Dissertations / Theses on the topic 'Droplet digital PCR'

The ability to quantify the amount of DNA in a sample is an essential technique in biology and is used in many fields of research. Droplet digital polymerase chain reaction (ddPCR) is an advanced technology developed for this purpose that enables more accurate and sensitive quantification than traditional real-time PCR. In ddPCR, nucleic acid (e.g., genomic DNA) within a sample is partitioned into thousands of droplets, along with the reagents needed to amplify and detect one or more DNA target sequences. After amplification takes place in all droplets, each droplet is individually read by a two-colour fluorescence detection system to determine whether or not it contains the target sequence. ddPCR experiments utilizing both fluorescence wavelengths are termed dual-channel, while simpler experiments can make use of only one fluorescence wavelength and are thus classified as single-channel. Droplets containing amplified product exhibit high fluorescence and are said to be positive, while those without product show little or no fluorescence and are considered negative. Using this binary, or digital, classification of droplets, the number of positive and negative droplets can be counted to allow for an absolute quantification of template abundance in the starting sample. ddPCR instruments are now available commercially and their use is growing. But, there are a very limited number of tools available for downstream data analysis. The key step in ddPCR data analysis is droplet gating: using the end-point fluorescence data to gate, or classify, droplets as either positive or negative for a given template. The proprietary software provided by BioRad Inc., a ddPCR instrument manufacturer, is currently the only program available to automatically analyze dual-channel ddPCR data. However, because this analysis tool often produces poor results, many ddPCR users resort to time-consuming and subjective manual data analyses, emphasizing the clear need for new ddPCR analysis tools. In this thesis, I devise an algorithm for automatic analysis of dual-channel ddPCR data that can objectively and reproducibly perform droplet gating. The proposed analysis method has been implemented in an R package and is also available as a web application online for easy and open access to any ddPCR user.
Science, Faculty of
Graduate

Afin de faire face aux conditions environnementales défavorables, certains animaux réduisent drastiquement leur activité métabolique et leur température grâce à des phases de torpeur hivernal. L’objectif de cette étude est d’établir une signature moléculaire de chacune des phases d’hibernation. Pour cela, les variations d’expression de 21 gènes impliqués dans le contrôle des fonctions saisonnières (horloge circadienne, hormones thyroïdiennes, récepteurs à la mélatonine) et le métabolisme ont été étudiées dans 8 organes. Les résultats ont mis en évidence une augmentation ubiquitaire de l’expression des gènes Périodes indiquant un possible réajustement de l’horloge au début de la phase de réveil. Ainsi qu’une régulation spécifique des déiodinases induisant une augmentation de la synthèse de thyroxine dans le tissu adipeux brun et l’hypothalamus pendant la torpeur et le réveil. Le récepteur MT2 du hamster d’Europe a été partiellement caractérisé génétiquement et pharmacologiquement. A la différence d’autres espèces de hamster dont le récepteur MT2 est tronqué, le récepteur étudié semble être fonctionnel pour la mélatonine et pourrait être critique durant l’hibernation
Living in the wild involves to cope with a variable seasonal environment availability. When winter is coming, animals use various strategies to adapt to hostile environment by limiting energy expenditure such as hibernation. In this study, expression of 21 selected genes was compared at different states of the hibernation cycle of the true hibernator European hamster. Level of mRNA encoding proteins involved in seasonal timing (melatonin receptors, thyroid metabolism, clock) and energy homeostasis were measured by digital droplet PCR in eight central and peripheral organs. During the arousal phase, Periods genes expression is increased in all organs indicating a possible resetting of body’s clocks at the beginning of the active period. The brown adipose tissue displays a specific regulation of deiodinases leading to increased synthesis of thyroxine during both torpor and arousal. The melatonin receptor MT2 of the European hamster had been partially cloned and pharmacologically characterized. While in most hamster species, MT2 is a natural knock out, the studied receptor seems to be functional and could be critical during hibernation

Le carcinome épidermoïde cutané (CEC) représente un enjeu important par sa fréquence et sa gravité potentielle.L’agressivité de ce cancer est liée à l’invasion péri-nerveuse (IPN), mode d’envahissement tumoral reconnu comme un facteur de mauvais pronostic.L’objectif de ce travail est de s’intéresser aux mécanismes favorisant l’IPN, en comparant 2 groupes appariés de CEC humains, avec et sans IPN.Pour cela nous avons réalisé une étude de facteurs et récepteurs neurotrophiques, de marqueurs de la transition épithélio-mésenchymateuse (TEM), et de la molécule NCAM1, par analyse immunohistochimique à partir de pièces chirurgicales de CEC et par analyse moléculaire en droplet digital PCR sur des cellules tumorales microdisséquées.L’analyse immunohistochimique a trouvé une forte expression de BDNF, TrkB, p75NGFR, Snail 1 et NCMA1 dans les cellules tumorales péri-nerveuses, contrastant avec une faible expression de ces marqueurs dans les cellules tumorales à distance du nerf. L’E-cadhérine était diminuée dans les cellules tumorales péri-nerveuses.L’analyse moléculaire en ddPCR montrait une diminution d’expression de l’E-cadhérine et une surexpression de BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 et NCAM1 dans les cellules tumorales péri-nerveuses par rapport aux cellules tumorales distantes du nerf.Nous avons démontré dans ce travail que l’invasion péri-nerveuse dans les CEC humains est liée aux neurotrophines, à la TEM et implique NCAM1
Cutaneous squamous cell carcinoma (SCC) is an important issue because of its frequency and potential severity.The aggressiveness of this cancer is related to perineural invasion (PNI), a mode of tumor dissemination recognized as a poor prognosis factor.The aim of this work is to study the mechanisms of PNI, comparing 2 matched- groups of human SCC with and without PNI.For this, we studied neurotrophins, epithelial-mesenchymal transition (EMT) markers, and the NCAM1 molecule, by immunohistochemistry analysis on surgical pieces of SCC and by molecular analysis with digital-droplet PCR on laser-microdissected tumor cells.Immunohistochemistry analysis found strong expression of BDNF, TrkB, p75NGFR, Snail 1 and NCMA1 in perineural tumor cells, contrasting with weak expression of these markers in tumor cells distant from the nerves. E-cadherin was decreased in perineural tumor cells.Molecular analysis in ddPCR showed decreased expression for E-cadherin and overexpression of BDNF, TrkB, p75NGFR, Snail1, Slug, Zeb2, Twist1 and NCAM1 in perineural tumor cells compared to tumor cells distant from the nerves.We have demonstrated in this work that PNI in human SCC is linked to neurotrophins and EMT, and involves NCAM1

The aim of this work is to establish novel strategies for the highly sensitive screening of cancer biomarkers in biological samples.To achieve this goal, we developed droplet-based microfluidic dPCR technique. Using a limiting dilution, individual DNA molecules are encapsulated within monodisperse droplets of a water-in-oil emulsion created with a microfluidic device. Fluorescent TaqMan® probes targeting the screened cancer biomarkers allow the detection of mutations. We focused on the mutations in the human KRAS gene for the development of our test. This method is also transposable in a multiplexed format for the parallel detection of multiple mutations in clinical samples.The developed technique allowed the precise quantification of a mutated KRAS gene in the presence of a 200,000-fold excess of un-mutated KRAS genes and enabled the determination of mutant allelic specific imbalance (MASI) in several cancer cell-lines. We validated our technique by screening for KRAS mutations in the blood plasma and tumor samples from patients with metastatic colorectal cancer.

IntroductionAcute myeloid leukemia (AML) is caused by a wide range of genetic aberrations, includingmutations within the genes that encode the enzymes isocitrate dehydrogenase 1 and 2(IDH1/2). Drugs that target mutant IDH1/2 are now available, which makes assessment of themutational status of IDH1/2 important in clinical diagnostics of AML. A promising method todetect these mutations is the droplet digital polymerase chain reaction (ddPCR), which showsadvantages of a high sensitivity and a simple workflow.AimTo evaluate ddPCR as method of choice to detect hotspot mutations in IDH1 (codon R132)and IDH2 (codon R140 and R172) in patients with AML.MethodsFifteen AML patients known to be positive for IDH1/2 diagnosed by a previously performednext generation sequencing (NGS) were selected for evaluation of ddPCR. Diagnosticsamples were tested for 14 patients, whereas follow-up samples were tested for one patient.ddPCR was performed using QX200™ Droplet Digital PCR system and data were presentedas fractional abundance of mutant allele.ResultsThe amount of mutant IDH1/2 in samples reported by ddPCR correlated well with the resultsfrom NGS when using probes that target their specific mutation. The detection limit formutant allele in the background of wild type IDH1/2 was 0,5% for IDH2 p.R140Q and 0.1%for IDH1 p.R132C/H.ConclusionddPCR that target specific mutations shows a great potential in measuring minimal residualdisease during follow-up. However, its use in screening for mutant IDH1/2 at the time ofdiagnosis is limited and alternative approaches should be considered.

Le sol est essentiel à toute société humaine notamment pour la production d'aliments. Son fonctionnement repose sur des réseaux d'interactions entre les éléments qui le composent et toute perturbation modifie ces réseaux. Les microorganismes eucaryotes représentent une composante importante de l'écosystème édaphique car ils sont impliqués dans des processus essentiels comme la régulation de populations de procaryotes. Mais paradoxalement, ils restent peu étudiés comparés aux bactéries notamment lorsqu'on s'intéresse aux cycles biogéochimiques autres que celui du carbone comme par exemple ceux des métaux. Suite à une pollution par des métaux, certains de ces microorganismes eucaryotes développent des mécanismes « de résistance » cellulaires. Dans ce contexte, le laboratoire d'accueil a isolé, directement à partir d'extraits d'ARN de sol, des gènes eucaryotes impliqués dans la résistance cellulaire au Cd. Ces nouveaux gènes forment une famille codant des protéines riches en cystéines dont les positions sont conservées au sein de cette famille. Mon projet de thèse a eu pour but de caractériser l'origine taxonomique et la fonction de cette famille de gènes. Dans un premier temps, la purification de cinq de ces protéines produites dans Escherichia coli et leurs caractérisations biochimiques par des méthodes spectrométriques ont permis de montrer que cette famille génique constitue une nouvelle famille de métallothionéines capables de chélater in vitro le Zn, le Cu et le Cd. Dans un second temps, une méthode de quantification par PCR quantitative de l'expression de ces gènes, extraits à partir de sol provenant de microcosmes, a été mise au point. Dans un troisième temps, nous avons tenté d'obtenir les régions génomiques bordant ces gènes environnementaux afin d'affilier les organismes qui les portent à un groupe taxonomique et d'analyser les régions promotrices de ces gènes par capture ciblée de gènes
Soil is essential to human societies, especially for food production. Its functioning relies on interaction networks sensitive to environmental alterations. Eukaryotic microorganisms are an important component of the soil ecosystem where they are involved in essential processes such as the regulation of prokaryotic populations. However, they remain poorly studied compared to bacteria, especially concerning their roles in biogeochemical cycles other than the carbon one such as metal cycles. In response to soil metal contamination, some of these eukaryotic microorganisms develop cellular "resistance" mechanisms. In this context, the host laboratory has previously isolated, directly from soils, eukaryotic genes able to confer Cd resistance. These genes form a family coding for cysteine-rich proteins whose cysteine positions are conserved within this sequences. My thesis project aimed at characterizing the function and taxonomic origin of this gene family. First, the purification of five of these proteins produced in Escherichia coli and their biochemical characterizations by spectrometric methods demonstrated that this gene family constitutes a new family of metallothioneins capable of chelating in vitro Zn, Cu and Cd. Some of these proteins are also able to confer Zn resistance when expressed in a sensitive yeast strain. In a second step, quantitative PCR methods for measuring expression levels of these genes in soil microcosms were developed. This will allow to evaluate the level of expression of these genes as a function of an increasing supply of exogenous metal. In a third step, we tried to obtain the genomic regions flanking these environmental genes in order to be able to associate the organisms from which they originate to a taxonomic group and to analyze the promoter regions of these genes using targeted capture

L’efficacité des traitements contre diverses pathologies dépend dans bien des cas de la précocité de la prise en charge des patients. Ce contexte pousse de nos jours les chercheurs à élaborer de nouvelles méthodes de diagnostic, généralement basées sur la détection de biomarqueurs spécifiques, permettant d’établir une corrélation entre un dérèglement moléculaire de l’organisme et la survenue d’une maladie. L’objectif de ces travaux était, par l’utilisation de la microfluidique digitale en gouttelettes, d’établir de nouvelles procédures simples et reproductibles, témoignant d’une sensibilité importante afin de déterminer d’infimes variations de l’état moléculaire de l’organisme à travers la recherche de biomarqueurs spécifiques. Pour cela nous avons élaboré une nouvelle gamme de tensioactifs fluorées adaptés aux applications biologiques en microfluidique digitale, ainsi que différentes stratégies d’étude des variations de l’expression de microARN extrait d’échantillons biologiques, basées respectivement sur les réactions induites par hybridation nucléotidique et sur la réaction de RT-PCR digitale
Efficiency of treatments for various diseases depends in many cases in precocity of patient management. Nowadays, this context urges researchers to develop new methods of diagnosis, generally based on the detection of specific biomarkers. These new methods allowing to establish correlations between physiological disorders and arisen of diseases states.The aim of this study was, by the use of droplet-based microfluidic, to work out a simple and reproducible procedure, with an increased sensitivity, to determine tiny variations of physiological state through the detection of specific biomarkers. Thus, we developed a new range of fluorinated surfactants fitted to biological applications in droplet-based microfluidics as well as various strategies to study variations of microRNA expressions in a biological sample. These methods, based on DNA-template reaction and digital PCR reaction, allows performing a substantial number of simultaneous reactions in micro-compartments (microdroplets) of picolitre volumes

L’ADN tumoral circulant (ADNtc) porte des altérations spécifiques de la tumeur des patients, qui sont détectables par un acte minimalement invasif. L’ADNtc représente donc un biomarqueur d’intérêt pour le suivi de l’évolution du cancer. Sa détection requière une technique hautement sensible et quantitative. Dans ce contexte, ce travail de thèse a porté sur la quantification et le suivi de l’ADNtc par PCR digitale en gouttelettes (PCRdg). Cet outil permet la détection d’altérations à l’échelle d’un ADN unique, offrant ainsi une sensibilité allant jusqu’à 0.001%. La détection de cet ADNtc a été réalisée par l’évaluation des biomarqueurs tels qu’une mutation spécifique de la tumeur, la fragmentation de l’ADNtc et l’hyperméthylation de séquences cibles. D’une part, nous avons observé que chez les patients atteints de cancer, l’ADN muté circulant est plus fragmenté que l’ADN non muté, et que cet ADN circulant de patients est globalement plus fragmenté que chez les sujets sains. D’autre part, une corrélation entre les pourcentages d’ADN muté et d’ADN hyperméthylé circulants a été observée au cours du suivi de patients. Ceci suggère la possibilité d’un suivi précis et quantitatif de l’ADNtc par l’évaluation de l’hyperméthylation en alternative à la détermination du statut mutationnel. Nous avons ensuite appliqué nos tests de détection de l’ADNtc dans le cadre de deux études cliniques. L’étude PLACOL, incluant 82 patients atteints de cancer colorectal métastatique, a permis de mettre en évidence deux facteurs pronostiques : un seuil de 0.1 ng/mL et la mesure de la pente de décroissance de la concentration en ADN muté ou hyperméthylé circulant. Dans la seconde étude, portant sur le mélanome métastatique dans le contexte d’une thérapie ciblée (vémurafenib), une corrélation inverse entre les concentrations d’ADNtc et de vémurafenib a été observée. Ces résultats suggèrent le potentiel clinique de l’ADNtc pour l’orientation thérapeutique des patients atteints de cancer avancé
Circulating tumor DNA (ctDNA) carries tumor-specific alterations that are detectable by minimally invasive sampling. It represents a highly pertinent marker for cancer monitoring during patients’ follow-up. CtDNA detection requires a highly sensitive and quantitative technique. In this context, this project focused on ctDNA quantification and monitoring by picoliter-droplet digital PCR. Thanks to the compartmentalization in millions of picoliter droplets, this tool allowed the detection of single DNA molecule with a sensitivity reaching 0.001%. Testing of ctDNA was performed through the evaluation of different potential biomarkers: specific mutations, ctDNA fragmentation, and hypermethylation of target sequences. On one hand, we observed in cancer patients that ctDNA is more fragmented than wild-type DNA, and, globally more fragmented than circulating DNA in healthy individuals. On the other hand, a strong correlation between percentages of hypermethylated and mutated DNA was observed during the follow-up of patients. Such results suggest the feasibility to precisely and quantitatively monitor ctDNA by the evaluation of hypermethylation as an alternative to the determination of mutational status. We have applied such ctDNA detection strategies in the context of two clinical studies. The PLACOL study, enrolling 82 metastatic colorectal cancer patients, allowed to highlight two prognostic factors: a ctDNA concentration threshold of 0.1 ng / mL, and the evaluation of ctDNA decreasing slope. In the second study, ctDNA was monitored in 11 melanoma patients in the context of a targeted therapy (vemurafenib). An inverse correlation between the concentrations of vemurafenib and ctDNA was demonstrated. These results suggest the clinical relevancy of ctDNA in advanced cancer patients, for the optimization of therapeutic management

Acute Lymphoblastic Leukemia (ALL) represents the most frequent cancer in childhood. Currently, more than 80% of children with ALL can be cured through intensive and risk-adapted chemotherapy protocols, but unfortunately, the remaining 20% ultimately relapse. Allogeneic hematopoietic stem cell transplantation (HSCT) is considered beneficial for approximately 10% of patients who are at high risk (HR) at frontline therapy according to the AIEOP-BFM protocol criteria, and for the majority of patients after ALL relapse. However, also after HSCT, relapse remains the leading cause of treatment failure in pediatric ALL. The strongest prognostic factor in childhood ALL is the monitoring of Minimal Residual Disease (MRD). MRD is defined as the persistence, in bone marrow (BM), of leukemic cells not identifiable through cyto-morphological methods. MRD diagnostics has been implemented into major frontline treatment protocols for pediatric ALL, in which it is routinely used to stratify patients into different risk classes: standard risk (SR), medium risk (MR) or high risk (HR) of relapse. The aim of MRD-based stratification is to refine therapy based on risk-class, maximizing cure and minimizing toxicities. Also for relapsed ALL patients and in patients undergoing HSCT, MRD assessment has been identified as one of the most relevant predictors of prognosis, useful to identify good and poor responders to the therapy. Nevertheless, the clinical significance of MRD in pediatric ALL patients given allogeneic HSCT has not yet been fully validated. The most widely used approach to detect MRD is represented by real-time quantitative PCR (RQ-PCR), a very sensitive and specific molecular assay. RQ-PCR is based on the patient-specific junctional regions of Immunoglobulin (Ig) and T-cell Receptor (TCR) genes rearrangements, detected on BM aspirates collected at diagnosis (or relapse) of ALL patient. In the first project of my PhD training (described in Chapter 1) we quantify MRD by RQ-PCR immediately before HSCT, in order to assess its clinical significance and impact on transplant outcome in a large cohort (119) of pediatric ALL patients in first, second or subsequent complete remission (respectively 1CR, 2CR or others CR). In addition, we consecutively analyzed MRD by RQ-PCR in 98/119 and 59/119 ALL patients, respectively during the first (post-HSCT1) and third (post-HSCT3) trimester after HSCT. The aim of these analyses was to address the question of whether MRD evaluation could provide further information to predict the risk of post-transplant leukemia recurrence. The overall 10-year event-free survival probability (EFSp) for patients with any level of positive MRD pre-HSCT was lower (39% for MRD < 1x10-3 and 18% for MRD ≥ 1x10-3) as compared with negative MRD patients (EFSp = 73%). When patients were analyzed according to the number of CR at HSCT, we observed that different levels of positivity had a different impact on EFSp: low-level MRD positivity had a negative impact only in patients transplanted in second or higher CR; while in first CR, only a high MRD positivity increased the risk of relapse. So pre-transplant MRD assessment confirmed to be a strong predictor of outcome and its effect was consistent throughout the different disease remissions. We also evaluated the EFSp according to the MRD assessment at post-HSCT1 and post-HSCT3. MRD negativity at early post-transplant was associated with a good EFSp (63%), that was even better when negativity was confirmed also at 3th trimester post-HSCT (pEFS = 84%). Also the variations of MRD levels over time were important. In particular the change between 1st and 3th trimester allowed to identify 2 categories of patients, with a dramatically different outcome: a group of patients with very poor prognosis (patients with an MRD increasing from post-HSCT1 to post-HSCT3) with an EFSp of only 8%, and a group of patients with very good prognosis (patients with unchanged negative MRD or decreasing to negative MRD and those with unchanged low-positive MRD) with an EFSp ≥ 80%. Overall, these results confirm that MRD assessment is important both before and after transplant, for early identification of patients with the highest risk of ALL recurrence and with a strong indication to a prompt immunological intervention and to adoption of new drugs. The second project (described in Chapter 2) was a preliminary study. We focused on a third generation PCR, the droplet digital PCR (ddPCR), that allows for an absolute quantification, with accurate concentration of target DNA. Instead, RQ-PCR allows for a relative quantification, since is based on the comparison with a calibration standard curve made with the diagnostic DNA of patient, for MRD level quantification in follow-up sample. Thus, availability of diagnostic sample can limit RQ-PCR assay. A broad spectrum of molecular markers has been yet interrogated using ddPCR for diagnostic purposes in various malignancies. Recently, the absolute method was evaluated for MRD quantification in lymphoproliferative disorders of adult, such as lymphomas and ALL; these reports showed a good correlation between quantitative PCR and ddPCR. However, there are still no studies in pediatric ALLs. In the light of this, we performed ddPCR analyses on BM samples of 65 pediatric ALL transplanted patients with the same primers and probes used for RQ-PCR and in the same reaction conditions. Comparing head-to-head the MRD results obtained with the two molecular approaches, we aimed to investigate the applicability of ddPCR for MRD assessment also in this context. First, we evaluated if positive but not-quantifiable (PNQ) MRD performed by RQ-PCR can be quantified by ddPCR; then we also evaluated the prognostic impact of pre-HSCT MRD levels assessed by ddPCR. A good level of concordance was found in results of both analyses (Pearson r = 0.98, P < 0.0001) and ddPCR was also able to quantify a various number of sample not-quantifiable by conventional RQ-PCR. Our results suggest that ddPCR has sensitivity, accuracy and reproducibility at least comparable with RQ-PCR. Statistical analyses have shown no significant differences in prognostic impact on outcome, if patients were stratified according to MRD levels detected by RQ-PCR and ddPCR, since EFSp of PNQ patients was very similar to that of MRD NEG by ddPCR (71% vs 68%, respectively). Despite this, the digital method was able to measure a positive and quantifiable value for 12 ALL patients who relapsed after HSCT, while RQ-PCR technique failed to identify relapse in advance. These preliminary data confirm that ddPCR may be an accurate and applicable tool for MRD evaluation also in the context of pediatric ALL clinical trials, but highlight the importance of extending the analysis on other retrospectively collected cases, to better define the role of ddPCR for prospective MRD evaluation in pediatric ALLs.
La Leucemia Linfoblastica Acuta (LLA) rappresenta la patologia tumorale più frequente in età pediatrica. Oltre l’80% dei bambini affetti da LLA viene trattato con successo grazie agli attuali protocolli di chemioterapia intensiva e basata sul rischio di ricaduta, ma sfortunatamente, il restante 20% ricade. Il trapianto di cellule staminali ematopoietiche (TCSE) ha un ruolo fondamentale nella guarigione di circa il 10% dei pazienti definiti ad alto rischio di ricaduta LLA in prima linea e per gran parte dei pazienti recidivati. Sfortunatamente, anche dopo il TCSE, la ricaduta si conferma come principale causa di fallimento terapeutico nelle LLA pediatriche. Il principale indicatore prognostico nelle LLA infantili è rappresentato dalla Malattia Residua Minima (MRM). La MRM è definita come la persistenza, all’interno del midollo osseo, di cellule leucemiche a livelli non identificabili attraverso esame citomorfologico. La valutazione della MRM è ormai parte integrante dei principali schemi terapeutici di prima linea, in cui viene usata per stratificare i pazienti in diverse classi di rischio di ricaduta (standard, intermedio o alto), con l'obiettivo di adattare la terapia al rischio individuale di ciascun paziente, ottimizzando le cure e riducendo al minimo la tossicità. Il monitoraggio della MRM è stato identificato come uno dei maggiori fattori predittivi di prognosi, anche per i pazienti ricaduti e per quelli trapiantati, in cui risulta ulteriormente vantaggioso per valutare la risposta alla terapia dei pazienti LLA. Ciononostante, il significato clinico della MRM nei pazienti sottoposti al TCSE non è ancora stato pienamente validato. L’approccio standard utilizzato per monitorare la MRM è attualmente rappresentato dalla real-time quantitative PCR (RQ-PCR), un saggio molecolare altamente sensibile e specifico, basato sulle regioni giunzionali dei riarrangiamenti dei geni delle immunoglobuline e del recettore dei linfociti T, identificati sugli aspirati midollari della diagnosi (o ricaduta) del paziente LLA. Nel primo progetto (descritto nel capitolo 1) del mio percorso di dottorato, abbiamo quantificato la MRM mediante PCR quantitativa immediatamente prima del TCSE, per valutare il suo significato clinico e l’impatto sull’outcome in una vasta coorte di pazienti pediatrici affetti da LLA (119), in prima remissione completa (1RC), seconda (2RC) o altre. Abbiamo poi analizzato MRM mediante RQ-PCR in 98/119 e 59/119 pazienti, rispettivamente durante il primo (post-TCSE1) e il terzo (post-TCSE3) trimestre dopo il trapianto. L’obiettivo di queste analisi è stato quello di capire se la valutazione MRM potesse fornire ulteriori informazioni, utili a identificare preventivamente i pazienti con maggior rischio di ricaduta leucemica dopo il trapianto. Dalle analisi di sopravvivenza in relazione ai livelli di MRM pre-TCSE nei pazienti LLA, qualsiasi livello di positività correla con un outcome sfavorevole (pEFS = 39% per MRM positiva < 1x10-3 e pEFS = 18% per MRM positiva ≥ 1x10-3), rispetto ai pazienti con MRM negativa (pEFS = 73%, P<0.0001). Inoltre, analizzando i pazienti in base al tipo di remissione al TCSE, livelli diversi di positività MRM correlano con un diverso impatto sulla pEFS: bassi livelli di positività MRM indicano infatti, una prognosi sfavorevole solo in pazienti trapiantati in seconda o altre RC, mentre in prima RC solo una positività alta si associa ad un aumentato rischio di ricaduta. Pertanto la MRM pre-TCSE si conferma come importante fattore predittivo di outcome e il suo effetto varia col variare del tipo di remissione al trapianto. È stata valutata, inoltre, la pEFS dei pazienti in base ai livelli di MRM post-TCSE1 e post-TCSE3; MRM negativa post-TCSE correla significativamente con un outcome favorevole, sia al 1° trimestre (pEFS = 63%), che ancor più se riscontrata al 3° trimestre (pEFS = 84%). Anche la valutazione prospettica del cambiamento di MRM è risultata significativa. In particolare, valutando la variazione di MRM dal 1° al 3° trimestre post-TCSE, i pazienti con MRM crescente hanno una prognosi sfavorevole (pEFS = 8%), mentre tutti gli altri gruppi correlano con una buona prognosi (pEFS ≥ 80%). Questi risultati confermano l’importanza del monitoraggio della MRM sia nel periodo precedente che successivo al TCSE, nell’identificare preventivamente pazienti ad alto rischio di ricaduta, possibili beneficiari di interventi immunologici preventivi. Il secondo progetto trattato (descritto nel capitolo 2) è stato uno studio preliminare, focalizzato su una PCR di terza generazione, la Droplet Digital PCR (ddPCR). Essa consente una quantifica di tipo assoluto, con un’accurata concentrazione del DNA target. La RQ-PCR fornisce, invece, una quantifica di tipo relativo, basata su una curva standard di calibrazione fatta con il DNA della diagnosi del paziente, per la quantificazione dei livelli di MRM di ciascun follow-up. Per cui, la PCR quantitativa può essere limitata dalla disponibilità di materiale diagnostico. Un ampio spettro di marcatori molecolari è già stato indagato mediante ddPCR per scopi diagnostici in varie patologie tumorali. Studi recenti hanno valutato l’applicabilità della ddPCR nell’ambito delle malattie linfoproliferative dell’adulto, come i linfomi e le LLA, mostrando una buona correlazione dei risultati fra le due metodiche in entrambi gli ambiti. Tuttavia, non sono ancora disponibili lavori che valutino questa correlazione nel campo delle leucemie pediatriche. Alla luce di questo, abbiamo eseguito analisi ddPCR sugli aspirati midollari di 65 pazienti pediatrici sottoposti a TCSE, utilizzando stessi primer e stesse sonde fluorescenti usati negli esperimenti RQ-PCR, nelle medesime condizioni di reazione. Mettendo a confronto i livelli di MRM emersi coi due approcci molecolari, si è investigata l’applicabilità della metodica assoluta per il monitoraggio della MRM anche in questo contesto. Inizialmente, sono stati valutati campioni risultati, mediante RQ-PCR, positivi ma non quantificabili (PNQ), per verificare se invece si potessero quantificare mediante ddPCR. Successivamente, è stato valutato anche l’impatto prognostico dei livelli MRM pre-TCSE ottenuti tramite ddPCR. Un buon livello di concordanza è emerso dai risultati ottenuti con entrambe le metodiche (Pearson r = 0.98, P < 0.0001); la ddPCR ha permesso, inoltre, di quantificare numerosi campioni risultati non quantificabili tramite RQ-PCR. I risultati suggeriscono che il metodo assoluto possieda sensibilità, accuratezza e riproducibilità almeno paragonabili alla PCR quantitativa convenzionale. I pazienti LLA analizzati sono stati stratificati sulla base dei livelli di MRD ottenuti con le due tecniche molecolari, ma nelle analisi di sopravvivenza non sono emerse differenze significative sulla prognosi. Infatti le pEFS dei pazienti con MRM negativa e positiva quantificabile per i due metodi risultano molto simili (rispettivamente 71% e 68%). Ciononostante, dal presente studio è emerso che col metodo digital sia stato possibile misurare un valore di MRM positivo e quantificabile per almeno 12 pazienti LLA che, in seguito al trapianto, hanno presentato una recidiva; viceversa, la RQ-PCR non era stata in grado di identificare anticipatamente la ricaduta di questi pazienti. Questi dati preliminari mostrano che la ddPCR possa essere un valido strumento per il monitoraggio della MRM e applicabile anche nel contesto dei trials clinici per pazienti LLA pediatrici. Tuttavia una prosecuzione dello studio ddPCR, con estensione della casistica analizzata, potrebbe essere utile a definire con precisione la significatività delle misurazioni con questa recente metodica.

This study has provided further understanding of the pathogenesis of EV71, one of the major etiological agents associated with significant mortality in Hand, Foot and Mouth disease. Elucidating the host-pathogen interaction and the mechanism that the virus uses to bypass host defence systems to establish infection will aid in the development of potential antiviral therapeutics against EV71.

Background: Currently, there is a lack of non-invasive tumour biomarkers with appropriate sensitivity and specificity to be used in routine clinical testing. The use of circulating microRNAs (miRNAs) as cancer biomarkers has been hypothesised based on their presence and stability in the circulation. Promising initial results for these tiny RNAs has been obtained in the field of breast cancer diagnostics. However, the accurate quantification of circulating miRNAs is more challenging than expected. In particular, several pre- and analytical variables have an impact on their final quantification, including the quantification method. Recently, a new droplet digital PCR (ddPCR) system that can be also used for microRNA quantification has been developed and proved to be very promising in liquid biopsy applications. Experimental design and findings: In order to develop a precise and accurate technique for miRNA quantification, we tested and compared the feasibility of quantifying circulating miRNAs with the new BioRad ddPCR system when used with EvaGreen dye– and TaqMan probe–based assays. In plasma and serum of patients with cancer and healthy controls, two circulating miRNAs and one added exogenous miRNA were quantified with both EvaGreen dye–based miRCURY LNA miRNA assays and TaqMan assays. The EvaGreen-based assay was precise, reproducible and sensitive. In comparison with TaqMan assays, high concordance was obtained for two endogenous miRNAs in serum and plasma. EvaGreen dye–based and TaqMan probe–based assays can be equally used with the ddPCR system to quantify circulating miRNAs. Afterwards, we selected a panel of six miRNAs (miR-10b-5p, miR-145-5p, miR-181a-5p, miR-148b-3p, miR-425-5p, miR-6523p) derived from microarray experiments or described in literature as potential circulating biomarkers for breast cancer. Then, we assessed their absolute levels in two independent cohorts of breast cancer patients and disease-free controls (Italy; n = 56, and USA; n = 94) using EvaGreen-based ddPCR. MiR-148b-3p, miR-181a-5p and miR-652-3p levels were significantly lower in the serum of breast cancer patients than in controls in both cohorts. For these three miRNAs, the stratification of breast cancer patients versus controls was confirmed by receiver operating characteristic curve analysis. Higher levels of serum miR-10b-5p were associated with clinico-pathological features of poor prognosis. These results confirmed the significant discrimination between breast cancer patients and healthy controls and the direction of down regulation. Conclusion: This study establishes the basis for using on a ddPCR for quantifying circulating miRNA biomarkers. Both study cohorts revealed very good agreement in terms of comparable absolute miRNA concentrations and consistent trends of dysregulation in breast cancer patients. This study finally powers the use of circulating miRNAs as cancer biomarkers and proposes miR181a-5p and miR-652-3p as diagnostic biomarker and miR10b-5p as prognostic biomarkers of breast cancer.
Stato dell’arte: L’assenza di marcatori tumorali non invasivi e con appropriata sensibilità e specificità per un uso clinico, rappresenta un problema fondamentale in ambito oncologico. L'utilizzo di microRNA (miRNA) circolanti come biomarker tumorali è stata ipotizzata sulla base della loro presenza e stabilità nel sangue e in altri fluidi biologici. Promettenti risultati preliminari sono stati ottenuti dall’utilizzo di questi piccoli RNA come biomarcatori del cancro al seno. Tuttavia è risultato fin da subito evidente che la quantificazione accurata dei miRNA circolanti è un processo molto complesso e influenzato da molteplici fattori. In particolare non esiste un accordo su quale sia il metodo di quantificazione migliore. E’ stato recentemente sviluppato un nuovo sistema di quantificazione di acidi nucleici chiamato Droplet Digital PCR (ddPCR), ma non era ancora stato testato per la quantificazione di miRNA circolanti. Disegno sperimentale e risultati: Al fine di sviluppare una tecnica precisa ed accurata per la quantificazione dei miRNA circolanti, abbiamo testato l’affidabilità del nuovo sistema ddPCR di BioRad (QX200) e confrontato i risultati della quantificazione dei miRNA circolanti con due chimiche diverse, una basata sull’intercalante EvaGreen e una basata su sonde TaqMan. Nel plasma e siero dei pazienti con cancro e controlli sani, due miRNA circolanti e un miRNA aggiunto esogenamente sono stati quantificati con saggi per miRNA basati su primer a LNA (Exiqon) combinati con EvaGreen o su sonde TaqMan (Applied Biosystem). Entrambi i saggi si sono dimostrati precisi, riproducibili e sensibili. La concordanza tra i dati di quantificazione ottenuti con le due metodiche è risultata essere molto buona. Abbiamo pertanto concluso che sia saggi basati su EvaGreen che sull’uso di sonde TaqMan possono essere ugualmente utilizzati con il sistema ddPCR per quantificare i miRNA circolanti. In seguito, abbiamo selezionato un gruppo di sei miRNA (miR-10b-5p, miR-145-5p, miR-181a-5p, miR-148b-3p, miR-425-5p, miR-652-3p) derivati da esperimenti microarray o descritti in letteratura come potenziali biomarker circolanti per il cancro al seno. Abbiamo quindi valutato i loro livelli assoluti (copie/µl) nel siero in due coorti indipendenti di pazienti con carcinoma mammario e controlli sani (Italia; n = 56, and USA; n = 94) utilizzando saggi ddPCR basati su primer a LNA e sistema EvaGreen. MiR-148b-3p, miR-181a-5p e miR-652-3p sono risultati significativamente più bassi nel siero dei pazienti con cancro al seno rispetto ai controlli in entrambe le coorti. Per questi tre miRNA la stratificazione dei pazienti con carcinoma mammario rispetto ai controlli è stata confermata tramite l’analisi di curve ROC. Inoltre, livelli sierici più elevati di miR-10b-5p sono stati associati con alcune caratteristiche clinico-biologiche a prognosi negativa degli stessi campioni. Conclusione: Questo studio stabilisce la base per l'utilizzo di test basati su ddPCR per la quantificazione di miRNA circolanti quali biomarcatori di tumore al seno. Entrambe le coorti studiate hanno rivelato un ottimo accordo in termini di concentrazioni assolute miRNA e tendenze coerenti di disregolazione in pazienti con cancro al seno rispetto ai controlli. Questo studio suggerisce pertanto l'uso di miRNA come biomarcatori tumorali circolanti e propone miR-181a-5p e miR-652-3p come biomarcatori diagnostici e miR10b-5p come biomarcatore prognostico del tumore al seno.

Nous avons pour but de développer un système microfluidique en gouttes, capable, à l’échelle de la cellule/bactérie unique, de détecter et de co-localiser plusieurs marqueurs génétiques, en utilisant une version digitale et multiplexée de la réaction de polymérisation en chaîne (PCR). Les systèmes de PCR digitale actuellement commercialisés ne le permettent toujours pas. Un tel prototype garantira la présence de multiples marqueurs à l’intérieur d’un même génome, ce qui permettra l’identification du pathogène avec précision et un taux de faux-positifs proche de zéro. Comme première application, nous démontrerons la possibilité de co-localiser quatre gènes de virulence de la souche O157:H7 d’Escherichia coli, un pathogène majeur, qui est détecté dans des échantillons alimentaires ou provenant de fèces cliniques pouvant aussi contenir des E. coli non pathogènes porteuses d’une partie des gènes de virulence. Avant de procéder à des tests TaqMan multicolores en point final, E. coli sera d’abord encapsulée dans des gouttes micrométriques et lysée par la chaleur in situ. Notre objectif est de démontrer que ce test peut être appliqué avec succès à un petit ensemble d’échantillons cliniques ou alimentaires
We aim to develop a prototype of droplet-based microfluidic system capable of detecting and colocalizing multiple genetic markers at the single cell/bacteria level, using the Polymerase Chain Reaction (PCR) in a digital multiplexed version. This cannot be achieved using current commercial digital PCR systems, and should increase the sensitivity and reliability of the detection of pathogens. Importantly, the system will guarantee the presence of multiple markers within the same genome and enable accurate identification, and bring the false positive rate close to zero. As a first application, we will demonstrate the possibility to co-localize 3 virulence genes in the E.coli strain O157:H7, a major foodborne pathogen, which has to be detected in clinical feces samples or food samples, which may also contain non pathogenic E. coli carrying only a subset of these virulence genes. E. Coli will be encapsulated in micrometric droplets, lysed by heating in situ prior performing a multicolour end-point Taqman assay. Our objective is to demonstrate that this test can be successfully applied to real clinical or food samples

Dans le contexte de la médecine régénérative cutanée, l’amplification massive en culture de précurseurs kératinocytaires est souvent requise mais elle peut s’accompagner d’une induction de la différenciation conduisant à une perte de potentiel régénératif. L’expansion ex vivo de précurseurs fonctionnels nécessite de contrôler deux processus : d’une part l’activation de la prolifération et d’autre part la préservation de l’immaturité. Mon laboratoire d’accueil a identifié les gènes KLF4 et MXD4 comme candidats potentiels dans le contrôle de ces processus. Mon travail de thèse a porté sur l’étude du rôle de KLF4 et MXD4 dans le contrôle des balances « quiescence/prolifération » et « immaturité/différenciation » au sein des précurseurs kératinocytaires de la peau humaine.Nous montrons que la répression de KLF4 comme celle de MXD4 inhibe la différenciation kératinocytaire et augmente la prolifération et la clonogénicité des précurseurs, aboutissant à un potentiel régénératif accru dans des modèles de reconstruction épidermique in vitro et pour KLF4 de xénogreffes in vivo. Les mécanismes des effets liés à KLF4 passent notamment par l’inhibition de la voie du TGFB1 et par la voie Wnt/β-caténine. Les effets liés à MXD4 sont contrôlés par la quantité de dimères c-MYC/MAX et MAD4/MAX, et impactent la voie TGF-β. Malgré des mécanismes partiellement similaires, l’intervention d’effecteurs épigénétiques non codants pourrait conférer des spécificités à chacun de ces deux gènes : des ARN long non-codants, qui pourraient réguler le caractère immature des précurseurs kératinocytaires, ont été identifiés.Notre concept a été démontré initialement en utilisant des outils de recherche, et notamment des inhibitions stables de l’expression de KLF4 et MXD4. Nous montrons également qu’une inhibition transitoire, donc applicable à un contexte clinique, est également fonctionnelle, en utilisant l’ARN interférence ou une inhibition moléculaire. La répression par la kenpaullone pourrait notamment constituer un outil prometteur pour promouvoir l’expansion de précurseurs épithéliaux fonctionnels
In the context of cutaneous regenerative medicine, massive amplification of keratinocyte precursor cells is usually required but can be accompanied with induction of differentiation, resulting in a loss of regenerative potential. Ex vivo expansion of functional precursors needs to control two processes : to activate proliferation in one hand, and to preserves immaturity in the other hand. My host laboratory has identified KLF4 and MXD4 genes as candidates that might control these processes. My thesis was focused on studying KLF4 and MXD4 roles in controlling the balance “quiescence/proliferation” and “immaturity/differentiation” of human skin keratinocytes precursors.We have shown that repression of KLF4 and MXD4 inhibits keratinocyte differentiation and increases proliferation and clonogenicity of precursors, resulting in enhanced regenerative potential in in vitro epidermis reconstruction assay, and for KLF4, in in vivo xenografts assays. Mechanisms of the effects linked to KLF4 include inhibition of TGFB1 pathway and Wnt/β-catenin pathway. Effects linked to MXD4 are controlled by the respective quantity of c-MYC/MAX and MAD4/MAX heterodimers, and also impact TGF-β pathway. Despite partially similar mechanisms, intervention of non-coding epigenetic factors might confer the specific regulation to each of these genes : long non-coding RNA, that might regulate immature character of keratinocytes precursor cells, have been identified.Our concept has been initially demonstrated using research tools, in particular stable repression of KLF4 and MXD4. We then show that a transient inhibition, applicable to a clinical context, is also functional, by using RNA interference or molecular inhibition. KLF4 and MXD4 transient repression by kenpaullone might constitutes a promising molecular tool to promotes ex vivo expansion of functional epithelial precursor cells

Dissertação de Mestrado em Segurança Alimentar apresentada à Faculdade de Farmácia
A verificação da autenticidade e da rotulagem alimentar fazem parte de um conjunto de procedimentos base levados a cabo pelas agências de controlo alimentar de cada país para assegurar um comércio justo, seguro e confiável. Com o objetivo de salvaguardar os interesses dos consumidores, entidades oficiais bem como laboratórios acreditados atestam a autenticidade, qualidade e conformidade no âmbito da rotulagem, efetuando para isso a identificação e, se for o caso, determinação da proporção em que os ingredientes se encontram num produto alimentar. Os métodos levados a cabo para atingir este propósito são fundamentalmente baseados na quantificação de DNA ou de proteínas. Neste trabalho foi aplicada a técnica de droplet digital PCR, técnica emergente em biologia molecular para a quantificação de suíno e bovino em amostras alimentares. A técnica de droplet digital PCR permite a deteção e quantificação absoluta do número de cópias de um determinado gene alvo, representando uma evolução significativa comparativamente com a técnica de PCR em Tempo-Real (método de referência para quantificação de espécies em amostras alimentares). Os resultados obtidos neste trabalho permitiram estabelecer métodos de quantificação para as espécies de bovino e suíno e demonstrar a adequabilidade de duas estratégias distintas para a quantificação destas espécies em análises de rotina de amostras de produtos cárneos. O combate à fraude alimentar e mais concretamente às adulterações nos produtos cárneos e ao incumprimento das regras gerais da rotulagem pode beneficiar das vantagens de quantificação absoluta de DNA da técnica de PCR Digital.
Verification of food authenticity and food labeling are part of a set of basic procedures carried out by the food control agencies of each country to ensure fair, secure and reliable trade.In order to safeguard the interests of consumers, official bodies as well as accredited laboratories attest the authenticity, quality and conformity in the labeling, by identifying and, where appropriate, determining the proportion of the ingredients in a food product. The methods carried out to achieve this purpose are fundamentally based on the quantification of DNA or proteins.In this work the droplet digital PCR technique, emerging technique in molecular biology, was applied for the quantification of swine and bovine in food samples. The droplet digital PCR technique allows the detection and absolute quantification of the number of copies of a given target gene, representing a significant evolution compared to the Real Time PCR technique (the current reference method for quantification of species in food samples). The results obtained in this work allowed the establishment of quantification methods for bovine and porcine species and demonstrate the suitability of two distinct strategies for the quantification of these species in routine analyzes of meat product samples. The fight against food fraud and more specifically adulteration of meat products and failure to comply with the general rules of labeling could benefit greatly from the absolute DNA quantification by the Digital PCR technique.

碩士
國立臺灣海洋大學
系統工程暨造船學系
105
In this paper, we report our study on developing an optical quantification method based on the imaging system of a smartphone for the application of digital PCR biochip. The system can automatically calibrate the difference effected by the light source and counts the number of positive wells by using the developed algorithm. Furthermore, to enhance the performance, this method can distinguish three different signals with difference colors, they are red, green, blue. To achieve this goal, the developed algorithm provided the capability to perform color calibration and spots counting. In the part of color calibration, a calibration transformation matrix was created by using a standard color card with red, green, blue patchs. Once color calibration was completed, information from three different color channels was converted into grayscale image separately. The top-hat transformation was used to perform background correction. Then, Otsu’s method was applied to identify target spots. The morphological operation was used to trim the edge of spots. Finally, the number of spots are counted and reported. Our experimental studies demonstrated that a simulated image of 3240 spots under a background interference can be identified. Furthermore, this computation method was also verified by using microfluidic droplet array.

For HIV-infected patients undergoing antiretroviral treatment (ART), the latent reservoir is a major barrier to cure. Formed in the acute stages of infection, the latent reservoir consists of cells in the host’s body where HIV can hide from the immune system and remain in a quiescent state. When a patient stops ART, the virus quickly rebounds, proliferates, and begins infecting more cells. The majority of assays available to measure the latent HIV reservoir measure HIV DNA and RNA using polymerase chain reaction (PCR). However, these methods can overestimate the reservoir size, because unintegrated and replication-incompetent DNA comprise a larger proportion of HIV nucleic acids than does replication competent HIV provirus. To measure integrated HIV DNA from infected donor samples provided by the HIV Reservoir Assay Validation and Evaluation Network (RAVEN) and The University of Texas Health Sciences Center at Houston (UTHealth), I applied pulsed-field gel electrophoresis (PFGE) to reduce the amount of unintegrated HIV DNA such as episomal 2-long-terminal-repeat (2-LTR) circles before quantitation with droplet digital PCR (ddPCR). This assay could prove useful to measure changes in the latent HIV reservoir, especially in clinical trials that aim to reduce its size through a variety of compounds.

Meine Untersuchungen zeigen, dass es sich bei der zellfreien DNA (cfDNA, engl. cell-free DNA) des Spenderorgans (GcfDNA, engl. graft-derived cell-free DNA) um einen klinisch vielversprechenden Biomarker zur direkten Ermittlung der Organschädigung im Sinne einer „flüssigen Biopsie“ handelt. Alles was dafür notwendig ist, ist eine Blutprobe des Empfängerpatienten. Im Gegensatz zu konventionellen Markern wird die Organschädigung unmittelbar, direkt und hochspezifisch angezeigt. Durch neue Entwicklungen in der Labordiagnostik lässt sich dieser Marker im routinemäßigen Einsatz mittels digital droplet PCR (ddPCR) bestimmen. Die Analyseergebnisse können bei verhältnismäßig niedrigen Kosten innerhalb eines Arbeitstages erstellt werden. Unmittelbar nach Transplantation ist bei allen Patienten eine sehr hohe Konzentration der GcfDNA messbar. Innerhalb von wenigen Tagen fallen die Werte schnell ab und erreichen die Größenordnung stabiler Transplantatempfänger, die in der Lebertransplantation unter 10% liegen. Dabei besteht keine signifikante Korrelation der initialen GcfDNA-Freisetzung mit der Ischämieschädigung des Spenderorgans, ermittelt durch die Dauer der kalten Ischämiezeit (WIZ). Bei unzureichender Immunsuppression ist eine erhöhte GcfDNA-Freisetzung zu beobachten. Mithilfe der GcfDNA als Marker der Organintegrität lässt sich auch der gemeinsame Effekt verschiedener Immunsuppressiva ermitteln. Die GcfDNA verhält sich dabei umgekehrt proportional zur immunsuppressiven Therapie. Patienten mit akuten Abstoßungen haben im Mittel GcfDNA-Werte oberhalb von 50%. Die GcfDNA-Werte sind bereits mehrere Tage vor einer klinisch manifestierten akuten Abstoßung erhöht. Auch eine virusassoziierte Transplantatschädigung durch Hepatitis C manifestiert sich in vergleichsweise höheren GcfDNA-Werten. Cholestasen gehen hingegen nicht mit erhöhten GcfDNA-Werten einher. Die immunsuppressive Therapie könnte sich durch den routinemäßigen Einsatz der GcfDNA sicherer, einfacher, zuverlässiger und individueller gestalten lassen. Unter-Immunsuppressionen und daraus resultierende Abstoßungen würden sich bereits in der subklinischen Phase erkennen lassen und die Therapie von der bloßen Reaktion auf klinische Ereignisse hin zur Prävention verschieben. Um das Ziel einer personalisierten Medizin zu erreichen, könnte die Immunsuppression für jeden Patienten auf das absolut notwendige und damit gegenüber der bisherigen Praxis optimale Maß festgelegt werden. Geringere Nebenwirkungen und eine Reduktion der Kosten für das Gesundheitswesen wären die Konsequenz. Dieser Marker könnte dazu beitragen, das finale Ziel, nämlich eine Verbesserung des Langzeiterfolges nach Organtransplantationen, zu erreichen. Multizentrische Studien zur Validierung dieses Markers vor dem routinemäßigen Einsatz laufen bereits.

Les travailleurs impliqués dans la gestion des matières résiduelles domestiques sont continuellement exposés à des bioaérosols pouvant entrainer des problèmes de santé de nature infectieux, allergène et cancérigène. Le but de cette étude était d’évaluer l’exposition aux bioaérosols des éboueurs et des chauffeurs de camion durant la collecte des déchets et des matières résiduelles à Montréal par une analyse multimétrique et multimédia. Les conditions de travail ont été évalués dans six camions (2 recyclages, 2 organiques et 2 domestiques). Pour chaque camion, des mesures ambiantes (cabine du chauffeur) et des mesures dans la zone respiratoire d’un collecteur et d’un chauffeur ont été réalisées. Des prélèvements sur la surface des sièges des chauffeurs ont aussi été effectués et les filtres à air de l’habitacle des camions ont été évalués. Les échantillons ont été analysés au laboratoire microbiologique de l’IRSST selon la méthode par culture microbienne et la méthode moléculaire Droplet Digital PCR. Les résultats indiquent que les collecteurs sont généralement les plus exposés aux bioaérosols. Les collecteurs des déchets domestiques étaient notamment les plus exposés avec des concentrations moyennes pour les bactéries (27 000 UFC/m3), les endotoxines (100 UE/m3) et les moisissures (5 900 UFC/m3) excédant les recommandations sanitaires. Néanmoins, les collecteurs du compost étaient les plus exposés aux moisissures (6 800 UFC/m3). E.coli et A.fumigatus ont été détectés dans tous nos échantillons avec des expositions plus importantes pour les travailleurs du domestique. De plus, les sièges des chauffeurs des camions domestiques avaient les niveaux de contamination les plus élevés. La concentration moyenne de A.fumigatus (2 500 UG/m3) dans l’air de ces camions était supérieure à celle des camions de recyclage et de compost. Les résultats des filtres n’ont pas permis de tirer de conclusions quant à l’exposition des chauffeurs aux bioaérosols. Les résultats de cette recherche ont permis de mettre en évidence que l’exposition des travailleurs lors de la collecte des déchets pourrait être influencée par le type de déchets, le poste de travail ainsi que les tâches exécutées. Cette étude confirme le potentiel élevé d’exposition aux bioaérosols des travailleurs attitrés au transport des matières résiduelles et des déchets. Elle valide aussi le besoin de réduire les expositions professionnelles par diverses stratégies, y compris les procédures de nettoyage de la cabine et l’utilisation d’équipement de protection respiratoire lors de tâches génératrices de bioaérosols (nettoyage des camions au jet, déchargement des camions).
Workers involved in the management of household residual materials are continuously exposed to bioaerosols which can cause them health problems of an infectious, allergenic and carcinogenic nature. The purpose of this study was to assess the exposure to bioaerosols of collectors and trucks drivers during the collection of domestic waste and residual materials in Montreal by a multimeric and multimedia analysis. A sampling campaign was conducted on six drivers and six collectors. A total of 6 trucks that collect recyclable (2), organic (2) and compost (2) waste were evaluated. Samples were also taken from the surface of the drivers' seats, and the cabin air filters were recovered. Samples were analyzed at the microbiology laboratory of the IRSST using the microbial culture method and the molecular method of Droplet Digital PCR (ddPCR). The results indicate that collectors are generally the most exposed to bioaerosols. Domestic waste collectors were in particular the most exposed with average concentrations for bacteria (27,000 CFU/m3), endotoxins (100 EU/m3) and fungi (5,900 CFU/m3) exceeding health recommendations. However, the compost collectors were the most exposed to fungi (6,800 CFU/m3). E. coli and A. fumigatus were detected in all of our samples, but exposures were greater for workers collecting domestic waste. In addition, the seats of drivers involved in domestic waste collection had the highest levels of contamination. The average concentration of A. fumigatus (2,500 UG/m3) in the air of the cabin of domestic waste trucks was higher than that of recycling and compost trucks. The results of the filters did not allow us to draw any conclusions on their role in terms of drivers exposure to bioaerosols. The results of this research highlighted that workers exposure to bioaerosols could be influenced by factors such as the type of waste, the workplace and workers’ tasks performed. This study confirms the high potential for exposure to bioaerosols of workers assigned to the transport of household residual materials and waste. It also validates the need to reduce workers exposures by various strategies including, cabin cleaning procedures and the use of respiratory protection equipment for specific tasks generating bioaerosols (water jet cleaning of trucks, unloading of trucks).

This work presents the results ofour research of cell-free nucleic acids (cfNA). The first part shows changes in methylation patterns of immune response genes promoters that are detectable in plasma during the hemodialysis sessions and also differences in methylation between patients and healthy subjects. Alterations include genes that play their role in the regulation of hematopoiesis and these changes are in close relation with the need of anemia therapy. In the other plasma cfNA study we detected miRNA signatures in patients with acute myeloid leukemia at diagnosis (6 highly abundant miRNAs found) and in remission achieved after standard chemotherapy (trend to n01malization, lower levels ofthese miRNAs). Another part of work presents data from the study of potential non-invasive biomarker of bladder cancer. The amounts of cfDNA in urine are higher in patients than in healthy subjects and there were found 5 down-regulated miRNAs. Simultaneously it was established set of 30 miRNAs that are constantly present in urine supematants independently on sex, age and healthy status of subjects. The last part presents analysis ofcell-free fetal DNA. We analyzed differences between a new quantification method - droplet digital PCR and real-time PCR which is used routinely nowadays. Slightly more precise was...