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Abstract In the Cape Fynbos of South Africa, Psoralea pinnata (L.) plants occur naturally in both wetland and welldrained soils and yet effectively fix N2 under the two contrasting conditions. In this study, nodule structure and functioning in P. pinnata plants from the two habitats were evaluated using light and transmission electron microscopy (TEM), as well as the 15N natural abundance technique. The results showed that, structurally, fully developed P. pinnata nodules were spherical in shape with six components (namely, lenticels, periderm, outer cortex, middle cortex, inner cortex, and a central bacteria-infected medulla region). Morphometric analysis revealed 44 and 84 % increase in cell area and volume of wetland nodules compared to those from upland. The percentage area of nodules occupied by the middle cortex in wetland nodules was twice that of upland nodules. As a result, the size of the medulla region in wetland nodules was significantly reduced compared to upland nodules. Additionally, the average area of medulla occupied by intercellular air spaces in wetland nodules was about five times that of upland nodules (about 431 % increase in wetland over upland nodules). TEM data also showed more bacteroids in symbiosomes of upland nodules when compared to wetland nodules. However, isotopic analysis of above-ground plant parts revealed no differences in symbiotic parameters such as N concentration, ∂15N and %Ndfa between wetland and upland P. pinnata plants. These results suggest that, under limiting O2 conditions especially in wetlands, nodules make structural and functional adjustments to meet the O2 demands of N2-fixing bacteroids.

Bipolar affective disorder and schizophrenia are highly heritable psychiatric illnesses and the leading causes of worldwide disability. The orbitofrontal cortex (OFC) is a region of the frontal lobe with wide spread connectivity with other brain areas involved in reward, motivation and emotion. Evidence from various neuroimaging, genetic, post-mortem and brain lesion studies suggest that orbitofrontal cortex may play a role in pathophysiology of mental illnesses. This thesis sought to investigate the pathogenesis of major psychiatric illnesses through the investigation of orbitofrontal morphology in schizophrenia and bipolar disorder and through its associations with brain structure and function. Orbitofrontal morphology and its structural and functional associations were examined in healthy controls, patients with schizophrenia or bipolar affective disorder, and those at high genetic risk using functional and structural MRI. In the first study we found that the orbitofrontal type III is more frequent and the orbitofrontal type I is less common in the right hemisphere in patients with schizophrenia while in patients with bipolar disorder type III appears more often in both left and right hemispheres. We then sought to examine the relationship of orbitofrontal morphology to disease risk in a study of 146 people at high risk of developing schizophrenia and 110 people at high risk of developing bipolar disorder. We discovered that in the unaffected high risk groups the orbitofrontal type III predicted the development of later psychiatric illnesses, when combined with anterior cingulate morphology. Finally we showed, in a further study, that OFC morphology was associated with measures of schizotypy, brain structure, brain function and cognition. In conclusion, orbitofrontal morphology is linked to major psychiatric disorder and has significant structural and functional associations. As orbitofrontal sulcogyral patterns are formed in early life a fuller awareness of their relevance to brain function holds out the prospect that we could use such measures as an indicator of vulnerability to the development of illness later in life. This work points to the potential for the foundation of a theory of predictive associations between morphological patterns and the development of psychosis.

The adrenal cortex is classically divided into three morphologically and biochemically distinct zones, covered by a thin, cellular capsule. The adult adrenal cortex is a dynamic tissue in which distinct regions of cell proliferation, movement and death have been identified. Several models for stem cell maintenance of the adult adrenal cortex have been proposed, but adrenocortical stem cells have not yet been identified. Adrenal cortices of 21OH/LacZ transgenic mice show similar mosaic patterns of β-galactosidase staining to X- inactivation mosaics and LacZ ↔ wildtype chimeras. 21OH/LacZ mice provide a tool for lineage analysis, which may help to i) identify clones of cells produced by stem cells in the adult, ii) determine when stem cells begin to function and iii) evaluate different models of how stem cells maintain the adrenal cortex. Analysis of 21OH/LacZ transgenic adrenal cortices showed that the randomly orientated clusters of fetal patches change progressively during the perinatal period to adult radial stripes. Correlation of changes in mosaic patterns and the locations of cell proliferation suggests that the stripes arise by edge-biased growth during the perinatal growth period. Although stem cells may not be involved in the initial formation of stripes, it seems likely that stem cells later maintain the stripes by producing clones of cells that move centripetally to displace the earlier fetal patterns and later replace aging cells. Various combinations of BrdU labelling and chase periods demonstrated that most cell division occurred in the outer 40% of the adrenal cortex, confirmed that cells moved towards the medulla and identified a population of label-retaining cells near the capsule, which could include stem cells. (Stem cells have been recognised as BrdU label-retaining cells in other tissues because they divide less frequently than their daughter cells so dilute the incorporated BrdU more slowly.) Stripe patterns in adult 21OH/LacZ transgenic adrenal cortices were examined to try to distinguish between various models proposed for stem cell maintenance of the adrenal cortex. The observed continuous radial stripe pattern favours the general hypothesis that a single population of stem cells in the periphery maintains the entire adrenal cortex, although other explanations are possible. Quantitative analysis of adult stripe patterns did not show the reduction in stripe number that might be predicted if an age-related decline in adrenocortical stem cell function occurs, as may happen in some other tissues.

The adrenal gland is an important endocrine organ, protecting the body against acute and chronic stress. The adrenal cortex consists of three morphologically and functionally distinct zones: the outer zona glomerulosa (zG), the zona fasciculata (zF), and the innermost zona reticularis (zR). In rodents, zG cells produce mineralocorticoids (mainly aldosterone), while zF cells secrete glucocorticoids (mainly corticosterone). The functions of zG and zF are defined by the mutually exclusive expression of Cyp11b2 and Cyp11b1 that encode the enzymes aldosterone synthase and 11β-hydroxylase, which catalyze the terminal reactions in the production of aldosterone and corticosterone, respectively. This thesis aims to investigate the maintenance of the definitive mouse adrenal cortex. This involves studies to identify the location of adrenal stem/progenitor cells, and the mechanisms by which differentiated adrenocortical cells are replenished in the adult mice. BrdU pulse-chase studies provided valuable information about cell division and cell fate under physiological or pathophysiological stimulations. The distribution of adrenocortical cells with nuclei stained positively for BrdU and/or Ki67 was identified. Ki67 labelling marked actively dividing cells and showed that adrenocortical cells originate at or around the zG/zF interface. BrdU labelling indicated that, following cell division, cells are displaced inwards and outwards. Acute angiotensin II treatment was shown to have no significant effects on the cell proliferation or turnover in any of the adrenocortical zones. The pathophysiological effects of long-term ACTH treatment were analyzed in a mouse model of congenital adrenal hyperplasia caused by a null mutation of Cyp11b1. Cell hypertrophy was evident in all regions of the adrenal cortex due to the impaired negative-feedback of the HPA axis. Adrenocortical cell proliferation was also increased particularly in the outer zona fasciculata at the border between zG and zF where adrenocortical stem/progenitor cells might be located. The intervening steps between cell proliferation and the final differentiation into steroidogenic zG and zF cells have yet to be discovered. A visual method of monitoring levels of Cyp11b2 and Cyp11b1would offer a convenient approach to track the stages of adult stem cell differentiation that lead to normal adrenal maintenance in vivo and in vitro. In the present study an AS-mCherry-11B-EGFP BAC construct was successfully engineered, in which Cyp11b2 and Cyp11b1 were substituted by mCherry and EGFP, respectively. This BAC construct was characterized in mouse adrenocortical Y1 cells. It was determined that EGFP faithfully recapitulated the expression of Cyp11b1. Forskolin or cAMP treatment induced a rapid cell rounding effect and caused the increased expression of EGFP transgene and endogenous Cyp11b1. An attempt was made to establish a transgenic mouse model, in which zG and zF cells would be marked with mCherry and EGFP respectively, allowing the differentiation of an adrenocortical stem cell to be traced. Following microinjection of the BAC into mouse zygotes, twoAS-mCherry-11B-EGFP transgenic founder mice were identified. Unfortunately, neither of them was able to transmit the transgene through germline, suggesting the mosaicism of transgene integration. Indeed, mosaicism of the transgenic adrenals was demonstrated by RT-PCR and immunostaining, which also revealed that the exogenous EGFP expression faithfully recapitulated the endogenous Cyp11b1 in adrenals. Although it is assumed that expression of Cyp11b2 and Cyp11b1 are mutually exclusive, zG and zF cells may have the plasticity to allow the transition from one cell type into another. The AS-mCherry-11B-EGFP BAC construct is a useful tool for studying in vitro ES cell differentiation towards the adrenocortical lineage. Transgenic AS-mCherry-11B-EGFP ES cells were successfully differentiated into mesenchymal stem cells, as identified by the expression of molecular markers for the mesenchymal lineage. It has been reported that steroidogenic factor (Sf1) can promote the differentiation of MSCs into steroidogenic cells, and Shh plays an important role in Sf1 expression and the consequent adrenal development. However, Shh treatment failed to achieve transformation of mesenchymal cells into adrenocortical cells. It is thought there might be a requirement for additional factors to combine with Shh in promoting the transdifferentiation of ESC-derived mesenchymal cells. Future studies will focus on the genetic control of Cyp11b1 and Cyp11b2 in transgenic AS-mCherry-11B-EGFP ES cells. In conclusion, the location and fate of the adrenocortical progenitor cells were demonstrated by the BrdU pulse-chase studies in different mouse models. An AS-mCherry-11B-EGFP BAC construct was generated, and used to study the mutual and differential controls of Cyp11b1 and Cyp11b2 expression in adrenocortical cells in vitro and in transgenic mice in vivo.

This thesis provides insights into the mechanism by which thalamocortical axons (TCAs) approach the cortex from their origin in the thalamus. Previous studies suggested that the reciprocal projections from the prethalamus and the ventral telencephalon guide TCAs to descend through the prethalamus and cross the diencephalic-telencephalic boundary (DTB), after which TCAs navigate through permissive corridor cells in the ventral telencephalon and cross the pallial-subpallial boundary (PSPB) before reaching their final targets in the cortex. The ‘Handshake Hypothesis’ proposed that pioneer axons from cortical preplate neurons guide TCAs into corresponding cortical areas. However, there is a lack of convincing evidence on whether TCAs need any guidance to cross the PSPB. In the current study, Adenomatous polyposis (Apc) gene is conditionally deleted from the cortex, by using Emx1Cre-APCloxP recombination technology. Apc is widely expressed in the nervous system including the cortical plate of the cortex and regulates axonal growth and neuronal differentiation. Deleting Apc may block neurite extension and/or affect the formation of attractive or repulsive cues in the cortex. By using DiI tracing as well as L1 immunohistochemistry techniques, I showed that in the Apc mutants cortical axons are absent and that TCAs initially navigate into the ventral telencephalon normally but fail to complete their journey into the cortex. They stop as they approach the PSPB, although the PSPB doesn’t seem to be directly affected by the mutation of Apc in the cortex. Additionally, Ig-Nrg1 (Neuregulin-1), the secreted protein that was suggested to play long-range roles in attracting TCAs towards the cortex, is present in the Apc mutant. This implies that Ig-Nrg1 is not sufficient for guiding TCAs into the cortex, and that additional guidance factors are needed. Moreover, my in vitro explant culture experiments show that the mutant cortex neither repel nor inhibit thalamic axonal outgrowth, indicating that the failure of TCAs in reaching the cortex is not due to the change of repulsive cues secreted by the mutant cortex. It rather indicates that the guidance factors for TCAs are likely to function through cell-cell contact mediated mechanisms. The Apc mutant cortex lacks these guidance factors, which might be the cortical axons. In conclusion, my data reveal a choice point for TCAs at the PSPB. Guidance factors from the cortex are needed for TCAs to cross the PSPB, which are absent in the Apc mutant. TCAs may need the direct contact with cortical axons and use them as an axonal scaffold to navigate into the cerebral cortex.

Hippocampus has an extended developmental trajectory, with refinements occurring in the trisynaptic circuit until adolescence. While structural change should suggest a protracted course in behavior, some studies find evidence of precocious hippocampal development in the first postnatal year and continuity in memory processes beyond. However, a number of memory functions, including binding and relational inference, can be cortically supported. Evidence from the animal literature suggests that tasks often associated with hippocampus (visual paired comparison, binding of a visuomotor response) can be mediated by structures external to hippocampus. Thus, a complete examination of memory development will have to rule out cortex as a source of early memory competency. We propose that early memory must show properties associated with full function of the trisynaptic circuit to reflect "adult-like" memory function, mainly (1) rapid encoding of contextual details of overlapping patterns, and (2) retention of these details over sleep-dependent delays. A wealth of evidence suggests that these functions are not apparent until 18-24 months, with behavioral discontinuities reflecting shifts in the neural structures subserving memory beginning approximately at this point in development. We discuss the implications of these observations for theories of memory and for identifying and measuring memory function in populations with typical and atypical hippocampal function. (C) 2015 The Authors. Published by Elsevier Ltd.

Previous research has investigated how the perirhinal cortex (PRC), hippocampus (HC) and temporal pole (TP) are involved in complex visual discrimination using a variety of stimuli. Results from these studies have shown that the PRC activates to a greater extent for object stimuli relative to scene stimuli and that familiar stimuli elicit greater activation than do novel stimuli. In contrast, the HC shows greater activation for scene stimuli relative to objects and has also been reported to show greater activation for familiar relative to novel stimuli. To our knowledge, however, no studies in humans have replicated the stimulus specificity findings reported for PRC and HC. Additionally, no studies have used a combination of perceptual difficulty and familiarity/novelty to investigate how varying these factors affects activation in PRC, HC and TP during visual discrimination tasks. Chapter 2 describes an fMRI study performed to investigate the PRC's and HC's involvement in object and scene visual discrimination. The results of this study showed that the PRC was activated similarly by scenes and objects and that the HC was activated similarly for objects and scenes. Chapter 3 describes an fMRI experiment that manipulated both familiarity and difficulty, measuring how this affected PRC, HC and TP activation. In PRC the results showed a significant interaction between novelty and level of difficulty, such that novel objects with high levels of overlapping features showed greater activation compared to all other conditions. In contrast, the HC only showed a main effect of difficulty, indicating that the stimuli with high, relative to low, levels of difficulty elicited greater activation regardless of familiarity. Cumulatively, the evidence above suggests that the involvement of the PRC and HC in visual discrimination is complex. We propose that PRC is engaged whenever visual discrimination is required for any stimuli with overlapping features not just objects, and is activated to a greater extent when stimuli are novel, while HC appears to respond to stimuli with overlapping features, regardless of familiarity.

published_or_final_version / Speech and Hearing Sciences / Doctoral / Doctor of Philosophy

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