It is a major challenge to understand functional neuronal circuits across the whole brain. Existing methods for observing neuronal activity represent a major bottleneck in addressing biological problems. In our lab, we developed Swept Confocally Aligned Planar Excitation (SCAPE) microscopy, which offers the ability to image a large 3D volume (e.g. 1000x800x250um) at speeds exceeding 10 volumes per second. Used with different genetically encoded fluorescent indicators, SCAPE enables us to observe neuronal activity across the whole brain of different small animal models, or a much larger volume of intact cortex/tissue compared to traditional approaches. The unique single objective design and flexible system layout of SCAPE makes it simple to image different samples without complex sample preparation and restraint. During this thesis work, I collaborated with biology and neuroscience labs to develop and optimize a range of novel in-vivo/in-vitro neuroimaging applications using SCAPE microscopy. In particular, my research has focused on using SCAPE to image freely crawling Drosophila Melanogaster larvae, intact mouse olfactory epithelium, head fixed behaving adult Drosophila, larval zebrafish brain and beating heart, and the neuronal system of behaving C. elegans, all in collaboration with experts in these models from Columbia University and other research institutions. I also developed and optimized different sample preparations and experimental procedures to take full advantage of the high-speed 3D imaging capabilities and flexibility of SCAPE microscopy. Finally, I optimized computational and image analysis techniques for large scale 5D SCAPE imaging datasets, including 3D cell tracking, large scale 3D data motion correction/registration, and cellular level neuronal activity extraction with different dimensionality reduction methods. The experiments I have performed in different animal models have enriched the long-term development of SCAPE by providing valuable feedback for system improvement and dissemination, and pushing the SCAPE design towards a more interchangeable platform with diverse capabilities suitable for routine uses by our collaborators and the wider neuroscience community.

Abstract not available

Systemic lupus erythematosus (SLE) and Sj?gren?s syndrome (SS) are both heterogeneous autoimmune diseases with strong B cell aspects. A proportion of SLE and SS patients exhibit elevated serum BAFF (B cell activating factor of the TNF family); BAFF plays a key role in B cell homeostasis, survival and tolerance. BAFF transgenic (Tg) mice develop nephritis and salivary gland destruction that resemble aspects of SLE and SS respectively. Autoimmune disease development in BAFF Tg mice correlates with marginal zone (MZ) B cell expansion and the abnormal presence of MZ-like B cells outside of the spleen. The role of MZ B cells in BAFF induced autoimmune disease was analysed by crossing BAFF Tg mice with Lymphotoxin-β knockout mice (creating LTβ-BTg mice) which lack most peripheral lymph nodes, and also lack MZ B cells as a result of disrupted splenic architecture. LTβ-BTg mice were not protected against nephritis but exhibited reduced salivary gland infiltration and destruction. Indicating that the development of sialadenitis but not nephritis in BAFF Tg mice is MZ B cell dependent. Nephritis development in LTβ-BTg mice was associated with the detection of B-1 B cells in the inflamed kidneys. As B-1a B cell survival is dependent on the spleen, the contribution of B-1a B cells to nephritis development in BAFF Tg mice was assessed by crossing BAFF Tg mice to congenitally asplenic Hox11-/- mice (creating Hox11 -BTg mice). The absence of a spleen and B-1a B cells in Hox11-BTg mice delayed the nephritis development. In contrast, splenectomy of BAFF Tg mice at 12 weeks of age did not alter nephritis onset. In these mice B-1a B cells persisted in the peritoneal cavity and MZ-like B cells were detected in the periphery 8 months after surgery. In summary, nephritis development in BAFF Tg mice is unaltered by the absence of MZ B cells, but delayed in the absence of a spleen, MZ and B-1a B cells. Thus, B-1a and B-1b B cells may be potential targets for the treatment of nephritis in SLE patients with elevated BAFF.

[Truncated abstract] The design and development of effective anti-viral immunotherapies requires a comprehensive understanding of the cellular and molecular processes that are involved in the generation and regulation of immune responses. The fundamental objective of the immune system is to successfully complete the task of eliminating/controlling the invading pathogen without causing overt pathology. Cytomegaloviruses (CMVs) are large DNA viruses that are able to evade immune attack and persist lifelong within the host. In a healthy host, CMV causes an asymptomatic infection, but in instances of decreased immune functions, such as in newborns, acquired immunodeficiency syndrome (AIDS) patients and transplant recipients, the infection can result in serious morbidity and mortality. Thus, human CMV (HCMV) is a clinically important pathogen and an understanding of the pathogenesis, mechanisms of immune subversion and, importantly the cascade of immune events that ensue following infection is highly relevant. The studies presented in this thesis have provided useful insight into various aspects of viral immunity and it is hoped that they will assist in the design of more effective therapies against viruses of clinical importance. Genetic variability in humans can greatly influence anti-viral immune responses and the outcome of viral infection. ... Furthermore, these studies provide novel evidence that NK cells are also crucial for the control of virus in some organs of susceptible mice during early acute infection. The data reveals that both NK cells and CD8+ T cells utilise perforin- and IFN-? dependent control of MCMV. Furthermore, these studies provide novel evidence that protection mediated by Ly49H+ NK cells in resistant mice is dependent on perforin. Chapter 3 focuses on the biological relevance of Grz during MCMV infection. These studies found that GrzA and GrzB are essential components of the machinery involved in limiting MCMV during acute infection. These analyses also provide the first evidence suggesting that GrzM plays a role, albeit minor, in controlling MCMV replication. Furthermore, the current studies suggest that Grz can mediate direct antiviral activities independent of the induction of cell death in conjunction with perforin. Interestingly, in the absence of both GrzA and GrzB (GrzAB), mice were as susceptible to MCMV infection as perforin-deficient mice. However, unlike perforin-deficient mice, GrzAB-deficient mice controlled and survived the infection. In Chapter 4 the roles of perforin, GrzA and GrzB in anti-viral immunity and immunopathology during MCMV infection were examined. These studies show that NK cell-derived perforin is required to eliminate infected targets as well as activated effector cells, suggesting that NK cells are crucial not only in defensive immunity but also in limiting the immune activation that follows MCMV infection. In summary, the studies presented in this thesis define the significant role played by specific effector molecules in limiting MCMV replication during different stages of this viral infection. Furthermore, these studies provide novel evidence that perforin, GrzA and GrzB play distinct roles in defensive immunity and limiting immunopathology during MCMV infection.

Lymphomas and leukemias are the most common cancer in children and young adults and in utero exposure to carcinogens may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/Kg b.w., gavage) on gestation day 17. Significant mortalities in young offspring were observed due to T-cell lymphoma. Lung and liver tumors also were observed in survivors at 10 months of age. To assess the role of the Aryl Hydrocarbon Receptor (AHR), we utilized crosses of B6129SF1/J (responsive) mice with strain 129S1/SvImJ (non-responsive). Offspring born to AHR non-responsive mothers had greater susceptibility to lymphoma, irrespective of offspring genotype. Responsive offspring displayed increased mortality if the mother was responsive. Lung adenomas showed Ki-ras mutations and exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. To examine the risk/benefit of maternal dietary phytochemical treatment against transplacental cancer, 2000 ppm indole-3-carbinol (I3C) was given to pregnant mice through diet from gestation day 9 till weaning. I3C significantly lowered mortality caused by lymphomas regardless of the maternal genotype, and also reduced lung tumor multiplicity in offspring born to AHR [superscript b-l/d] dams. Distribution of I3C in most maternal and fetal tissues was quantified following a single gavage of [¹⁴C]-I3C to the pregnant mice. DBP-DNA adducts were observed in both maternal and fetal tissues by ³³P postlabeling and HPLC analysis and were modulated by I3C and AHR genotype. I3C also modulated phase I and phase II enzyme protein expression in dams and gene expression in newborn thymus. I3C chemoprotection may involve modification of the bioavailability of DBP to the fetus and/or modulation of gene expression in the fetus as well. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice. These results raise the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults and, that the addition of chemoprotective agents to the maternal diet may reduce cancer risk among offspring. / Graduation date: 2006

Antibodies neutralize and eliminate pathogens, malignancies, and toxins by acting either alone or in association with Fc receptors which, once engaged, activate the elimination mechanisms of phagocytic cells. Based on structural differences, antibodies are divided into functionally distinct classes (IgM, IgD, IgG, IgE and IgA). Structure-function relationships within these classes are not well characterized. In addition, animal models for the assessment of potential therapeutic strategies for the modulation of the interaction between antibodies and Fc receptors are not established. Nonhuman primates are widely used to model human diseases and, represent excellent in vivo systems for this assessment. Therefore, we have studied nonhuman primate IgD as well as IgG and IgA specific Fc receptors in rhesus macaques, cynomolgus macaques, baboons and sooty mangabeys. IgD genes had not been identified in nonhuman primates nor the IgD receptors characterized in any species. We characterized IgD genes of the four monkey species, as well as chimpanzees and dogs. In contrast to other antibody classes, the IgD hinge regions are highly conserved between human and nonhuman primates, thus indicating a role in Fc receptor binding. In humans, Fc receptors CD16a (natural killer cells) and CD16b (neutrophils) bind IgG1 and IgG3, and CD89 (myeloid cells) binds IgA. To assess ligand binding and glycosylation properties of nonhuman primate CD16a, CD16b, and CD89, we sequenced, cloned, and generated recombinant molecules in a mammalian expression system. Our results verify the presence of CD16a, but not CD16b in nonhuman primates. CD16a is expressed on monocytes and a subpopulation of lymphocytes. In sooty mangabeys, CD16 is also expressed on neutrophils. Recombinant sooty mangabey/baboon CD16a binds to human IgG1 and IgG2, but not IgG3 and IgG4. Monkey CD89 has the same peripheral blood leukocyte expression profiles as humans, and binds human and recombinant macaque IgA. Blocking of N-glycans inhibited expression of CD89, but only marginally CD16a expression. Although extensive similarities of antibody/Fc receptor interactions exist between human and nonhuman primates, several differences must be considered when evaluating therapeutic strategies. However, these differences can be exploited to further characterize the structure-function relationships existing within antibody molecules and respective receptors.

The activities of phosphohexose isomerase (EC. 5.3.1.9), isocitrate dehydrogenase (EC. 1.1.1.42) and lactate dehydrogenase (EC. 1.1.1.27) were measured in 14 normal and 21 tumorous mouse mammary tissue samples. Methods of tissue extraction and activity determination in this study employed equipment found in most clinical laboratories.For each of the three enzymes there was a statistically significant (p <.05) elevation of the tumor sample group's activities. The activities of the normal tissues were: PHI x = 9.6 and SD = 4.6, ICD x = 13.2 and SD = 5.3, and IDH x = 10.9 and SD = 5.3. The activities of the tumorous tissues were: PHI x = 55.2 and SD = 29.9, ICD x = 40.5 and SD = 23.8, and IDH x = 55.8 and SD = 31.4.Using values of 20, 27, and 21 for the upper limit of normal activity (x + 2SD) for PHI, ICD, and LDH respectively, the tissue samples can be divided into normal and tumorous groups with l00% sensitivity and 85% specificity.Ball State UniversityMuncie, IN 47306

Lawsonia intracellularis causes proliferative enteropathies in juvenile mammals. The porcine (PPE) and equine (EPE) diseases are worldwide. Rabbits and hamsters are naturally susceptible, the latter being a classic modeling-host for PPE. None is known for EPE, besides foals. An in vitro evaluation of antimicrobial efficacy against L. intracellularis is difficult. This study aimed to validate a laboratory animal EPE model and to investigate pharmacokinetics (PK) and efficacy of gallium maltolate (GaM) as an alternative antimicrobial therapy. Infected animals were inoculated with cell-cultured L. intracellularis and infection was verified with clinically utilized diagnostic tests. Initially, 2 groups of EPE-infected rabbits were compared to 1 uninfected group. After inoculation (PI), EPE-infected rabbits showed mild clinical signs; detectable seroconversion, fecal shedding, gross lesions in intestinal tissues (IT), and early immuno-histochemistry labeling of L. intracellularis antigen. Thus, a humane EPE-rabbit model was achieved. Subsequently, EPE-infected hamsters were compared to uninfected and PPE-infected hamsters; whereas, PPE-infected rabbits were compared to EPE-infected rabbits. EPE-hamsters did not develop infection, unlike PPE-infected controls; and PPE-rabbits did not develop IT lesions or seroconversion comparable to EPE-rabbits. Therefore rabbits were chosen as the EPE modeling-host for the GaM studies. First, GaM PK and IT concentrations of Ga and Fe were measured. Then, GaM efficacy was compared to a current EPE antimicrobial treatment. During sampling, the intra-arterial catheters in the rabbits’ ears were protected with a novel moleskin-cover, allowing repeated sampling while minimally restrained. The PK study was based on the comparison of EPE-infected and uninfected rabbits, after a single treatment with GaM, collection of serial blood samples and IT samples. The only differing PK parameter, between groups, was a decrease in the terminal phase rate constant of the EPE-rabbits, so a 48h dosing interval was chosen for the efficacy study. In the efficacy study, 3 groups of EPE-infected rabbits were treated with GaM, doxycycline and a placebo, respectively. No differences were noted between treatments, in terms of lesions and fecal shedding. GaM appears no more efficacious than doxycycline in EPE- rabbits. In conclusion, albeit GaM tolerance appeared adequate in rabbits, results do not support its use in EPE-infected animals.

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences