Doctor of Philosophy (PhD) / BACKGROUND: The accumulation of abnormal extracellular deposits beneath the retinal pigment epithelium characterises the pathology of early age-related macular degeneration. However, the histopathological threshold at which age-related changes become early AMD is not defined, and the effect of each of the deposits (basal laminar deposit and membranous debris) on disease progression is poorly understood. Evidence suggests that macrophages play a key role in the development of AMD lesions, but the influence of basal laminar deposit (BLamD) and membranous debris on the recruitment and programming of local macrophages has not been explored. Although evidence also suggests that inflammation and innate immunity are involved in AMD, the significance of anti-retinal autoantibodies to disesase pathogenesis is not known. AIMS: (i) To determine the histopathological threshold that distinguishes normal ageing from early AMD; (ii) to determine the influence of BLamD and membranous debris on disease progression; (iii) to examine whether distinct early AMD phenotypes exist based on clinicopathological evidence; (iv) to determine the histopathological context in which Bruch’s membrane macrophages first found; (v) to examine the relationship between Bruch’s membrane macrophages and subclinical neovascularisation; (vi) to determine if the progressive accumulation of BLamD and membranous debris alters the immunophenotype of Bruch’s membrane macrophages and/or resident choroidal macrophages; (vii) to determine if the anti-retinal autoantibody profile differs significantly between normal individuals and those with early AMD, neovascular AMD or geographic atrophy; (viii) to examine whether baseline anti-retinal autoantibodies can predict progression to advanced AMD in individuals with early AMD; and (ix) to examine whether baseline anti-retinal autoantibodies can predict vision loss in individuals with neovascular AMD. METHODS:Clinicopathological studies were performed to correlate progressive accumulation of BLamD and membranous debris to fundus characteristics and visual acuity, as well as to sub-macular Bruch’s membrane macrophage count. Immunohistochemical studies were perfomed to determine whether the presence of BLamD and membranous debris altered the programming of Bruch’s membrane or resident choroidal macrophages. The presence of serum anti-retinal autoantibodies was determined by western blotting, and the association with disease progression examined in early and neovascular AMD. RESULTS: The presence of both basal linear deposit (BLinD) and a continuous layer of BLamD represents threshold early AMD histopathologically, which was seen clinically as a normal fundus in the majority of cases. Membranous debris accumulation appeared to influence the pathway of progression from early AMD to advanced AMD. Bruch’s membrane macrophages were first noted when a continuous layer of BLamD and clinical evidence of early AMD were present, and increased with the amount of membranous debris in eyes with thin BLamD. Eyes with subclinical CNV had high macrophage counts and there was some evidence of altered resident choroidal macrophage programming in the presence of BLamD and membranous debris. Serum anti-retinal autoantibodies were found in a higher proportion of early AMD participants compared with both controls and participants with neovascular AMD, and in a higher proportion of individuals with atrophic AMD compared to those with neovascular AMD. The presence of baseline anti-retinal autoantibodies in participants with early AMD was not associated with progression to advanced AMD. Participants with neovascular AMD lost more vision over 24 months if they had IgG autoantibodies at baseline compared to autoantibody negative participants. CONCLUSIONS: The finding that eyes with threshold early AMD appear clinically normal underscores the need to utilise more sophisticated tests to enable earlier disease detection. Clinicopathological evidence suggests two distinct early AMD phenotypes, which follow two pathways of AMD progression. Macrophage recruitment and programming may be altered by the presence of BLamD and membranous debris, highlighting the need to further characterise the biology of human resident choroidal macropahges. Anti-retinal autoantibodies can be found in both control and AMD sera, and future approaches that allow the examination of subtle changes in complex repertoires will determine whether they are involved in AMD disease pathogenesis.

Doctor of Philosophy (PhD) / BACKGROUND: The accumulation of abnormal extracellular deposits beneath the retinal pigment epithelium characterises the pathology of early age-related macular degeneration. However, the histopathological threshold at which age-related changes become early AMD is not defined, and the effect of each of the deposits (basal laminar deposit and membranous debris) on disease progression is poorly understood. Evidence suggests that macrophages play a key role in the development of AMD lesions, but the influence of basal laminar deposit (BLamD) and membranous debris on the recruitment and programming of local macrophages has not been explored. Although evidence also suggests that inflammation and innate immunity are involved in AMD, the significance of anti-retinal autoantibodies to disesase pathogenesis is not known. AIMS: (i) To determine the histopathological threshold that distinguishes normal ageing from early AMD; (ii) to determine the influence of BLamD and membranous debris on disease progression; (iii) to examine whether distinct early AMD phenotypes exist based on clinicopathological evidence; (iv) to determine the histopathological context in which Bruch’s membrane macrophages first found; (v) to examine the relationship between Bruch’s membrane macrophages and subclinical neovascularisation; (vi) to determine if the progressive accumulation of BLamD and membranous debris alters the immunophenotype of Bruch’s membrane macrophages and/or resident choroidal macrophages; (vii) to determine if the anti-retinal autoantibody profile differs significantly between normal individuals and those with early AMD, neovascular AMD or geographic atrophy; (viii) to examine whether baseline anti-retinal autoantibodies can predict progression to advanced AMD in individuals with early AMD; and (ix) to examine whether baseline anti-retinal autoantibodies can predict vision loss in individuals with neovascular AMD. METHODS:Clinicopathological studies were performed to correlate progressive accumulation of BLamD and membranous debris to fundus characteristics and visual acuity, as well as to sub-macular Bruch’s membrane macrophage count. Immunohistochemical studies were perfomed to determine whether the presence of BLamD and membranous debris altered the programming of Bruch’s membrane or resident choroidal macrophages. The presence of serum anti-retinal autoantibodies was determined by western blotting, and the association with disease progression examined in early and neovascular AMD. RESULTS: The presence of both basal linear deposit (BLinD) and a continuous layer of BLamD represents threshold early AMD histopathologically, which was seen clinically as a normal fundus in the majority of cases. Membranous debris accumulation appeared to influence the pathway of progression from early AMD to advanced AMD. Bruch’s membrane macrophages were first noted when a continuous layer of BLamD and clinical evidence of early AMD were present, and increased with the amount of membranous debris in eyes with thin BLamD. Eyes with subclinical CNV had high macrophage counts and there was some evidence of altered resident choroidal macrophage programming in the presence of BLamD and membranous debris. Serum anti-retinal autoantibodies were found in a higher proportion of early AMD participants compared with both controls and participants with neovascular AMD, and in a higher proportion of individuals with atrophic AMD compared to those with neovascular AMD. The presence of baseline anti-retinal autoantibodies in participants with early AMD was not associated with progression to advanced AMD. Participants with neovascular AMD lost more vision over 24 months if they had IgG autoantibodies at baseline compared to autoantibody negative participants. CONCLUSIONS: The finding that eyes with threshold early AMD appear clinically normal underscores the need to utilise more sophisticated tests to enable earlier disease detection. Clinicopathological evidence suggests two distinct early AMD phenotypes, which follow two pathways of AMD progression. Macrophage recruitment and programming may be altered by the presence of BLamD and membranous debris, highlighting the need to further characterise the biology of human resident choroidal macropahges. Anti-retinal autoantibodies can be found in both control and AMD sera, and future approaches that allow the examination of subtle changes in complex repertoires will determine whether they are involved in AMD disease pathogenesis.

<p>Bronchiolitis Obliterans (BO) is a major cause of chronic airway dysfunction after toxic chemical inhalation. The pathophysiology of BO is not well understood, but epithelial cell injury has been closely associated with the development of fibrotic lesions in human studies and in animal models of both toxin- and transplant-induced BO. However, while almost all cases and models of BO include epithelial injury, not all instances of epithelial injury result in BO, suggesting that epithelial damage per se is not the critical event leading to the development of BO. In this dissertation, we describe a model of chlorine (Cl2)-induced BO in which mice develop tracheal and large airway obliterative lesions within 10 days of exposure to high (350 ppm), but not low (200 ppm), concentrations of Cl2 gas. Lesions develop in a series of well-demarcated pathological changes that include epithelial denudation, inflammatory cell infiltration by day 2 after exposure, fibroblast infiltration and collagen deposition by day 5, and in-growth of blood vessels by day 7, ultimately leading to lethal airway obstruction by days 9-12. Using this model, we were able to test our hypothesis that loss of epithelial progenitor cells is a critical factor leading to the development of obliterative airway lesions after chemical inhalation. Indeed, these lesions arise only under conditions and in areas in which basal cells, the resident progenitor cells for large airway epithelium, are eliminated by Cl2 exposure. </p><p>The molecular pathways contributing to BO development are not well understood. Mechanisms of epithelial injury differ across BO models, but we hypothesized that after the inciting injury, BO models share common pathways. We compared microarray analysis from day 5 non-BO- and BO-inducing chemical injuries and subsequently identified biological pathways that may contribute to BO pathogenesis. Our findings add support to pathways previously implicated in BO development and more importantly, suggest potential new pathways and molecular mediators of BO. Furthermore, we evaluated the efficacy of therapeutic inhibition of neovascularization or inflammation to prevent Cl2-induced BO. To date, our therapeutic interventions were ineffective. Nonetheless, our findings suggest that in the context of Cl2-induced BO, vascular endothelial growth factor receptor 2 (a mediator of neovascularization) and inducible nitric oxide synthase (a mediator of inflammation) are not critical in BO pathogenesis.</p><p>In sum, our work introduces and characterizes a novel Cl2-induced murine model of BO. Using this model we demonstrated that in the absence of basal cells, epithelial regeneration does not occur and regions of epithelial denudation persist from which an aberrant repair process is initiated, leading to obliterative airway lesions. Our findings suggest that, irrespective of the cause, loss of epithelial progenitor cells may be a critical factor leading to the development of BO. Furthermore, our gene expression analysis implicates novel mediators and signaling pathways in the development of BO. Our analysis lays the foundation for more rigorous exploration of these targets in the pathogenesis of BO.</p> / Dissertation

Located in the mesopontine tegmentum, the pedunculopontine tegmental nucleus (PPTg) is comprised principally of glutamatergic, cholinergic and GABAergic neurons. In addition to being fully integrated into basal ganglia, PPTg projects to thalamus and motor output sites in the brainstem. Previous studies have shown a range of behavioural changes after PPTg manipulation. Prominent amongst these is an apparent deficit in the ability to learn the consequences of actions. PPTg is divisible into a posterior component (pPPTg) in receipt of rapid polymodal sensory input and projecting into VTA/SNc dopamine neurons and an anterior component (aPPTg) in receipt of basal ganglia outflow and projecting into SNc and lower brainstem structures. The research described here assesses the role of the pPPTg in instrumental learning. Using a contingency degradation paradigm, it was shown that inactivation of the pPPTg (by muscimol microinfusion) specifically blocked the updating of associations between actions and outcomes, without the affecting the ability to re-execute previously learned instrumental actions. Selective bilateral destruction of pPPTg cholinergic neurons (with the fusion toxin diphtheria toxin – urotensin II [Dtx-UII]) resulted in >90% loss of pPPTg cholinergic neurons. These lesions produced no detectable changes on any measured aspect of an instrumental learning task consisting of various fixed and variable ratio schedules of reinforcement and extinction. Subsequent experiments found that the same selective cholinergic pPPTg lesions also produced no changes in the locomotor response to nicotine or rate of nicotine sensitisation. These results are the first to demonstrate a brainstem role in action-outcome learning. Results support the view that PPTg performs a ‘first pass' analysis on incoming sensory data and interfaces salient aspects of this with appropriate basal ganglia and brainstem circuitry, with glutamatergic pPPTg projections sending an essential signal and cholinergic projections performing as part of a wider modulatory system.

Contemporary Spanish basal readers, published for use in elementary bilingual Spanish/English classrooms in the United States, reflect a technological view of curriculum, a behavioristic view of learning, and a part to whole view of reading. Although teacher's guides and promotional materials for the basal series make reference to recent reading theory and research, the basal materials themselves, when examined from a theoretical perspective, demonstrate little understanding of the reading process. Six basal reading programs, published since 1980, were studied: Addison Wesley's Hagamos Caminos; Scott Foresman's Focus: Leer para triunfar; Houghton Mifflin's Programa de lectura en espanol de Houghton Mifflin; Macmillan's Mil Maravillas; Economy's Economy Spanish Reading Program; and Santillana's Lectura en dos idiomas. Each series was surveyed using the Program Profile Continuum Survey and then analyzed in depth with the Spanish Program Profile Instrument. Both evaluation instruments focus on reading, language, learning, and teaching theory. The results of the study of the six series suggest the programs are more alike than different. These similarities can be summarized as follows: (1) Despite the fact that the series approach print differently, the ultimate goal of skill exercises in all the series is word identification. (2) Recent comprehension theory is discussed in the teacher's guides, but comprehension questions reflect the idea that comprehension is a product rather than a process. (3) Student text is carefully controlled and often repetitious because the assumption is that language is habit. (4) The language of the majority of the student text in all of the series is adapted. (5) The materials reflect the view that the teacher is a technician leading the passive learner. (6) The scope and sequence of the programs sets many students up for failure because each step is dependent upon mastery of the previous steps. The materials of the Spanish basal reading programs studied do not reflect the current state of knowledge about the reading process in Spanish. Alternate programs using a whole language approach to learning would allow Hispanic students to choose their own reading, write their own stories, and become literate in their first language.

This research is a content analysis of six recently published basal reader series, each of which begins with a readiness/priming sequence that starts with letters, words, connected strings of words, and a few pages of activities which might be considered general readiness. While this concept of readiness for reading reflects a word recognition view of reading, marketing promotions include eclectic statements regarding philosophy, materials, and methods which reflect an early childhood view of child development and meaning construction. Publishers imply that reading is a process of constructing meaning, but they supply materials for both students and teachers which neither encourage nor allow for meaningfulness. While descriptions of the series studied promise special and unique approaches to readiness, they are very similar to one another in every aspect of readiness. Each provides an overwhelming proportion of activities and workbook pages with the major instructional focus on letters and words. There are minor differences in connected text among the various series, but those differences are primarily in regard to the specific type of vowel control used. Considerations about the syntactic and semantic language systems follow the same formulae throughout the industry. Publishers of basal readers hire professional educators to quote research and lend credibility to their "scientifically" designed programs, but the published materials often contradict statements made by these professionals. While there is some indication in the manuals and promotional materials that suggest knowledge on the part of editors and authors concerning research in the areas of emergent literacy and psycholinguistic theory, there is little within the materials and methods that reflects this knowledge.

Over the years there have been many criticisms of the contents of basal reading series. This study sought to describe the contents of basal readers by analyzing the student reading materials contained in the books for grades one, three, and five of three current basal readers, published by D. C. Heath, Houghton Mifflin, and Silver, Burdett, and Ginn. It determined the amount of material written for the basals and the amount of material originating in other literature sources. It also described the amount and types of adaptation to the text and visual displays in material that originated elsewhere. Further, it calculated the amount of material that was classified into each of seven literature genres and six writing types using number of selections, pages, and words as units of analysis. While other researchers have each investigated some of the points examined here, none has explored all and none has used all three units of analysis to describe the contents of basal readers. Each of the student reading selections was analyzed as to the number of pages and words and then classified into the following categories: written for the basal or taken from other literature sources, one of seven literature genres or an added reading instruction category, and one of six writing types. Material that had its origins outside the basal reader was located and compared to the basal version with notations for each adaptation to text or visual display. The adaptations were classified into twenty two different types of text adaptation or six different types of visual display adaptation. While less than half of the reading material was created expressly for the basal readers examined, most of the material from other literature sources was adapted in some way. The largest category of adaptation was deletions. Overall, while there were selections in each of the categories for literature genre and writing type, the distribution was uneven. For almost all of the points examined, individual differences were found among the three reading series and the grade levels in all of the means of analysis.

<p>Finding and consuming food and water are among the most critical functions for an animal's survival. Food seeking (e.g., exploration and approach) and consummatory (e.g., licking, chewing, swallowing) behaviors are usually highly controlled, resulting in stable food intake, body mass, and fat stores in humans and laboratory animals. These variables are thought to be governed by homeostatic control systems that closely regulate many aspects of feeding behavior. However, the homeostatic mechanisms underlying these processes are often disrupted in humans, resulting in either hyperphagia or hypophagia. Despite many decades of investigations into the regulatory circuits of animals and humans, the neural circuits that underlie voluntary feeding are unclear. There have been considerable advances into understanding how the brain is able to broadly regulate food consumption (e.g., the role of circulating hormones on food intake and body weight). As much work has focused on hypothalamic mechanisms, relatively little is known about how other neural systems contribute to specific aspects of food seeking and consumption. </p><p> The basal ganglia have been implicated in many aspects of motivated behavior including appetitive and consummatory processes. However, the precise role that basal ganglia pathways play in these motivated behaviors remain largely unknown. One reason for this is that the basal ganglia are functionally and anatomically heterogeneous, with distinct functional circuit elements being embedded within overlapping tissue. Until recently, tools permitting identification and manipulation of molecularly defined neuron populations were unavailable. </p><p> The following experiments were designed to assess the role of the basal ganglia in regulating appetitive and consummatory behavior in mice. The first experiment (Chapter 2) examines the relationship between neural activity in the substantia nigra¬, a¬ major output nucleus of the basal ganglia, and an animal's motivational state. Both dopaminergic and GABAergic neurons show bursts of action potentials in response to a cue that predicts a food reward in hungry mice. The magnitude of this burst response is bidirectionally modulated by the animal's motivational state. When mice are sated prior to testing, or when no pellets can be consumed, both motivational state and bidirectional modulation of the cue response are unchanging. </p><p> The second set of experiments (Chapter 3 and 4) utilizes a mouse model of hyperdopaminergia: Dopamine transporter knockout mice. These mice have persistently elevated synaptic dopamine. Consistent with a role of dopamine in motivation, hyperdopaminergic mice exhibit enhanced food seeking behavior that is dissociable from general hyperactivity. Lentiviral restoration of the dopamine transporter into either the dorsolateral striatum or the nucleus accumbens, but not the dorsomedial striatum, is sufficient to selectively reduce excessive food seeking. The dopamine transporter knockout model of hyperdopaminergia was then used to test the role of dopamine in consummatory processes, specifically, licking for sucrose solution. Hyperdopaminergic mice have higher rates of licking, which was due to increased perseveration of licking in a bout. By contrast, they have increased individual lick durations, and reduced inter-lick-intervals. During extinction, both knockout and control mice transiently increase variability in lick pattern generation while reducing licking rate. Yet they show very different behavioral patterns. Control mice gradually increase lick duration as well as variability in extinction. By contrast, dopamine transporter knockout mice exhibited more immediate (within 10 licks) adjustments--an immediate increase in lick duration variability, as well as more rapid extinction. These results suggest that the level of dopamine can modulate the persistence and pattern generation of a highly stereotyped consummatory behavior like licking, as well as new learning in response to changes in environmental feedback. </p><p> The final set of experiments was designed to test the relationship between consummatory behavior and the activity of GABAergic basal ganglia output neurons projecting from the substantia nigra pars reticulata to the superior colliculus, an area that has been implicated in regulating orofacial behavior. Electrophysiological recording from mice during voluntary drinking showed that activity of GABAergic output neurons of the substantia nigra pars reticulata reflect the microstructure of consummatory licking. These neurons exhibit oscillatory bursts of activity, which are usually in phase with the lick cycle, peaking near the time of tongue protrusion. Dopaminergic neurons, in contrast, did not reflect lick microstructure, but instead signaled the boundaries of a bout of licking. Neurons located in the lateral part of the superior colliculus, a region that receives direct input from GABAergic projection neurons in the substantia nigra pars reticulata, also reflected the microstructure of licking with rhythmic oscillations. These neurons, however, showed a generally opposing pattern of activity relative to the substantia nigra neurons, pausing their firing when the tongue is extended. To test whether perturbation of the nigrotectal pathway could influence licking behavior, channelrhodopsin-2 was selectively expressed in GABAergic neurons of the substantia nigra and the axon terminals within the superior colliculus were targeted with optic fibers. Activation of nigrotectal neurons disrupted licking in a frequency-dependent manner. Using optrode recordings, I demonstrate that nigrotectal activation inhibits neurons in the superior colliculus to disrupt the pattern of licking. </p><p> Taken together, these results demonstrate that the basal ganglia are involved in both appetitive and consummatory behaviors. The present data argue for a role of striatonigral dopamine in regulating general appetitive responding: persistence of food-seeking. Nigraltectal GABA neurons appear to be critical for consummatory orofacial motor output.</p> / Dissertation

Ciliopathies are a group of disorders that arise from ciliary dysfunction. Meckelin (MKS3 or TMEM67) is a conserved transmembrane protein found at the transition zone of ciliated cells. In humans MKS3 is one of 3 genes linked to the ciliopathy Meckel Syndrome. This disease is characterized by occipital meningioencephalocoele, polycystic kidneys, fibrotic changes to the liver, postnatal polydactyly and situs inversus. Paramecium tetraurelia is a single celled ciliated eukaryote. Its surface is organized of a meshwork of cortical units that run the length of the cell. At the center of the cortical units are either one or two basal bodies. In two basal body units only the posterior basal body is ciliated. From the ciliated basal body, three rootlets project in stereotypical orientations: the post-ciliary rootlet projects posteriorly, the transverse microtubule projects toward the adjacent basal body row and the striated rootlet projects anteriorly. Both the post-ciliary rootlet and transverse microtubule are microtubule-based structures. The striated rootlet is composed of multiple subunits that are predicted to have conserved segmented coiled coil domains known as SF-Assemblin domains. In Picariello at al., 2014, we showed that MKS3 is present in the transition zone of Paramecium tetraurelia and that RNAi for MKS3 leads to global ciliary loss. Additionally, RNAi for MKS3 results in the disorganization of the basal body rows. Within the areas of disorganization, the basal bodies along with their striated rootlets, post-ciliary rootlets and transverse microtubules are rotated away from their expected orientation. Interestingly, the post-ciliary rootlet and transverse microtubule are still attached at the expected angles relative to each other within the areas of disorganization. Initial GST pull-down experiments using the coiled coil domain of MKS3 suggest a potential interaction between MKS3 and the striated rootlet family members KdC1 and KdB2. To test potential interactions between MKS3 and the striated rootlet we identified 27 potential striated rootlet family members in Paramecium. Full-length sequences for 13 of these genes were marked at their N-terminus with a 3x FLAG sequence. Components with a conserved SF-Assemblin domain were distributed uniformly within the striated rootlet. Components lacking the SF-Assemblin domain were found in various cellular locations, but not within the striated rootlet. GST pull-down experiments utilizing the MKS3 C-terminus as bait were performed using cells expressing the FLAG-tagged striated rootlet family members. Unfortunately a clear interaction between MKS3 and the striated rootlet remains elusive. The organized nature of the surface of Paramecium has allowed us to identify a previously unrealized function for MKS3. Our immunofluorescence data suggest that MKS3 functions outside the transition zone to maintain basal body row organization by potentially contributing to a link between the basal body and the striated rootlet. Without the link, the migrating basal bodies are free to rotate and project their rootlets in the wrong directions. Although the nature of the link remains elusive, the identification of disorganized basal body rows upon MKS3 reduction suggests that, in addition to ciliary dysfunction, basal body polarity defects may contribute to the development of MKS.

There have been few studies investigating the eye movement behavior of Parkinson’s disease patients during fixation. This study objectively measured the eye movements of 36 patients with Parkinson’s disease, and 20 age matched controls. Stimuli consisted of ten standardized text passages first organized by Miller and Coleman. In addition, subjects followed a randomly displaced step jump target motion. Pendular nystagmus was found in all Parkinson’s subjects, with an average frequency of 7.44 Hz. Saccadic peak velocity and duration along the main sequence were not statistically different from controls. A slower rate of reading was also noted in the Parkinson’s group in terms of characters per minute, but with no more regressions than normal. Rate of square wave jerks was also found to be normal. This suggests that the hallmark feature of eye movements in Parkinson’s disease is a pendular nystagmus during fixation, and all saccadic activity to be normal.