There is extensive research regarding the neural mechanisms that control satiety and meal termination; in contrast, there is very limited understanding of how the central nervous system regulates meal onset and thus the duration of the postprandial intermeal interval (ppIMI) and meal frequency. Based on emerging evidence, we hypothesize that dorsal hippocampal neurons, which are critical for episodic memory, form a memory of a meal and inhibit meal onset during the ppIMI. To test whether hippocampal neurons form a memory of a meal, we first determined that ingesting sucrose or isopreferred concentrations of the non-caloric sweetener saccharin increased the expression of the plasticity-related immediate early gene activity-regulated cytoskeleton-associated protein (Arc) in dorsal CA1 hippocampal (dCA1) neurons in Sprague-Dawley rats. Furthermore, repeated exposure to the sucrose meal attenuated the ability of the sucrose to induce Arc expression. Together, these data indicate that orosensory stimulation produced by a sweet taste is sufficient to induce synaptic plasticity in dCA1 neurons in an experience-dependent manner. Second, we showed that reversibly inactivating dorsal hippocampal neurons with infusions of the GABAA agonist muscimol after the end of a sucrose meal accelerated the onset of the next meal, indicating that dorsal hippocampal neurons inhibit meal onset. Lastly, using a clinically-relevant animal model of early life inflammatory injury, we found that neonatal injury (1) impairs hippocampal-dependent memory, (2) decreases the ppIMI and increases sucrose intake, (3) increases body mass, (4) attenuates sucrose-induced Arc expression in dCA1 neurons, and that (5) blocking inflammatory pain with morphine at the time of injury reverses the effects of injury on memory, energy intake and Arc expression. Collectively, the findings of this dissertation support the overarching hypothesis that dorsal hippocampal neurons inhibit meal onset during the ppIMI and suggest that dorsal hippocampal dysfunction may contribute to the development and/or maintenance of diet-induced obesity.

Cotton fiber development is a fundamental biological phenomenon. In spite of its economical importance, a large proportion of cotton fiber initiation is unknown. A naked seed mutant (N1N1) was compared with its isogenic lines of cotton (Gossypium hirsutum, TM-1) using a 70-mer oligonucleotide microarray that contained 1,536 features designed from a subset of cotton fiber ESTs. Statistical analysis and quantitative RT-PCR identified 23 "fiber-associated" genes. The annotation suggested that the temporal regulation of genes involved in transcriptional and translational regulation, signal transduction, and cell differentiation during early stages of fiber development. To get a large view of fiber initiation, a new cotton oligonucleotide microarray was developed containing sequences from an ovule EST library from Gossypium hirsutum L. T̲M̲-1 immature o̲vules (GH_TMO), a set from Jonathan Wendel's lab at Iowa State University, and the pilot set of oligos used for previous study. Global gene expression studies were performed with microdissected fiber initials (or epidermis) and inner ovules to investigate fiber preferentially expressed genes. Laser capture microdissection and antisense RNA (aRNA) amplification allowed us to collect fiber initials (0 DPA and 2 DPA) or epidermal layers (-2 DPA) from whole ovule tissues. The gene expression profiles of fiber initials showed up-regulation of fiber proteins, myb transcription factors, and hormonal regulators as well as trichome related factors during fiber initiation. In each developmental stage, different sets of gene categories in molecular function or biological processes were over- or under-represented, suggesting temporal regulation of genes during fiber development. One of the possible "fiber associated genes" found in microarray analyses, RD22 like gene (GhRDL), was highly enriched in the epidermis of cotton ovules during fiber initiation. The function of GhRDL was studied with the Arabidopsis trichome system which shares many similarities with fiber development. Overexpression of 35S::GhRDL into Arabidopsis thaliana Columia-0 induced seed hairs (or seed trichomes) and pRDL:GUS was localized in Arabidopsis seeds. This suggests that GhRDL plays an important role in the seed trichome development and can be a key player in cell differentiation and fiber development. / text

The mechanism by which prostate cancer progresses from androgen dependence (AD) to androgen independence/castration resistance (AI/CR) is currently a major focus of prostate cancer-related research. Prostate cancers that progress to a state of AI/CR are typically resistant to most standard types of treatments. Due to its primary role in driving normal prostate cell growth and proliferation, the androgen receptor (AR) is believed to play a key role in progression. Coregulators, or any proteins which may either enhance or abrogate AR activity, are considered to be one of the potential mechanisms by which AR function may become impaired. Cyclin G-associated kinase (GAK) was initially identified as a potential coregulator of AR in a Tup 1 repressed transactivation system. A LNCaP cDNA library was screened for proteins which interacted with the NH2-terminus of AR. GAK was isolated from three independent library clones using two different AR baits (AR 1-549 and AR 1-646). This interaction was confirmed via GST pulldown and coimmunoprecipitation experiments, and preliminary luciferase assays suggested that GAK activates AR in a hormone dependent manner. In this study, my objectives were to validate GAK’s role as a coregulator of AR and to determine if overexpressing GAK affects progression to AI. In vitro luciferase assays whereby GAK was either overexpressed or knocked down in both LNCaP and PC3 cells did not significantly affect AR activity. Xenograft experiments utilizing a doxycycline (DOX) inducible lentiviral LNCaP-GAK overexpressing stable cell line demonstrated that while GAK may not play a significant role in modulating AR activity, it may adopt a more subtle role enhancing tumour take and tumour volume growth rate in vivo. While these results could not confirm GAK to be a direct coregulator of AR, it is entirely possible that GAK may influence prostate cancer progression, albeit indirectly. Recent publications report a growing amount of evidence suggesting GAK’s involvement in the critical cellular process of clathrin coated vesicle endocytosis, the dysregulation of which could potentially indirectly affect AR regulated genes.

In this paper, we study primary decomposition of any proper submodule N of a module M over a noetherian ring R. We start by briefly discussing basic facts about the very well known case where M is a finitely generated module over a Noetherian ring R, then we proceed to discuss the general case where M is any module over a Noetherian ring R. We put a lot of focus on the associated primes that occur with the primary decomposition, essentially studying their uniqueness and their relation to the associated primes of M/N.

In this thesis, I present a model of multidomain evolution with associated algorithms and software for phylogenetic analysis of multidomain families, as well as applications of this novel methodology to case-studies and the human genome. Phylogenetic analysis is of central importance to understanding the origins and evolution of life on earth. In biomedical research, molecular phylogenetics has proved an essential tool for practical applications. Current molecular phylogenetic methods are not equipped, however, to model many of the unique characteristics of multidomain families. Genes that encode this large and important class of proteins are characterized by a mosaic of sequence fragments that encode structural or functional modules, called domains. Multidomain families evolve via domain shuffling, a process that includes insertion, internal duplication, and deletion of domains. This versatile evolutionary mechanism played a transformative role in major evolutionary transitions, including the emergence of multicellular animals and the vertebrate immune system. Multidomain families are ill-suited to current methods for phylogeny reconstruction due to their mosaic composition. Different regions of the same protein may have different evolutionary histories. Moreover, a protein may contain domains that also occur in otherwise unrelated proteins. These attributes pose substantial obstacles for phylogenetic methods that require a multiple sequence alignment as input. In addition, current methods do not incorporate a model of domain shuffling and hence, cannot infer the events that occurred in the history of the family. I address this problem by treating a multidomain family as a set of co-evolving domains, each with its own history. If the family is evolving by vertical descent from a conserved set of ancestral domains, then all constituent domains will have the same phylogenetic history. Disagreement between domain tree topologies is evidence that the family evolved through processes other than speciation and gene duplication. My algorithms exploit this information to reconstruct the history of domain shuffling in the family, as well as the timing of these events and the ancestral domain composition. I have implemented these algorithms in software that outputs the most parsimonious history of events for each domain family. The software also reconstructs a composite family history, including duplications, insertions and losses of all constituent domains and ancestral domain composition. My approach is capable of more detailed and accurate reconstructions than the widely used domain architecture model, which ignores sequence variation between domain instances. In contrast, my approach is based on an explicit model of events and captures sequence variation between domain instances. I demonstrate the utility of this method through case studies of notch-related proteins, protein tyrosine kinases, and membrane-associated guanylate kinases. I further present a largescale analysis of domain shuffling processes through comparison of all pairs of domain families that co-occur in a protein in the human genome. These analyses suggest that (1) a remarkably greater amount of domain shuffling may have occurred than previously thought and (2) that it is not uncommon for the same domain architecture to arise more than once through independent events. This stands in contrast to earlier reports that convergent evolution of domain architecture is rare and suggests that incorporating sequence variation in evolutionary analyses of multidomain families is a crucial requirement for accurate inference.

Human chromosome 13q14 has been identified as one of the hotspots of deletion in prostate cancer, multiple myeloma, and chronic lymphocytic leukemia. Chromosome Condensation 1-like (CHC1L) is an uncharacterized gene in this region. CHC1L is found within the smallest common region of loss of heterozygosity in prostate cancer, and its decreased expression is linked to pathogenesis and progression of both prostate cancer and multiple myeloma. In the present study, we have generated Chc1L gene knockout mice and demonstrated that loss of this gene increases tumorigenesis in two year old mice. Knockout and heterozygous mice are predisposed to development of Histiocytic Sarcoma and Histiocyte-Associated Lymphoma. Bone marrow and splenic cells from 8-12 week old knockout mice have elevated viability ex vivo. These data provide the first direct evidence that CHC1L is a tumor suppressor gene involved in suppression of histiocyte-rich neoplasms.

Members of the membrane-spanning four-domains, subfamily A (MS4A) family are small polypeptides that share the structural features of four-transmembrane domains and unevenly sized extracellular loops. The family includes CD20, FcεRIβ and HtM4, plus a number of relatively uncharacterised proteins / predicted proteins. MS4A proteins are discussed in relation to other protein families, such as the tetraspanins, that are also characterised by four-transmembrane domains. The aim of this study was to identify the cell and tissue distribution, subcellular localisation, and function of a newly discovered member of the MS4A family, hepatocellular carcinoma-associated antigen 112 (HCA112). At a subcellular level, HCA112 was found on the plasma membrane of transfected COS-7 cells, and also within the Golgi complex, trans-Golgi-network, and early endosomes. The molecule is orientated such that the large loop is extracellular and the Nand C-terminal domains are cytoplasmic. The presence of HCA112 associated with components of the endocytic pathway raised the question of whether some originated from the surface membrane. Antibody was used to label a HA epitope tag engineered into the large extracellular loop of HCA112, and the bound antibody was tracked through early endosomes to the recycling compartment. Here it co-localised with internalised transferrin, indicating strongly that HCA112 is internalised via clathrin-dependent mechanisms. Several endocytic sorting motifs within the intracellular domains of HCA112 were investigated for their ability to direct internalisation of HCA112. Deletion of a di-leucine motif was found to slow but not prevent endocytosis, suggesting that it is involved in endocytosis of HCA112, although not essential for the process. When HCA112 expression constructs featuring N- and C-terminal domain truncations were examined, it was found that the N-terminal tail does not affect the subcellular localisation or trafficking of HCA112, while deletion of the C-terminal intracellular domain resulted in retention of the mutant protein in the ER. HCA112 has a wide tissue distribution and is highly expressed in the lining/covering and parenchymal epithelium of some tissues, proximal renal tubules, ductal epithelium in a number of organs, endothelial cells, some steroidogenic endocrine cells, adipocytes, smooth muscle cells, follicular dendritic cells and macrophages. The expression of HCA112 by a wide range of cell types suggests that its function(s) has general importance and is not limited to any specific cell type(s). After reflection on the functions of the HCA112-expressing cells, a common theme that emerged was one of endocytic activity. This lead to speculate that one function of HCA112 might be related to uptake of macromolecules, for instance, in antigen processing and presentation. This might be a general function, such as facilitating uptake of other cell membrane proteins, or directing the traffic of endocytic vesicles. It was noted that HCA112 has a similar cell and tissue distribution to the scavenger receptor and fatty acid translocase FAT/CD36 (Zhang et al., 2003). Furthermore, in cells co-transfected with HCA112 and FAT/CD36, the two molecules co-localise in early endosomes and co-immunoprecipitate, suggesting that the molecules physically and spatially associate. Thus, HCA112 could be involved with (or complement) FAT/CD36 in its functions as a long chain fatty acid transporter and scavenger receptor. A proteomics study of proteins that co-immunoprecipitated with HCA112 detected putative interactions with a number of proteins. These included LR8, transferrin receptor, interferon induced transmembrane proteins 2 and 3, Calpain-6, stomatin, PDGF α receptor, and heat shock 70 kDa protein 8 (HSPA8, formerly known as clathrin un-coating ATPase). Of these, LR8 and the transferrin receptor were investigated in more detail. The results provide strong evidence that HCA112 forms a novel complex with LR8, and that this may be involved in macromolecule internalisation or trafficking of membrane proteins, such as FAT/CD36 or the transferrin receptor. In the case of the transferrin receptor, this traffic appears to involve the clathrin-dependent pathway, but it is possible that when HCA112 is associated with FAT/CD36, it functions within lipid raft domains. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1375454 / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2009

Doctor of Health Sciences / A common and clinically important complication of late stage human immunodeficiency virus Type 1 (HIV-1) infection is HIV-associated neurocognitive disorder (HAND). HAND encompasses three syndromes, HIV-associated asymptomatic neurocognitive impairment (ANI), HIV-1- associated mild neurocognitive disorder (MND), and HIV-1-associated dementia (HAD). It is estimated that 30-60% of all HIV-1 infected individuals will have at least mild neurocognitive impairment (MND), and 10-15% of those will develop HAD. Research conducted outside medicine has focused on identifying the type and pattern of neuropsychological impairments present in people with HAND, and to correlate impairments identified from neuropsychological testing with scores on laboratory-based tests of everyday task performance. Typically, the performance of tasks and routines in daily life occurs in naturalistic contexts, and is orchestrated around the achievement of personally meaningful, needed and/or desired performance goals. It requires that one uses cognitive strategies to attend, perceive, remember, decide, plan and act on intentions within real-world contexts. Little is known about the impact of cognitive information processing strategy application impairments on the performance of meaningful tasks and routines carried out by people with HAND in contexts where performance would naturally occur. This research addressed this gap by investigating the real-world impact of information strategy application disorder in a sample of 30 men diagnosed with HAD, the most severe form of HAND. The home contexts of those in the sample consisted of home, supported living and residential care. The criterion-referenced Perceive, Recall, Plan and Perform (PRPP) System of Task Analysis was used to identify the level of task performance mastery demonstrated by men in the sample (Stage One), and the information processing strategy application errors that impacted on their performances (Stage Two). The Clinical Staging of AIDS Dementia Complex (CSADC) scale was used to identify the level of severity of HAD. A total of seventy one task performances were assessed across the sample in a variety of naturalistic contexts. None of the men in the sample demonstrated mastery of task performance. The mean Mastery score was 30.07%. The predominant type of error made by men as they performed daily life tasks was Timing; they spent too much time completing tasks. This was followed by errors of Accuracy; they made mistakes in what they did. Descriptive analysis of the PRPP Stage Two scores revealed that these men had difficulties across all domains of information processing strategy application but most notably with Plan Quadrant (Mean 30.75%) and Perceive Quadrant (Mean 53.49%) strategy application behaviours. Rasch calibration of the ordinal PRPP Stage Two strategy application scores produced an interval-level linear hierarchy of information processing strategy application difficulties experienced by the group. Men in the sample demonstrated problems sequencing complex tasks, choosing plans and actions, analysing problems encountered, and monitoring sensory changes during performances. Problems were also identified in their abilities to contextualise their performances to fit within time constraints (Contextualises to Duration), and enact plans in a fluid manner (Flows). Differences in performances between men with mild dementia versus those with moderate/severe dementia identified using a 2 x 4 repeated measures ANOVA carried out on the Rasch-calibrated PRPP Stage Two scores revealed similarities in performance across Perceive, Recall, Plan and Perform Quadrants but those with mild dementia performed better overall. Further analyses revealed specific differences in performance between those with mild versus those with moderate/severe dementia. Most striking about the findings was that men at both ends of the dementia spectrum had relatively good Recall Quadrant strategy application capacities (Mean 75.30%). Even those with the lowest total PRPP Stage Two scores, could recognize and use objects, and recall the procedures of known tasks. A statistically significant predictive correlation was found between Plan Quadrant disorders and severity of dementia. This pilot study demonstrated the utility of the PRPP System, a criterion-referenced, occupation-embedded, ecological method of identifying task performance skill and information processing strategy application disorders impacting on performance, for use with people living with HIV/AIDS who have HAD. Identifying the specific impact of information processing strategy application disorders on real-world task performance provides occupational therapists with information necessary to more specifically tailor therapy to the individual performance and participation needs of people with HIV-1-associated dementia.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.