Although feeding is important for optimal development and growth in insects, there are several points during the insect life cycle that are non-feeding: metamorphosis, pupation, and overwintering. Non-feeding periods also occur in response to internal cues, such as feedback from nutrient thresholds and immune responses being activated. Additionally, as an insect goes through different developmental stages, its nutritional requirements change in response to or in preparation for non-feeding periods. Most physiological responses like feeding are regulated through an interconnection of pathways, but how these networks change in response to different energy demands, such as immune challenges or changes in metabolism, is poorly understood. One significant pathway that is involved in regulating several physiological processes is the insulin signaling pathway. In my dissertation research, I tested hypotheses explaining the regulation of physiological processes during non-feeding periods in two agriculturally relevant insects, Manduca sexta and Megachile rotundata. First, I investigated how internal cues such as dietary lipid content and immune challenges cause non-feeding periods in M. sexta. Then, I investigated how insulin signaling regulates development during a non-feeding period like overwintering changes in M. rotundata. Since the insulin signaling (IIS) pathway is critical for development and growth, I focused on testing if this pathway plays a role in regulating non-feeding periods. My research showed that increased dietary lipid content causes a cessation of feeding, which suggests there is a possible lipid threshold that when reached, causes M. sexta to switch from lipid consumption for storage to lipid excretion. When looking at another cue like immune challenges, my results showed that during a bacterial infection, a Toll-mediated suppression of IIS pathway may be regulating feeding and causing a non-feeding period exhibited as sickness-induced anorexia. Lastly, my results also showed that the IIS pathway is suppressed in overwintering M. rotundata, and that this process can change in response to temperature. Overall, my dissertation research showed that the insulin signaling pathway and nutrient content play a vital role in regulating non-feeding periods. Investigating insulin signaling, lipid metabolism, and innate immunity in these species closes a gap in knowledge of invertebrate development.

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No description available.

Streptococcus pneumoniae (Spn) is the most common etiology of bacterial pneumonia, which is one of the leading causes of death in children and the elderly worldwide. During non-lethal infections with Spn, immune cells accumulate in the lungs and protect against reinfection with more lethal strains, this protection is termed heterotypic immunity. Lymphocyte populations such as resident memory T cells and resident memory B cells are known to be crucial for heterotypic immunity, but their diversity remains understudied. Here, we aimed to elucidate resident lymphocyte heterogeneity in the lungs after recovery from pneumococcal pneumonia, and their contributions to heterotypic immune protection. We developed a comprehensive immunophenotyping panel for full-spectrum flow cytometry (FSFC) to identify novel subsets of lymphocytes and combined it with an unbiased analysis approach. With this tool we discovered that murine lungs were enriched for unexpected subsets of resident lymphocytes, and we defined CD73 as a potential lymphocyte residence marker. We discovered a novel subset of CD4+ T cells defined by the phenotype CD11a+CD69+GL7+, which corresponded to a significant proportion of lung CD4+ TRM cells. Initial analyses demonstrated GL7+ T cells resembled CD4+ TRM cells. Functional studies revealed that unlike GL7- TRM subsets that were mostly RORT+, GL7+ TRM cells were also Gata-3+ and/or T-bet+ could secrete type 2 or type 1 cytokines, suggesting they were poised to be TH2 or TH1-like in function. This study emphasizes the use of a multiparameter panel for FSFC as tool to identify novel lymphocyte subsets. We conclude that the environment of pneumonia-recovered lungs contains heterogeneous subsets of resident lymphocytes, including GL7+ TRM cells. We propose these subsets may contribute to lung immunity in unique ways and may be important players in serotype-independent protection to pneumococcal pneumonia. / 2024-01-23T00:00:00Z

Plants have several defense systems used as protection from environmental stressors, and the plant cell wall (CW) is considered the “frontline” during attack. The CW is a complex structure essential for growth and development and is composed of components such as cellulose, polysaccharides, pectin, and lignin. Lignin is the central source of strength for the CW and helps form a water-impermeable barrier for defense. The extent of plant CW defense, immunity, and the components involved still need further investigation. Integrin-Linked Kinases (ILKs), a subfamily of Raf-like kinases (RAFs), are thought to participate in signal-processing pathways of the CW and plasma membrane via integrin-like receptors (ILRs). Histological characterization was used to observe how ILKs participate in CW surveillance, and an analysis revealed that ILK1 and ILK4 are negative regulators of isoxaben-induced lignin accumulation, while ILK5ox is positive. Collectively, ILK1, ILK4, and ILK5 play important roles in the modulation of CW structure.

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No description available.

No description available.

Bacillus anthracis is a facultative extracellular bacterial pathogen that causes cutaneous, gastrointestinal or respiratory disease in many vertebrates, including humans. Commercially available anthrax vaccines for immunization of humans are known to provide protection of limited duration and may not protect against the respiratory form of the disease. Commercially available live vaccines for animals have been shown to cause disease in certain species. Brucella abortus is a facultative intracellular bacterium that causes chronic infection in animals and humans. As with other intracellular pathogens, cell mediated immune responses (CMI) are crucial in affording protection against brucellosis. B. abortus strain RB51 has been shown to be useful in eliciting protective CMI and antibody responses against Brucella in cattle and other animal species. Since the protective antigen (PA) of B. anthracis is known to induce antibodies, the pag gene encoding PA was expressed in B. abortus RB51, producing a dual vaccine to protect against both brucellosis and anthrax. In a previous study, the entire pag gene was expressed in strain RB51 and following immunization the vaccine induced antibodies against PA in A/J mice. However, PA stability and protective efficacy were less than desirable as only 1/6 were protected. The studies in this dissertation involved synthesizing a gene corresponding to domain 4 (PA4) of the pag gene utilizing the native codon usage of Brucella. The PA4 domain was fused to Brucella signal sequences of Brucella 18kDa protein, superoxide dismutase or no signal sequence to localize the PA4 to the outside cell envelope, periplasmic space or cytosol respectively. Comparisons of the expression level and stability of the native and synthetic PA4 in B. abortus strain RB51 were assessed by immunoblot. The protective efficacy of PA4 expressed in Brucella was assessed by immunization and protection studies in A/J mice against a live challenge with either B. abortus or B. anthracis Sterne spores. ELISA and western blot indicate the induction of PA specific antibodies by these recombinant strain RB51 vaccine constructs. Results based on subisotype antibody ELISA (IgG, IgG1, IgG2a and IgM) and CMI assays (cytokine ELISA of IL-4 and INF-g, and LPA) suggest a Th1 based immune response to strain RB51 and PA. B. abortus strain RB51 expressing PA4 fused to the signal sequence of Brucella 18kDa protein was able to induce 50% protection, while strain RB51 expressing PA4 with no signal sequence gave 17% protection against B. anthracis Stern spore challenge. Mice were boosted with an intraperitoneal injection of purified PA after an initial immunization with Brucella vaccine candidates, sterile saline or pure PA. Protection assessed by live challenge with B. anthracis Sterne spores increased following boosting with PA in 4 cases. Immunization with purified PA, and 3 strain RB51/PA vaccines and a PA boost gave protection against a spore challenge ranging from partial to full. This study suggests that additional work is needed to define the antigens of B. anthracis involved in the induction of specific CMI. / Ph. D.

Two studies were conducted on endurance horses predominantly of Arabian breeding participating in an 80 km ride dedicated to research in April 2001 (Trial 1) and April 2002 (Trial 2). Objectives were to determine effects of endurance exercise, antioxidant supplementation, and a feed rich in fiber and fat vs. a high fat sweet feed on immunoglobulin A and G concentrations as well as identify selective IgA deficiency in endurance horses of Arabian breeding. There were no effects of distance in Trial 1 on IgA (P = 0.73) or IgG (P = 0.18) concentrations. In Trial 2, IgA concentrations increased (P = 0.05) and IgG concentrations increased (P = 0.006) after the start of the race. There were no effects of antioxidant supplementation on IgA (P = 0.16), IgG (P = 0.16), and IgM (P = 0.70) concentrations. There were no diet effects on IgA (P = 0.80), IgG (P = 0.59), and IgM (P = 0.54) concentrations. There were horses in both trials that were deficient in IgA only. Concentrations of IgG and IgM were within normal ranges, and there were no differences in training, performance and transportation variables, IgG concentrations, antioxidant supplementation, and feed supplementation compared to the horses with normal IgA concentrations. The concentration of IgM was higher in IgA deficient horses in Trial 1 (P = 0.035) and Trial 2 (P = 0.017). Horses with deficient IgA tended to be associated with health problems commonly found in humans and dogs affected with selective IgA deficiency. / Master of Science