Aluminium applications can be found in the vast majority of industries – particularly the automotive, aerospace and building sectors. Light weight, good corrosion resistance, high strength with good machining and weldability has led 6xxx series alloy to be the most widely used for extrusion products. Semi-continuous direct-chill (DC) casting is a well established process and the most widely used in the production of wrought aluminium extrusion billets. The techniques have continuously evolved since its invention in the 1930s. To ensure high productivity and a quality billet by DC casting, grain refiners are added during casting prior to solidification. It is efficient, cost effective and considered optimized in modern production techniques. However, some problems still persist, for example, macrosegregation, centerline cracking, porosity, hot tearing, etc. For surface finish critical products, particles in added grain refiners may cause surface defects during downstream processing. Additions of grain refiners are also not desirable for recycling of the end use products. As a novel DC casting technology, the melt conditioned DC casting (MC-DC) technology is developed to achieve uniform fine equiaxed grains without deliberate additions of grain refiners. The MC-DC process is implemented by submerging a rotor-stator high shear device into the mould assembly of a conventional hot-top vertical DC caster. In this work, the fundamentals of MC-DC process has been investigated by studying the flow patterns in the sump using computer modelling in combination with thermal field measurement and delineation of the sump profile. Followed is the microstructural evolution of the MC-DC castings. Then the formation of Fe-bearing intermetallics which are critical to the arrangement of homogenisation treatment are presented. The grain refining mechanism by MC-DC is due to enhanced heterogeneous nucleation on dispersed oxides and grain fragments by intensive melt shearing, in combination with dendrite fragmentation and transportation in a uniform temperature and solute field. By optimising MC-DC parameters, alleviation of macrosegregation can be achieved even compared with DC-GR castings. Another finding is the correlation between grain structure and the distribution of the Fe-intermetallic particles. It has been demonstrated that equiaxed dendritic grains with fine secondary dendritic arm spacings achieved in MC-DC are preferred rather than finer granular grains in grain refined material. MC-DC also promotes the formation of α- Fe-bearing intermetallics. All these offer the potential for the reduction of homogenisation practices currently required as part of the DC process.

abstract: Specific dendritic morphologies are a hallmark of neuronal identity, circuit assembly, and behaviorally relevant function. Despite the importance of dendrites in brain health and disease, the functional consequences of dendritic shape remain largely unknown. This dissertation addresses two fundamental and interrelated aspects of dendrite neurobiology. First, by utilizing the genetic power of Drosophila melanogaster, these studies assess the developmental mechanisms underlying single neuron morphology, and subsequently investigate the functional and behavioral consequences resulting from developmental irregularity. Significant insights into the molecular mechanisms that contribute to dendrite development come from studies of Down syndrome cell adhesion molecule (Dscam). While these findings have been garnered primarily from sensory neurons whose arbors innervate a two-dimensional plane, it is likely that the principles apply in three-dimensional central neurons that provide the structural substrate for synaptic input and neural circuit formation. As such, this dissertation supports the hypothesis that neuron type impacts the realization of Dscam function. In fact, in Drosophila motoneurons, Dscam serves a previously unknown cell-autonomous function in dendrite growth. Dscam manipulations produced a range of dendritic phenotypes with alteration in branch number and length. Subsequent experiments exploited the dendritic alterations produced by Dscam manipulations in order to correlate dendritic structure with the suggested function of these neurons. These data indicate that basic motoneuron function and behavior are maintained even in the absence of all adult dendrites within the same neuron. By contrast, dendrites are required for adjusting motoneuron responses to specific challenging behavioral requirements. Here, I establish a direct link between dendritic structure and neuronal function at the level of the single cell, thus defining the structural substrates necessary for conferring various aspects of functional motor output. Taken together, information gathered from these studies can inform the quest in deciphering how complex cell morphologies and networks form and are precisely linked to their function. / Dissertation/Thesis / Ph.D. Neuroscience 2013

abstract: Patients with schizophrenia have impaired cognitive flexibility, as evidenced by behaviors of perseveration. Cognitive impairments may be due to dysregulation of glutamate and/or loss of neuronal plasticity in the medial prefrontal cortex (mPFC). The purpose of these studies was to examine the effects of mGluR5 positive allosteric modulators (PAMs) alone and in combination with the NMDAR antagonist MK-801, a pharmacological model of schizophrenia. An operant-based cognitive set-shifting task was utilized to assess cognitive flexibility, in vivo microdialysis procedures to measure extracellular glutamate levels in the mPFC, and diolistic labeling to assess the effects on dendritic spine density and morphology in the mPFC. Results revealed that chronic administration of the mGluR5 PAM CDPPB was able to significantly reduce the effects of chronically administered MK-801 on both behavioral perseveration and glutamate neurotransmission. Results also showed that CDPPB had no evidence of an effect on dendritic spine density or morphology, but the mGluR5 negative allosteric modulator fenobam caused significant increases in spine density and the frequency of occurrence of spines with smaller head diameters. Conclusions include that CDPPB is able to reverse MK-801 induced cognitive deficits as well as alterations in mPFC glutamate neurochemistry. The culmination of these studies add further support for targeting mGluR5 with PAMs as a novel mechanism to alleviate cognitive impairments in patients with schizophrenia. / Dissertation/Thesis / Doctoral Dissertation Psychology 2014

The ubiquitously expressed plasma membrane Na⁺/H⁺ exchanger isoform 1 (NHE1) plays an important role in directed cell migration in non-neuronal cells, an effect which requires both the ion translocation and actin cytoskeleton anchoring functions of the protein. In the present study, an analogous role for NHE1 as a modulator of neurite outgrowth was evaluated in vitro utilizing NGF-differentiated PC12 cells as well as mouse neocortical neurons in primary culture. Examined at 3 d.i.v., endogenous NHE1 was found to be expressed in growth cones, where it gave rise to an elevated intracellular pH in actively-extending neurites. Application of the NHE inhibitor cariporide at an NHE1-selective concentration (1 μM) resulted in reductions in neurite extension and elaboration while application of 100 μM cariporide, to inhibit all known plasmalemmal NHE isoforms, failed to exert additional inhibitory effects, suggesting a dominant role for the NHE1 isoform in modulating neurite outgrowth. In addition, whereas transient overexpression of full-length NHE1 enhanced neurite outgrowth in a cariporide-sensitive manner in both NGF-differentiated PC12 cells and WT neocortical neurons, neurite outgrowth was reduced in NGF-differentiated PC12 cells overexpressing NHE1 mutants deficient in either ion translocation activity or actin cytoskeleton anchoring, suggesting that both functional domains of NHE1 are important for modulating neurite elaboration. A role for NHE1 in modulating neurite outgrowth was confirmed in neocortical neurons obtained from NHE1-/- mice which displayed reduced neurite outgrowth when compared to neurons obtained from their NHE1⁺/⁺ littermates. Further, neurite outgrowth in NHE1-/- neurons was rescued by transient overexpression of full-length NHE1 but not with mutant NHE1 constructs again suggesting that both functional domains of NHE1 are important for modulating neurite outgrowth. Finally, the growth promoting effects of netrin-1 but not BDNF or IGF-1 were abolished by cariporide in WT neocortical neurons and while both BDNF and IGF-1 were able to promote neurite outgrowth in NHE1-/- neurons, netrin-1 was unable to elicit this effect. Taken together, these results indicate that NHE1 is a permissive regulator of early neurite morphogenesis and also plays a novel role in netrin-1-stimulated neurite outgrowth. / Medicine, Faculty of / Graduate

Nicotine from smoking or from other products containing nicotine has adverse effects on the fetus during pregnancy, such as respiratory problems. Our laboratory has previously shown that exposure to nicotine during development (DNE) alters hypoglossal motor neuron (XII MN) function, including decreased excitatory synaptic input, desensitized nicotinic acetylcholine receptors, increased input resistance, and differences in the precision and reliability of spike timing in XIIMNs. Evidence of DNE effects on XIIMN function prompted us to test the hypothesis that DNE will affect the development of primary dendrites and the soma. Brainstem slices were collected from neonates and motoneurons were filled with neurobiotin via whole-cell patch clamp. Filled cells were visualized with heavy metal intensified-3,3'-Diaminobenzidine (DAB) reaction. DAB-stained cells were analyzed using Neurolucida hardware and software. On average, the maximum soma diameter of more rostral XIIMNs was larger than that in more caudal cells. Also, caudal XIIMNs had more primary nodes than rostral XIIMNs, and there was a significant treatment effect on minimum soma diameter (Control, 13.76 ± 0.71 µm; DNE, 18.09 ± 1.22 µm). The results from this study uncovered potential effects of nicotine on XIIMNs found in rostral and caudal regions of the hypoglossal nucleus.

Drug-associated stimuli trigger craving and relapse in addiction. Murine morphine conditioned place preference (CPP) was used to model learning of opioid associations. We examined how morphine and learning interact to alter neuron morphology in the nucleus accumbens (NAc) core and shell after acquisition and extinction of CPP. Conditioning with morphine dose-dependently increased place preference compared to saline. In comparison to those from saline conditioned and morphine non-conditioned controls, neurons from the NAc core of morphine conditioned mice had increased dendritic complexity, as defined by increased dendritic length, number, and Sholl intersections. This effect is due to the combination of morphine and learning, which is different from effects of morphine or conditioning alone. Morphine administration without conditioning was associated with increased spine density in the core, which was reversed by CPP acquisition. Control conditioning with saline produced no morphology changes. Morphine CPP extinction was associated with decreased dendritic complexity, reversing the increased complexity seen after acquisition. Mice that extinguished CPP had similar dendritic complexity to saline conditioned mice, in terms of dendritic count and intersections, but less dendritic complexity than non-extinguished mice that retained CPP. Since dopamine release imbues salience to stimuli that coincide with drug use, and the dopamine D1 receptor mediates CPP acquisition, we tested the effect of SKF81297 D1 receptor agonist on CPP extinction and associated accumbal neuron morphology. SKF81297 (0.8 mg/kg) injected after each extinction training session impeded extinction, and produced increased dendritic complexity compared to controls. SKF81297 may have sustained conditioned associations, disrupted consolidation of extinction, and/or disrupted the decline in dopamine levels that may occur throughout extinction sessions. We hypothesize that changes occurred in the NAc core because this region mediates how stimuli and drug effects direct motor action. Since D1 receptors oppose extinction of drug-cue-induced behavior, they play a role in reinforcing opioid addiction. Acquisition and extinction may be opposite processes in the brain, as in behavior. Extinction may include some reversal of acquisition learning as well as being new learning with its own pathway. Interventions that target D1 receptors or that selectively reduce NAc core dendritic complexity may contribute to opioid addiction treatment.

Yukako Hattori, Tadao Usui, Daisuke Satoh, Sanefumi Moriyama, Kohei Shimono, Takehiko Itoh, Katsuhiko Shirahige, Tadashi Uemura, Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier, Developmental Cell, Volume 27, Issue 5, 9 December 2013, Pages 530-544, ISSN 1534-5807 / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18418号 / 生博第298号 / 新制||生||39(附属図書館) / 31276 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 西田 栄介, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Dendrite growth is the primary form of crystal growth observed in laser deposition process of most commercial metallic alloys. The properties of metallic alloys strongly depend on their microstructure; that is the shape, size, orientation and composition of the dendrite matrix formed during solidification. Understanding and controlling the dendrite growth is vital in order to predict and achieve the desired microstructure and hence properties of the laser deposition metals. A two dimensional (2D) model combining the finite element method (FE) and the cellular automaton technique (CA) was developed to simulate the dendrite growth both for cubic and for hexagonal close-packed (HCP) crystal structure material. The application of this model to dendrite growth occurring in the molten pool during the Laser Engineered Net Shaping (LENS®) process was discussed. Based on the simulation results and the previously published experimental data, the expressions describing the relationship between the cooling rate and the dendrite arm spacing (DAS), were proposed. In addition, the influence of LENS process parameters, such as the moving speed of the laser beam and the layer thickness, on the DAS was also discussed. Different dendrite morphologies calculated at different locations were explained based on local solidification conditions. And the influence of convection on dendrite growth was discussed. The simulation results showed a good agreement with previously published experiments. This work contributes to the understanding of microstructure formation and resulting mechanical properties of LENS-built parts as well as provides a fundamental basis for optimization of the LENS process.

Advanced breast cancer predominantly metastasizes to the skeleton, at which point patients suffer bone loss, pain, heightened fracture risk and their prognosis significantly declines. The skeleton is sensitive to its highly dynamic mechanical environment whereby bone mass is increased when applied loads are increased whereas bone loss occurs when applied loads are reduced. Increased mechanical loading inhibited bone metastatic tumor formation and progression in vivo, but the underlying mechanisms are currently under investigation. Here, we focus on the osteocyte, a specialized bone cell well-known as the primary mechanosensor and director of remodeling in the skeleton. Osteocytic dendrites are important for mechanosensing, and their number and length increase with applied loading. Dendrite connections are also known to suppress breast cancer growth and bone metastasis. How breast cancer cells affect loading-induced changes in dendrites, or downstream effects on mechanosensing and remodeling, is unknown. To examine how breast cancer cells modulate osteocyte function, we exposed osteocytes (MLO-Y4 cells) to medium conditioned by breast cancer cells (MDA-MB231 cells) and to fluid shear stress using a rocker platform (2 hrs per day for 3 days, shear stress 1.1 Pa). In the absence of loading, treatment with tumor conditioned media alone did not alter dendrite number per MLO or overall cell number. When loading was applied to MLOs treated with conditioned media, dendrite formation increased in MLOs despite the presence of tumor-derived factors. Conditioned media and loading together reduced MLO cell number, suggesting that the combination of these two factors may be stimulating apoptosis and dendrite formation increases in the remaining viable cells. Next, to model the more physiological situation in which both cell populations undergo loading, we exposed MLOs to loading as well as media conditioned by breast cancers that were also similarly loaded. When control (nonloaded) MLOs were treated with conditioned media from loaded breast cancer cells, their dendrite formation increased in a manner similar to that observed due to loading alone. When MLOs simultaneously underwent loading and treatment with loaded conditioned media, dendrite formation was greatest. Loaded conditioned media also decreased MLO cell number independent of MLO loading status. The results suggested that loading stimulates breast cancer cells to induce both osteocyte dendrite formation and apoptosis, which is a possible mechanism for the inhibitory role of applied loading on bone metastasis.

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