The objective of this research is to develop automated and integrated microsystems for high-resolution imaging and high-throughput phenotyping / laser ablation of C. elegans. These microsystems take advantage of microfluidic technology for precisely handling animals and computer-aid automation for high-throughput processing. We demonstrated automated and high-throughput imaging / sorting and laser ablation of C. elegans. This thesis work is divided into four parts: development of a microsystem for imaging and sorting, development of a microsystem for laser cell ablation, development of a novel temperature measurement method, and development of pressure measurement method in microchannels. First, a microsystem was developed for high-throughput microscopy at high resolution and sorting. The microfluidic chip integrates novel microfluidic components to trap, position, immobilize, and sort/release animals. To characterize device operation and aid design of the device numerical models were developed. The experimental results demonstrate that the device operates robustly in a completely automatable manner. Additionally, a sophisticated control algorithm developed by Matthew Crane (Dr. Hang Lu¡¯s lab) automates the entire process of image acquisition, analysis, and sorting, which allows the system to operate without human intervention. This microsystem sorted worms based on their fluorescent expression pattern with over 95% accuracy per round at a rate of several hundred worms per hour. Secondly, the technologies developed for the imaging/sorting system were adapted and further improved to develop a microsystem for high-throughput cell laser ablation of C. elegans. The multiplex ablation module combined with the embryo trap module enables robust manipulation of embryos/L1-stage C. elegans. In addition, software for image processing and automation was developed to allow high-throughput cell ablations. This system performed ablation of a large number of animals and demonstrated accurate ablation by showing behavioral defects of the ablated worms in a chemotaxis avoidance assay. Thirdly, to aid future development of the microdevices, a novel in situ method for three-dimensionally resolved temperature measurement in microchannels was developed. This method uses video-microscopy in combination with image analysis software (developed by Jaekyu Cho in Dr. Victor Breedveld¡¯s group) to measure Brownian diffusion of nanoparticles that is correlated to temperature. This method offers superior reproducibility and reduced systematic errors. In addition, we demonstrated that this method can be used to measure spatial temperature variations in three dimensions in situ. Lastly, a method for pressure measurement in microdevices was also developed through collaboration with Hyewon Lee (Dr. Hang Lu¡¯s lab) to aid further device optimization. These micro pressure-sensors are composed of two flow layers with a polydimethylsiloxane (PDMS) membrane in between. The membrane deforms as a function of pressure and its deformation is quantified by a simple image-based method. These sensors offer high-precision pressure measurement in broad sensing ranges. In addition, a pressure transduction scheme combined with imaging-based method enables multiplex pressure measurement for simultaneously detecting pressures in multiple locations in a microsystem. Overall, the technologies developed in this thesis will establish a solid basis for continuous improvement of the microsystems for multi-cellular model organisms. This high-throughput technology will facilitate a broad range of biological and medical research.

Due to the large extent to which important biological mechanisms are conserved evolutionarily, the study of a simple soil nematode, C. elegans, has provided the template for significant advances in biology. Use of this model organism has accelerated in recent years as developments of advanced reagents such as synapse localized fluorescent markers have provided powerful tools to study the complex process of synapse formation and remodeling. Even as much routine biology work, such as sequencing, has become faster and easier, imaging protocols have remained essentially unchanged over the past forty years of research. This, coupled with the ability to visualize small, complex features as a result of new fluorescent reagents, has resulted in genetic screens in C. elegans becoming increasingly labor intensive and slow because microscopy mainly relies on manual mounting of animals and phenotyping is usually visually done by experts. Genetic screens have become the rate limiting factor for much of modern C. elegans research. Furthermore, phenotyping of fluorescent expression has remained a primarily qualitative process which has prevented statistical analysis of subtle features. To address these issues, a comprehensive system to allow autonomous screening for novel mutants was created. This was done by developing novel microfluidic devices to enable high-throughput screening, systems-level components to allow automated operation, and a computer vision framework for identification and quantitative phenotyping of synaptic patterns. The microfluidic platform allows for imaging and sorting of thousands of animals at high-magnification within hours. The computer vision framework employs a two-stage feature extraction to incorporate local and regional features and allows for synapse identification in near real-time with an extremely low error rate. Using this system thousands of mutagenized animals were screened to indentify numerous novel mutants expressing altered synaptic placement and development. Fully automated screening and analysis of subtle fluorescent phenotypes will allow large scale RNAi and drug screens. Combining microfluidics and computer vision approaches will have a significant impact on the biological community by removing a significant bottleneck and allowing large-scale screens that would have previously been too labor intensive to attempt.

Cette étude sur la locomotion de C. elegans vise à mieux comprendre le fonctionnement de son système nerveux et à apporter des éléments nouveaux de réflexion pour la conception de modèles ou d'objet biomimétiques. Ce travail débute par une description du ver, de sa physiologie ainsi que des principaux modes de locomotion connus : la nage (en milieu liquide) et la reptation (sur gel aqueux). Puis dans le cas de la nage, nous mettons en évidence une dissymétrie du mouvement, nécessaire pour la progression en milieu visqueux. L'analyse des vitesses des déplacements locaux permet de faire un bilan des forces exercées sur le ver, en admettant que celles-ci sont visqueuses. On montre ainsi que les coefficients de friction transverse et longitudinale peuvent être assimilés à ceux d'un ellipsoïde allongé. Dans le cas du mode reptation, on observe une diminution de l'amplitude de la tête vers la queue. L'interaction ver-substrat est abordée d'abord théoriquement (hypothèse de lubrification). Il en résulte des prédictions pour les coefficients de friction en désaccord avec les résultats expérimentaux. Ce désaccord est expliqué par la mise en évidence de seuils de friction statique. On mesure aussi la rigidité passive d'un ver. Un confinement vertical du ver en milieu liquide permet d'observer une transition continue de la nage vers la reptation. On montre que la période du mouvement ainsi que le déphasage entre les mouvements de la tête et la queue augmentent avec le placage. Un confinement horizontal du ver sur substrat permet de contraindre l'amplitude. On montre que la longueur d'onde diminue avec l'amplitude.

Heat shock factor 1 (HSF1) is the master transcriptional regulator of the heat shock response (HSR), an evolutionarily conserved cellular stress response. HSF1 promotes the expression of a variety of molecular chaperones that aid in restoring protein homeostasis upon exposure to proteoxic stress. However, all of the proteins responsible for regulating the HSR together with HSF1 are unknown. A genome-wide yeast two hybrid screen was performed to identify new S. cerevisiae Hsf1 protein interacting partners. Two GAL4 DNA binding domain-Hsf1 fusion proteins (baits) were constructed with mutations in the Hsf1 C-terminal activation domain to dampen Hsf1 mediated auto-activation of the reporter gene. Each haploid bait strain was mated with a haploid prey strain containing one of ~6,000 S. cerevisiae open reading frames fused to the GAL4 activation domain (prey). Interaction between the bait and prey reconstituted the GAL4 protein enabling it to bind to a GAL4 DNA binding site and activate the HIS3 reporter gene. The identified proteins from 4 screens were pooled generating 240 putative Hsf1 interacting partners. This list was narrowed to 38 candidates by selecting the 15 strongest interactions identified based on colony size and 33 candidates conserved in C. elegans. Hsf1 interactions with the 14 candidates in which protein expression was confirmed were then re-tested by a manual yeast two-hybrid assay. Hsf1 interactions with Sti1, Rim2 and Prp46 were repeatable in this manual assay. A study of the impact of knockdown of each of their C. elegans homolog on the HSR was performed using RNAi in an hsp70-promoter::GFP reporter strain of C. elegans. Preliminary results suggest that knockdown of Sti1 may impact the HSR in the worm. Further study of Sti1 and other potential Hsf1 interacting partners identified in this screen is warranted.

The heat shock response (HSR) is the cell's molecular reaction to protein damaging stress and is critical in the management of denatured proteins. Activation of HSF1, the master transcriptional regulator of the HSR, results in the induction of molecular chaperones called heat shock proteins (HSPs). Transcription of hsp genes is promoted by the hyperphosphorylation of HSF1, while the attenuation of the HSR is regulated by a dual mechanism involving negative feedback inhibition from HSPs and acetylation at a critical lysine residue within the DNA binding domain of HSF1, which results in a loss of affinity for DNA. SIRT1 is a NAD+-dependent histone deacetylase that has been reported to deacetylate HSF1, thus promoting stress-induced HSF1 DNA binding ability and increasing HSP expression (Westerheide, Anckar et al. 2009). While an abundance of research is aimed to investigate SIRT1 substrate regulation, the mechanism in which SIRT1 itself is regulated is less understood (Haigis and Sinclair 2010). Positive and negative modulators of SIRT1 include AROS and DBC1, respectively, and have yet to be investigated in relation to SIRT1-dependent regulation of the HSR. In addition, metabolic stress such as caloric restriction has been shown to modulate SIRT1 activity in yeast (Rahat, Maoz et al. 2011), but the effect of caloric restriction on the HSR is unknown. Using cell-based assays, we have investigated how the HSR may be controlled by factors influencing SIRT1 activity. We found that heat shock results in an increase in the cellular NAD+/NADH ratio and an increase in recruitment of SIRT1 to the hsp70 promoter. Furthermore, we found that the SIRT1 modulators, AROS and DBC1, impact hsp70 transcription, HSF1 acetylation status, and HSF1 recruitment to the hsp70 promoter. The nematode Caenorhabditis elegans is a useful model organism for testing the relationship between the HSR and metabolism, as these animals can easily be calorically-restricted via bacterial limitation and possess the mammalian SIRT1 homolog, Sir2.1. Using C. elegans, we demonstrate that caloric restriction and heat shock have a synergistic effect on the HSR in a sir2.1-dependent manner. We show that caloric restriction increases the ability of heat shock to promote thermotolerance and fitness in wild-type animals and to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1. These studies provide insight into SIRT1-dependent regulation of the HSR and the impact of metabolism on this response. We highlight the SIRT1 modulators AROS and DBC1 as two new targets available for therapeutic regulation of the HSR and add caloric restriction as another HSR activator that can synergize with heat shock.

Surgery of axons in C. elegans using ultrafast laser pulses, and observing their subsequent regrowth opens a new frontier in neuroscience, since such research holds a great potential for the development of novel therapies and cures to neurodegenerative diseases. In order to make the required large-scale genetic screenings in C. elegans possible and thus obtain statistically significant biological data, an automated laser axotomy system needs to be developed. Microfluidic devices hold the promise of improved throughput by integrating different functional modules into a single chip. The first step to developing a microfluidic device for laser axotomy is to devise an on-chip worm trapping method, which maintains a high degree of immobilization to sever axons without using anesthetics. In this thesis, we present a novel method that uses a thin, deflectable PDMS membrane that individually traps worms in a microfluidic device. Axons can successfully be severed with the same accuracy as those using conventional paralyzing techniques. This device also incorporates recovery chambers for housing worms after surgery and for time-lapse imaging of axonal regrowth without the repeated use of anesthetics. Towards accomplishing an automated, high-throughput laser axotomy system, we developed an improved microfluidic design based on the same mechanical immobilization technique. This second generation device allows for serially processing of a large quantity of worms rapidly using a semi-automated system. Integrated to the opto-mechanical platform, a software program utilizing image processing techniques is developed. This semi-automated program can automatically identify the location of worms, their neuronal cell bodies, focus on the axons of interest, and align the laser beam with the axon via a PID based viso-servo feedback algorithm. Statistic data demonstrate that there is no significant difference in axonal reconnection rates between surgeries performed on-chip and using anesthetics. To improve flow control, a three-dimensional novel microfluidic valve structure is designed and fabricated. This novel valve structure allows for a complete sealing of the flow channel, without degrading optical conditions for imaging and laser ablation in the trapping area. Finally, we developed a prototypical microfluidic assembly that will eventually be able to interface a well-plate to automatically deliver population of worms from individual wells to the automated chip for axotomy. This interface consists of a microfluidic multiplexer to significantly reduce the number of solenoid valves needed to individually address each well. / text

This work presents optogenetics and real-time computer vision techniques to non-invasively manipulate and monitor neural activity with high spatiotemporal resolution in awake behaving Caenorhabditis elegans. These methods were employed to dissect the nematode's mechanosensory and motor circuits and to elucidate the neural control of wave propagation during forward locomotion. Additionally, similar computer vision methods were used to automatically detect and decode fluorescing DNA origami nanobarcodes, a new class of fluorescent reporter constructs.

The ability of a neuron to alter its synaptic connections during development is essential to circuit assembly. Synapse remodeling or refinement has been observed in many species and many neuronal circuits, yet the mechanisms defining which neurons undergo remodeling are unclear. Moreover, the molecules that execute the process of remodeling are also obscure. To address this issue, we sought to identify targets of the transcription factor unc-55 COUP-TF, which acts as a cell-specific repressor of synapse remodeling in C. elegans. unc-55 COUP-TF is expressed in VD neurons, where it prevents synapse remodeling. DD neurons can remodel synapses because they do not express unc-55 COUP-TF. Ectopic expression of unc-55 COUP-TF in DD neurons prevents remodeling. We identified the transcription factor Hunchback-like hbl-1 as a target of UNC-55 COUP-TF repression. Differential expression of hbl-1 explains the cell-type specificity of remodeling. hbl-1 is expressed in the DD neurons that are capable of remodeling, and is not expressed in the VD neurons that do not remodel. In unc-55 mutants, hbl-1 expression increases in VD neurons where it promotes ectopic remodeling. Moreover, hbl-1 expression levels bidirectionally regulate the timing of DD remodeling, as increases in hbl-1 cause precocious remodeling while decreases in hbl-1 cause remodeling delays. Finally, hbl-1 coordinates heterochronic microRNA and neuronal activity pathways to regulate the timing of remodeling. Increases or decreases in circuit activity cause increases or decreases in hbl-1 expression, and consequently early or delayed remodeling. Thus, convergent regulation of hbl-1 expression defines a genetic mechanism that patterns activity-dependent synaptic remodeling across cell types and across developmental time. We identified other targets of UNC-55 COUP-TF regulation using gene expression profiling, and implicate some of these factors in the regulation of remodeling using functional genomic screens. Our work suggests roles for conserved networks of transcription factors in the regulation of remodeling. We propose a model in which hbl-1 and other targets of unc-55 COUP-TF transcriptional repression are responsible for regulating synapse remodeling in C. elegans.

Dietary restriction (DR), or the reduction of food consumption without malnutrition, is the most conserved method of preventing or reversing age associated diseases. It is also the most conserved method of increasing lifespan, across model organisms. We developed a robust liquid DR method for C. elegans, to investigate requirements for stress defense and NAD\(^+\)-associated mechanisms in mediating DR induced longevity. We found that DR lifespan extension depended upon stress defense regulators that act downstream of TORC1 and growth pathways, as well as SIR-2.1/SIRT1 and the NAD\(^+\) salvage pathway enzyme PNC-1. Surprisingly, PNC-1 was not required for improvement in two measures of healthspan, or the period of life spent free from disease. This suggests that the genetic regulation of DR effects can be uncoupled from one another, and that increased healthspan does not always indicate increased lifespan.

The Caenorhabditis elegans Eph RTK, VAB-1, has known roles in neuronal and epidermal morphogenesis as well as oocyte maturation through interaction with its ephrin ligands. In humans, Eph receptors are involved in nervous and vascular system development and have been implicated in cancer formation and progression. DAF-18, a C. elegans ortholog of the human tumour suppressor gene, PTEN, has been identified as an interacting partner with the Eph RTK, VAB-1. Mutations in human PTEN have been associated with numerous cancers and in the worm, DAF-18 is a well studied member of the DAF-2/Insulin receptor-like signaling pathway which has roles in dauer formation, thermotolerance and adult longevity. Our lab has previously shown that VAB-1/EphR binds DAF-18. To further investigate the significance of this interaction as well as offer additional function to the proteins involved, I have shown that VAB-1/EphR is a negative regulator of DAF-18/PTEN at the protein level. Western blotting reveals that endogenous expression of DAF-18/PTEN is low in wild-type animals and expression is increased in a vab-1/ephR mutant. Additionally, VAB-1/EphR and DAF-18/PTEN are expressed in head neurons, oocytes and the germline precursor cells, Z2/Z3. vab-1/ephR mutants show increases longevity and sensitivity to dauer conditions which is consistent with increased DAF-18/PTEN activity. Lastly, daf-18(ok480) is able to suppress the oocyte maturation phenotype and increased MAPK expression displayed by vab-1(dx31) animals, providing genetic evidence of an interaction. By identifying the tissues where these proteins are co-expressed and substantiating the interaction with multiple analyses, novel roles may be proposed for each: VAB-1/EphR in DAF-2/Insulin signaling and DAF-18/PTEN in oocyte maturation downstream of VAB-1/EphR signaling. This work provides further understanding of how an organism coordinates complex developmental processes and reiterates the notion that cellular signaling is a complex network of interacting players. As many signaling pathways are evolutionarily conserved, my research in C. elegans may provide a mechanism on how Eph RTKs and PTEN are regulated in more complex organisms, including humans. / Thesis (Master, Biology) -- Queen's University, 2009-02-27 17:09:10.582