux essais d��velopp��s, la sensibilit��, s��lectivit��, limite de d��tection, l���optimisation de la gamme dynamique et l���analyse du MTX dans les milieux complexes ont ��t�� inspect��s. De plus, le prototype de la plateforme SPR miniaturis��e fut valid�� par sa performance ��quivalente aux syst��mes SPR existants ainsi que son utilit�� pour analyser les ��chantillons cliniques des patients sous chimioth��rapie du MTX. Les concentrations de MTX obtenues par le prototype furent compar��es avec des techniques standards, soit un essai immunologique bas�� sur la polarisation en fluorescence (FPIA) et la chromatographie liquide coupl��e avec de la spectrom��trie de masse en tandem (LC-MS/MS) pour valider l���utilit�� du prototype comme un outil clinique pour les tests rapides de quantification du MTX. En dernier lieu, le d��ploiement du prototype �� un laboratoire de biochimie dans un h��pital d��montre l�����norme potentiel des biocapteurs SPR pour utilisation en milieux clinique.

Inhibition r��versible et photomarquage de la transglutaminase tissulaire

La transglutaminase tissulaire est une enzyme d��pendante du calcium qui catalyse la formation de liens isopeptidiques, entre les cha��nes lat��rales de r��sidus glutamine et lysine, permettant, par le fait m��me, la r��ticulation des prot��ines dans les syst��mes biologiques. Elle joue un r��le, entre autres, dans l���endocytose, la r��gulation du d��veloppement des cellules, et m��me dans l���apoptose. N��anmoins, une d��r��gulation de l���activit�� biologique de cette enzyme peut entrainer diff��rentes pathologies, comme la formation de cataractes, de plaques amylo��des dans la maladie d���Alzheimer, ou encore peut mener au d��veloppement de la maladie c��liaque. C���est pourquoi une meilleure connaissance du m��canisme d���action de cette enzyme et la possibilit�� de r��guler son action �� l���aide de substrats ou d���inhibiteurs sont n��cessaires. Dans cette optique, une m��thode d���expression et de purification de la transglutaminase humaine a ��t�� d��velopp��e, permettant de travailler directement avec la cible pharmacologique d��sir��e. De plus, une ��tude du mode d���inhibition et de liaison d���une classe d���inhibiteurs r��versibles pr��c��demment d��couverte dans le groupe, soit la famille des trans-cinnamoyles, a permis d���identifier que la puissance de ces mol��cules est influenc��e par la pr��sence du calcium et qu���une inhibition d��pendante du temps est observ��e, en lien avec un potentiel ��quilibre conformationnel lent de la transglutaminase. D���un autre c��t��, la susceptibilit�� �� une attaque nucl��ophile par des thiols de cette classe de mol��cule rend leur potentiel pharmacologique grandement diminu��, et c���est pourquoi une nouvelle famille de mol��cules a ��t�� identifi��e, bas��e sur un squelette ynone, avec une valeur d���IC50 tr��s prometteuse de 2,6 ��M, en faisant un des meilleurs inhibiteurs r��versibles de la transglutaminase d��velopp��s �� ce jour. Finalement, une strat��gie de photomarquage jumel��e �� une analyse de spectrom��trie de masse en tandem a ��t�� d��velopp��e pour la d��couverte du site de liaison du substrat d��riv�� de la lysine, dans le but de mieux comprendre le m��canisme complexe de cette enzyme.