Fragile X Syndrome (FXS) is a neurodevelopmental disorder and the most common heritable single gene cause of Autism Spectrum Disorder (ASD). The Fragile X (FMR1-KO) mouse model has been used to understand the pathophysiology of the disease. However, the majority of studies have been done in adult mice and early life outcomes have yet to be explored. Therefore, in order to contribute to the knowledge of the neurodevelopmental processes associated with brain disorders, this thesis examines postnatal outcomes in the Fragile X Mouse Model: early life behaviours, the developmental trajectory of a set of ASD risk genes, and neuroanatomical phenotype. The first study examined ultrasonic vocalizations in pups and showed a transient increase in calls in FMR1-KO mice. To understand the relationship between early life behaviours, the second study examined outcomes in the pre-pubertal period in these mice when challenged with lipopolysaccharide and maternal separation. The results showed genotype and treatment interactions affecting sexually dimorphic behavioural outcomes and developmental milestones. In the third study, possible underpinnings of behavioural differences were explored by examining mRNA expression of the neuroligins and neurexins. In FMR1-KO mice, changes were transient and sex-specific, suggesting these as molecular effectors in the disease. Lastly, using structural brain imaging, the fourth study examined regional volume differences that may be related to behavioural differences. Differences in regions affected in FXS patients were observed and genetic background was shown to affect the neuroanatomical phenotype. Overall, this thesis demonstrates that the FXS model recapitulates some outcomes in other ASD mouse models and shows that this single gene has multiple interactions with sex, strain, and postnatal challenge which manifests at specific ages at molecular, brain structure and behavioural levels. This work contributes to the efforts elucidating the neurobiology of ASD and reverse translation approaches to identify therapeutic targets for neurodevelopment disorders. / Dissertation / Doctor of Philosophy (PhD) / Autism spectrum disorder (ASD) is a diagnosis based on observed behaviours: impaired communication and repetitive actions. However, there are genetic and other behavioural differences in ASD patients that are not shared among the group. It is important to tease apart this group since current treatments for ASD do not target the biological problems or the core impairments. This thesis focuses on Fragile X Syndrome, the leading genetic condition that results in ASD in order to understand the biological basis of ASD. Using a mouse model, compared to healthy mice, these studies report changes in behaviours, in the size of different brain regions, and in molecules involved in connecting brain cells during development. These findings shed light on the molecular story underlying ASD. By understanding the nature of influences on the developing brain, the type and timing of interventions can be designed to keep the brain on a healthy trajectory.

Since the discovery of the p120ctn binding partner, Kaiso, a BTB/POZ transcription factor, several studies have implicated the protein in both development and tumourigenesis. Most information about Kaiso’s function in vertebrate development has been gleaned from studies in Xenopus laevis embryos where Kaiso negatively regulates the Wnt signalling pathway. Since the Wnt signalling pathway is crucial in intestinal development, intestinal-specific Kaiso overexpressing mice were generated and characterized to elucidate Kaiso’s role in a mammalian context. Kaiso transgenic (KaisoTg/+) mice were viable and fertile but developed gross histopathological changes in the small intestine. The KaisoTg/+ mice exhibited enlarged crypts accompanied by increased secretory cell differentiation reminiscent of inhibition of the Notch pathway. Indeed, the Notch effector protein, HES1, is decreased in KaisoTg/+ mice. Additionally, KaisoTg/+ mice display a neutrophil-specific intestinal inflammation reminiscent of the knockdown of p120ctn. Interestingly, the KaisoTg/+ mice display decreased p120ctn localization at the membranes and an increase in the neutrophil adhesion molecule, ICAM-1, both of which induce neutrophilia. Notably, the KaisoTg/+ mice developed multiple crypt abscesses over time due to massive neutrophil infiltration of the epithelial cell layers. This is the first study to examine the in vivo roles of Kaiso in a mammalian context and our findings suggest a regulatory role for Kaiso in the inflammatory and Notch signalling pathways. / Thesis / Candidate in Philosophy

Fine-grained descriptions of brain connectivity are required to understand how neural information is processed and relayed across spatial scales. Prior investigations of the mouse brain connectome have employed discrete anatomical parcellations, limiting spatial resolution and potentially concealing network attributes critical to connectome organization. In this work, we provide a voxel-level description of the network and hierarchical structure of the directed mouse connectome, unconstrained by regional partitioning. We found that hub regions and core network components of the voxel-wise mouse connectome exhibit a rich topography encompassing key cortical and subcortical relay regions. We also typified regional substrates based on their directional topology into sink or source regions, and reported a previously unappreciated role of modulatory nuclei as critical effectors of inter-modular and network communicability. Finally, we demonstrated a close spatial correspondence between the mesoscale topography of the mouse connectome and its functional macroscale organization, showing that, like in primates and humans, the mouse cortical connectome is organized along two major topographical axes that can be linked to hierarchical patterns of laminar connectivity, and shape the topography of fMRI dynamic states, respectively. This investigation was paralleled by further studies aimed to more closely relate structural connectome features to the corresponding large scale functional networks of the mouse brain. We first focused on the mouse default mode network (DMN), describing its axonal substrates with sublaminar precision and cell-type specificity. We found that regions of the mouse DMN are predominantly located within the isocortex and exhibit preferential connectivity. Dedicated tract tracing experiments carried out by the Allen Brain Institute revealed that layer 2/3 DMN neurons projected mostly in the DMN, whereas layer 5 neurons project both in and out. Further analyses revealed the presence of separate in-DMN and out-DMN-projecting cell types with distinct genetic profiles. Lastly, we carried out a fine-grained comparison of functional topography and dynamic organization of large-scale fMRI networks in wakeful and anesthetized mice, relating the corresponding functional networks to the underlying architecture of structural connectivity. Recapitulating prior observations in conscious primates, we found that the awake mouse brain is subjected to a profound topological reconfiguration such to maximize cross-talk between cortical and subcortical neural systems, departing from the underlying structure of the axonal connectome. Taken together, these results advance our understanding of the foundational wiring principles of the mammalian connectome, and create opportunities for identifying targets of interventions to modulate brain function and its network structure in a physiologically-accessible species.

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PEA3 is a member of the expanding Ets family of transcription factors. In the adult mouse, PEA3 mRNA is expressed at highest levels in the brain, epididymis and at lower levels in the mammary gland, testes, ovary and uterus. PEA3 mRNA is expressed differentially during mouse embryogenesis and is down-regulated following retinoic acid induced differentiation in mouse embryonal carcinoma cell lines. PEA3 is overexpressed at the transcriptional level in 93% of all HER2/neu positive human breast tumors. The molecular basis for differential transcription of the PEA3 gene is not known. Sequence analysis revealed that the upstream region of the PEA3 gene has characteristics of a CpG island and does not possess a recognizable "TATA" element. Rapid amplification of 5' eDNA ends (5'RACE) reveals that transcription initiates from multiple sites, consistent with the absence of TATA elements. To localize cis-acting sequences required for PEA3 expression, deletions of the putative promoter were placed upstream of a luciferase reporter gene and tested for activity in the FM3A cell line. FM3A cells express substantial levels of PEA3 mRNA and protein, which suggests that all of the factors required for transcription are present in the cells. Transient transfections of 5' and 3' deletion mutants of the PEA3 promoter indicated that the efficiency of the PEA3 promoter depended on both negative and positive cis-elements, located upstream and downstream of the transcription start sites. A DNA fragment containing a region from -3 to +676, relative to the major start site of transcription, was sufficient for maximal promoter activity. Luciferase reporter plasmids containing more 5' flanking sequence had lower activity indicating the presence of silencer elements. To aid the identification of critical sequence elements within the minimal PEA3 promoter, we cloned and sequenced the putative human PEA3 promoter. Comparison of the mouse and human PEA3 DNAs revealed that sequences required for maximal promoter activity in the mouse were highly conserved in the human gene. Furthermore, these conserved sequences corresponded to a variety of consensus binding sites: 6 Sp1, 8 c-ets-1, 3 PEA3, 3 AP-2, 3 MZF-1, 2 MyoD, 2 Ik-1, 2 c/EBPB, 2 oEF-1/USF, 2 HSFI and one of each of the following: AP-4, Ik-2, SRY, CP2, HEN-I, CREB andE47. / Thesis / Master of Science (MS)

The American Cancer Society predicted that 230,480 women would be diagnosed with, and 39,520 women would die from breast cancer (BC) in the United States in 2011. While the incidence of female BC has been decreasing, BC remains the second leading cause of cancer death among women in the United States. Cancer cachexia, the cancer-related loss of muscle, affects up to 25% of BC patients and is associated with poor prognosis and decreased quality of life. Alterations to the dystrophin glycoprotein complex (DGC), a transmembrane, multi-subunit protein complex with structural and signaling roles, have been reported in mammary tumors of BC patients and skeletal muscles of cachectic cancer patients. However, this complex is most frequently studied for its role in Duchenne muscular dystrophy (DMD), a severe, progressive muscle wasting disease. Despite the similar alterations reported in these diseases, it is unclear whether alterations in the DGC in one tissue can impact the progression of disease in another. Purpose: The purpose of the studies described in this dissertation was to identify differences in body composition, energy expenditure and plasma cytokine content between the C57BL/10ScSn-Dmdmdx/J (mdx) mouse model of DMD and C57BL/10ScSnJ (BL/10) control mice and to determine whether systemic alteration of the DGC (as observed in the mdx mouse) alters the growth of E0771 murine mammary tumors. Results: There were differences in body composition and plasma cytokine profiles between mdx and BL/10 mice. We also found that, relative to controls, the tumor–induced increase in cytokines that promote invasion and metastasis was not as severe in mdx mice. Conclusions: This study revealed several differences between mdx and BL/10 mice and provides support for the suggestion that the mdx mouse may not be an accurate model of DMD. In addition, the improved cytokine profile of tumor-bearing mdx mice suggests that the acute phase of DMD may be protective against BC invasion and metastasis. Further research should confirm this effect and determine whether alterations in the DGC of the mdx mouse are directly or indirectly responsible. / Ph. D.

Ambient temperature is known to drive changes in the thermal physiology of mammals, such as an increase in the capacity for thermogenesis in winter. Previous work has shown that acclimation to chronic cold increases the capacity for non-shivering thermogenesis (NST) and thermogenic capacity (cold-induced maximal oxygen consumption, V̇O2max) in deer mice (Peromyscus maniculatus). Photoperiod, is an important driver of seasonal changes in physiology. In the wild, animals are attuned to seasonal changes in temperature and photoperiod. However, the independent and combined effects of temperature and photoperiod on the capacity for NST in small mammals, such as the deer mouse, are still not fully understood. To address this, we acclimated deer mice to long or short photoperiods (12h or 8h light), in either thermoneutral or cold conditions (30°C or 5°C). To simulate the fall conditions in their natural habitat (Nebraska) we gradually reduced either daylength, temperature, or both over 4 weeks and acclimated mice an additional 4 weeks at those conditions. After the 8 weeks of acclimation, we determined NST and V̇O2max. We found that cold and short photoperiod were necessary to increase NST, and either short photoperiod or cold was sufficient to increase V̇O2max. There were no corresponding differences in iBAT mass, lipid droplet morphology, nor in mitochondrial content. However, there was a corresponding increase in UCP1 content per unit mitochondria. These data highlight the importance of both photoperiod and temperature as cues to prepare thermogenic responses beneficial as winter approaches. / Thesis / Master of Science (MSc)

Emotion can influence important user behaviors, including purchasing decisions, technology use, and customer loyalty. The ability to easily assess users' emotion during live system use therefore has practical significance for the design and improvement of information systems. In this paper, we discuss using human-computer interaction input devices to infer emotion. Specifically, we utilize attentional control theory to explain how movement captured via a computer mouse (i.e., mouse cursor movements) can be a real-time indicator of negative emotion. We report three studies. In Study 1, an experiment with 65 participants from Amazon's Mechanical Turk, we randomly manipulated negative emotion and then monitored participants' mouse cursor movements as they completed a number-ordering task. We found that negative emotion increases the distance and reduces the speed of mouse cursor movements during the task. In Study 2, an experiment with 126 participants from a U.S. university, we randomly manipulated negative emotion and then monitored participants' mouse cursor movements while they interacted with a mock e-commerce site. We found that mouse cursor distance and speed can be used to infer the presence of negative emotion with an overall accuracy rate of 81.7 percent. In Study 3, an observational study with 80 participants from universities in Germany and Hong Kong, we monitored mouse cursor movements while participants interacted with an online product configurator. Participants reported their level of emotion after each step in the configuration process. We found that mouse cursor distance and speed can be used to infer the level of negative emotion with an out-of-sample R-2 of 0.17. The results enable researchers to assess negative emotional reactions during live system use, examine emotional reactions with more temporal precision, conduct multimethod emotion research, and create more unobtrusive affective and adaptive systems.

Deciphering the mechanisms that underlie circuit function in the hippocampal formation is a key challenge for neuroscience. This region, which includes the medial entorhinal cortex (MEC), is critical for spatial learning and episodic memory in humans. Spatially modulated cells in the MEC, the grid cells, provide a topographical representation of space, but we are yet to establish the neuronal properties that underlie this or the contribution that particular cells in different regions of the MEC and hippocampus make to circuit function. This is partially because the specific targeting of the network with genetic tools is complicated by a multitude of cell types with predominantly unknown molecular profiles. To address our limited understanding of the molecular organisation of the MEC, I have characterised how the expression of genes is distributed throughout different layers of the MEC, using a custom-designed resource that facilitates analysis of in situ hybridisation data from the Allen Brain Atlas. Through simultaneous extraction of gene expression data across thousands of 2D aligned images, I reveal striking differences between layers within MEC, demonstrating that layer II contains the highest proportion of genes enriched in a single layer, whereas gene expression is very rarely confined to layer III. Of particular interest, layer II of MEC is highly enriched for Alzheimer’s disease pathway genes, providing insight into its vulnerability as one of the first brain regions to show pathology. I also identify over 1000 genes that are expressed with a dorso-ventral gradient that maps onto the topographic organisation of MEC connectivity, grid cell spatial resolution and synaptic integrative properties of cells. An intriguing group of genes that closely relate circuit activity to gene expression, the plasticity-related activity-dependent genes, often show this pattern of expression. Focussing on the activity-dependent expression of one such activity-regulated, plasticity-related gene, Arc, I provide a novel view of MEC function. During simple novel exploration, Arc expression is up-regulated to a much greater extent in the deep layers of dorsal MEC than in the grid cell-rich superficial layers. By selectively disrupting the predominant hippocampal input to dorsal MEC, which terminates in the deep layers, I show that the significance of this up-regulation is independent of hippocampal inputs. Thus, although research addressing MEC function is particularly focussed on the superficial layers, during the exploratory behaviour that potentially primes the system for representing an environment, important plasticity may be occurring at the synapses onto deep layer neurons. In summary, my investigations of baseline and activity-dependent gene expression in MEC have revealed a molecular organisation both across different layers and along a functionally relevant gradient. This may be important for specifically targeting microcircuits in MEC and for characterising how laminar and regional differences contribute to the encoding of space in the hippocampal formation.