Sleep or sleep-like states are highly conserved phenomena across the animal kingdom. It is a rapidly reversible period of inactivity, marked by an increased threshold for sensory arousal. While sleep is a critical physiological process essential for survival, and chronic sleep deprivation has detrimental effects, its full range of functions remains not fully understood. Sleep interacts closely with several physiological processes, including tissue growth and repair, stem cell regeneration, synaptic pruning, hormonal regulation, attention, focus, immune function, learning, and memory consolidation. 

My PhD research focuses on understanding the role of sleep in modulating learning and memory in Drosophila melanogaster. I am studying the immune function mutants with disrupted sleep patterns caused by mutations in antimicrobial peptide (AMP) genes. These mutations impair both immune function and sleep, making them a valuable model to investigate the role of AMPs in sleep regulation and their broader impact on cognition. To explore how sleep regulates spatial learning and memory, I am developing a thermal-visual arena in which flies can be trained to complete spatial tasks using visual cues in an LED-lit environment. Using a split-GAL4 approach, I can precisely target and manipulate specific subsets of neurons to uncover the circuitry involved in spatial learning and memory. 

I also aim to understand the neural mechanisms behind spatial learning and how they decline with age. Using two-photon microscopy, I will study how aging affects neuronal activity during spatial learning and investigate whether sleep restoration can reverse age-related cognitive decline.

The focus of my MSc research was to understand the pattern of subcellular localization of eomesoderminA (eomesA) mRNA in the early developing zebrafish embryo. For this project, I standardized a single-molecule fluorescent in situ hybridization method (Stellaris) to probe the high-resolution localization of eomesA transcripts. I found that eomesA mimics the localization pattern of germplasm RNPs at the cleavage furrow and cortical periphery of cells during early embryogenesis. 

To investigate whether eomesA granules co-localize with germplasm RNPs, I performed fluorescence in situ hybridization (FISH) for eomesA in combination with immunofluorescence staining for a germplasm marker—non-muscle myosin protein (NMII-p)—to visualize both in the same embryo. It was challenging but crucial to standardize a protocol that combined both techniques. High-magnification image analysis revealed the nature of eomesA–germplasm co-localization during early development and suggested that eomesA may depend on the same cytoskeletal elements for its localization as germplasm RNPs. To test this, I used pharmacological perturbations targeting cytoskeletal elements such as microtubules, actin, and myosin to assess the effects on the transport and localization patterns of both eomesA transcripts and germplasm RNPs. These perturbations revealed that eomesA relies on the same machinery used by germplasm RNPs for their transport and localization. A dynamic microtubule and actin network is essential for the proper localization of both eomesA and germplasm RNP granules. 

I also predicted the secondary structures of the complete eomesA RNA and its 3'-UTR and 5'-UTR using an in silico approach with the Vienna RNAfold server, alongside other conserved germplasm marker RNAs, including bicoid, nanos, deadend, and vasa. These secondary structures may be used for comparative studies to identify conserved localization signal motifs in RNAs that co-localize with germplasm. Once identified, gRNAs targeting the localization sequences will be synthesized and co-injected with Cas9 protein into embryos to produce maternal-zygotic mutants that disrupt eomesA localization without affecting protein function. This approach will help us understand the role and significance of eomesA localization in early zebrafish development and its association with germplasm, given their co-localization and shared dependency on a dynamic microtubule–actin network. 

In addition to my thesis project, I also participated in the lab’s efforts to explore the relevance of cell size during zebrafish embryogenesis. We found that altered cell sizes affect early cell migration movements, resulting in gastrulation defects. To study this, we generated haploid and tetraploid zebrafish embryos and demonstrated that haploid embryos have smaller cells while tetraploid embryos have larger cells compared to diploids. I performed qPCR analysis of genes in the Planar Cell Polarity (PCP) pathway to examine their expression profiles around the developmental stages when these defects emerge. The qPCR results indicated that the observed defects were caused solely by altered cell size, not by other effects of altered ploidy.