RET is a tyrosine kinase receptor that induces cell survival, proliferation and migration. When bound by its ligand, GDNF (glial derived neurotrophic factor) and its co-receptor, GFRalpha-1 (GDNF family receptor alpha-1), tyrosine residues in the intracellular domain of the receptor are phosphorylated as part of signal transduction. In the kidney, RET is required for branching of the ureteric bud and it also regulates the maturation of the ureter in the urinary tract. Although it is well established in mice that the absence or overexpression of RET leads to severe kidney abnormalities, the cellular basis for these defects remains unexplored. To determine whether the overexpression of RET affected the development of the urinary tract, HoxB7/RET mice were tested for the presence of vesico-ureteric reflux (VUR) in which urine flows retrogradely from the bladder back up the ureter. To further understand how the urinary tract is formed, we looked for the presence of VUR in HoxB7/RET and wildtype mice at E17. These results provide a new understanding of how combined kidney and urinary tract malformations occur in humans.

The chondroitin sulphate epitope 846 of aggrecan is abundant in foetal cartilage, barely detectable in normal adult cartilage, but reappears in the cartilage and body fluids of arthritic patients. This epitope has been proposed to be a marker of aggrecan synthesis during new cartilage formation and repair. The purpose of the present studies was to investigate, in vitro, whether the epitope was truly reflective of aggrecan synthesis, and to understand its role in the cartilage repair process. Foetal bovine chondrocyte cultures were established to study aggrecan synthesis in order to investigate whether the epitope 846 was present on these molecules. These studies showed that the epitope was indeed present on the newly synthesised aggrecan molecules and that these were preferentially retained within the extracellular matrix. The larger size and higher epitope density of the matrix molecules, compared to those molecules which were released into culture medium, suggested a role for the 846-epitope bearing molecules in the formation of new cartilage and during repair. Normal adult articular cartilage cultures were established to investigate whether the epitope could be synthesised by this tissue under conditions where the tissue had been stimulated to repair a damaged matrix, by trypsin-treatment of the cartilage. In these studies, an increase in abundance of the epitope on newly synthesised proteoglycans was observed, further indicating a role for the epitope-bearing molecules in cartilage repair. Explant cultures of osteoarthritic cartilage demonstrated that the release of the 846-bearing molecules from the cartilage was accompanied by the release of some of the newly synthesised 35S-sulphate labelled proteoglycans from the cartilage, and that these molecules contained, in part at least, the epitope. In addition, the release of 846-bearing molecules from the cartilage into culture medium or synovial fluid was reflective of the epitope content of the tissue. The stru

The pharmacokinetics of verapamil were evaluated in the young and elderly after steady state administration of a sustained release formulation. The objective was to determine whether the interindividual metabolic variability of verapamil and its main metabolites could be correlated with the enzymatic activity of CYP3A4 by using dextromethorphan (DM) as an in vivo probe of CYP3A4 activity. / Pharmacokinetic analysis showed a significant age related increase in the clearance of verapamil. Verapamil interindividual variability could not be correlated with formation of 3-methoxy-morphinan. Our results suggest that verapamil metabolism may be significantly mediated by other cytochromes.

Treatment of chick myoblasts with the glucosidase inhibitors bromoconduritol (BCD) or N-methyl-1-deoxynojirimycin (MDJN), but not the mannosidase I inhibitor 1-deoxymannojirimycin (ManDJN), decreased the rate of cell-adhesion to fibronectin and laminin and increased the rate of adhesion to collagen. The adhesion of chick myoblasts is predominantly mediated by integrin(s), as judged by inhibition of adhesion by an anti-integrin antibody (JG22). The effects of BCD, MDJN and ManDJN on myoblast integrin detectable at the myoblast cell surface with JG22 antibody correlated well with their effects on adhesion to fibronectin and laminin, and paralleled the previously reported effects of these agents on myogenesis. Interaction of integrin with the extracellular matrix appears critical for terminal differentiation. Mn$ sp{2+}$ ions increased myoblast adhesion to ECM proteins, and antagonized the effect of BCD and MDJN on myoblast differentiation. / Finally BCD and MDJN were found to act at an early stage in myoblast differentiation, down regulating the expression of both myogenin and muscle $ alpha$-actin mRNA in L$ sb6$ myoblasts.

Idiopathic central precocious puberty may compromise adult height. Gonadotrophin-releasing hormone analogues (GnRHa) suppress puberty and increase final height prediction, but their influence on final height is unclear, especially in girls with pubertal onset just below eight years of age, the traditional limit of normal. / At seven children's hospitals, we identified 53 treated and 24 untreated patients (24 and 7 to final height, respectively) whose pubertal onset was between ages 5--8 years. At baseline, bone age advancement and predicted adult height were similar in the two groups. In both groups, the predicted adult height slightly overestimated the final height. What role, if any, GnRHa therapy played in preventing a shorter adult height is uncertain in these borderline cases. The substantial intra- and inter-observer variability in bone age readings compromised the utility of the height prediction method. / The methodological challenges inherent to this study are identified and discussed.

The intervertebral disc has been identified as a possible contributor to two very common back disorders: degenerative disc disease and scoliotic deformity. Little is known about the composition and turnover of extracellular matrix in the human intervertebral disc and end-plate with these two pathologies. / We studied 121 intervertebral discs representing all age groups and degeneration grades. In addition, fifteen scoliotic discs and seventeen control discs were analyzed. / Synthesis in intervertebral discs, end-plates, and scoliotic tissues were measured by immunoassay measuring the content of a putative aggrecan biosynthesis marker (846) and contents of types I and II procollagen markers (CPI & CPII). The percentage of denatured type II collagen was assessed by the presence of an epitope that is exposed with cleavage of type II collagen. / We identified three matrix turnover phases in the intervertebral disc and end-plate. Phase I (growth) is characterized by active synthesis of matrix molecules and active denaturation of type II collagen. Phase II (maturation and ageing phase) is distinguished by progressive drops in synthetic activity and denaturation of type II collagen. Phase III (degeneration and fibrotic phase) is illustrated by decreased synthesis of aggrecan and type II procollagen and an increase in collagen type II denaturation and type I procollagen synthesis, the later dependent on age and grade of tissue degeneration. / The results of the scoliotic study indicate that synthesis of type II procollagen in the end-plate and annulus was higher in scoliotic discs. Aggrecan synthesis showed a similar trend in the nucleus pulposus of scoliotic disc. By contrast, the contents of hydroxyproline, glycosaminoglycan, and water were significantly lower in scoliotic discs while total protein content was higher in scoliotic discs, indicating that non-collagenous protein content was increased in scoliotic discs. This elevated synthetic activity without an associated collagen denaturation reflects an overall synthetic response without an increased matrix turnover, suggesting that scoliotic changes are due to an altered and ineffective synthetic response to a pathologic mechanical environment. / This thesis represents the first in situ quantitation of human lumbar intervertebral disc matrix composition and turnover with ageing, degeneration, and scoliotic change. These observations are an important advancement in our biochemical understanding of disc ageing, disc degeneration, and adolescent idiopathic scoliosis.

The replacement of the hyaline cartilage model by bone tissue during osseous development involves an extensive resorption of cartilage. The current study examines the role played by matrix metalloproteinases (MMPs) in cartilage resorption in the tibial epiphysis of 8 and 10 day old rats. The entry of cellular, highly vascularized channels requires the degradation of cartilage components. As a first step, aggrecan is cleaved by an unidentified MMP along the edges of the channel walls, as detected by an antiserum directed to a cleavage fragment retained in the tissue; the presence of an active MMP at the channel edge is confirmed by the binding of a recombinant MMP inhibitor, TIMP-2. Second, using histozymography, the collagen component of cartilage is degraded by another MMP, identified as gelatinase B by inhibition studies, at similar channel sites. Thus, two proteinases combine their efforts to produce the resorption associated with channel invasion preceding the appearance of the secondary ossification center in the rat tibial epiphysis.

Genetic and non-shared environmental factors (factors experienced by one child to the exclusion of his siblings) have been strongly implicated in the aetiology of Attention Deficit Hyperactivity Disorder (ADHD). Shared environmental factors were not implicated. Pregnancy, labour/delivery and neonatal complications (PLDNC) were often associated to ADHD. However, no investigations aimed at delineating the shared/non-shared nature of these factors were reported. In order to identify PLDNC which are more likely to be non-shared, we recruited 65 children with ADHD and assessed their history for PLDNC. 45 of them had non-affected siblings that were also assessed for PLDNC. Compared to their non-affected siblings, ADHD children had significantly higher neonatal complications (p = 0.01). Furthermore, neonatal complications were associated with higher motor activity (p = 0.04) and attention deficits (p = 0.03) in ADHD diagnosed children, suggesting that neonatal complications are more likely to be non-shared environmental factors of etiological relevance to ADHD.

<p> There are two main patterns of development within animals: direct and indirect. Direct developers are animals such as humans, whose offspring are basically smaller versions of the adult. Indirect developers have a larval stage that can be dramatically different than the adult, and consequently go through a transformation known as metamorphosis. Frogs are a well-known example of vertebrate indirect development, developing first into an aquatic, herbivorous tadpole that later transforms into a terrestrial, carnivorous frog. This transformation is largely regulated by a single hormone&mdash;thyroid hormone (TH). Changes in TH play a vital role in tissue transformations such as, differentiation and growth of the limbs, remodeling of the gut tube and liver, as well as resorption of larval features such as the tail. In addition, the timing of metamorphic changes can differ substantially among species. For instance, the average rate of metamorphosis for a majority of frogs/toads (anurans) is between 3-5 weeks, yet there are a multitude of frogs and toads that take several months or even years to go through this process (Duellman and Trueb, 1994; Gilbert, 2010; Petranka, 2007; Provenzano and Boone, 2009). At the other extreme are frogs that have reduced or even eliminated the free-living larval period such as the Eastern Spadefoot toad, <i>Scaphiopus holbrookii, </i> which has one of the shortest larval periods found in metamorphosing frogs or direct developing frogs, like <i>Eleutherodactylus coqui</i> which have no free-living larval stage. This diversity of developmental patterns sparks many questions about the precise molecular and developmental roles TH has on metamorphosis in frogs. How can a single regulator produce such a wide range of responses, not only between species that differ in metamorphic timing, but also among tissues within a single individual? The vast majority of recent studies that examine tissue specific responses to TH have focused on the genes that code for TH binding proteins or for TH receptors (Hollar et al., 2011; Buchholz et al., 2011). However, a very important aspect is being overlooked in these studies, which is the actual level of the TR proteins themselves. </p><p> There are three distinct mechanisms that regulate tissue responses to TH: deiodinase enzymes, cytosolic thyroid hormone binding proteins (CTHBPs), and thyroid hormone receptors (TRs) (Morvan-Dubois <i>et al.,</i> 2008: Buchholz <i>et al.,</i> 2006). The overall objective of the current study is to develop a technique that will allow protein level analysis of two of the three components implicated in the regulation of tissue specific responses to TH during tadpole metamorphosis: CTHBPs and TRs. I hypothesize that changes in the affinity and/or capacity of thyroid hormone receptors and cytosolic thyroid hormone binding proteins to bind TH throughout metamorphosis underlie the timing and extent of tissue remodeling. In fresh tissue, saturation binding assays suggest a difference in binding capacity among tissues.</p>

<p> Schizophrenia is a prevalent neurological disorder characterized by disrupted neuronal circuitry. Antipsychotic drugs (APDs) are capable of ameliorating the symptoms of schizophrenia with varying efficacy. Clozapine, the "gold-standard" for antipsychotic drug treatment, has been shown by this lab to induce the outgrowth of mediodorsal thalamic (MDT) dendritic arbor in rodents, a brain region which has altered function and decreased regional volume in schizophrenic patients. These studies further explored the ability of APD treatment to restructure dendrite arbor and the mechanisms of clozapine's ability to elaborate MDT arbor. Additionally, glutamate hypofunction is thought to contribute to the schizophrenic disease state. Using a novel model of perinatal glutamate hypofunction, we examined the long-term effects on dendritic architecture of developmental glutamate signaling disruption. </p><p> MDT dysfunction is hypothesized to contribute to cognitive symptoms of schizophrenia. Clozapine has increased efficacy in ameliorating these symptoms. To further understand clozapine's actions to remodel MDT dendritic architecture, we examined whether clozapine-induced morphological alterations are limited to the thalamus or if they also occur in additional regions associated with cognitive schizophrenic pathology, the hippocampus and striatum. We found that clozapine can induce dendritic remodeling in the hippocampus, but the not to the amplitude of remodeling seen in the thalamus, indicating that the MDT is uniquely altered by clozapine treatment and may be an important locus of clozapine's action. </p><p> The mechanisms of clozapine's remodeling of MDT arbor, we examined changes to mRNA and miRNA expression and calcium dynamics in the MDT in response to APD treatment. Clozapine-treatment altered the expression of genes involved in cytoskeletal remodeling, external membrane receptors, and calcium dynamics, as well as increased the rate of calcium influx into thalamic neurons. </p><p> Disruption to glutamate signaling has been hypothesized to contribute to schizophrenic pathology. Disruption to perinatal vesicular glutamate packaging along the corticolimbic axis has long term effects for neuronal morphology and function. Interestingly, we find that disruption along the corticolimbic axis also has downstream effects on MDT dendritic architecture. </p><p> These studies show that the MDT is an important locus of action for clozapine and is capable of remodeling dendritic architecture in response to afferent circuitry dysfunction.</p>