Immunity, behavior, and circadian regulation are important ways that animals maintain homeostasis. Defects in these physiologies often lead to disease or even death, yet many questions remain about how these physiologies are related. I explored the interactions between innate immunity, behavior, and circadian regulation by using Drosophila melanogaster, a convenient, genetically tractable model organism with both functionally and molecularly conserved innate immune and circadian clock systems. In the first chapter, I show that feeding, a circadian-regulated behavior, increases immunity to a sepsis-like infection. In the second, I present evidence suggesting that aging-related changes in immunity may be linked to circadian defects. Finally, I use a novel automated method to demonstrate that reduced grooming is a conserved sickness behavior in Drosophila. The feeding project ultimately showed that mutating TORC2 components could increase the host’s ability to kill and clear a bacterial infection, as well as survive the pathogenic effects of infection. Therefore we have identified a possible drug target to create host-based therapies for sepsis patients. We also have established Drosophila as a model system for studying a conserved sickness behavior: reduced grooming. This experimental paradigm will allow researchers to isolate mutants that do not show reduced grooming, and investigate whether this sickness behavior is adaptive or not.

A major pursuit of Developmental Biology is to determine how organisms composed of cells containing a single genome generate stereotyped body plans with diverse, complex morphologies. The development of these patterns is often determined by gradients of secreted factors known as morphogens, which activate cascades of gene expression to subdivide fields of cells into increasingly complex patterns. In many animals, including Drosophila, a rudimentary anterior-posterior (A-P) and dorsal-ventral (D-V) axes of the body plan are already established in the zygote, but the proximal-distal (P-D) axis of any appendages must be generated and patterned seperately. The spatio-temporal information responsible for activating gene expression and cell signalling that establishes this new axis is integrated at DNA regulatory elements often referred to as enhancers. The segmented leg of the insect Drosophila melanogaster offers an ideal system for studying how signalling pathways control P-D axis establishment and patterning. In addition to the fact that flies are a particularly genetically tractable model organism, many of the signals required for leg patterning have already been identified. A number of signalling pathways, including Wingless (Wg), Decapentaplegic (Dpp) and Epidermal Growth Factor Receptor (EGFR), are important for proper P-D axis patterning in a dynamic fashion during embryonic and larval development. The leg primordia are fist specified in the embryo and then patterned throughout development as intercalated circles and rings of gene expression are established in the leg imaginal disc. The radius of these domains corresponds to the P-D axis of the adult appendage. A rudimentary P-D axis is established in the embryo and the larval leg imaginal disc by the expression of the transcription factors Distalless, Dachshund and Homothorax in distal, medial and proximal domains, respectively. The P-D axis is further refined by activation of EGFR signalling in the presumptive tarsus, the distal-most portion of the fly leg, during the early third larval instar. As well as slightly later, in medial and proximal rings. EGFR signalling is a ubiquitous pathway with numerous roles throughout fly development as well as across metazoan taxa. Its activation produces diverse cellular outcomes such as growth, differentiation, or regulation of apoptosis depending on the precise regulation of its inputs and modulation of intracellular signalling components in a tissue-specific manner. The precise mechanism by which EGFR signalling is activated during tarsal patterning is the focus of this dissertation. As a crucial first step in the detailed characterization of EGFR activation in the leg, we have identified leg-specific enhancers of the genes encoding the neuregulin-like EGF ligand Vein and the ligand-activating protease Rhomboid and performed genetic and site-specific mutagenesis experiments to characterize the factors necessary to activate expression of vein and rho in the distal leg. While the enhancers of vein and rho (vnE and rhoE, respectively) employ similar transcriptional programs to activate target gene expression, there are some key differences. Both enhancers require Dll for their expression throughout leg development, however vnE requires Wg and Dpp only early and later becomes independent from these signals while rhoE requires them until much later in development. Further, vnE requires Sp1 while rhoE does not. These differences may be important for the precise timing of expression of these genes, with vn expression coming on several hours earlier than that of rho. It has been proposed that the distal source of EGFR ligand may act as a long-range morphogen to pattern the entire tarsus in a graded manner (Campbell, 2002; Galindo et al., 2005). Our analysis indicates that vnE and rhoE represent the only sources of EGFR ligand in the distal leg. Therefore, in order to determine the importance of distal of EGFR signalling for tarsal patterning we carried out CRISPR targeting to delete vnE and rhoE. Because these deletions produce only mild distal leg truncations and cannot be worsened by removal of other candidate EGFR inputs (for example the Rho homolog, Roughoid) we conclude that the long-range distal gradient model for P-D patterning by EGFR must be revised. Instead we propose that the tarsal segments are patterned by the combined action of a local, distal gradient of EGFR supplied by vnE and rhoE combined with secondary, more medial sources of EGFR signal. Our analysis of the mechanism by which EGFR patterns the distal leg segments improves our understanding not only of leg development, but also of how the EGFR pathway is regulated in general. Our conclusions have important evolutionary implications, as receptor tyrosine kinase signalling, of which EGFR is an example, is involved in limb patterning in taxa whose limbs themselves are not thought to be structurally homologous to fly legs (Panganiban et al., 1997; Pires-daSilva and Sommer, 2003). Further, the components of the EGFR pathway assessed in this work are highly conserved signalling molecules, involved in cell proliferation and are therefore often misregulated in tumors. A nuanced understanding of the ways in which EGFR signalling is activated, particularly via regulation at non-protein-coding loci, could motivate new therapeutic approaches.

Virus-host interactions determine virus transmissibility and virulence, and underlie coevolution that shapes interesting biological phenomena such as the genetic architecture of host resistance and host range. Characterization of the virus factors that exert selective pressure on the host, and the host genes which underlie resistance and adaptation against viruses will help to define the mechanistic pathways embroiled in host-virus coevolution. In this thesis, I describe the viral causes and host consequences of host-virus coevolution. These include genomic signatures consistent with antagonistic coevolution in antiviral RNA interference pathway genes such as high rates of positive selection and polymorphism, loci that underlie genetic variation in resistance to virus infection, and apparent conflict between NF-κB signalling and DNA virus infection. The RNA interference (RNAi) pathway is the most general innate immune pathway in insects, underlined by the observation that many viruses encode suppressors of RNAi (VSRs). The relationship between RNAi and VSRs has garnered attention as a plausible battleground for host-virus antagonistic coevolution, and genomic patterns in Drosophila support this hypothesis. However, genomic patterns in the N-terminal domain of the key RNAi effector gene, Argonaute-2, have not been described. In Chapter 2, I sequence the Argonaute-2 N-terminal domain using PacBio long-read sequencing technology to describe variation within and across Drosophila species, and test whether this variation is associated with resistance to Drosophila C Virus. The RNAi pathway evolves adaptively in Drosophila, but this has not been formally extended across invertebrate species. In Chapter 3, I quantify rates of adaptive protein evolution and describe evidence for selective sweeps in RNAi pathway genes using population genomic data from 8 insect and nematode species. These analyses indicate that RNAi genes involved in suppression of transposable elements and defence against viruses evolve rapidly across invertebrates, and I identify genes with signatures of elevated adaptation in multiple insect species. Host genes that underlie host-virus interactions have been described in RNA virus infection of Drosophila, however substantially less attention has focussed on the host response to DNA viruses, primarily because no DNA viruses have been isolated from Drosophila. In Chapter 4, I describe the isolation of Kallithea virus, a Drosophila dsDNA nudivirus, and characterise the host response to infection and genetic variation in resistance. I find that Kallithea virus infection causes early male-specific lethality, a cessation of oogenesis, and induction of undescribed virus-responsive genes. Further, I describe genetic variation in resistance and tolerance to Kallithea virus infection, and identify a potential causal variant for virus-induced mortality in Cip4. Insect viruses commonly encode viral suppressors of RNAi, however there are a multitude of antiviral immune mechanisms besides RNAi which may select for viral-encoded inhibitors. In Chapter 5, I describe the requirement for RNAi and NF-κB in immunity against Kallithea virus, and map gp83 as a virus-encoded inhibitor of NF-κB signalling. I find that gp83 inhibits Toll signalling at the level of, or downstream of NF-κB transcription factors, and that this immunosuppressive function is conserved in other nudiviruses.

No description available.

Under most conditions, meiotic recombination is essential for ensuring that organisms adapt to ever changing biotic and abiotic conditions and, as such, it shapes evolutionary change within and between species. The interplay between selection and recombination plays a role shaping levels diversity within populations. Remarkably, recombination is itself an evolving trait that varies at many levels: between distant species of eukaryotes, between closely related species and among populations (and individuals) of the same species. Recombination rates also vary across genomes. Most of the causes and mechanisms of this plasticity in recombination rates and distribution are not clearly understood. Also, our understanding of how this variability in recombination rates influences levels of diversity within populations and across genomes is incomplete. Here, I present a study combining molecular genetics with bioinformatic techniques to characterize recombination landscapes in Drosophila melanogaster. I present a model that accounts for a significant fraction of the variation in crossover rates across the genome of Drosophila melanogaster. Our predictive model suggests that crossover distribution is influenced by both meiosis-specific chromatin dynamics and very local constitutively open chromatin associated with DNA motifs that prevent nucleosome stabilization. I also present a novel method for genomic scans to identify recent events of adaptation in using nucleotide diversity data. In addition, I characterized variability in recombination rates in different populations of D. melanogaster and detected that the highest degree of variability in recombination rates across the genome is associated with intermediate genomic scales, and that this intermediate scale also plays a major role in explaining differences in recombination among populations. Our report is the first linking variation in recombination rates across genomes (genomic) and among populations (evolutionary), possibly suggesting a common mechanistic/genomic cause. Finally, I present preliminary data of the first large-scale project to study the effects of multiple environmental conditions in recombination rates at genome-wide level. In conclusion, these studies provide a new framework to investigate variation in recombination rates and to understand the genomic causes and evolutionary consequences.

The nervous system integrates multiple sources of sensory information that ultimately controls motor neurons to generate complex movements. Motor neurons form topographically organised ‘myotopic maps’ between the nerve cord and muscles in the periphery to ensure that correct pre-motor inputs into motor dendrites are relayed through corresponding axons to the appropriate muscle groups. Therefore understanding the development and assembly of motor neuronss is crucial for understanding how animals execute various motor outputs. In adult Drosophila, ~50 motor neurons are topographically organized between each leg and the nerve cord in a highly stereotyped manner (Baek and Mann, 2009). In this thesis, I describe a novel group of transcription factors that act in a combinatorial manner to specify the projections of distinct Drosophila leg motor neurons. Our studies suggest that morphological transcription factors regulate various downstream cell-surface genes that are involved in the assembly of motor circuitry. Using in vivo live imaging I describe the developmental steps involved in Drosophila leg motor neuron axon targeting during metamorphosis and the spatial expression patterns of a novel hetero-binding Ig domain transmembrane protein family – the DIPs and Dprs (Ozkan et al., 2013) in leg neuro-musculature. I further describe a function between interacting partners DIP-alpha and Dpr10, expressed in subsets of leg motor neurons and muscles respectively, in establishing the final stereotyped terminal axon branching of corresponding motor neurons. The combinations of such interactions throughout development between leg motor neurons, not only with muscles in the periphery, but also among themselves, with leg sensory neurons and other components in the central nervous system may ultimately lead to synaptic specificity and stereotyped morphologies of Drosophila leg motor neurons.

Diseases such as epilepsy, pain, and neurodegenerative disorders are associated with changes in neuronal dysfunction due to an imbalance of excitation and inhibition. This work details a novel electroconvulsive seizure assay for C. elegans using the well characterized cholinergic and GABAergic excitation and inhibition of the body wall muscles and the resulting locomotion patterns to better understand neuronal excitability. The time to recover normal locomotion from an electroconvulsive seizure could be modulated by increasing and decreasing inhibition. GABAergic deficits and a chemical proconvulsant resulted in an increased recovery time while anti-epileptic drugs decreased seizure duration. Successful modulation of excitation and inhibition in the new assay led to the investigation of a cGMP-dependent protein kinase (PKG) which modulates potassium (K+) channels, affecting neuronal excitability, and determined that increasing PKG activity decreases the time to recovery from an electroconvulsive seizure. The new assay was used as a forward genetic screening tool using C. elegans and several potential genes that affect seizure susceptibility were found to take longer to recover from a seizure. A naturally occurring polymorphism for PKG in D. melanogaster confirmed that both genetic and pharmacological manipulation of PKG influences seizure duration. PKG has been implicated in stress tolerance, which can be affected by changes in neuronal excitability associated with aging, so stress tolerance and locomotor behavior in senescent flies was investigated. For the first time, PKG has been implicated in aging phenotypes with high levels of PKG resulting in reduced locomotion and lifespan in senescent flies. The results suggest a potential new role for PKG in seizure susceptibility and aging. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Insect grooming has various functions, including defense against parasites and pathogens, cleaning of dust particles, and maintenance of sensory receptors. The hierarchy of grooming behavior suggests that cleaning one body part is more crucial than the other, the priority order more specifically being eyes, antennae, abdomen, then wings, followed by the thorax. Histamine is an extensively studied neurotransmitter found in the central nervous system of many animals. In Drosophila, histamine is found in both the peripheral and central nervous systems and is necessary for visual and mechanosensory behaviors. Histamine-gated chloride channel 1 (HisCl1) and Ora transientless (Ort) are two characterized histamine receptors, both of which are vital for visual signaling in the fly. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

Periodontal diseases (PD) affect 46% of American adults over age 30. These diseases cause symptoms including bleeding and swelling of the gums, bone resorption, and tooth loss, that affect quality of life and have a high economic burden. Periodontal diseases are caused by an imbalance in the oral microbiome, from a healthy state that contains anti-inflammatory commensals like Streptococcus gordonii and mitis, to a diseased state that has pro-inflammatory anaerobic pathogens including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. The latter initiate disease progression in the oral cavity. However, it’s the host immune response that causes a majority of the symptoms. Ideally, treatment for PD would be approached from both sides to reduce the numbers of pro-inflammatory bacterial cells in the oral cavity but also reduce the host immune response. A novel therapeutic, amixicile, has been created, which specifically targets anaerobes through the pyruvate:ferredoxin oxidoreductase (PFOR) system, the mechanism of energy metabolism found in anaerobic organisms. Our studies show that amixicile inhibits in vitro growth of oral anaerobes in monospecies cultures at concentrations as low as 0.5 µg/mL in broth and 1 µg/mL in biofilms, without affecting the Gram-positive commensal species. In multispecies cultures, amixicile specifically inhibited anaerobes, even in biofilms, with the concentration as low as 5 µg/mL in broth and 10 µg/mL in biofilms. By not affecting the commensal bacteria, we think this treatment could restore a healthy oral microbiome. Aside from the bacterial presence, the host response, particularly the innate immune response is not well understood. Using a Drosophila melanogaster infection model, we elucidated the innate immune response to both mono- and multispecies infections. The 7-Species infection included bacteria mentioned above and Aggregatibacter actinomycetemcomitans in order to replicate in vivo-like disease conditions. We determined that both Drosophila Toll and Imd pathways, which mimic TLR/IL-1 and TNF signaling pathways of mammalian innate immunity respectively, respond to the 7-Species challenge. We also verified virulent bacteria in Drosophila, including P. gingivalis and P. intermedia. Future directions include RNA sequencing to determine the full scope of immune gene expression and using human immune cells to further clarify the response.

Two manifestations of gene regulation-- dosage compensation and ontogenic regulation--were examined in normally positioned and relocated v+ genotypes in Drosophila melanogaster to determine the role of gene position in these control functions. Enzyme assays, used as criteria of gene activity, were performed on various genotypes containing different doses of v+ in normal and relocated positions in male and female flies. The results indicate that although differently positioned v+ genes may specify different tryptophan pyrrolase activities, they still show dosage compensation. In each case, the enzyme activity associated with each gene, either on the X, Y, or third chromosome, is twice as much in males as it is in females. This indicates that dosage compensation is not confined to the gene when located on the X chromosome. In order to determine if the pattern of activity of the gene during ontogeny is altered by relocation, T(l; 3)rasv genotypes and wild type controls were assayed at the same stages of development. The experimental design allowed a comparison of the ontogenic expression of three different genes--v+, Zw, and Pgd--through the activities of their associated enzymes. The results indicate that changing the gene's position may alter its ontogenic expression. Animals with v+ on the third chromosome have a unique peak of tryptophan pyrrolase activity in larvae which is not present in wild type. The activity in this peak is at l east 10 times higher than that observed in 72-hour wild type larvae, in fact, higher than that observed in any normal genotype at any time during development. With the exception of this peak, the developmental curves of enzyme activity are similar, although the relocated genes specify consistently lower enzyme activities than do normally positioned genes. The unique peak is not the result of a general physiological effect since the patterns of Zw and Pgd activity appear to be the same in wild type and translocated v+ genotypes. The relevance of the data to earlier studies and to models for gene regulation is discussed.