eScholarship@McGill

    Regulation of the sodium potassium adenosine triphosphatase (Na,K-ATPPase) by FXYD 2

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    The Na,K-ATPase, or Na+ pump is an integral membrane protein found in the cells of virtually all higher eukaryotes and is one of the most important systems in cellular energy transduction. Na,K-ATPase catalyzes the electrogenic exchange of three intracellular Na+ for two extracellular K+ ions coupled to the hydrolysis of one molecule of ATP. The research described in this thesis concerns the regulation of the Na,K-ATPase by FXYD 2, a member of the FXYD family of small single transmembrane proteins. FXYD 2, commonly known as the gamma modulator, is located primarily in the kidney and has a role in modulating the enzyme's apparent affinities for ligands. This study has addressed several aspects of gamma structure and function, namely its function in intact cells, the function of the gamma transmembrane domain, and delineation of regions of the enzyme's catalytic alpha subunit with which gamma interacts. Transport assays using intact transfected HeLa cells showed that the two gamma variants, gammaa and gammab, cause (i) an increase in K+/Na+ antagonism, seen as an increase in K'Na at high K+ concentration, and (ii) an increase in apparent ATP affinity seen as an increase in ouabain-sensitive K+ influx as a function of ATP concentration. These results are consistent with those obtained earlier with unsided membrane preparations. The present study also showed a gamma-mediated increase in steady-state intracellular Na+ concentration and, in contrast to assays using permeabilized membranes, a gamma-mediated increase in apparent affinity for extracellular K+. Experiments with synthetic gamma transmembrane (gamma-TM) peptides provided insight into the role of the TM region such that incubation of these peptides with membranes containing alphabeta pumps modulated K'Na similarly to transfected full-length gamma, indicating that the TM domain alone can cause an increase in K'Na at high K+ concentration. Results with gamma-TM bearing the Gly41→Arg missense mutation associated with familial renal hypomagnesemia provided direct evidence that this mutation prevents gamma association with alphabeta pumps. In a study aimed to identify regions of alpha critical for the functional effects of gamma, interactions of alpha1/alpha2 (and the reverse alpha2/alpha1) chimeras with gamma showed the importance of the carboxy terminus, particularly TM 9. The chimera data also indicate that interactions of transmembrane regions of the catalytic alpha subunit with FXYD proteins are not necessarily the sole determinants of the kinetic effects of gamma on Na+ affinity since the extramembranous L7/8 loop of a appears to modulate intramembranous alpha-gamma interactions that mediated the increase in K+/Na+ antagonism

    Developmental study of normative ritualistic

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    The high prevalence of compulsive-like behaviours in normal childhood suggests that these behaviours may play an important role in development. Furthermore, the similarities between the typical compulsive behaviours of childhood and the clinically significant behaviours that characterise Obsessive Compulsive Disorder (OCD) suggest that these two phenotypes may share similar neuropsychological profiles. In this study, two theories are investigated; one, that the neuropsychological deficits attributed to the pathogenesis of OCD also play a role in the manifestation of typical compulsive behaviours of childhood. Two, that compulsive behaviour supports the advancement of adaptive behaviour.The participants were 48 elementary school children (19 males) between the ages of 72 and 152 months (M= 106.5, SD = 24.49). Parents rated the frequency of typical compulsive behaviour on the Childhood Routines Inventory (CRI; Evans et al., 1997). Adaptive behaviour was assessed with the Vineland Adaptive Behavior Scales---Survey Form (VABS; Sparrow, Balla, & Cicchetti, 1984). The measure of set shifting was the manual 64-card version of the Wisconsin Card Sort Task (WCST; Kongs, Thompson, Iverson, & Heaton, 1981). Response inhibition was tested with the Conners' Continuous Performance Test (CPT; Conners, 2000) and the Tapping Test (Diamond & Taylor, 1996). Stepwise multiple regressions were performed to examine the relationships amongst variables of interest. The participants were later divided into three groups (seven years and younger, seven to ten years, ten years and older) to examine relationships by age. Adaptive behaviour and response inhibition were predictive of levels of typical compulsive behaviour across the age range. Together, coping skills and play and leisure skills as assessed on the VABS emerged as the most important predictors of the repetitive compulsive behaviours. Response latency on the tapping task was the strongest predictor of increased levels of just right compulsive behaviour. Set shifting did not predict levels of compulsive behaviour although positive correlations were found in the youngest group. Overall, the data support the notions that clinical OCD and the typical compulsive and ritualistic behaviour of childhood share similar neuropsychological profiles, and that compulsive behaviour in childhood supports the development of increasingly advanced adaptive behaviour

    Magnetar-like x-ray bursts from anomalous x-ray pulsars

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    The leading model for understanding the energetics of anomalous X-ray pulsars (AXPs) is that they are "magnetars"---young, isolated neutron stars powered by the decay of their enormous magnetic fields. The identification of AXPs as magnetars is motivated by the similarity of AXPs to another enigmatic class of sources, the Soft Gamma Repeaters (SGRs). We report on long-term monitoring of AXPs using the Rossi X-ray Timing Explorer (RXTE). We monitor AXPs with RXTE to study their rotational stability, to search for variations in their pulsed flux and pulsed morphology. During our regular monitoring observations we discovered multiple bursts from two AXPs: this was the first time such behavior has been observed from these sources. Prior to our monitoring campaign, the detection of bursts solely from SGRs was the principle difference between AXPs and SGRs. The first detection of AXP bursts came in Fall 2001 when we discovered two SCR-like X-ray bursts from the direction of AXP 1E 1048.1--5937. Due to RXTE's large (1° x 1°) field-of-view we could not unambiguously identify the AXP as the burster. Recently, we detected a third burst from the direction of this source as well as a simultaneous increase in the pulsar's pulsed emission---this clearly identified the AXP as the burster. The most outstanding demonstration of AXP bursting behavior came on 2002 June 18 when AXP 1E 2259+586 underwent a major outburst involving 80 bursts and several changes in the persistent and pulsed emission, including a huge pulsed flux enhancement, a pulse morphology change and a rotational glitch. We also find variations in the persistent emission of AXPs in the absence of an obvious outburst. For example, we discovered two pulsed flux flares from AXP 1E 1048.1--5937. Both flares lasted several months and had well resolved few-week-long rises. The long rise tunes of the flares is a phenomenon not previously reported for this class of object, but has a clear explanation within the context of the magnetar model. All these results imply a close relationship between AXPs and SGRs, which we now believe are both magnetars, and have posed significant challenges to competing models

    Force field adaptation in speech production

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    Although audition may appear to be the dominant sensory modality in speech production, the capacity for intelligible speech following severe hearing loss suggests that other sensory information - for example, somatosensory feedback - may also contribute to the achievement of speech targets. The aim of this thesis is to explore the role of somatosensory feedback in speech produced by healthy adults.The first study aimed at providing a test of whether somatosensory feedback plays a role in speech production beyond the language acquisition period in early childhood. In order to achieve this goal, we designed a pattern of forces that affects jaw movements during speech production, but at the same time has no measurable acoustic effect. We found that subjects compensated for such a distortion in speech movement trajectories, even though it had no impact on the sounds. In contrast, no adaptation was observed in matched non-speech jaw movements, indicating that this was not an inevitable consequence of exposing the orofacial apparatus to this pattern of forces. This is the first demonstration that somatosensory information on its own drives the achievement of articulator positions in speech.In study one, it was observed that subjects only adapted to the loads in the opening phase of the jaw movement. In order to elucidate this somewhat unexpected finding, we carried out experiments in which we manipulated the linguistic content of the training utterance. We found that subjects compensated for the perturbations only in portions of the movement that contained a vowel-to-vowel transition. It was suggested that the required kinematic precision during a transition between two vocal tract shapes associated with vowels is higher than during transitions between a consonant and a vowel. It also points to the speech-like nature of the observed adaptation.The third study aimed at investigating the extent to which speech motor learning generalizes across acoustic contexts. We trained subjects to produce utterances while exposed to a velocity dependent force field. After learning, the subjects were tested with new utterances, matched on dynamics to the ones used in training. Note that even if the acoustic contents of the test and the training utterances were different, the loads had a similar effect on both speech movements. We showed that learning did not transfer to the test utterances; therefore adaptation was restricted to the specific training context. These results point to the specificity of speech motor learning

    Actual versus best practices for young children with cerebral palsy : a survey of pediatric occupational therapists and physical therapists in Quebec, Canada

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    Background and rationale. Cerebral palsy (CP) is one of the most common disabling disorders of childhood and constitutes a substantial portion of pediatric rehabilitation. This condition demands comprehensive rehabilitation using age-appropriate tasks and activities and encompassing aspects of body function and structure, activity and participation, and personal and environmental factors. Yet little is known regarding actual occupational therapy (OT) and physical therapy (PT) practices.Objective. The primary objective of this doctoral thesis was to describe OT and PT practices for young children with CP in the Province of Quebec, Canada.Methods. This was a cross-sectional, multi-centered survey. All eligible and consenting pediatric occupational therapists (OTs) and physical therapists (PTs) were interviewed using a structured telephone interview based on vignettes of two typical children with CP at two age points---18 months and 4 years. Reported practices were grouped according to the International Classification of Functioning, Disability, and Health (ICF). Literature review of pediatric assessments and interventions potentially used for CP was done to determine their level of evidence of effectiveness. In addition, two expert groups provided, for each vignette, a best practice problem identification list and a best practice intervention list.Results. A total of 62 PTs (83.8% participation rate) and 85 OTs (91.4% participation rate) participated in the study. Overall, 91.9% of PTs and 67.1% of OTs reported using at least one standardized pediatric assessment for at least one vignette. OT and PT interventions focused primarily on impairments and primary function, with less attention to interventions related to play and recreation/leisure. Clinicians reported the need for more training and education specific to CP and to the use of research findings in clinical practice. Wide variations and gaps were identified in therapists' responses suggesting the need for a basic standard of PT and OT management as well as strategies to encourage knowledge dissemination regarding current best practice. Further, implementation of evidence-based practice necessitates more collaboration between researchers, clinicians and administrators

    Fatty acid biomarker analysis to characterize soil microbial communities in soybean agroecosystems with Sclerotinia stem rot disease

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    Soybean (Glycine max (L.) Merr.) is one the major crops produced worldwide. However, soybean is susceptible to many diseases. Sclerotinia stem rot (SSR) disease caused by Sclerotinia sclerotiorum (Lib.) de Bary is considered one of the most important fungal diseases of soybean. It can be controlled by chemicals (e.g. fungicides), by breeding cultivars with disease resistance and by cultural control (e.g. increasing the width between rows, reducing plant populations). A promising and complementary method of controlling SSR disease in the field is the application of biological control agents. Biological control agents introduced in a soil environment will interact with other soil food web organisms, as do the pathogenic organism and infected plants, which may change the genetic and functional diversity in soil microbial communities. Profiling these changes may lead to an improved understanding of the interactions between these players (biological control agents, pathogens, soil biota and plants) in the biological control phenomenom, permiting us to exploit naturally-occurring ecological relationships and develop more sustainable approaches to control soybean diseases. Fatty acid biomarkers analysis was used to profile microbial communities in soils. Two laboratory studies were conducted to evaluate the methods used for extraction and profiling the fatty acid biomarkers from soil samples with a range of soil properties (clay content, organic matter content), The first study investigated the best solvent mixture for recovering fatty acid biomarkers from soil using an automated pressurized solvent extraction (PSE) system. Solvent mixtures containing chloroform and methanol were more efficient at extracting fatty acids from agricultural soils than hexane:2-propanol and acetone. The second study presented an exploratory pyrolysis-mass spectrometry technique to rapidly fingerprint soil lipids extracted from different agroecosystems. Pyrolysis-mass spectrometry discriminated among soils and crop production systems in the same way as the fatty acid profiling. I also report on the efficicacy of biological control agents to control Sclerotinia stem rot disease in soybean. A two-year study was conducted in soybean fields under conventional or no tillage to determine whether Trichoderma virens (SoilGard(TM)) and arbuscular mycorrhizal fungi (a mixture of Glomus intraradices and G. mosseae ), used alone or in combination, could reduce sclerotinia stem rot (SSR) disease incidence. Generally, SSR disease indicators, as well as the soybean yield, were not affected significantly by the biological control treatments. I then studied whether changes in microbial community composition were related to the inoculation of the biological control agents and the disease incidence in soybean fields. Inoculation of biological control agents changes the expression of many soil fatty acids during both years of the trial. Also, in the plots with severely diseased plants, fatty acids biomarkers of gram positive and actinomycetes bacteria were significantly greater than in plots with healthy plants. I conclude that further improvement in laboratory techniques and procedures will permit researchers to efficiently extract and characterize soil lipids, providing new insight into soil organic matter dynamics and soil microbial ecology. Further study will be needed to verify the efficacy and optimize the application method, dose and timing of biocontrol agents to provide protection against SSR disease in soybean fields

    Characterization of the nod and sdh operons in the legume symbionts Bradyrhizobium japonicum and Sinorhizobium meliloti

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    This study was undertaken to characterize the nod and sdh operons of Bradyrhizobium japonicum and Sinorhizobium meliloti. Ten putative B. japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. The mutants were found to have higher nodY expression than the wild-type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1muM) and/or temperature (15°C). The expression of nodY gene induction was determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochitooligosaccharide than the wild-type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. Based on nodY gene expression and symbiotic phenotype the B. japonicum mutants were classified into three groups.A molecular genetic approach was taken to investigate the regulation of expression of succinate dehydrogenase (SDH) in S. meliloti. The sdhCDAB genes encoding SDH were shown by RT-PCR to be co-transcribed and thus constitute an operon. The transcriptional start site and putative promoter region of the first gene in the operon, sdhC , were identified by 5'-RACE and DNA sequence analysis. Transcriptional lacZ fusions to sdhC indicated that expression of the operon is regulated by carbon source in the growth medium but not by growth phase. The highest expression of the sdh operon was observed in cells grown with acetate, arabinose and glutamate, as sole carbon sources, and the lowest expression was observed in cells grown with glucose and pyruvate as sole carbon sources.Also presented is the isolation and characterization of the first defined sdh mutant in a rhizobial species. The mutants helped demonstrate that the total lack of SDH activity would be lethal to S. meliloti cells. Symbiotic phenotype of the mutants indicated that SDH is required for N2-fixation

    Proteomics and genetic studies of dystroglycan function in the nervous system

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    Muscular dystrophies are a group of diseases that are often caused by loss-of-function mutations affecting the dystrophin glycoprotein complex (DGC). The common feature of the diseases is muscle degeneration, which is often associated with mental retardation and various retinal defects, including ones of synaptic transmission. However, the mechanisms of the disease remain largely unknown, especially those in the central nervous system. I have focused on dystroglycan (DG), the transmembrane protein in the DGC that links the cytoskeleton to the extracellular matrix and is essential for muscle survival and brain development. I have used proteomics and Drosophila genetics to study DG function in the brain and retina.Using proteomics I found that beta-DG is directly associated with the GTPase dynamin 1 in the retina and in the brain together with alpha-DG and Grb2, and immunohistochemically beta-DG was colocalized with dynamin 1 in the outer plexiform layer where photoreceptor terminals are localized. Moreover, loss of DG in differentiated DG-null embryonic stem cells significantly increases dynamin-mediated transferrin-uptake and re-expression of DG in null cells by infection with an adenovirus containing DG reduced transferrin uptake to levels seen in wild-type cells. This result implies that one of mechanisms in muscular dystrophy might be the altered synaptic vesicle endocytosis, especially in the retina where synaptic transmission defect has been known for decades.Muscular dystrophies show not only impaired retinal synaptic transmission and several DG-related congenital muscular dystrophies also display retinal structural defects. To further understand the roles of DG in the retina, I used Drosophila eye as a model and demonstrated for the first time that DG is required cell-autonomously for photoreceptor morphogenesis in the developing visual system. Deficiency of DG in the eye causes severe disruption of retinal structure, aberrant lens formation and abolition of electroretinogram in the adult fly eye. These adult defects appear derived from autonomous photoreceptor cell (PRC) defects in the early pupa including size arrest, loss of polarity and progressive degeneration. All defects in the eye, however, can be reversed by re-expression of wild type DG in DG-deficient PRCs, suggesting DG functions cell-autonomously in PRCs and non-autonomously for lens. In the 3rd instar larvae DG is present in the apical tips and the basal membranes of PRCs, two polarized locations opposing the extracellular matrix. At the pupal stage it continues to mainly distribute at the apical rhabdomere and basal membrane of PRCs. Over-expression of DG leads to larger ommatidia but the PRC number remains unchanged, suggesting that DG is both necessary for and sufficient to promote PRC expansion. By rescue experiments, I demonstrated that the extracellular DG alone could not rescue DG-deficient eye defects, whereas the intracellular DG can substantially ameliorate PRC degeneration and structural defects while some PRCs remain disorganized, a sign of disrupted PRC planar polarity in absence of the extracellular DG. Therefore, our data suggest that the degeneration and planar polarity disruption in DG-deficient PRCs are two independent processes that appear to require the respective function of intracellular and extracellular DG. In summary, our experiments demonstrated several novel findings and provided the basis for future investigations on DG function and the molecular mechanisms of nervous system defects in muscular dystrophies

    Location and expression of genes related to the cytoplasmic male sterility system of Brassica napus

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    Cytoplasrnic male sterility (CMS) is a maternally inherited defect in the production of pollen, the male gamete of the flower. This sterility can be suppressed by nuclear Restorer of Fertility (Rf) genes that normally downregulate the expression of the CMS-associated novel mitochondrial gene. In Brassica napus, nap CMS and pol CMS are associated with related chimeric mitochondrial genes orf222 and orf224, respectively. CMS in both nap and pol is associated with a polar loss of locule development, loss of synchronous locule development and clumping of sporogenous tissue away from the tapetal cell layer, as well as secondary effects on petal and bud formation. In nap CMS, early accumulation of orf222 transcripts in the locule regions of developing anthers is associated with sterility, while the absence of orf222 transcripts from the locules is associated with fertility restoration. Accumulation of novel antisense transcripts of atp6 in a cell specific manner which matches that of sense transcripts of orf222 and atp6 in nap CMS anthers may be indicative of a post-transcriptional regulatory mechanism associated with CMS in flower buds.Restoration of fertility in Brassica napus nap and pol CMS is associated with nuclearly encoded genes Rfn and Rfp, respectively. These restorers are very closely linked to one another, and may be allelic. Further efforts to isolate Rfp have narrowed the genomic region to approximately 105 kb of a syntenic region in Arabidopsis thaliana. Cosmid clones isolated from a library of Brassica rapa genomic DNA introgressed with Rfp have been successfully sorted into contigs through the application of the amplified fragment length polymorphism technique. The region to which Rfp is mapped is syntenic to a region of Arabidopsis DNA that is a duplication of a second location at the 23 megabase region of chromosome 1 of that genome. This region contains pentatricopeptide (PPR) motif-encoding genes that are highly related to other restorers of fertility of other species. By inference, Rfp from Brassica napus may encode PPR motifs. The PPR genes related to these previously characterized restorers of fertility are often found alongside the restorer genes existing as mini-clusters of several PPR-encoding genes. This is likely caused by selective pressure acting on PPR-encoding genes that resulted in diversification and multiplication of these genes. In addition, the PPR genes of this duplicated region are not syntenically located, whereas the non-PPR-encoding genes maintain their syntenic locations. The same is true for orthologous comparisons between Arabidopsis and other plant species. PPR genes are therefore malleable and capable of alteration in response to changing environmental pressures, such as the evolution of sterility inducing genes

    The impact of the chemotherapeutic drug cyclophosphamide on rat spermiogenic chromatin remodeling /

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    Male reproductive health is of growing concern, as male toxicant exposure can affect progeny outcome; sperm chromatin structure integrity may be a contributing factor. The formation of mature sperm involves the expression of numerous proteins involved in organizing and packaging the chromatin in a specific manner; this ensures transmission and participation of the paternal genome in embryogenesis. Exposure of male rats to cyclophosphamide as spermatid chromatin is remodeled has an adverse effect on embryo development. The hypothesis of this thesis is that cyclophosphamide exposure causes genetic damage and alters the sperm proteome, thus disrupting components of chromatin condensation and organization during spermiogenesis. The first objective was to assess the phase specificity of the susceptibility of spermiogenic germ cells to cyclophosphamide-induced DNA damage. Spermatozoa were analyzed for DNA strand breaks using the comet assay. Cyclophosphamide-induced DNA damage was dose-related and accumulated over time. Germ cell phase-specific damage was maximal during midspermiogenesis; this reflects an increased susceptibility of step 9-14 spermatids at a key point in sperm chromatin remodeling, the histone-protamine exchange. The second objective was to examine the sperm chromatin structure and basic proteome. Multiple assays demonstrated that the effects of cyclophosphamide on sperm chromatin structure were also germ cell-phase specific; midspermiogenic spermatids were most sensitive. Sperm were less condensed with reduced total thiol and protamine contents. The sperm basic proteome was also altered; identified proteins are involved in a variety of spermiogenic and fertilization events. The nuclear matrix organizes chromatin into loop domains, and various components of somatic cell matrices are targets for chemotherapeutic agents. Therefore the last objective of this study was to assess the effect of cyclophosphamide exposure on the protein profile of the sperm nuclear matrix. The expression of several nuclear matrix protein components, a number of which were identified for the first time, was altered following drug exposure. Together these results show that cyclophosphamide alters male germ cell chromatin remodeling at both the DNA and protein level; this could alter sperm function and thus explain the adverse effects on early embryo development
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