The zebrafish belladonna (bel) mutation was identified in a large-scale mutagenesis screen to identify genes involved in retino-tectal pathfinding in Tubingen, Germany. In bel mutants, after exiting the eye, retinal axons grow ipsilaterally instead of crossing the midline to form optic chiasm. bel mutants are semi-viable and live bel embryos at 5 days show a "dilated pupil" phenotype after which the mutation was named. Later work showed that bel mutants have functional eyes although the optokinetic response is reversed in the mutants. Previous work in our lab showed that most retinal axons in the mutants initially grow towards the midline but later turn ipsilaterally. Also, two major forebrain commissures, the anterior commissure (AC) and the post-optic commissure (POC) also failed to form in bel mutants. These studies showed that bel defects are restricted to forebrain. Detailed analysis of eye sections showed defects in bel eye morphology during embryonic and adult stages. Initial work also mapped the bel locus on chromosome 8 and finer mapping linked one z-marker on either side of bel locus (z24272 and z44909). My dissertation project was to clone the bel gene and understand its role in forebrain patterning and axon guidance. I identified that bel locus encodes a zebrafish lim-homeodomain transcription factor, Lhx2. To further understand how bel(lhx2) might affect axon guidance, I first showed that bel mutants have subtle defects in forebrain patterning in the regions where axons cross the midline. I also showed that these forebrain patterning defects lead to defects in expression of proper cellular and molecular axon guidance cues at the midline in bel mutants. Finally, I showed that bel(lhx2) is required for cell proliferation in the diencephalon. Thus my detailed analysis of bel mutants has revealed new roles for lhx2 in diencephalon patterning and axon guidance.

The ability of established synaptic connections to strengthen and weaken (synapse plasticity) underlies higher order behavior such as learning and memory. Several gaps remain in our understanding of the cellular and molecular changes in pre- and postsynaptic cells associated with synapse plasticity. Using the Drosophila larval neuromuscular junction as a convenient model system throughout my dissertation work, I have concentrated on understanding various molecular mechanisms that underlie this phenomenon. Specifically, I have addressed fundamental issues governing the postsynaptic cell in these processes, such as: (i) what are the crucial proteins required to scaffold the postsynaptic apparatus during plasticity? (ii) what are the molecules that are brought together at this scaffold and how are their levels regulated during plasticity? and finally (iii) what are the signals that initiate the formation of these scaffolds during plasticity? More specifically, I have uncovered a mechanism by which a novel synaptic protein, Guk-holder (Gukh), coordinates the formation of a tripartite complex at the synapse with two proteins containing multiple protein-protein interaction domains: Discs Large (DLG) and Scribble (Scrib) (Mathew et al., 2002). By bringing DLG and Scrib together at the synapse, Gukh likely coordinates the function of these two complexes. As stated above Fasciclin II (FasII) is one of several molecules that is localized to the synapse by Dig (Thomas et al., 1997a), and its levels and symmetric distribution at the synapse have been found to be important for regulating synapse plasticity (Ashley et al., 2005; Schuster et al., 1996b). I showed that FasII also undergoes fast recycling at the synaptic membrane and that this recycling is regulated by the Drosophila homolog of Amphiphysin (Amph)-(Mathew et al., 2003). Such a mechanism would be compatible with a potential activity dependent regulation of FasII levels during plasticity. To understand the initial signaling mechanisms by which synaptic scaffolds are organized during synapse formation, I further investigated the role of the Wingless receptor Dfrizzled2 (DFz2) in this process. Wg signaling has been previously implicated in synapse development and proper localization of postsynaptic proteins (Packard et al., 2002). In these studies I uncovered an unconventional mechanism involved in transducing the Wg signal in the muscle cell. I show that DFz2 is cleaved at the synaptic plasma membrane, and the c-terminus of the molecule traffics to the nucleus. This nuclear import of DFz2 is dependent upon Wg signaling and is important to transduce the downstream effects of Wg signaling at synapses (Mathew et al., 2005 in press). (Abstract shortened by UMI.)

The peripubertal period is a period during which significant brain re-organization occurs, and during which changes in the neuroendocrine milieu normally lead to the onset of fertility. Shipping female mice during the peripubertal period causes a long-lasting behavioral defeminization of feminine sexual behavior. Because shipping is a stressor, it was suggested that peripubertal stress exposure might interfere with the regulation of feminine sexual behaviors in adult female mice. As a result, the goal of this research was to examine the effects of peripubertal stress exposure on neuroendocrine regulation of the hypothalamo-pituitary-gonadal (HPG) and the hypothalamo-pituitary-adrenal (HPA) axis. However, although peripubertally exposing female mice to shipment stress caused a significant decrease in sexual receptivity, peripubertal exposure to restraint stress, food deprivation, social stress and instability, or multiple stressors (heat, light, and restraint) did not decrease sexual receptivity during adulthood. Only exposure to a high dose of bacterial endotoxin during the peripubertal period duplicated the defeminizing effect of shipping during the peripubertal period on adult mouse feminine sexual behavior. Interestingly, significant long-term effects of peripubertal stress exposure were also observed in the regulation of the HPA axis, where exposure to peripubertal shipping and LPS led to blunted stress-induced corticosterone responses in adult female mice. Peripubertal exposure to shipping and LPS had long-lasting effects on steroid receptor expression in brain areas relevant to female reproduction. Nevertheless, peripubertal exposure to LPS did not interfere with the ability of estradiol to induce the release of LH. Moreover, although peripubertal exposure to shipping decreased masculine sexual behavior in male, but not female, mice, peripubertal exposure to LPS was without effect on masculine sexual behavior. These results suggest that exposure to some, but not all, peripubertal stressors can induce long-lasting changes in reproductive behaviors, as well as changes in the expression of sex steroid receptors known to regulate these behaviors.

Background  Patients in the intensive care unit often receive broad spectrum antibiotic (e.g., gentamicin) treatment that are mostly eliminated through the renal route. Therefore, the kidney function is a crucial factor to establish a good dosage regimen especially in patients with acute kidney injuries. Old-fashioned estimation formulas are used today to estimate the kidney function and there is room for improvement. A publication from 2009 have presented that clearance for gentamicin is a good enough estimation compared to the golden standard measurement methods.  Aim  The aim of the study was to first find and investigate already developed equations to estimate non-steady-state kidney function. Thereafter, the aim was to identify which equations, both suited for steady-state and non-steady-state situations, that could best correlate to CLg in adult ICU patients.  Methods  PubMed was used to gather data about non-steady-state kidney function. The equations were then applied on Excel and a dataset containing 100 patients was used to calculate kidney function estimates. Clearance for gentamicin were then compared to the estimates using linear regression, Bland-Altman plots and P30 values.  Results and discussion  The Cockcroft-Gault formula had the highest P30-value, while Grubb’s cystatin C formula generated the best Bland-Altman plot as well as the best linear regression. However, neither of these were good enough to be used in the clinical work. Surprisingly, the Jelliffe equation was the poorest to estimate kidney function.  Conclusion  None of the evaluated estimations were clinically good enough to replace the gentamicin clearance method.

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Call number: LD2668 .T4 1978 P38 / Master of Science

Alzheimer’s disease (AD) is a neurodegenerative disease which affects over 500,000 people in the UK. Worldwide 44 million people are affected by AD and other dementias. Most cases occur over the age of 65 and is characterised by gradual and increasing loss of cognitive function and behavioural abnormalities. The main causes are a build-up of the toxic protein amyloid-β (Aβ) and hyperphosphorylation of the microtubule stabilising protein: tau, leading to neurofibrillary tangles (NFT). These two hallmarks of disease result in neuronal damage and cell death causing associated symptoms and eventually death. Orexins (OX) are neuropeptides which function to regulate the sleep-wake cycle and feeding behaviour. They are produced from a prepro-orexin (PPO) molecule and cleaved into two isoforms: orexin-A (OXA) and orexin-B (OXB). OXA and OXB are the ligands for two G-protein coupled receptors (GPCR): orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). 50-80,000 OX producing neurons project to many areas of the brain including the lateral hypothalamus (LHA), locus coeruleus (LC), tuberomammillary nucleus (TMN), paraventricular nucleus (PVN) and raphe nuclei and from these areas regulate feeding and appetite and the sleep wake cycle through their receptors. QRFP is a newly discovered neuropeptide which exerts similar orexigenic activity including the control of feeding behaviour. It is the ligand for the GPCR GPR103, both of which are widely expressed in the brain and also in the retina, testes, thyroid, pituitary and prostate. GPR103 also shares 48 and 47% protein sequence homology with OX1R and OX2R respectively. It is in these tissues where it can exert other physiological functions including regulation of feeding, control of the gonadotropic axis and bone formation. The exact expression and signalling characteristics and physiological actions of QRFP/GPR103 are still poorly understood. It is through the physiological functions of the orexigenic system and the clinical symptoms observed in AD which suggests a possible link between the two. For example, in AD one of the main reasons for institutionalisation is the severely dysregulated sleep pattern that is experienced by sufferers. They experience increased nocturnal activity and early awakenings as well as hypersomnia and excessive daytime sleepiness; all of which is beyond what someone of the same age experiences. As well as this AD patients suffer from significant weight loss and a significant negative correlation has been identified between progression of disease and appetite. All of this points towards an involvement of the orexigenic system in AD. AD patients have been found to have a 40% loss of immunoreactive OX neurons and have severe reductions in circulating OXA. This led us to believe that the OX system is of vital importance in AD and could be targeted to ameliorate symptoms. Studies have implicated OX and OXR in memory processes, appetite regulation, and severe disturbances of the sleep-wake cycle all of which are phenotypes of AD. Given that they play a key role in energy homeostasis and physiological behaviour, we hypothesise that OXs and their receptors are implicated in the pathophysiology of AD. Therefore, in this study we will investigate the detailed expression and signalling characteristics of OXR and GPR103 in vitro and in clinical samples In this study we neuronally differentiated two human neuroblastoma cell lines: IMR32 and SH-SY5Y. Neuronally acquired phenotype was confirmed through increased neurite length, increased expression of key neuronal proteins and increases in microtubule-associated protein tau (MAPT), neurogenin1 (NG1) and neuron-specific enolase (NSE) as well as a reduction in the neuronal marker of immaturity; nestin (NES). OXR and GPR103 were confirmed in both cell lines after differentiation at mRNA and protein level and were shown to be fully functional through phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2). We also identified possible cross talk of GPR103 with the OXR though addition of selective OXR antagonists, which blocked QRFP induced ERK1/2 phosphorylation. We show for the first time that addition of Aβ42 and zinc sulphate to mimic AD in vitro, results in a significant reduction of OX1R and GPR103 in the cell lines SH-SY5Y and we have performed the first comprehensive study in clinical AD patients which demonstrate a loss of OX1R, OX2R and GPR103 at mRNA and protein level compared to age matched controls in the hippocampus. We performed microarray analysis which identified many genes and pathways regulated by the OXA, OXB and QRFP; including corticotropin-releasing hormone receptor (CRHR1), regulated in development and DNA damage responses 1 (REDD1), erythropoietin (EPO), Bcl-2-like protein 1 (BCL2L11), myb proto-oncogene protein (c-myb), vasoactive intestinal peptide (VIP), endothelin 1 (EDN1) as well as the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and hypoxia-inducible factor-1α (HIF-1α) pathways. These genes are all implicated in neuroprotection, particularly in AD. This represents the first comprehensive gene expression data in a neuroblastoma cell line for these orexigenic proteins. Collectively these data suggest a potential role of the orexigenic system in neuroprotection and a functional loss of the receptors in AD patients which could confer a loss of neuroprotection through the orexigenic system. Pharmacological intervention directed at the orexigenic system may prove to be an attractive avenue towards the discovery of novel therapeutics for diseases such as AD and improving neuroprotective signalling pathways.