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.

A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 muL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10min. Following extraction, the 1500bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10min for 30cycles. The total assay time was 23min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 muL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

Philosophiae Doctor - PhD / Sequencing an HIV quasispecies with next generation sequencing technologies yields a dataset with significant amplification bias and errors resulting from both the PCR and sequencing steps. Both the amplification bias and sequencing error can be reduced by labelling each cDNA (generated during the reverse transcription of the viral RNA to DNA prior to PCR) with a random sequence tag called a Primer ID (PID). Processing PID data requires additional computational steps, presenting a barrier to the uptake of this method. MotifBinner is an R package designed to handle PID data with a focus on resolving potential problems in the dataset. MotifBinner groups sequences into bins by their PID tags, identifies and removes false unique bins, produced from sequencing errors in the PID tags, as well as removing outlier sequences from within a bin. MotifBinner produces a consensus sequence for each bin, as well as a detailed report for the dataset, detailing the number of sequences per bin, the number of outlying sequences per bin, rates of chimerism, the number of degenerate letters in the final consensus sequences and the most divergent consensus sequences (potential contaminants). We characterized the ability of the PID approach to reduce the effect of sequencing error, to detect minority variants in viral quasispecies and to reduce the rates of PCR induced recombination. We produced reference samples with known variants at known frequencies to study the effectiveness of increasing PCR elongation time, decreasing the number of PCR cycles, and sample partitioning, by means of dPCR (droplet PCR), on PCR induced recombination. After sequencing these artificial samples with the PID approach, each consensus sequence was compared to the known variants. There are complex relationships between the sample preparation protocol and the characteristics of the resulting dataset. We produce a set of recommendations that can be used to inform sample preparation that is the most useful the particular study. The AMP trial infuses HIV-negative patients with the VRC01 antibody and monitors for HIV infections. Accurately timing the infection event and reconstructing the founder viruses of these infections are critical for relating infection risk to antibody titer and homology between the founder virus and antibody binding sites. Dr. Paul Edlefsen at the Fred Hutch Cancer Research Institute developed a pipeline that performs infection timing and founder reconstruction. Here, we document a portion of the pipeline, produce detailed tests for that portion of the pipeline and investigate the robustness of some of the tools used in the pipeline to violations of their assumptions.

Neisseria meningitidis är en bakterie som kan leda till invasiv sjukdom eller endast orsaka bärarskap i nasopharynx. Bakterien delas in i olika serogrupper och klonala komplex. Vissa av dessa grupper och klonala komplex förekommer endast hos invasiva isolat och andra bland bärare, vilket tyder på att det finns genetiska skillnader mellan invasiva och bärarisolat. I denna studie undersöktes genen pilE som kodar för PilE proteinet och ingår i bakteriens pili. Proteinet finns i två klasser, klass 1 och klass 2. Metoden som användes för att studera eventuella skillnader i förekomst och uttryck av pilE genen var digital droplet PCR (ddPCR). Både DNA och RNA kvantifierades med ddPCR för att undersöka antalet kopior av pilE genen (DNA) samt dess uttryck (RNA) mellan invasiva isolat och bärarisolat, mellan klass 1 pilE isolat och klass 2 samt fördelning av klasserna i isolattyperna. Principen för ddPCR är att dela ett prov till tiotusentals nanodroppar där individuella droppar genomgår PCR för en senare avläsning av flourescens från prober. Resultatet visade skillnad mellan bärarisolat och invasiva isolat där de invasiva isolaten visade mindre uttryck av pilE än bärarisolat. Klass 2 isolat hade signifikant färre genuttryck än klass 1 isolat och invasiva isolat visade klass 2 i högre utsträckning än bärarisolat. / Neisseria meningitidis is a bacterium that can cause invasive disease or carriage in the nasopharynx. The bacteria are divided into different serogroups and clonal complexes. Specific serogroups and clonal complexes are more frequent or are only found among invasive isolates or in carriage isolates. This study has investigated pilE, a gene that encodes the PilE protein located in the bacteria´s pilin and the protein is found in either class 1 or class 2. digital droplet PCR was used to investigate differences in presence and expression of the gene pilE. Both DNA and RNA was quantified to study the difference in copy number of the pilE gene (DNA) and its expression (RNA) between invasive isolates and carriage isolates but also between class 1 isolates and class 2 isolates. The distribution of classes between the isolate types was also investigated. The ddPCR method divides a sample into thousands of nanodroplets and a PCR reaction occurs in each droplet followed by droplet reading where fluoroscens from probes is measured. The difference that could be seen was that the invasive isolates expressed pilE in lower copies. Class 2 isolates had a significantly lower gene expression than class 1 isolates and invasive isolates expressed class 2 in a higher frequency.

This work introduces technology for rapid pathogen detection using handheld optical immunoassay and wire-guided droplet PCR systems. There have been a number of cases of foodborne or waterborne illness among humans that are caused by pathogens such as Escherichia coli O157:H7, Salmonella typhimurium, Influenza A H1N1, and the norovirus. The current practices to detect such pathogenic agents are: cell/viral culturing, immunoassays, or polymerase chain reactions (PCRs). These methods are essentially laboratory-based methods that are not at all real-time and thus unavailable for early-monitoring of such pathogens. They are also very difficult to be implemented in field, preventing early detection opportunities. This dissertation is divided into three papers that present methodologies towards the expeditious detections of infectious pathogens and the miniaturization of these detection systems towards field-deployable and point-of-care applications. Specifically, the work presented focuses on two methodologies: (1) light scatter detection using immunoagglutination assays with optimized Mie light scatter parameters in a real biological matrix consisting of plant tissue, and (2) wire-guided droplet manipulations for rapid and improved sample analysis, preparation, and PCR thermocycling. Both of these methods carry a collective objective towards providing high impact technologies for addressing the issues of food-related outbreaks and overall public safety. In the first paper, the direct and sensitive detection of foodborne pathogens from fresh produce samples was accomplished using a handheld lab-on-a-chip device, requiring little to no sample processing and enrichment steps for a near-real-time detection and truly field-deployable device. The detection of Escherichia coli K12 and O157:H7 in iceberg lettuce was achieved utilizing optimized Mie light scatter parameters with a latex particle immunoagglutination assay. The system exhibited good sensitivity, with a limit of detection of 10 CFU mL⁻¹ and an assay time of <6 min. Minimal pretreatment with no detrimental effects on assay sensitivity and reproducibility was accomplished with a simple and cost-effective KimWipes filter and disposable syringe. Mie simulations were used to determine the optimal parameters (particle size d, wavelength λ, and scatter angle θ) for the assay that maximize light scatter intensity of agglutinated latex microparticles and minimize light scatter intensity of the tissue fragments of iceberg lettuce, which were experimentally validated. This introduces a powerful method for detecting foodborne pathogens in fresh produce and other potential sample matrices. The integration of a multi-channel microfluidic chip allowed for differential detection of the agglutinated particles in the presence of the antigen, revealing a true field-deployable detection system with decreased assay time and improved robustness over comparable benchtop systems. In the second paper, we demonstrate a novel method of wire-guided droplet manipulations towards very quick RT-PCR. Because typical RT-PCR assays take about 1–2 h for thermocycling, there is a growing need to further speed up the thermocycling to less than 30 min. Additionally, the PCR assay system should be made portable as a point- of-care detection tool. Rapid movements of droplets (immersed in oil) over three different temperature zones make very quick PCR possible, as heating/cooling will be made by convective heat transfer, whose heat transfer coefficients are much higher than that of conduction, the latter of which is used in most conventional PCR systems. A 30-cycle PCR of a 160 bp gene sequence amplified from 2009 H1N1 influenza A (human origin) was successfully demonstrates in 6 min and 50 sec for a very large 10 μL droplet (with additional 4 min for reverse transcription). The proposed system has a potential to become a rapid, portable, point-of-care tool for detecting influenza A. In the third paper, a wire-guided CNC apparatus was used to perform droplet centrifugation, DNA extraction, and VQ-PCR thermocycling on a single superhydrophobic surface measuring 25 mm by 55 mm and a multi-chambered PCB heater. This methodology exhibited no limitations on the complexity and configuration of procedures that it can perform, making it versatile and far-reaching in its applications. The only modification required for adding or implementing changes for a new protocol is through simple pre-defined programming. The highly adaptive and flexible system was used to execute easily pre-programmed droplet movements and manipulations for the rapid detection of Escherichia coli from PCR detection. Serial dilutions were performed to simulate a diluted field sample with a high level of accuracy. Centrifugation of the diluted sample containing E. coli demonstrated a novel approach to sample pre-treatment. Furthermore, the extraction of DNA from the sample droplet containing E. coli was also performed on the same superhydrophobic surface as the previous 2 steps, requiring less than 10 min. Following extraction, the genetic material was amplified using wire-guided droplet PCR thermocycling, successfully completing 30 cycles of Peptidase D (a long 1500 bp sequence) in 10 min. The droplet centrifugation process was determined to greatly improve the positive band intensity over the non-centrifuged sample. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of accuracy and automation.

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.

Droplet microfluidics is an emerging technology for analysis of single cells and biomolecules at high throughput. The controlled encapsulation of particles along with the surrounding microenvironment in discrete droplets, which acts as miniaturized reaction vessels, allows millions of particles to be screened in parallel. By utilizing the unit operations developed to generate, manipulate and analyze droplets, this technology platform has been used to miniaturize a wide range of complex biological assays including, but not limited to, directed evolution, rare cell detection, single cell transcriptomics, rare mutation detection and drug screening. The aim of this thesis is to develop droplet microfluidics based methods for analysis of single cells and nucleic acids. In Paper I, a method for time-series analysis of mammalian cells, using automated fluorescence microscopy and image analysis technique is presented. The cell-containing droplets were trapped on-chip and imaged continuously to assess the viability of hundreds of isolated individual cells over time. This method can be used for studying the dynamic behavior of cells. In Paper II, the influence of droplet size on cell division and viability of mammalian cell factories during cultivation in droplets is presented. The ability to achieve continuous cell division in droplets will enable development of mammalian cell factory screening assays in droplets. In Paper III, a workflow for detecting the outcome of droplet PCR assay using fluorescently color-coded beads is presented. This workflow was used to detect the presence of DNA biomarkers associated with poultry pathogens in a sample. The use of color-coded detection beads will help to improve the scalability of the detection panel, to detect multiple targets in a sample. In Paper IV, a novel unit operation for label-free enrichment of particles in droplets using acoustophoresis is presented. This technique will be useful for developing droplet-based assays that require label-free enrichment of cells/particles and removal of droplet content. In general, droplet microfluidics has proven to be a versatile tool for biological analysis. In the years to come, droplet microfluidics could potentially be used to improve clinical diagnostics and bio-based production processes. / <p>QC 20160926</p>

Miniaturization of biological analysis is a trend in the field of biotechnology aiming to increase resolution and sensitivity in biological assays. Decreasing the reaction volumes to analyze fewer analytes in each reaction vessel enables the detection of rare analytes in a vast background of more common variants. Droplet microfluidics is a high throughput technology for the generation, manipulation and analysis of picoliter scale water droplets an in immiscible oil. The capacity for high throughput processing of discrete reaction vessels makes droplet microfluidics a valuable tool for miniaturization of biological analysis. In the first paper, detection methods compatible with droplet microfluidics was expanded to include SiNR FET sensors. An integrated droplet microfluidics SiNR FET sensor device capable of extracting droplet contents, transferring a train of droplets to the SiNR to measure pH was implemented and tested. In paper II, a workflow was developed for scalable and target flexible multiplex droplet PCR using fluorescently color-coded beads for target detection. The workflow was verified for concurrent detection of two microorganisms infecting poultry. The detection panel was increased to multiple targets in one assay by the use of target specific capture probes on color-coded detection beads.   In paper III, droplet microfluidics has been successfully applied to single cell processing, demonstrated in paper III, where reverse transcription was performed on 65000 individually encapsulated mammalian cells. cDNA yield was approximately equivalent for reactions performed in droplets and in microliter scale. This workflow was further developed in paper IV to perform reverse transcription PCR in microfluidic droplets for detection of exosomes based on 18S RNA content. The identification of single exosomes based on RNA content can be further developed to detect specific RNA biomarkers for disease diagnostics. Droplet microfluidics has great potential for increasing resolution in biological analysis and to become a standard tool in disease diagnostics and clinical research. / <p>QC 20171024</p>

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.

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...