Acoustophoresis in Variously Shaped Liquid Droplets

The ability to precisely trap, transport and manipulate micrometer-sized objects, including biological cells, DNA-coated microspheres and microorganisms, is very important in life science studies and biomedical applications. In this study, acoustic radiation force in an ultrasonic standing wave field is used for micro-objects manipulation, a technique termed as acoustophoresis. Free surfaces of liquid droplets are used as sound reflectors to confine sound waves inside the droplets. Two techniques were developed for precise control of droplet shapes: edge pinning and hydrophilic/hydrophobic interface pinning. For all tested droplet shapes, including circular, annular and rectangular, our experiments show that polymer micro particles can be manipulated by ultrasound and form into a variety of patterns, for example, concentric rings and radial lines in an annular droplet. The complexity of the pattern increases with increasing frequency, and the observations are in line with simulation results. The acoustic manipulation technique developed here has the potential to be integrated into a more complex on-chip microfluidic circuit. Especially because our method is well compatible with electrowetting technology, which is a powerful tool for manipulating droplets with free surfaces, the combination of the two methods can provide more versatile manipulation abilities and may bring a wealth of novel applications. In the end, we demonstrate for the first time that acoustophoresis can be used for manipulating Caenorhabditis elegans

Imaging Immune Surveillance of Individual Natural Killer Cells Confined in Microwell Arrays

New markers are constantly emerging that identify smaller and smaller subpopulations of immune cells. However, there is a growing awareness that even within very small populations, there is a marked functional heterogeneity and that measurements at the population level only gives an average estimate of the behaviour of that pool of cells. New techniques to analyze single immune cells over time are needed to overcome this limitation. For that purpose, we have designed and evaluated microwell array systems made from two materials, polydimethylsiloxane (PDMS) and silicon, for high-resolution imaging of individual natural killer (NK) cell responses. Both materials were suitable for short-term studies (<4 hours) but only silicon wells allowed long-term studies (several days). Time-lapse imaging of NK cell cytotoxicity in these microwell arrays revealed that roughly 30% of the target cells died much more rapidly than the rest upon NK cell encounter. This unexpected heterogeneity may reflect either separate mechanisms of killing or different killing efficiency by individual NK cells. Furthermore, we show that high-resolution imaging of inhibitory synapse formation, defined by clustering of MHC class I at the interface between NK and target cells, is possible in these microwells. We conclude that live cell imaging of NK-target cell interactions in multi-well microstructures are possible. The technique enables novel types of assays and allow data collection at a level of resolution not previously obtained. Furthermore, due to the large number of wells that can be simultaneously imaged, new statistical information is obtained that will lead to a better understanding of the function and regulation of the immune system at the single cell level

Radial Sizing of Lipid Nanotubes Using Membrane Displacement Analysis

We report a novel method for the measurement of lipid nanotube radii. Membrane translocation is monitored between two nanotube-connected vesicles, during the expansion of a receiving vesicle, by observing a photobleached region of the nanotube. We elucidate nanotube radii, extracted from SPE vesicles, enabling quantification of membrane composition and lamellarity. Variances of nanotube radii were measured, showing a growth of 40-56 nm, upon increasing cholesterol content from 0 to 20%

Contribution à la compréhension de l'immunopathogénie de l'aspergillose nasosinusale chez le chien

Sinonasal aspergillosis is a sever fungal rhinosinusitis mainly affecting large breed dogs in their mid ages. Its most common causal agent is A. fumigatus, a fungus that is largely spread in the atmosphere. As of today, the diagnostic and treatment for this disease remain a challenge for the practicing veterinary doctor. Very little data is available to explain why such a ubiquitous fungus induces a sever rhinosinusitis in otherwise healthy dogs, while other dogs do not present any sign of fungal infection. The authors of a study analysing the expression of mRNA encoding for certain cytokines and chimiokines in the nasal mucosa of SNA affected dogs, propose the assumption that dogs develop a protective immunity (Th1) against A. fumigatus, but that it could be blocked by an excessively intense regulating immunity (massive production of IL-10). Indeed, it is commonly described that in humans affected by invasive aspergillosis, as well as in mice-based models, the production of immunoregulating cytokines (IL-10) should be considered as a sign of the escalation of the sickness and an absence of its remission. The objective of this thesis was to investigate the adaptive immunity reaction of SNA affected dogs based on the assumption that sick subjects develop a protective immunity that is antagonised by a disproportional regulating immunity. Three axes of analysis have been considered to answer this objective. The first looked into the difference of expression and/or production of cytokines and transcriptor factors prototypics of the different adaptative and regulatory immunological paths: Th1 (IFN-γ and Tbet) – Th2 (IL-4 and GATA3) – Th17 (IL-17A and RORc) and Treg (IL-10 and FoxP3) in PBMC of affected and healthy dogs after A. fumigatus stimulation. Secondly, an analysis of genes by microarray has been carried on nasal mucosa biopsies of affected and healthy dogs. Thirdly, the promotor zone of the gene encoding IL-10 in dogs has been analysed by sequencing. This study has been done within three cohortes of dogs: Rottweiler-Labrador and Golden containing affected and healthy dogs. The objective was to investigate, as it is the case in human medicine, the possibility of a genetic modification as a factor susceptible of leading to SNA development. The results of the first study revealed that: (1) the PBMC of half the controls dogs and every affected dogs expressed a relevant overexpression of IFN-γ. This increase was significantly more important within PBMC of affected dogs. The analysis of IFN-γ production in culture supernatants was in accordance with these last observations. A significant increase in the expression of mRNA coding for Tbet was also observed in half of the PBMC of affected dogs. (2) a significant increase in expression of mRNA encoding IL-4 was observed in the PBMC of most of the affected and healthy dogs. This increase was significantly higher in the PBMC of affected dogs than in the healthy dogs. (3) the PBMC of most control and affected dogs also revealed an increase in expression of mRNA encoding IL-17A. This increase was statistically more important in the PBMC of affected dogs than in the healthy dogs. (4) a relevant decrease in mRNA expression encoding IL-10 was observed in the PBMC of more than half of the affected dogs. The expression of the mRNA encoding IL-10 was significantly smaller in the PBMC of affected dogs than in the healthy dogs. The microarray analysis showed that: (1) amongst the 49 overexpressed biological groups, 13 were associated to the immunological or inflammatory process; (2) the nasal mucosa of affected dogs presented an increase in the expression of genes encoding for molecules involved directly (IFN-γ and STAT4) and indirectly (IL-16, CCL3, CCL4, and CXCL10) in the development of protective Th1 immunity, as well as molecules involved in the regulatory branch of the immune response (IL-16 and Ikaros). The sequencing analysis of the promotor region of the gene encoding IL-10 revealed the presence of polymorphisms. Three polymorphisms were observed more frequently in clones belonging to the three studied cohorts, excepted for the clones belonging to SNA affected Rottweiler. The polymorphisms observed in dogs were not similar to those described in humans. The first study showed an increase in the expression of mRNa encoding IFN-γ - Tbet – IL-4 and IL-17A in most of the PBMC of the affected dogs, and a decrease in the expression of IL-10 in comparison with the PBMC of healthy dogs. Similar results were observed in mice repeatedly affected by A. fumigatus. The suggested hypothesis was that an intense Th17 immunity resulted in a massive inflammatory reaction leading to a favourable environment were A. fumigatus was able to proliferate as hyphae. In return, hyphae would lead to the development of a non-protective Th2 immunity. It is tempting to suggest that the same hypothesis could be made for dogs affected by SNA. In order to reinforce this hypothesis, we should compare the expression of the different molecules involved in the Th17 immunity inside the nasal mucosa of affected and healthy dogs. Additionally, we should be running a kinetic study based on the expression of prototypical cytokines in parallel with the analysis of the production of these cytokines in culture supernatants. Ideally these studies should use DC and lymphocytes isolated from the nasal mucosa of affected and healthy dogs. In conclusion, an new hypothesis could be formulated: the possibility that not the overstimulation of the regulatory branch of the immunity response but an overstimulation of the Th17 branch of the immune response could be the cornerstone of the incapacity of dogs to clear from their SNA. The results of the microarray study were partially in accordance to the starting hypothesis. Indeed, the results showed an overexpression of genes involved in in the development of the protective Th1 (IFN-γ, STAT4, IL-16, CCL3, CCL4, and CXCL10) as well as genes involved in the regulatory path of the adaptive immunity (IL-16 and Ikaros). But the results of this study did not show an increase in IL-10. No conclusion could be drawn from these results; indeed, they were only the reflection of a fixed image at a given moment and we cannot consider qPCR results as the exact replica of the production of cytokines in the microenvironment. Nevertheless this study pointed out new possible areas of research. The results obtained after the sequencing of the promotor zone of the gene encoding IL-10 did not show any clear difference between affected and healthy dogs. However, this study was undertaken with a very limited number of dogs. In order to further assess the possibility of a genetic modification as the cornerstone of the development of SNA, more dogs should be analysed and the sequencing analysis should be run in parallel with an ELISA analysis

Intercellular transfer and organization of proteins at the natural killer cell immunological synapse

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

La tumeur faciale transmissible du Diable de Tasmanie

The Tasmanian Devil is the largest carnivorous marsupial of Australia. Since 1996, this species faces an emerging transmissible and fatal disease that is characterized by eruption of tumors on the face and the neck. Having carried 75.000 animals off in only 8 years, the Tasmanian Devil’s transmissible tumor dramatically threatens the survival of the species, which would have disastrous ecological consequences because of its key role as a predator and as a scavenger. The following text gathers the information available on the features of the disease and brings to the light its astonishing etiology and mode of transmissionLe Diable de Tasmanie est le plus grand marsupial carnivore d’Australie. Depuis 1996, cette espèce est confrontée à une maladie émergente, transmissible et fatale, caractérisée par l’apparition de tumeurs au niveau de la face et du cou. Ayant causé la mort de 75.000 animaux en huit ans seulement, la tumeur transmissible du Diable de Tasmanie représente aujourd’hui une menace susceptible d’entraîner l’extinction de l’espèce, ce qui aurait des conséquences écologiques désastreuses en raison de son rôle essentiel en tant que grand prédateur et charognard. Le texte qui suit fait le point sur la maladie, son étonnante étiologie et son mode de transmission par allogreffe