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Estrogen is thought to protect against the development of chronic kidney disease, and menopause increases the development and severity of diabetic kidney disease. In this study, we used streptozotocin (STZ) to induce diabetes in the 4-vinylcyclohexene diepoxide (VCD)-treated mouse model of menopause. DNA microarrays were used to identify gene expression changes in the diabetic kidney postmenopause. An ANOVA model, CARMA, was used to isolate the menopause effect between two groups of diabetic mice, diabetic menopausal (STZ/VCD) and diabetic cycling (STZ). In this diabetic study, 8,864 genes of the possible 15,600 genes on the array were included in the ANOVA; 99 genes were identified as demonstrating a >1.5-fold up- or downregulation between the STZ/VCD and STZ groups. We randomly selected genes for confirmation by real-time PCR; midkine (Mdk), immediate early response gene 3 (IEX-1), mitogen-inducible gene 6 (Mig6), and ubiquitin-specific protease 2 (USP2) were significantly increased in the kidneys of STZ/VCD compared with STZ mice. Western blot analysis confirmed that Mdk and IEX-1 protein abundance was significantly increased in the kidney cortex of STZ/VCD compared with STZ mice. In a separate study, DNA microarrays and CARMA analysis were used to identify the effect of menopause on the nondiabetic kidney; VCD-treated mice were compared with cycling mice. Of the possible 15,600 genes on the array, 9,142 genes were included in the ANOVA; 20 genes were identified as demonstrating a >1.5-fold up- or downregulation; histidine decarboxylase and vanin 1 were among the genes identified as differentially expressed in the postmenopausal nondiabetic kidney. These data expand our understanding of how hormone status correlates with the development of diabetic kidney disease and identify several target genes for further studies.

To examine the effect of a central noradrenergic lesion on the reactivity of the 5-HT1B receptor we compared intact male rats with rats in which noradrenergic nerve terminals were largely destroyed with the neurotoxin DSP-4 (50 mg/kg × 2, on the 1st and 3rd days of postnatal life). When rats attained 10 weeks of age, control and DSP-4 rats were divided into two subgroups receiving either saline or the serotonin (5-HT) synthesis inhibitor (p-chlorophenylalanine; p-CPA; 100 mg/kg). Employing an elevated plus maze test, we demonstrated that CP 94,253 (5-propoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-pyrrolo[3,2-b]pyridine hydrochloride)(4.0 mg/kg; 5-HT1B agonist) induced an anxiogenic-like action in control rats; however, it failed to elicit this effect in the DSP-4 group. Surprisingly, in p-CPApretreated rats anxiogenic-like activity was observed both in control and DSP-4 treated rats. CP 94,253 significantly attenuated 5-HT synthesis in the medial prefrontal cortex (mPFC) of control rats, and SB 216641 (N-{3-[3-(dimethylamino) ethoxy]-4-methoxyphenyl}-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-[1,1'-biphenyl]-4-carboxamide hydrochloride) (4.0 mg/kg; 5-HT1B antagonist) was able to antagonize this effect. Conversely, CP 94,253 failed to significantly inhibit the 5-HT synthesis rate in DSP-4-treated rats. In the microdialysis study CP 94,253 induced long-lasting attenuation of 5-HT release in the mPFC of control rats but had no effect in DSP-4 rats. These data lead to the proposal that presynaptic 5-HT1B autoreceptors underwent desensitization in DSP-4 treated rats.

The anterior neuroectoderm (ANE) is progressively restricted along the anterior-posterior axis to a territory around the anterior pole during the blastula stages in sea urchin embryos. Information obtained from Wnt/beta-catenin, Wnt/JNK and Wnt/PKC signaling pathways is responsible for this positioning process. Several secreted Wnt modulators of the Dickkopf and Secreted Frizzled Related Protein families (Dkk1, Dkk3, and sFRP1/5) are expressed within the ANE and play important roles in the Wnt signaling network during this process. In this study, we have characterized the function of another secreted Wnt modulator, sFRP3/4, within this Wnt signaling network. Here we have shown that the maternal sFRP3/4 is necessary for the early ANE restriction mechanism during cleavage stage. We also found that Fzl1/2/7 activates zygotic sFRP3/4 during blastula stage, which possibly antagonizes ANE restriction mechanism. During the final phase of ANE restriction mechanism sFRP3/4 and Dkk1 likely works together define the final ANE territory.

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This thesis is an exploration of method demonstrated in a series of mechanical drawings of the elements. An investigation of methods of drawing and of how different questions can be asked through drawing. Furthermore, this thesis asks the question of place in the elements of fire, earth, and sky in the formal relationship between walls, joints, trusses. These architectural elements protect against the natural ones, forming place focused around the hearth. This thesis traces the formal relationship between the elements of architecture stemming from the German thinker Gottfried Semper's treatise on architecture, but diverges from Semper's fixation with materiality and delves into questions of form, appearance, and construction through drawing. The drawings address questions about the elements; questions about form, tectonics, and the relation of part to whole. The whole becomes a building focused on forming a place and the relation of the elements to one another. / Master of Architecture / "Throughout all phases of society the hearth formed that sacred focus around which the whole took order and shape. It is the first and most important, the moral element of architecture. Around it were grouped the three other elements: the roof, the enclosure, and the mound, the protecting negations or defenders of the Hearth's flame against the three hostile elements of nature" (Semper 102) This reading, The Four Elements of Architecture by Gottfried Semper, and this quote were the origin of my thesis, which became a series of studies of these elements as parts and their relations to one another. Through mechanical investigative drawings I explored the traits and qualities assigned to these elements by Semper and developed my own understanding of them. Each element has a tectonic quality and architectural characteristic that defines it. The tectonic quality is "different technical skills of man used to construct the elements: ceramics and afterwards metal work around the hearth, water and masonry works around the mound, carpentry around the roof and its accessories… Wickerwork was the essence of the wall" (Semper 103-104). The mound forms the base of the building connecting it to the hearth, and also joins to the hearth and raises it. The roof as a dominant element protects from above. The wall is a spatial enclosure that forms the protected space. The hearth is the central and social element of the building, protected from nature by the other elements. In addition to an investigation of each of the elements, I also had an interest in constructing a building informed by these investigations, forming a place of gathering that focused around the hearth. How would the protective elements form and appear in space, and how would the hearth form the place around it. The wall, defined as woven and enclosing, led to a series of questions regarding its form, material, and connection to the other elements. Semper's understanding of woven extends to being a dressed wall or a wall of a pattern of parts, such as brick or wood. How can variation of size, depth, and material be used to form patterns in a wall? How do hierarchies of panels, verticality, and horizontality differ from one another? How can the wall connect to the mound and emphasize the existing character of the building? The roof, defined as dominant and of carpentry, led to a series of questions regarding frames and joints. My main interest in the roof was its structure, and the interior spatial quality it provided. How can the joint and the frame inform one another? What are the qualities of trusses? What are different types of joints and how do they display themselves? How do joints form a truss and the truss form a frame? The mound, defined as earthen and meeting, was investigated in conjunction with other elements. It forms the base that protects the hearth from the earth and acts as a base to the other elements. Its original use as a structural base for houses in marsh lands is no longer prevalent and its role lessened. Semper understood this evolution, "some elements [become] more developed while others [recede] into the background" (Semper 103). The hearth, defined as central and of worked metal, led to a series of questions regarding material, form, and relation to its surroundings. The hearth acts as a single focus that forms areas of repose protected by the other elements from nature. How should it connect to the roof and the floor? Benefits of a hanging hearth versus traditional? How can it act as more by storing wood and the other tools it needs to be worked? How can it relate to other elements through material and form? As Semper stated, "some elements [become] more developed" and I found some of these progressions through my studies. One of these developments is the hearth creating areas of transition in the interior and the need to connect this transitioning to the exterior. The frame of the hearth is repeated as a threshold, forming an area of transition between the exterior and the interior. Another development is the roof and the chimney belonging to the sky. As the mound recedes from importance, the sky and its connection to the roof develops further. These boards each developed from a series of questions, and led to further questions. They ask about the part and then about the part in relation to other parts. Each question can stand on its own, but it is only when you keep asking questions that you begin to find something. Each part is its own, but when formed together it creates something new. And that was my thesis, a series of questions and the joining of parts.

Myocardial infarction (MI) occurs following acute thrombotic occlusion of a coronary artery, and triggers a robust inflammatory response. Within hours, neutrophils are recruited to the infarcted myocardium followed by the infiltration of pro-inflammatory Ly6Chi monocytes. Transition from the pro-inflammatory macrophage phenotype (M1) to an anti-inflammatory, pro-resolution phenotype (M2-like) is critical to successful infarct healing. Interventions that polarize macrophages towards an anti-inflammatory 'M2-like' phenotype improve infarct healing in the experimental MI mouse model and reduce subsequent adverse remodelling of the myocardium, but the endogenous mechanisms that regulate repair are not well understood. Furthermore, differences in the resolution of inflammation in C57BL/6 and BALB/c mice, which are two of the commonly used wild-type mouse strains in experimental MI have not been characterised. We previously found that low peripheral blood eosinophil count is associated with increased short-term risk of mortality in low-intermediate risk patients with ischaemic heart disease. This suggests that eosinophils may have a role in the successful remodelling and repair of the heart following myocardial infarction. Eosinophils express a number of immuno-modulating cytokines and lipid mediators implicated in the resolution of inflammation. Increasingly prominent is interleukin-4 (IL-4), a cytokine that has been found to maintain the anti-inflammatory M2-like phenotype in macrophages. We therefore hypothesised that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are key in regulating the subsequent inflammatory response and scar tissue formation during infarct repair and cardiac remodelling. Experimental MI was induced by permanent left anterior descending artery ligation in isofluorane anaesthetized 12-15 week-old male wild-type (WT) BALB/c, WT C57BL/6, IL4Rα-/-, IL-4Rαflox/-, IL-4Rαflox/-LysMCre mice and eosinophil-deficient ΔdblGATA mice. Cardiac function was characterised by high-resolution ultrasound and immune cell infiltration by flow cytometry of single cell infarct and remote zone tissue digests. Blood eosinophil count and 6-month all-cause mortality were assessed in 732 consecutive patients undergoing primary percutaneous coronary intervention for ST-segment elevation myocardial infarction (STEMI). The rate of mortality due to cardiac rupture was significantly higher in C57BL/6 mice in comparison with BALB/c mice at Day 7 post-MI. This was associated with a higher proportion of pro-inflammatory Ly-6Chi macrophages infiltrating the infarct zone tissue of C57BL/6 mice following MI. An accompanying reduction in the number of splenic Ly-6Chi monocytes post-MI, suggestive of splenic monocyte mobilisation, was seen in C57BL/6 mice but not found in BALB/c mice. Furthermore, C57BL/6 mice had a delayed transition in macrophage polarisation towards an anti-inflammatory phenotype. Disruption of IL4Rα signalling, in mice null for the IL4Rα gene, resulted in increased F4/80+ macrophage and pro-inflammatory Ly6Chi macrophage infiltration of the infarct zone and reduced expression of the anti-inflammatory macrophage marker CD206, compared to wild-type controls. Furthermore, expression of GATA3 and ST2, both associated with the immunosuppressive function of (CD4+ Foxp3+) regulatory T cells, was reduced in infarct zone regulatory T cells from IL4Rα-/- mice. These findings were associated with defective wound healing with impaired angiogenesis, increased scar size, disarrayed infarct zone collagen deposition, accompanied by modified expression of plod2 that encodes the collagen cross-linking enzyme lysyl hydroxylase 2. Resulting in greater left ventricular dilatation and loss of cardiac function, as well as a higher 7- day mortality due to cardiac rupture in IL4Rα-/- mice. This indicates that successful infarct repair requires the engagement of IL-4Rα signalling to facilitate the accumulation of anti-inflammatory macrophages and highly immunosuppressive ST2+ regulatory T cells in the heart following MI. Resident cardiac macrophages from naïve hearts of IL-4Rαflox/-LysMCre mice failed to undergo LysMCre-mediated deletion of the IL-4Rα gene, potentially because low or absent expression of Lyz2 (encoding lysozyme M). In both ST-elevation MI (STEMI) patients and mice after acute MI, there was a decline in peripheral blood eosinophil count, with activated eosinophils being recruited to the infarct zone and paracardial adipose tissue of mice. The transcription factors GATA-1 plays a role in the differentiation of eosinophils from eosinophil progenitor cells. Deletion of GATA-1 results in loss of the eosinophil lineage and has been exploited to develop the eosinophil-deficient ΔdblGATA mouse. ΔdblGATA mice were used to address the role of eosinophils in cardiac remodelling following MI. ΔdblGATA mice had increased left ventricular dilatation and reduced ejection fraction after induction of MI, relative to wild-type mice. ΔdblGATA mice had increased scar size with disarrayed infarct zone collagen deposition, accompanied by modified expression of the genes plod2 and lox, which are associated with collagen cross-linking. The proportion of CD206+ anti-inflammatory macrophages was less in the infarct zone of ΔdblGATA mice, but was restored by adoptive transfer of eosinophils from WT mice. Furthermore, adverse cardiac remodelling in eosinophil-deficient ΔdblGATA mice was rescued by provision of IL-4 complex following MI. In conclusion, an enhanced inflammatory response following MI underlies the increased risk of cardiac rupture seen with WT C57BL/6 mice in comparison to WT BALB/c mice. WT BALB/c mice are protected from cardiac rupture, which was associated with an absence of splenic monocyte mobilisation following ischaemic injury. The resolution of inflammation was found to be dependent on IL4Rα signalling which is crucial for cardiac repair and remodelling, through modulating inflammatory cell recruitment and phenotype, as well as scar formation. Eosinophils are recruited to the heart post-MI and are essential for regulating cardiac repair and remodelling, likely through provision of IL-4. Therefore, we were able to show that IL-4Rα signalling and recruitment of eosinophils to the myocardium following infarction are both key in regulating the subsequent inflammatory response and scar tissue formation during infarct healing and cardiac remodelling.