In situ hybridization (ISH) has played a crucial role in developing a spatial transcriptomic understanding of emerging model organisms in the past, but advancing high-throughput RNA-sequencing (RNA-seq) technology has pushed this method into the shadows, leading to a loss of data resolution. This shift in research towards the exclusive use of RNA-seq neglects essential considerations for transcriptomic studies including the spatial and temporal expression of transcripts, available budget, experimental design needs, and validation of data. A synergy of spatiotemporal transcriptomic techniques is needed, using the bulk and unbiased analysis of RNA-seq and the visual validation and spatiotemporal resolution of ISH. Integration of this synergistic approach can improve our molecular understanding of non-model organisms and establish the background data needed for advancing research techniques. A prime example lies within an emerging model of the marine science and symbiosis fields, where I present a case study on a threatened coral reef keystone – the cnidarian-dinoflagellate symbiosis. Establishing a whole-mount ISH protocol for the emerging cnidarian model Aiptasia (sea anemone) will help future studies reveal the gene regulation underpinning the establishment, persistence, and breakdown of this complex symbiotic relationship.

Many researchers across a wide range of disciplines have turned to gene expression anal- ysis to aid in predicting and understanding biological outcomes and mechanisms. Because genes are known to work in a dependent manner, it’s common for researchers to first group genes in biologically meaningful sets and then test each gene set for differential expression. Comparisons are made across different treatment/condition groups. The meta-analytic method for testing differential activity of gene sets, termed multi-variate gene set testing (mvGST), will be used to provide context for two persistent and problematic issues in gene set testing. These are: 1) gathering organism specific annotation for non-model organisms and 2) handling gene annotation ambiguities. The primary purpose of this thesis is to explore different gene annotation gathering methods in the building of gene set lists and to address the problem of gene annotation ambiguity. Using an example study, three different annotation gathering methods are proposed to construct GO gene set lists. These lists are directly compared, as are the subsequent results from mvGST analysis. In a separate study, an optimization algorithm is proposed as a solution for handling gene annotation ambiguities.

Identifying homologous genes, that is genes from a common ancestor, is important in comparative genomic studies for understanding gene annotation and the predicted function of a gene. Several pieces of software, of which the most well-known is BLAST, have been developed for identifying homologues, but this can be challenging in non-model organisms where sometimes poor quality of genome assemblies and lack of annotation make it difficult to robustly identify homologues. The aim of this project was to build a bioinformatic framework for homology detection using genomes from non-model organisms. The approach developed used genome annotations, annotated polypeptide sequences and genome assembly sequences to detect homologous genes.The framework was applied to identify Drosophila melanogaster homologous wing development genes in the genomes of nine other insect species with the aim to understand the evolution of loss of wings. To identify changes related to wing loss, the homologous protein sequences obtained were aligned and phylogenetic trees were built from them. The aim of creating the multiple protein alignments and phylogenetic trees was to shed light on whether changes in gene sequences can be related to presence or absence of wings. From the set of 21 candidate wing development genes identified with literature and subsequent database searches, I tested eight and was successful in identifying homologues for all of them in eight of the 10 in sectgenomes. This was done using a combination of text searches in genome annotations, searches with Exonerate v. 2.4.0 alignment program in annotated polypeptide sequences and in genome assemblies. The eight genes chosen for testing the framework were based on initial finding of putative homologues in the eight insect genomes when using the first two steps of the framework. For the set of homologous wing development genes examined I was not able to identify any conclusive pattern of potential protein coding changes that correlated with loss of wings in these species. Improvement to the current pipeline could include using query sequences from closer relatives of the 8 test species than D. melanogaster and, of course, testing of the remaining wing development genes as well as further literature study of wing development genes. Together these could improve future studies on the evolution of wing loss in insects.

Mechanical forces have been observed to affect various aspects of life, for example, cell differentiation, cell migration, locomotion and behavior of multicellular organisms etc. Such forces are generated either by external entities such as mechanical touch, fluid flow, electric and magnetic fields or by the living organisms themselves. Study of forces sensed and applied by living organisms is important to understand the interactions between organisms and their environment. Such studies may reveal molecular mechanisms involved in mechanosensation and locomotion. Several techniques have been successfully applied to measure forces exerted by single cells and cell monolayers. The earliest technique made use of functionalized soft surfaces and membranes as substrates on which cell monolayers were grown. The forces exerted by the cells could be measured by observing deformation of the substrates. Atomic Force Microscope (AFM) is another sensitive instrument that allows one to exert and measure forces in pico-Newton range. Advances in micromachining technology have enabled development of miniature force sensors and actuators. Latest techniques for mechanical force application and measurement use micromachined Silicon cantilevers in single as well as array form and micropillar arrays. Micropillar arrays fabricated using soft lithography enabled the use of biocompatible materials for force sensors. Together, these techniques provide access to a wide range of forces, from sub micro-Newton to milli-Newton. In the present work, types of forces experienced in biological systems and various force measurement and actuation techniques will be introduced. This will be followed by in depth description of the two major contributions of this thesis, 1) ―Colored polydimethylsiloxane micropillar arrays for high throughput measurements of forces applied by genetic model organisms‖. Biomicrofluidics, January 29, 2015. doi: 10.1063/1.4906905 2) ―Air microjet system for non-contact force application and the actuation of micro-structures‖. Journal of micromechanics and microengineering, December 15, 2015. doi: 10.1088/0960-1317/26/1/017001 Device developed for force measurement consists of an array of micropillars made of a biocompatible polymer Poly Dimethyl Siloxane (PDMS). Such devices have been used by researchers to measure traction forces exerted by single cells and also by nematode worm Caenorhabditis elegans (C. elegans). C. elegans is allowed to move in between the micropillars and the locomotion is video recorded. Deflection of the micropillar tips as the worm moves is converted into force exerted. Transparent appearance of C. elegans and PDMS poses difficulties in distinguishing micropillars from the worm, thus making it challenging to automate the analysis process. We address this problem by developing a technique to color the micropillars selectively. This enabled us to develop a semi-automated graphical user interface (GUI) for high throughput data extraction and analysis, reducing the analysis time for each worm to minutes. Moreover, increased contrast because of the color also delivered better images. Addition of color changed the Young‘s modulus of PDMS. Thus the dye-PDMS composite was characterized using hyper-elastic model. The micropillars were also calibrated using commercial force sensor. Analysis of forces exerted by wild type and mutant C. elegans moving on an agarose surface was performed. Wild type C. elegans exerted a total average force of 7.68 µN and an average force of ~1 µN on an individual pillar. We show that the middle of C. elegans exerts more force than its extremities. We find that C. elegans mutants with defective body wall muscles apply significantly lower force on individual pillars, while mutants defective in sensing externally applied mechanical forces still apply the same average force per pillar compared to wild type animals. Average forces applied per pillar are independent of the length, diameter, or cuticle stiffness of the animal. It was also observed that the motility of the worms with mechanosensation defects, lower cuticle stiffness, and body wall muscle defects was reduced with worms that have defective body wall muscle having the largest degree. Thus, we conclude that while reduced ability to apply forces affects the locomotion of the worm in the micropillar array, the reduced motility/locomotion may not indicate that the worm has reduced ability to apply forces on the micropillars. We also used the colored micropillar array for the first time to measure forces exerted by Drosophila larvae. Our device successfully captured the peristaltic rhythm of the body wall muscles of the larva and allowed us to measure the forces applied on each deflected pillar during this motion. Average force exerted by 1st instar wild type Drosophila larvae was measured to be ~ 1.5 µN per pillar. We demonstrated that a microjet of air can be used to apply forces in micro-Newton range. We developed a standalone system to generate a controlled air microjet. Microjet was generated using a controlled electromagnetic actuation of a diaphragm. With a nozzle diameter of 150 µm, the microjet diameter was maintained to a maximum of 1 mm at a distance of 5 mm from the nozzle. The force generated by the microjet was measured using a commercial force sensor to determine the velocity profile of the jet. Axial flow velocities of up to 25 m/s were obtained at distances as long as 6 mm. The microjet exerted a force up to 1 µN on a poly dimethyl siloxane (PDMS) micropillar (50 µm in diameter, 157 µm in height) and 415 µN on a PDMS membrane (3 mm in diameter, 28 µm thick). We also demonstrate that from a distance of 6 mm our microjet can exert a peak pressure of 187 Pa with a total force of about 84 µN on a flat surface with 8 V operating voltage. Next, we demonstrated that the response of C. elegans worms to the impinging air microjet is similar to the response evoked using a manual gentle touch. This contactless actuation tool avoids contamination and mechanical damage to the samples. Out of the cleanroom fabrication and robust design make this system cost effective and durable. Magnetic micropillars have been used as actuators. We fabricated magnetic micropillar arrays and designed actuation mechanisms using permanent magnet and a pulsed electromagnet. Force of about 19 µN was achievable using a permanent magnet actuation. In a pulsed electromagnetic field micropillar exerted a force of about 10 µN on a commercial force sensor. These techniques have promising applications when actuation needs to be controlled from long distances.

Spinal muscular atrophy (SMA) and Charcot-Marie-Tooth disease type 2D (CMT2D)/distal SMA type V (dSMAV) are two incurable neuromuscular disorders that predominantly manifest during childhood and adolescence. Both conditions are caused by mutations in widely and constitutively expressed genes that encode proteins with essential housekeeping functions, yet display specific lower motor neuron pathology. SMA results from recessive inactivating mutations in the survival motor neuron 1 (SMN1) gene, while CMT2D/dSMAV manifests due to dominant point mutations in the glycyl-tRNA synthetase (GlyRS) gene, GARS. Using a number of different model systems, ranging from Caenorhabditis elegans to the mouse, this thesis aimed to identify potential novel therapeutic compounds for SMA, and to increase our understanding of the mechanisms underlying both diseases. I characterised a novel C. elegans allele, which possesses a point mutation in the worm SMN1 orthologue, smn-1, and showed its potential for large-scale screening by highlighting 4-aminopyridine in a screen for compounds able to improve the mutant motility defect. Previously, the gene encoding three isoforms of chondrolectin (Chodl) was shown to be alternatively spliced in the spinal cord of SMA mice before disease onset. I performed functional analyses of the three isoforms in neuronal cells with experimentally reduced Smn levels, and determined that the dysregulation of Chodl likely reflects a combination of compensatory mechanism and contributor to pathology, rather than mis-splicing. Finally, working with two Gars mutant mice and a new Drosophila model, I have implicated semaphorin-plexin pathways and axonal guidance in the GlyRS toxic gain-of-function disease mechanism of CMT2D/dSMAV.