Microfluidic culture platforms for C. elegans bacterial interaction and digestion studies at single-organism resolution

Vittorio Viri
EPFL, 2021
Thèse EPFL

Résumé

Increasing interest in understanding fundamental biological processes, among which aging and nutrition, has led to the study of simple and powerful model organisms, as the round-worm Caenorhabditis elegans (C. elegans). The nematode, selected by Sydney Brenner , shares slightly over one-third of its genetic base code with human beings, making it an optimal model organism for many human related studies. It has been established that bacteria, besides being the principal nutrition source for these nematodes, play an active role in their aging process. In later life stage, i.e. after the larval development, the bacterial grinder of the worms loses efficiency permitting viable bacteria to reach and colonize the gut of the nematodes . Intestinal persistency of bacteria is highly related both to the strain of the hosted organism and of the host; in the first case the pathogenicity of bacteria determines colonization whereas in the second case the speed of the ingestion and digestion mechanism adopted by the host is one of the causes that can determine the occurrence of a condition of illness. Conventional methods commonly employed by biologists for C. elegans observation, widely applied in studies of interaction between worms and bacteria, food intake and processing analysis, result in some cases to be time consuming and difficult to operate. In order to solve these study related issues, such as observations of digestion related pheno-types on individual worms during the entire developmental time and reproducible food stimulation, microfluidics techniques, based on control of microliters of fluid in a laminar flow regime conditions, revealed to be useful for a consistent increase of the overall accuracy. Technical improvement in research and new analytical tools allowed single worm reversible immobilization and imaging and high throughput analysis, but gathering phenotypic information at single-cell or individual organism level, with higher throughput and better precision than conventional methods, still remains challenging. Combining the multiple technical advantages brought by microfluidics to the study of C. elegans, we focus our attention in developing new devices and protocols aiming to investigate how microbiota interact with the host organism. In particular, we intend to develop a novel microfluidic approach for performing long-term live imaging, bacterial processing and standardized food intake assays in an automated way. Worms sorting and immobilization by passive PDMS structures and accurate food concentration dispens-ing will be accomplished. We intend to obtain quantitative measurement of C. elegans food digestion, intake and development during the adult stage first and then in all stages, showing how digestion process, food intake and bacterial hosting is highly correlated with multiple characterizing phenotypes. To obtain food absorption measurement at single worm resolution, we apply an optical method of quantification using green fluorescent protein (GFP) labeled E. coli bacteria together with fluorescent microscopy techniques. Feeding behaviour and physiological digestion response under different exposure conditions will be tested in order to investigate how these factors impact on the development of the organism.

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https://infoscience.epfl.ch/record/287069?ln=fr

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Vittorio Viri
EPFL, 2021
Thèse EPFL

Résumé

Official source

https://infoscience.epfl.ch/record/287069?ln=fr

À propos de ce résultat

Concepts associés (16)

Concepts associés

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Publications associées (9)

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In this thesis a new approach for single molecule detection and analysis is explored. This approach is based on the combination of two well established methods, fluorescence correlation spectroscopy (FCS) and total internal reflection fluorescence microscopy (TIRFM). In contrast to most existing fluorescence spectroscopy techniques, the subject of primary interest in FCS is not the fluorescence intensity itself but the random intensity fluctuation around the mean value. Intensity fluctuations are induced by thermal noise in a minute observation volume, which is in classical FCS the confocal volume of a confocal microscope. E.g. FCS is commonly utilized to investigate diffusion. In this case, diffusing fluorescent molecules entering or leaving the observation volume cause intensity fluctuations, which are analyzed by calculating the temporal autocorrelation of the observed signal. The autocorrelation is a measure for the self-similarity of a signal and contains information about the average fluctuation strength and duration. The confocal observation volume, i.e. the measurement volume that is actually seen by the detector is approximately given by the product of the optical transfer function with the fluorescence exciting intensity distribution of a focused laser beam. To achieve a high signal-to-background ratio a small observation volume is absolutely essential, first of all because the background from e.g. scattered light increases with the size of the observation volume. Second, a small volume assures for a small average number of fluorophores inside the observation volume and therefore for a high fluctuation amplitude i.e. FCS signal. This thesis proposes and discusses an alternative to confocal FCS specially conceived for measurements on surface-bound molecular systems, such as biological receptors or immobilized enzymes. In contrast to confocal FCS, fluorescence is excited within an evanescent field generated by total internal reflection (TIR) of a laser beam at the interface between a microscope coverslip and the sample. This is achieved by focusing the laser beam off-axis at the back focal plane of a high NA oil-immersion objective. The collimated beam that emerges from the objective is incident at an oblique angle at the coverslip-sample interface and totally internal reflected. In contrast to confocal FCS, the generated observation volume is completely confined to the surface and background fluorescence as well as scattered light from the bulk is efficiently suppressed. Our method, called objective-type TIR-FCS in the following, features an increased collection efficiency compared to existing techniques that combine evanescent wave excitation and FCS. Existing techniques use total internal reflection on the surface of a prism to generate an evanescent field. This leads to a configuration where the choice of objectives is limited to air or water-immersion objectives. In our system we use a high NA oil-immersion objective, specially conceived for TIR applications, which collects light efficiently. The collection efficiency is further enhanced by a naturally occurring change of the emission properties of fluorophores close to interfaces between dielectric media. The presence of the interface favors emission into the optically denser medium so that about 60% of the emitted light can be collected. These factors, together with a reduced observation volume lead to a very sensitive method with a high potential for applications in single molecule detection and analysis. The performance of the proposed method was experimentally shown for measurements on molecules subject to Brownian motion and binding to modified coverslips. In particular, it was experimentally shown that objective-type TIR-FCS features high signal-to-background ratio on a single molecule level. In this thesis, concise derivations of analytical expressions for autocorrelation functions for diffusion and most important, the case of ligands reversibly binding to a single and localized binding site are presented. The derived model allows for the quantitative determination of binding rates for a single receptor. We strongly believe that the application of these results in the context of investigations of receptor-ligand binding kinetics will allow for deeper understanding of cellular signaling. Moreover, this thesis discusses the applicability of the proposed method in enzymology. Enzymes, as most proteins are subject to continuous changes of their structure or conformation. These conformational changes are correlated with the function of the enzyme. In the discussed example the enzyme catalyzes an oxidation where the product is fluorescent but the substrate is not. The function of the enzyme i.e. the recurring product formation leads to observable intensity fluctuations. Since function and conformational states are correlated, conformational fluctuations can be investigated by means of FCS as was already shown for confocal FCS. A technique closely related to FCS is fluorescence lifetime spectroscopy (where lifetime refers to the mean lifetime of the electronic excited state). Whereas in FCS the relaxation after random deviations from thermal equilibrium is investigated, relaxation of excited fluorophores towards their electronic ground-state is investigated in lifetime spectroscopy. The technique is used for e.g. discrimination between fluorophores with different lifetimes. Lifetime spectroscopy combined with imaging is used in many domains of life-science, including microarray reading and medical diagnostics. In preliminary work we developed a novel approach to perform lifetime imaging that is based on a multiplexing technique. The proposed method requires no mechanical scanning stage and only a single-point detector. Furthermore, noise is reduced under certain circumstances if the signal is low. Characteristics of this technique as well as advantages and disadvantages are shortly discussed.

EPFL2006

Chargement

Development and application of experimental tools to elucidate the gene regulatory network underlying adipogenesis

Carine Delattre-Gubelmann

Excess fat mass in obese individuals, which is characterized by an increase of white adipocyte cell size and number, significantly raises the risk of developing metabolic syndrome symptoms as well as cancer. A detailed understanding of the transcriptional mechanisms mediating white adipocyte differentiation (i.e., adipogenesis) is required to counteract the growing obesity epidemics. In this thesis, we have implemented two combinatory approaches to detect new protein-DNA interactions in the mouse, one of which targets the adipogenic gene regulatory network (GRN) that underlies the white adipocyte differentiation process. As a first approach to shed light on GRNs, we have developed and validated a powerful approach that combines a high-throughput yeast one-hybrid-based (Y1H) system to detect transcription factors (TFs)-DNA element interactions, together with a novel microfluidics-based technique, enabling the identification and characterization of individual binding sites for detected TFs within the respective regulatory sequences at unprecedented throughput and resolution. Using well-described regulatory elements as well as an uncharacterized enhancer which can play also a role in adipogenesis, we show that our approach in a first phase uncovers many known as well as novel TF-DNA interactions, of which a large proportion were independently validated. In addition, we further demonstrate how our approach can be used in a second phase to characterize detected interactions by enabling the generation of a relative DNA occupancy landscape over the length of the respective DNA element. Furthermore, we provide evidence that this system enables the detection of novel interactions that may have in vivo relevance. Given the strict requirements for direct and well-characterized protein-DNA interaction data to generate and model gene regulatory networks, the presented two-tiered approach should be of great value to enhance our understanding of the regulatory principles underlying mouse gene expression. To identify transcription factors involved in white adipocyte differentiation, we overexpressed TFs in preadipocytes and quantified their effect on differentiation. To this end, we have created a mouse open-reading frame resource consisting of more than 750 fully sequence-verified TF clones. We transferred these TF clones to a Tet-On lentiviral vector and used them to infect white preadipocytes. Based on microscopy-assisted quantification of lipids after induction of differentiation, we identified putatively novel TF candidates that strongly enhanced differentiation based on the increased lipid accumulation, indicating a potential role in adipogenesis. Gene expression and ChIP-seq experiments have been performed for the three top candidates (ZEB1, ZFP30 and ZFP277) to further study their function within the adipogenic regulatory network. To assess whether the effect of these TFs on adipogenesis could be reproduced in vivo, murine adipose stromal-vascular fraction containing adipocyte precursor cells were implanted into mice fed a high-fat diet, after each of the three TF candidates were knockdown or overexpressed in these cells. This experiment and the RNA-seq analyses are currently being processed while this thesis is being written. Furthermore, due to the absence of quantitative real-time PCR (qPCR) primer designing tools that would allow for gene-specific expression profiling in a high-throughput fashion, we design a user-friendly platform: GETPrime. This platform uniquely combines and automates several features critical to optimal qPCR primer design for all annotated Homo sapiens, Mus musculus, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio genes in the Ensembl database. GETPrime primers have been extensively validated experimentally, demonstrating their high quality as well as high transcript specificity in complex samples.

EPFL2013

Chargement

Magnetic model in multiferroic NdFe3(BO3)(4) investigated by inelastic neutron scattering

Henrik Moodysson Rønnow

We performed inelastic neutron scattering measurements on single crystals of NdFe3((BO3)-B-11)(4) to explore the magnetic excitations, to establish the underlying Hamiltonian, and to reveal the detailed nature of hybridization between the 4f and 3d magnetism. The observed spectra exhibiting a couple of key features, i.e., anticrossing of Nd and Fe excitations and anisotropy gap at the antiferromagnetic zone center, are explained by the magnetic model including spin interaction in the framework of weakly coupled Fe3+ chains, interaction between the Fe3+ and Nd3+ moments, and single-ion anisotropy derived from the Nd3+ crystal field. The combination of the measurements and calculations reveals that the hybridization between 4f and 3d magnetism propagates the local magnetic anisotropy of the Nd3+ moment to the Fe3+ network, leading to the determination of the bulk structure of both electric polarization and magnetic moment in the multiferroics of the spin-dependent metal-ligand hybridization type.

2015

Chargement

EPFL2006

Development and application of experimental tools to elucidate the gene regulatory network underlying adipogenesis

Carine Delattre-Gubelmann

EPFL2013