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Von, Schnakenburg Claus Christian. « Molecular analysis of the AGXT gene and linkage studies in primary hyperoxaluria type 1 ». Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299831.
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DEMARTA, JOCELYNE. « L'hyperoxalurie primaire de type i de revelation tardive chez l'adulte : a propos de deux cas ». Reims, 1992. http://www.theses.fr/1992REIMM081.
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Newbound, Garret C. « Transcriptional control of human t-cell leukemia virus type-1 in primary lymphocytes / ». The Ohio State University, 1997. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487948440826361.
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Hayman, Anna. « The biodiversity of human immunodeficiency virus type 1 : evolution during primary infection and transmission ». Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.252357.
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Estève, Julie. « Transfert de gènes dans les cellules souches pluripotentes induites : application à la thérapie génique de l'hyperoxalurie primitive de type 1 ». Thesis, Bordeaux, 2018. http://www.theses.fr/2018BORD0280/document.
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L’hyperoxalurie primitive de type 1 (ou HP1) est une maladie héréditaire du métabolisme liée à un déficit en enzyme hépatocytaire AGT (alanine:glyoxylate aminotransférase), codée par le gène AGXT. Ce déficit entraîne, chez les patients atteints d’HP1, une excrétion hépatique accrue d’oxalate ; celui-ci est ensuite éliminé dans les urines où il se complexe avec le calcium pour former des néphrolithiases oxalo-calciques massives, pouvant conduire à une insuffisance rénale chronique. Le seul traitement curatif disponible pour cette pathologie est la greffe allogénique combinée hépatorénale, actuellement limitée par la disponibilité des donneurs de greffons, une morbi-mortalité significative et la nécessité d’un traitement immunosuppresseur au long cours. L’objectif du projet de recherche est de développer une thérapie génique de l’HP1 par greffe de cellules hépatiques autologues génétiquement corrigées. La faible disponibilité et la difficulté d’amplification in vitro des hépatocytes adultes nous a conduit à explorer la piste des cellules souches pluripotentes induites (iPSCs) pour produire des cellules hépatiques humaines utilisables en médecine régénérative. Nous avons dérivé et caractérisé des lignées de cellules iPSCs à partir de fibroblastes de patients atteints d’HP1, après expression transitoire des facteurs de reprogrammation par des vecteurs Sendai. Nous avons développé deux stratégies de thérapie génique additive par insertion d’un minigène codant une séquence optimisée de l’ADNc AGXT au moyen (1) d’un vecteur lentiviral à expression hépato-spécifique et (2) d’un processus de recombinaison homologue au locus AAVS1 facilité par le système de clivage ciblé de l’ADN « CRISPR/Cas9 ». Enfin, nous avons mis en évidence l’expression de la cassette thérapeutique après différenciation hépatocytaire des iPSCs génétiquement corrigées. Ces résultats ouvrent de nouvelles perspectives de médecine régénérative pour l’HP1 par transplantation de cellules hépatocytaires autologues génétiquement corrigées dérivées d’iPSCs de patientsPrimary hyperoxaluria type 1 (or PH1) is an inherited metabolic disorder related to the deficiency of the hepatic AGT enzyme (alanine:glyoxylate aminotransferase), which is encoded by the AGXT gene. In PH1 patients, this deficiency leads to oxalate overexcretion by liver, followed by urine filtration and complexation with calcium to form massive calcium-oxalate nephrolithiasis potentially leading to chronic renal failure. The only available curative treatment is combined hepatorenal allogeneic engraftment, which is currently limited by the availability of transplant donors, significant morbidity and mortality, and the need for long-term immunosuppressive treatment. The aim of our research project is to develop gene therapy for PH1, consisting in engraftment of genetically corrected autologous liver cells. Considering that adult hepatocytes are hardly available and expandable in vitro, we chose to explore the use of induced pluripotent stem cells (iPSCs) to produce human liver cells for application in regenerative medicine. We derived and characterized iPSC lines from PH1 patient fibroblasts after transient expression of reprogramming factors delivered by Sendai virus vectors. We developed two additive gene therapy strategies by inserting a minigene encoding an optimized AGXT cDNA sequence using (1) a lentiviral vector designed for liver-specific expression and (2) homologous recombination process at the AAVS1 locus favoured by the targeted DNA cutting system “CRISPR/Cas9”. Finally, we highlighted therapeutic cassette expression after hepatic differentiation of genetically corrected iPSCs. These results pave the way for regenerative medicine for PH1 by transplantation of genetically modified autologous hepatocyte-like cells derived from patient-specific iPSCs
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Hildick, Keri. « Mechanisms underlying the trafficking and distribution of cannabinoid receptor type 1 in primary hippocampal neurons ». Thesis, University of Bristol, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654590.
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coupled receptors (GPCRs) in the central nervous system, where it is concentrated at the axonal plasma membrane of specific neurons. Whilst the , therapeutic and psychotropic effects of cannabinoid receptor stimulation have been recognised for over 4000 years, through recreational and medicinal Cannabis use, ,it is only in the last few decades that our scientific understanding of both exogenous and endogenous cannabinoid system regulation has significantly advanced. At the presynaptic terminal, CB1R activation mediates neurotransmission through retrograde messengers, which are released from the post-synaptic terminal in response to activity-dependent signal transduction. Consequently the CB1R can be considered as a master regulator of synaptic transmission. However, compared to other GPCRs, the field of CB1R trafficking is in its infancy and the pathways and mechanisms of CB1R regulation are at best, incomplete. Using N-terminal fluorescent-tagged CB1R constructs, I have investigated the trafficking and diffusional mobility of CB1Rs in cultured hippocampal neurons. My work has revealed a crucial role for the C-terminal tail of the CB1R (ctCB1R) in regulating both its plasma membrane dynamics and surface localization. Specifically, using truncation and deletion mutagenesis I identified a 21 amino acid stretch in the ct-CB1R, which is critical for maintaining its axonal polarized surface distribution in hippocampal neurons. This region corresponds to a putative helical motif, H9, for which no known functional role has previously been identified. Moreover, I have demonstrated that the ct-CB1R . is sufficient to promote an axonal fate, presumably through interactions with specific scaffolding and adaptor proteins. Accordingly, I have performed an unbiased proteomics analysis using affinity purification mass spectrometry and have identified several candidate interactors, including a cluster of proteins associated with clathrin-mediated 'endocytosis, which , provide additional mechanistic insight into the underlying biochemical machinery regulating CB1R distribution in neurons .
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Raker, Verena [Verfasser]. « Herpes simplex virus type 1 (HSV-1) infection alters growth factor signaling in primary cortical brain cells / Verena Raker ». Hannover : Bibliothek der Tierärztlichen Hochschule Hannover, 2017. http://d-nb.info/1150340320/34.
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Loro, Emanuele Loro. « Normal myogenesis and increased apoptosis in myotonic dystrophy type-1 muscle cells ». Doctoral thesis, Università degli studi di Padova, 2010. http://hdl.handle.net/11577/3423200.
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Myotonic dystrophy type 1 (DM1) is caused by (CTG)n expansion in the 3’-untranslated region of DMPK gene. Mutant transcripts are retained in nuclear RNA foci, which sequester RNA binding proteins thereby misregulating their functions (i.e. splicing regulation). Controversy still surrounds the pathogenesis of the DM1 muscle distress, characterized by myotonia, weakness and wasting with distal muscle atrophy.
Eight primary human cell lines from adult-onset (DM1) and congenital (cDM1) patients, (CTG)n range 90-1800, were successfully differentiated into aneural-immature and contracting-innervated-mature myotubes. Morphological, immunohistochemical, RT-PCR and Western blotting analyses of several markers of myogenesis indicated that in vitro differentiation-maturation of DM1 myotubes was comparable to age-matched controls. In all pathological muscle cells, (CTG)n expansions were confirmed by long PCR and RNA fluorescence in-situ hybridization. Moreover, the DM1 myotubes displayed the splicing alteration of insulin receptor and MBNL1 genes associated to the DM1 phenotype.
Considerable myotube loss and atrophy of 15-day-differentiated DM1 myotubes indicated activated catabolic pathways, as confirmed by the presence of apoptotic (caspase-3 activation, cytochrome c release, chromatin fragmentation) and autophagic (P62/LC3) markers. Treatment with the pancaspase inhibitor Z-VAD significantly reduced the decrease in myonuclei number and in average width in15-day-differentiated DM1 myotubes. We thus propose that the muscle wasting typical in DM1 is due to impairment of muscle mass maintenance-regeneration, through premature apoptotic-autophagic activation, rather than altered myogenesis.La distrofia miotonica di tipo 1 (DM1) è causata dall'espansione (CTG)n nella regione trascritta ma non tradotta al 3' del gene DMPK. I trascritti mutati sono trattenuti in foci nucleari, i quali sequestrano diverse proteine leganti RNA spesso alterandone le funzioni (i.e. regolazione dello splicing). A livello del muscolo, i meccanismi patogenetici che portano a miotonia, debolezza e perdita di massa dei muscoli distali, non sono ad oggi chiari. Otto linee di mioblasti primari umani, ottenuti da biopsie di pazienti affetti da DM1 nelle forme adulta e congenita (range di espansione tra 90 e 1800 CTG), sono state differenziate ed innervate con successo, ottenendo miotubi in grado i contrarre. L'analisi morfologica e la quantificazione di diversi marker di miogenesi mediante RT-PCR e Western blotting, hanno indicato che il diferenziamento in vitro dei mioblasti primari DM1 è indistinguibile da quello ottenuto con mioblasti di controllo. In ciascuna linea DM1 è stata confermata l'espansione (CTG)n mediante long-PCR ed ibridizzazione in situ. Inoltre, nei miotubi DM1 è stato rilevata l'alterazione dello splicing del recettore per l'insulina e di MBNL1, caratteristica del fenotipo DM1. A 15 giorni di differenziamento, una considerevole perdita di miotubi DM1 ha suggerito l'attivazione di pathways catabolici, come confermato dalla presenza di marker di apoptosi (taglio proteolitico della caspasi 3, rilascio di citocromo c dai mitocondri, frammentazione della cromatina) e di autofagia (aumento dei livelli di LC3 lipidato e di P62). Il trattamento con l'inibitore delle caspasi Z-VAD si è dimostrato efficace nell'attenuare la riduzione del numero di mionuclei e del calibro medio dei miotubi a 15 giorni di differenziamento. Proponiamo quindi che la compromissione muscolare tipica della DM1 sia dovuta, più che ad un'alterata miogenesi, a problemi nei meccanismi di mantenimento/rigenerazione, che si esplicano attraverso la prematura attivazione di apoptosi e/o autofagia
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Villablanca, Andrea. « Genetic background of familial primary hyperparathyroidism / ». Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-520-4/.
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Gaston, Andrea Michelle Marshall. « Adolescents with type 1 diabetes mellitus : an exploration of patients' and their primary caregivers' illness representations ». Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.410941.
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Vierimaa, O. (Outi). « Multiple Endocrine Neoplasia Type 1 (MEN1) and Pituitary Adenoma Predisposition (PAP) in Northern Finland ». Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514288227.
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Abstract
Multiple endocrine neoplasia type 1 (MEN1) is an inherited syndrome characterized by parathyroid, gastroenteropancreatic and pituitary neuroendocrine tumours. In Northern Finland, two founder mutations of the MEN1 gene (1466del12, 1657insC) accounting for the majority of the MEN1 cases, have common ancestors born in the 18th and 19th centuries, respectively. Three small clusters of familial pituitary adenoma have also been detected, two of which could be linked by genealogy to a common ancestral couple born in the 18th century.
Clinical evaluation of 82 MEN1 mutation carriers showed that age was a risk factor for most of the MEN1-related manifestations. In the whole group, nonfunctional pancreatic tumour (NFPT) was more common in the frameshift/nonsense mutation carriers (odds ratio 3.26; 95% confidence interval 1.27–8.33, P = 0.014), whereas gastrinoma was more common in the in-frame/missense mutation carriers (OR 6.77, CI 1.31–35.0, P = 0.022). In the founder mutation carriers, the 1657insC mutation predicted the risk for NFPT (OR 3.56, CI 1.29–9.83, P = 0.015), while the 1466del12 mutation was associated with the risk for gastrinoma (OR 15.1, CI 1.73–131.9, P = 0.014).
The mean ages at death of the 32 obligatory MEN1 founder mutation carriers born between 1728 and 1929 were compared to those of the 29 spouses and sex-matched life expectancy estimates derived from Finnish national statistics. The ages at death of the mutation carrier males (61.1 ± 12.0 years) and females (67.2 ± 10.7 years) did not differ from the control groups.
PAP (pituitary adenoma predisposition) locus was mapped in the chromosome region 11q12–11q13 by whole-genome single-nucleotide polymorphism genotyping. Combining the linkage and the gene expression array data, AIP (aryl hydrocarbon receptor interacting protein) was chosen for sequencing. The nonsense mutation Q14X was identified in the affected (acromegaly, gigantism, prolactinoma) family members and in four other patients. Loss of heterozygosity was detected in pituitary adenomas of AIP mutation carriers.
Mutation analysis of MEN1, HRPT2 (hyperparathyroidism 2), CASR (calcium-sensing receptor), CDKN1B (cyclin-dependent kinase inhibitor 1B) and AIP genes was performed in primary hyperparathyroidism patients with features of inherited predisposition. One out of 29 patients was found to have the 1466del12 mutation, while no mutations were detected in other genes.
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Garlington, Jennifer Erin, et Jennifer Erin Garlington. « Exploring Family Perceptions About Primary Care Management Following Diagnosis of Type 1 Diabetes in Preschool-Age Children ». Diss., The University of Arizona, 2016. http://hdl.handle.net/10150/621004.
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Purpose: To describe family perceptions about pediatric primary care management following diagnosis of type 1 diabetes mellitus (T1DM) in preschool-aged children living in the Pacific Northwest region of the United States. Study Design and Method: Mothers of children diagnosed with T1DM before the fifth birthday and within the past two years were recruited anonymously through two regional support groups. Perceptions about pediatric primary care management following T1DM diagnosis were elicited through an anonymous 30-item online survey. Demographic characteristics of mother and child were obtained as well as information about five important domains of health care management for a young child with T1DM: (1) multidisciplinary, (2) holistic and compassionate, (3) accessible and communicative, (4) uses current standards and technology, and (5) actively promotes safe self-management. Results: Twenty-one biological mothers participated in this study, each on behalf of a child diagnosed with T1DM who fit inclusion criteria. Overall mothers held positive perceptions about care management by PCPs and endocrinologists within context of each of the five domains. Most mothers felt included in care planning, valued periodic well-child exams, and believed the child's providers were accessible, communicated effectively, and usually demonstrated consideration/compassion for the family. Although a majority of mothers at least somewhat agreed that the PCP used current standards and technology to care for the child, and functioned as the center of his/her health care coordination, these domains elicited a slightly greater number of responses indicating uncertainty or disagreement. Clinical Implications: Nurses and pediatric practitioners can use findings from this study to plan continued exploration into the perceptions and care management needs of families following diagnosis of a very young child with T1DM. The domains of care used to assess mothers' perceptions about care management-based on tenets of the Chronic Care Model (CCM) and Patient Centered Medical Home (PCMH)-can be used by pediatric PCPs and endocrinologists to dialogue with patients and staff about how care management may be improved for these families. Providing opportunities for feedback to the families of young children with T1DM should be encouraged so future research can examine relationships between care management variables and clinical outcomes.
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Jagannathan, Ram. « Identification of biomarkers for type 2 diabetes : analysis of a primary prevention study among Asian Indians with impaired glucose tolerance ». Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/32118.
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Primary prevention of type 2 diabetes (T2DM) is an important strategy for curbing its rising global burden. Though lifestyle modification has provided an effective method of preventing/delaying incidence of diabetes in high-risk individuals, it has not been widely implemented even in developed countries due to its high-cost, need for expertise and difficulties in translating the benefits of lifestyle intervention to the community at large. Hence, there is an urgent need to identify an alternate mode of delivery to transmit healthy lifestyle information to high-risk individuals. In this trial, we sought to determine whether lifestyle advice through mobile phone text messaging could reduce incident diabetes compared to standard lifestyle advice in Asian Indian men with prediabetes. The study showed for the first time that mobile phone messaging is an effective and acceptable method to deliver advice and support towards lifestyle modification to prevent T2DM in men at high risk. The identification of novel predictors for T2DM is an arduous task. The glycaemic markers of diabetes (fasting plasma glucose, 2hr post glucose load and HbA1c) are, in fact, risk factors for microvascular complications of diabetes and it was on this basis that diagnostic cut-offs for diabetes were arrived at. Nevertheless, elevated levels of glycaemic markers in the sub-clinical, or pre-diabetic, range are associated with increased risk of progression to diabetes. However, there is already considerable deterioration of beta cell function by the time diabetic dysglycaemia occurs. The way forward is clearly to identify biomarkers that serve as reliable predictors of progression to diabetes rather than simply reflecting accompanying levels of glycaemia. In this thesis using the database of the above mentioned trial it was aimed to identify the predictors of T2DM in Asian Indian cohort with prediabetes at baseline. The classical risk factors studied here are: 1) increased prevalence of the hypertriglyceridemic waist phenotype, 2) a combination of HbA1c and gamma glutamyl transferase and 3) a measure of beta cell compensation (disposition index) predicted incident diabetes. Among these, the disposition index was the most powerful predictor in the cohort. In addition to these classical risk factors mentioned above, in a small nested-case control, cross sectional study, the association of adipokines (adiponectin, leptin, interleukin-6 (IL-6), retinol-binding protein4 (RBP4)) and vitamin D3 were assessed to study the mechanistic link of novel biomarkers with diabetes. In this cohort, lower levels of baseline adiponectin, and higher IL-6 and RBP4 were associated with diabetes. Though, many of these provided a novel mechanistic pathogenic link with diabetes they did not improve prediction over and above that of glycaemic measures in identifying individuals with diabetes. However, the non-glycaemic biomarkers appear to have a role in the underlying pathogenesis of diabetes.
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Gomez, Alejandro Martin. « Study of the HIV-1-mediated induction of BAFF in primary human monocytes and monocyte-derived macrophages ». Doctoral thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/26298.
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L’infection par le VIH-1 (Virus de l’Immunodéficience Humaine de type I) est caractérisée par une réplication virale persistante, une activation chronique du système immunitaire, une déplétion des cellules T CD4+ et de plusieurs dysfonctionnements immunitaires qui sont observés même chez les cellules qui ne sont pas ciblées par le virus telles que les cellules B. Plusieurs cytokines et facteurs de croissance, qui sont augmentées dans le sérum des individus infectés par le VIH-1, ont été suggérés de déclencher directement et/ou indirectement l'activation des cellules B, et l'un d'eux est BAFF (B cell activating factor). BAFF est un composant essentiel de l'homéostasie des lymphocytes B mais sa surproduction résulte en une hyperplasie des cellules B, lymphoprolifération, une hypergammaglobulinémie et des symptômes d’autoimmunité. Les différentes études présentées dans cette thèse convergent vers l'objectif général de mieux caractériser les mécanismes qui mènent à la surproduction de BAFF par les monocytes primaires humains et les macrophages dérivés des monocytes dans le contexte de l’infection par le VIH-1. Nous démontrons que le VIH-1 cause la sécrétion de BAFF dans les monocytes par un processus dépendant de l’interféron (IFN) de type-I. De plus, nous avons identifié les cellules dendritiques plasmacytoïdes (CDP) comme la source de l’IFN de type-I dans nos cultures de monocytes, ce qui démontre que l’interaction entre ces cellules est nécessaire pour la production de BAFF générée par le VIH-1. En plus, nous avons aussi démontré que le VIH-1 régule la production de BAFF dans les macrophages et ce processus nécessite une infection productive par le virus et est influencé par le statut du phénotype cellulaire mais est indépendant de la transduction de signal par les récepteurs de type Toll, l’IFN de type-I ainsi que l'action de la protéine virale Nef. En résumé, cette thèse offre de nouvelles connaissances dans les mécanismes d'augmentation de BAFF dans le contexte de l’infection par le VIH-1. Celles-ci pourraient être pertinentes dans le développement de thérapies qui pourraient aider à restaurer la fonctionnalité normale du compartiment des cellules B chez les sujets infectés par le VIH-1.HIV-1 (Human Immunodeficiency Virus I) infection is characterized by persistent viral replication, chronic immune activation, CD4+ T cell depletion and several immune dysfunctions that are observed even in cells that are not targeted by the virus such as B lymphocytes. Some B-cell abnormalities observed in HIV-1-infected individuals include hypergammaglobulinemia, nonspecific B-cell activation, class switching, increased cell turnover, breakage of tolerance as well as a loss of the capacity to generate and maintain memory, among others. Several cytokines and growth factors that are increased in the serum of HIV-1-infected individuals have been suggested to directly and/or indirectly trigger B-cell activation, and one of these is the B-cell-activating factor (BAFF). BAFF is an essential component of B-cell homeostasis but excess production results in B-cell hyperplasia, lymphoproliferation, hypergammaglobulinemia, and symptoms of autoimmunity. The mechanisms of BAFF upregulation in the context of HIV-1 infection are not fully understood and no previous studies have addressed the ability of fully competent HIV-1 to induce BAFF production by myeloid cells. The different studies presented in this thesis converge to the general objective of better characterizing the mechanisms underlying BAFF upregulation by primary human monocytes and monocyte-derived macrophages in the context of HIV-1 infection. We show here that HIV-1 drives BAFF secretion in monocytes by a type-I interferon (IFN)-dependent process. Moreover, we identified plasmacytoid dendritic cells (pDCs) as the cellular source of this type-I IFN-directed modulatory effect in our monocyte cultures, demonstrating that a pDC/monocyte interplay is required for the HIV-1-induced BAFF production. In addition, we provide evidence that HIV-1 upregulates BAFF production in monocyte-derived macrophages and this process relies on productive virus infection, which is itself influenced by the cell phenotype status, and is independent of Toll-like receptors and type-I IFN signal transduction as well as the action of Nef. Altogether, this doctoral project provides new insights for the increased BAFF levels observed during HIV-1 infection. These findings might be relevant for the design of therapies that could help restore the functionality of the B-cell compartment in HIV-1-infected individuals.
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Dong-Newsom, Phing. « Social Stress-Induced Modulation of Primary and Recurrent HSV-1 Infections in Balb/c Mice ». The Ohio State University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=osu1241716184.
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Sundaram, Selvam. « A text messaging approach to behavioural change, tailored using the transtheoretical model, in the primary prevention of type 2 diabetes mellitus in Asian Indians ». Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/32146.
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Landmark diabetes prevention trials have shown that lifestyle modification is effective in preventing type 2 diabetes (T2D). In general, information technology with short message service (SMS) and interventions based on behavioral theories of individualized motivation are more effective than non-tailored intervention. The Transtheoretical Model (TTM) is a combination of behavioral theory and stage-based theory. We used TTM-tailored healthy lifestyle and motivating SMS messages as an intervention strategy in a 2-year prospective randomized controlled diabetes prevention trial with two groups (control n=266: intervention n=271) of men with impaired glucose tolerance. The main objectives were: 1. To study the utility and acceptability of TTM-based SMS in reducing the incidence of T2D. 2. To analyse the change in TTM stages in the study groups in lifestyle factors (diet and physical activity practices) with respect to incidence of diabetes at the end of the study. 3. To analyse the effects of TTM tailored SMS intervention on quality of life and general health and diabetes awareness. Evaluation tools included: 1.TTM stages of change inventory for diet and physical activity practices. 2. SMS acceptability questionnaire. 3. WHOQOL-BREF (quality of life) questionnaire. 4. General health and diabetes awareness questionnaire. Questionnaires 1 and 2 were study-specific and developed by the candidate whereas 3 and 4 were standardized. Dietary and physical activity questionnaires had been used in previous diabetes prevention studies in India. The TTM-based SMS intervention reduced the incidence of T2D (50 [18%] intervention group; 73 [27%], control group (absolute risk reduction 9%)). Significant differences in TTM stage were observed in the intervention group (p<0.05) at six and twenty-four months for dietary practices. Significant differences (p<0.05) were observed in physical activity practices only at six months. SMS helped improve quality of life. Significant improvement (p<0.001) was observed in general health awareness and diabetes awareness in the intervention group at the end of the study. The TTM-based SMS intervention was associated with reduced incidence of T2D. The intervention helped improve dietary habits. It also helped improve QOL and general health and diabetes awareness level.
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Aljehany, Buthaina. « The impacts of a health education programme on primary school teachers' knowledge and attitudes towards type 1 diabetes mellitus in children in Saudi Arabia ». Thesis, University of Salford, 2016. http://usir.salford.ac.uk/39345/.
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Introduction The incidence of T1DM in the Kingdom of Saudi Arabia (KSA) is particularly high at 36.99 per 100,000. The number of newly diagnosed cases in children (0-14 years) is estimated at 10,700 per year, constituting a major public health problem. Schools are important for secondary prevention, treatment and management of Type 1 Diabetes Mellitus (T1DM). Teachers need to be knowledgeable about diabetic emergences. Aim The aim of this study was to assess the impact of a health education programme on primary school teachers' knowledge and attitudes towards T1DM in children attending schools in Jeddah City, KSA. Methods A repeated measures non-equivalent groups design was adopted, with testing of the teachers at baseline, three month, and six month stages. Data was collected from 2013 to 2014. A structured, self-administered questionnaire was employed in Arabic. A total of 540 teachers were recruited in equal numbers by gender, of which 318 completed all test stages. The intervention consisted of a new and specially designed educational programme comprised of lectures and activities, with additional reading materials. SPSS was used for quantitative analysis of the data, and paired samples t-test and ANOVA were used to test for differences within and between experimental and control groups. Results No significant differences were found at the baseline pre-test stage between the groups in teachers' knowledge of T1DM in children, or their attitudes towards managing T1DM. The mean knowledge scores in the experimental group increased significantly at the post-test stage (three months), and again at the post-test 2 stage (six months). The mean knowledge scores of the control group fluctuated to some extent at the post-test and post-test 2 stages. The mean attitude scores in the experimental group showed significant increases at the post-test and post-test 2 stages, but there was no significant change in the mean attitude scores of the control group. Conclusion The results clearly demonstrate the effectiveness of the intervention in improving teachers' knowledge and attitudes about managing T1DM in schools. The inexpensive programme could be integrated into the Saudi national child health programme, and policy and practice recommendations are proposed to the Ministry of Education.
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Coats, Charles Jason. « Development of primary neuronal culture of embryonic rabbit dorsal root ganglia for microfluidic chamber analysis of axon mediated neuronal spread of Bovine Herpesvirus type 1 ». Thesis, Manhattan, Kan. : Kansas State University, 2010. http://hdl.handle.net/2097/4115.
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Rousseau, Antoine. « Cinétique des effecteurs immunologiques impliqués dans la protection contre le virus Herpès simplex type 1 (HSV1) après primo-infection par une autre souche non neurovirulente : vers un modèle vaccinal ». Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS339.
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Chez l’homme, la primo-infection par le vírus Herpès simplex de type 1 (HSV-1) a lieu au niveau de la muqueuse oro-pharyngée. Après une phase de réplication, les virions pénètrent les terminaisons axonales et migrent vers le ganglion trijumeau ipsilatéral (TG), puis vers le TG controlatéral. Le virus entre alors dans un état de latence dans les 2 TG. Les réactivations d’HSV-1 dans ces neurones sont responsables des kératites herpétiques (KH), unilatérales et survenant toujours du même côté chez un patient donné.En utilisant un modèle murin oro-oculaire, qui reproduit les aspects essentiels de la maladie chez l’homme, nous avons précédemment démontré que l’inoculation virale latéralisée d’un côté de la bouche entraine une réplication virale dans la lèvre, suivie d’une KH ipsilatérale à l’inoculation 6 jours plus tard. De manière concomitante, le virus se propage aux 2 TG, mais les réactivations ne surviennent que du côté ipsilatéral à l’inoculation. Nous avons également observé qu’après une primo-infection dans une lèvre avec une souche d’HSV-1 non-neurovirulente, les souris étaient protégées contre les signes de la maladie et contre la réactivation d’une souche sauvage pleinement virulente inoculée secondairement dans l’autre lèvre (souche ré-infectante). Afin de comprendre les mécanismes immunitaires en jeu dans cet état de protection, nous avons combiné une analyse en cytométrie en flux multiparamétrique à des tests immunologiques, pour quantifier et définir l’infiltrat immunitaire hématopoïétique et les chémokines inflammatoires au site d’inoculation et dans les TG. Nous avons démontré qu’après une inoculation unique avec la souche sauvage virulente, un infiltrat immun riche en cellules pro-inflammatoires, survenaient de manière retardée dans les lèvres inoculées, et persistaient dans les TG. A l’opposé, l’infiltrat immunitaire était plus précoce dans les tissus réinfectés (après une primo-infection par la souche non neurovirulente), plus riche en cellules de l’immunité adaptative, et associé à des concentrations moindres de chémokines inflammatoires. En outre, cet infiltrat s’estompait plus rapidement, avec une disparition concomitante des chémokines inflammatoires. Ces données permettent de mieux comprendre la nature et la cinétique de la réponse immunitaire anti-HSV-1, et pourront être utiles pour le développement futur de stratégies vaccinales anti-HSV-1In humans, Herpes simplex virus type 1 (HSV-1), primary infection occurs in the oral mucocutaneous tissues. Virions replicated here penetrate sensitive neuronal axons, migrate to both trigeminal ganglion (TG) where it established a lifelong latency. Reactivations of HSV-1 in the TG neurons induce clinical recurrences in the connected peripheral tissues. This process is involved in herpes simplex keratitis (HSK), a condition that, strikingly, occurs almost exclusively in the same eye for a given patient. Based on an experimental oro-ocular (OO) model of HSV-1 infection, that recapitulates most of these human clinical features, we previously demonstrated that a virus inoculation on one side of the mouth, leads to viral replication in the lip, followed by HSK. Virus concomitantly disseminates to both TG, but reactivation only occurs in the TG ipsilateral to the inoculation site. We also observed that after a primary inoculation with a non-neurovirulent strain of HSV-1 in one lip, mice are protected against both acute phase disease and reactivation after a superinfection with a fully virulent wild-type strain of HSV-1 in the contralateral lip.In order to understand the underlying mechanisms involved in this state of protection, we combined high resolution flow cytometry and bead-based immunoassays, to quantify hematopoietic subsets and inflammatory chemokines in the site of inoculation and in the TG. We demonstrated that after a single inoculation with the wild-type strain, a delayed immune infiltrate, boasting more proinflammatory subsets, occurred in the lip and persisted in the TG. In contrast, the immune infiltrate occurred earlier in the superinfected lip and ipsilateral TG, with less inflammatory chemokines but more adaptive immune subsets. Moreover, cellular infiltrate resolved faster, correlating with nullification of inflammatory chemokines locally. These data show that immune response kinetics influence the development of natural immunity to HSV-1, and can be harnessed to protect against disease and reactivations
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Lespiau, Florence. « Logique sans peine ? : comment nous sommes plus performants et motivés pour raisonner logiquement à propos des connaissances primaires ». Thesis, Toulouse 2, 2017. http://www.theses.fr/2017TOU20101/document.
Texte intégralRésumé :
L’apprentissage donne souvent l’impression d’être un processus long et difficile, notamment quand il fait penser à l’école et à la difficulté que tout le monde a déjà ressentie pour maintenir sa motivation pour telle ou telle matière. Pourtant, il y a des choses que l’on apprend sans enseignement. Par exemple, apprendre à parler sa langue maternelle se fait naturellement sans effort conscient. Les connaissances primaires et secondaires sont une façon de distinguer ce qui est facile ou difficile à apprendre. Les connaissances primaires sont celles pour lesquelles nos mécanismes cognitifs auraient évolué, permettant une acquisition sans effort, intuitive et rapide alors que les connaissances secondaires sont apparues récemment : ce sont celles pour lesquelles nous n’aurions pas eu le temps d’évoluer et dont l’acquisition serait longue et coûteuse. Les écoles se focalisent essentiellement sur ce deuxième type de connaissances. Leur défi est de permettre ces apprentissages longs et coûteux, et, pour cela, de maintenir la motivation des apprenants. Une piste de recherche s’appuie sur le fait que les connaissances secondaires sont construites sur la base des connaissances primaires. En effet, personne n’est capable d’enseigner « initialement » une langue maternelle alors que l’apprentissage des langues étrangères s’appuie sur cette première langue. Le présent travail explore le caractère motivant et peu coûteux des connaissances primaires pour faciliter l’apprentissage de la logique en tant que connaissance secondaire. En modifiant la présentation de problèmes logiques avec des habillages liés aux connaissances primaires (e.g., nourriture et caractéristiques d’animaux) ou secondaires (e.g., règles de grammaire, mathématiques), huit premières expériences ont permis de mettre en avant les effets positifs des connaissances primaires que le contenu soit familier ou non. Les résultats montrent que les connaissances primaires favorisent la performance, l’investissement émotionnel, la confiance dans les réponses et diminuent la charge cognitive perçue. Quant aux connaissances secondaires, elles semblent miner la motivation des participants et générer une sensation de conflit parasite. De plus, présenter des problèmes avec un habillage de connaissances primaires en premier permettrait de réduire les effets délétères des connaissances secondaires présentées ensuite et aurait un impact positif global. Trois autres expériences ont alors mis ces résultats à l’épreuve de tâches d’apprentissage afin de proposer une approche qui favorise l’engagement des apprenants et leur apprentissage. Ces découvertes tendent à montrer que les recherches sur l’apprentissage bénéficieraient à prendre en considération les connaissances primaires plutôt que de les négliger car elles sont « déjà apprises »Learning often gives the impression of being a long and difficult process, especially when it reminds us of school and the difficulty that everyone has already experienced in maintaining motivation for a particular subject. Yet there are things we learn without teaching. For example, learning to speak one’s mother tongue is a natural process without conscious effort. Primary and secondary knowledge is a way of distinguishing what is easy or difficult to learn. Primary knowledge is the knowledge for which our cognitive mechanisms have evolved, allowing effortless, intuitive and rapid acquisition, whereas secondary knowledge has recently emerged: it is the knowledge for which we would not have had time to evolve and for which acquisition would be long and costly. Schools focus mainly on this second type of knowledge. Their challenge is to enable this lengthy and costly learning, and to do so, to maintain the motivation of learners. A research path is based on the fact that secondary knowledge is built on the basis of primary knowledge. Indeed, no one is able to teach a mother tongue “initially”, whereas foreign language learning is based on that first language. This work explores the motivational and inexpensive nature of primary knowledge to facilitate the learning of logic as secondary knowledge. By varying the content of logical problems with primary (e.g., food and animals’ features) or secondary knowledge (e.g., grammar rules, mathematics), the first eight experiments highlighted the positive effects of primary knowledge, whether or not the content was familiar. The results showed that primary knowledge promoted performance, emotional investment, confidence in responses and decreased perceived cognitive load. Secondary knowledge seemed to undermine participants’ motivation and generated a feeling of parasitic conflict. In addition, presenting primary knowledge content first reduced the deleterious effects of secondary knowledge presented second and would have an overall positive impact. Three other experiments then tested these results on learning tasks in order to propose an approach that fosters learners’ engagement and learning. These findings tend to show that research about learning would benefit from taking primary knowledge into account rather than neglecting it because it is “already learned”
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Coutinho, Flavia Lima. « Avaliação da densidade mineral óssea em pacientes com hiperparatireoidismo primário hereditário associado à neoplasia endócrina múltipla tipo 1, antes e após paratireoidectomia ». Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-16062009-171933/.
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INTRODUÇÃO: Hiperparatireoidismo primário (HPT) é uma doença endócrina relativamente comum, caracterizada por hipercalcemia associada a concentrações de PTH elevadas ou inapropriadamente normais. A maioria dos pacientes (90%-95%) apresenta a forma esporádica da doença, enquanto a forma familiar pode ocorrer associada à neoplasia endócrina múltipla tipo 1 (NEM1) e tipo 2, HPT-tumor de mandíbula, HPT neonatal severo e HPT isolada familiar. HPT associado com NEM1 (HPT/NEM1) difere da forma esporádica em vários aspectos, entre eles: acometimento multiglandular das paratireóides (hiperplasia x adenoma); início da doença mais precoce (20 x 40 anos); afeta homens e mulheres em proporção semelhante (1:1), em contraste a 1:3 no HPT esporádico; diferentes tratamentos cirúrgicos (paratireoidectomia total ou subtotal x adenomectomia); maior taxa de recorrência após paratireoidectomia (PTx); e tende a ser menos agressivo que o HPT esporádico. No HPT esporádico, o perfil da perda mineral óssea e o impacto do tratamento cirúrgico na densidade mineral óssea (DMO) estão bem definidos. Por outro lado, dados sobre perda óssea no HPT/NEM1 e sua potencial recuperação após PTx são escassamente relatados. O objetivo deste estudo é avaliar o perfil densitométrico e o impacto do tratamento cirúrgico na DMO em pacientes com HPT/NEM1. MÉTODOS: Neste estudo, avaliamos inicialmente 36 pacientes (18 homens e 18 mulheres) com diagnóstico de HPT/NEM1 (média de idade ao diagnóstico de HPT de 38,99 ± 14.46 anos, 20-74 anos). Estes pacientes pertenciam a oito famílias não relacionadas previamente caracterizadas clinicamente e portadoras de mutações germinativas MEN1. Avaliamos a DMO no terço proximal do rádio distal (1/3 RD), fêmur (colo do fêmur e fêmur total) e coluna lombar (L1-L4) destes 36 pacientes. A DMO foi medida pela densitometria óssea de dupla emissão com fonte de raios X (DXA) e os valores expressos em índice T, índice Z e em valores absolutos (g/cm2). Após esta avaliação da DMO, vinte e quatro pacientes foram submetidos à paratireoidectomia total seguida por auto-implante em antebraço não dominante. Em um grupo selecionado de 16 pacientes foi avaliada a densidade mineral óssea antes e após (período médio de 15 meses) o tratamento cirúrgico. RESULTADOS: Desmineralização óssea (osteoporose/osteopenia) foi observada no 1/3 RD (28/34, 79,4%); colo do fêmur (26/36, 72,7%) e na coluna lombar (25/36, 69,4%). Osteopenia foi principalmente observada no colo do fêmur (19/36, 52,8%), seguida pelo 1/3 RD (14/34, 41,2%) e coluna lombar (11/36, 30,5%). Osteoporose foi observada principalmente na coluna lombar (14/36, 38,9%) e 1/3 RD (14/34, 41,2%); enquanto no colo do fêmur (7/36, 19,4%) a prevalência foi menor . Valores médios de índice T estavam severamente reduzidos no 1/3 RD (- 2,46±1,436 DP), seguido pela coluna lombar (-2,05±1,539 DP). O colo do fêmur foi o menos afetado (-1,60±1,138 DP). Nos 16 pacientes submetidos ao tratamento cirúrgico, no período médio de 15 meses após PTx, a DMO (g/cm2) aumentou significativamente na coluna lombar de 0,843 para 0,909 g/cm2 (+ 8,4%; p=0,001). A DMO (g/cm2) no colo do fêmur também aumentou significativamente de 0,745 para 0,798 g/cm2 (+ 7,7%; p=0.0001). No 1/3 RD não houve modificação estatisticamente significante da DMO (0,627 ± 0,089 para 0,622 ± 0,075; p=0,76). CONCLUSÃO: Nossos dados demonstraram que o rádio distal é o sítio ósseo preferencial para desmineralização óssea e que a coluna lombar pode não estar relativamente protegida na HPT/MEN1, como descrito no HPT esporádico. Um aumento significante foi observado na coluna lombar e no colo do fêmur em pacientes com HPT/NEM1, em um período médio de 15 meses após paratireoidectomia; enquanto no terço proximal do radio distal, não houve melhora significativa durante este estudoINTRODUTION: Primary hyperparathyroidism (HPT) is a relatively common endocrine disorder, which is characterized by hypercalcemia and elevated or inappropriately normal levels of PTH. Most patients (90-95%) present with the sporadic form of the disease, whereas familial cases may occur associated with multiple endocrine neoplasias type 1 (MEN1) and type 2, jaw tumours, as well as severe neonatal form and familial isolated HPT. HPT associated with MEN1 (HPT/MEN1) differs from sporadic primary HPT (s- HPT) in the following aspects: it presents as a multiglandular parathyroid neoplasia (hyperplasia vs adenoma); it has an earlier disease onset (20 vs. 40 years of age); there is a sex ratio of 1:1 in contrast to the 1:3 ratio for s- HPT; different surgical treatment (total or subtotal parathyroidectomy x adenomectomy); there are higher recurrence rates after a parathyroidectomy (PTx); and it frequently tends to be less aggressive than s-HPT. In s-HPT, the bone loss profile and the impact of parathyroid surgery are well defined. In contrast, data on bone losses in HPT/MEN1 and the potential bone recovery after PTx have been scarcely reported. The aim of this study is to evaluate the bone mineral status and the impact of surgical treatment on bone mineral density (BMD) in HPT/MEN1 patients. METHODS: We studied 36 cases (18 males and 18 females) diagnosed with HPT/MEN1 (average age at the HPT diagnosis of 38.9 ± 14.46 years; range, 20-74 years). These patients belonged to eight unrelated MEN1 families previously clinically characterized and harboring germline MEN1 mutations. We have assessed the values of BMD in the proximal one third distal radius (1/3 distal radius), femoral (femoral neck and total) and lumbar spine (L1-L4) of these 36 HPT/MEN1 cases. BMD values were measured by dual-energy X-ray absorptiometry and the values expressed in T, Z-score and in absolute values. After BMD analyses, twenty four out of them were submitted to total parathyroidectomy followed by autoimplant in the non-dominant forearm. BMD measurements were evaluated before and in a mean period of 15 months after surgery, in a subset of 16 patients. RESULTS: Bone demineralization (osteoporosis/osteopenia) was seen at the proximal third of distal radius (28/34, 79.4%); femoral neck (26/36, 72.7%) and in the lumbar spine (25/36, 69.4%). Osteopenia was mostly found in femoral neck (19/36, 52.8%), whereas 1/3 distal radius (14/34, 41.2%) and lumbar spine (11/36, 30.5%) were also represented. Osteoporosis was mostly marked at lumbar spine (14/36, 38.9%) and 1/3 DR (14/34, 41.2%), but femoral neck (7/36, 19.4%) was also affected. Mean T score values at the 1/3 DR were severely reduced (-2.46±1.436 SD), followed by lumbar spine (-2.05 ± 1.539 SD). The femoral neck was the least affected site (-1. 60 ± 1.138 SD). In the 16 cases submitted to surgical treatment, in a mean period of 15 months after PTX, BMD (g/cm2) significantly increased at the lumbar spine from 0.843 to 0.909 g/cm2 (+ 8.4%; p=0.001). Femoral neck BMD (g/cm2) also increased significantly from 0.745 to 0.798 g/cm2 (+ 7.7%; p=0.0001). In the proximal one third of distal radius, BMD (g/cm2) remained unchanged (baseline, 0.627 ± 0.089 to 0.622 ± 0.075; p=0.76). CONCLUSION: Our data confirmed distal radius as the preferential site of bone demineralization and that lumbar spine may not be relatively protected in HPT/MEN1, as related in the s-HPT. A significant increase in the BMD has been verified in the lumbar spine and femoral neck BMD in 16 patients with HPT/MEN1, in a mean period of 15 months after parathyroidectomy. However, the proximal one third of distal radius BMD did not present significant improvement during this study
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Damianse, Sabrina da Silva Pereira. « Frequência de neoplasia endócrina múltipla tipo 1 em pacientes portadores de adenomas hipofisários ». Universidade Federal do Maranhão, 2016. http://tedebc.ufma.br:8080/jspui/handle/tede/1431.
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The multiple endocrine neoplasia type 1 (MEN1) is a genetic syndrome with autosomal dominant transmission, characterized by tumors of the parathyroid, anterior pituitary and pancreas. Primary hyperparathyroidism is the most common clinical presentation in MEN1 and evaluation of patients with pituitary adenomas for the presence of hyperparathyroidism could identify patients with this syndrome. The aim of this study was to identify the frequency of MEN1 by serum calcium and parathyroid hormone measurement in patients with pituitary adenomas followed at the Endocrinology Service of the Hospital Universitário da Universidade Federal do Maranhão (HUUFMA). This is a descriptive study with data collected from the patients' medical charts in june 2015 to may 2016. We evaluated 300 patients with pituitary adenoma subtypes (128 prolactinomas, 67 acromegaly, 22 corticotropinomas, 3 gonadotropinomas and 80 adenomas clinically nonfunctioning) finding a frequency of 1.3% of MEN1 patients among patients with adenomas pituitary. Patients with MEN1 were mostly female and the average age at diagnosis of pituitary adenoma was 42.7 years, ranging between 24 and 57 years old. Pituitary tumors of these patients were more often macroadenoma and the predominant subtype was somatotropinoma. At initial diagnosis, our patients had apparently sporadic pituitary lesions, however, or were confirmed with MEN1 early because they already have signs and/or symptoms of hyperparathyroidism, or have been diagnosed very late caused mild symptoms of parathyroid disease. Therefore, screening measures serum calcium and PTH in patients with pituitary adenomas are recommended, primarily, because these tests are necessary to identify the most common disease in MEN1, primary hyperparathyroidism. The study contributed to the identification of new patients with MEN 1 in those patients with pituitary adenomas with the benefit of early diagnosis, appropriate therapeutic approach and genetic counseling in family forms.
A neoplasia endócrina múltipla tipo 1 (NEM1) é uma síndrome genética, com transmissão autossômica dominante, caracterizada por tumores da paratireóide, da hipófise anterior e do pâncreas. O hiperparatireoidismo primário é apresentação clínica mais frequente na NEM1 e a avaliação dos pacientes com diagnóstico de adenomas hipofisários quanto à presença de hiperparatireoidismo poderia identificar pacientes com esta síndrome. O objetivo deste estudo foi identificar a frequência de NEM1 a partir das dosagens séricas de cálcio e paratormônio em pacientes portadores de adenomas hipofisários acompanhados no Serviço de Endocrinologia do Hospital Universitário da Universidade Federal do Maranhão (HUUFMA). Trata-se de um estudo descritivo com dados coletados a partir dos prontuários de atendimento dos pacientes no período de junho de 2015 a maio de 2016. Foram avaliados 300 pacientes com diagnóstico de adenoma hipofisário de diferentes subtipos (128 prolactinomas, 67 acromegálicos, 22 corticotropinomas, 3 gonadotropinomas e 80 adenomas clinicamente não-funcionantes) encontrando-se uma frequência de 1,3% de pacientes NEM1 dentre os portadores de adenomas hipofisários. Os pacientes com NEM1 eram, em sua maioria, do sexo feminino e a média de idade ao diagnóstico da lesão hipofisária foi de 42,7 anos, variando entre 24 e 57 anos de idade. Os tumores hipofisários desses pacientes eram mais frequentemente macroadenomas e o subtipo predominante foi somatotropinoma. Ao diagnóstico inicial, dos pacientes eram, aparentemente, portadores de lesões pituitárias esporádicas, no entanto, ou foram confirmados precocemente com NEM1, pois já possuíam sinais e/ou sintomas relacionados ao hiperparatireoidismo, ou foram diagnosticados muito tardiamente devidos sintomas leves da doença paratireoidiana. Portanto, o rastreio com dosagens de cálcio e PTH séricos em pacientes portadores de adenomas hipofisários é recomendado, principalmente, por serem exames necessários para identificar a doença mais frequente na NEM1, o hiperparatireoidismo primário. O estudo contribuiu para identificação de novos pacientes com NEM 1, naqueles portadores de adenomas hipofisários com o benefício do diagnóstico precoce, abordagem terapêutica adequada e aconselhamento genético nas formas familiares.
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Miloudi, Hadjer. « Étude des anomalies de XPO1 dans les lymphomes B Anomalies de l’exportine 1 dans les he´mopathies malignes : des mutations au ciblage the´rapeutique Cytoplasmic cyclin D1 controls the migration and invasiveness of mantle lymphoma cells STAT6 is a cargo of exportin 1 : Biological relevance in primary mediastinal B-cell lymphoma Mutant E571K and wild-type XPO1 effects are balanced in B-cell lymphoma ». Thesis, Normandie, 2020. http://www.theses.fr/2020NORMC409.
Texte intégralRésumé :
L'exportine-1 (ou XPO1) joue un rôle clé dans le transport de nombreux ARN et près de 200 protéines cargos. La mutation « hot-spot » XPO1-E571K est présente chez près de 25% des patients atteints par le lymphome primitif du médiastin (PMBL) et la forme classique du lymphome de hodgkin (cHL), et à plus faible fréquence dans la leucémie lymphoïde chronique (LLC) (3%). Des mutations touchant la voie JAK2/STAT6 sont aussi retrouvées dans le PMBL et le cHL. C’est pourquoi nous avons étudié l’implication de XPO1 dans la voie JAK2/STAT6. Nous avons montré que STAT6 est une protéine cargo de XPO1 par les technique immunofluorescence et PLA (Proximity Ligation Assay). Nous avons montré que le traitement au selinexor induit une accumulation nucléaire de STAT6 sauvage et mutée et que STAT6 peut interagir avec les formes mutée et sauvage de XPO1.Afin de rechercher l’impact fonctionnel de la mutation E571K de XPO1 nous avons comparé plusieurs paramètres physiologiques entre les trois lignées de PMBL. Aucune différence n’a été observée. En effet, la présence de l’amplification de l’allèle sauvage dans la lignée présentant la mutation E571K (MedB1) pourrait masquer les éventuels effets de la mutation. De plus, dans la cohorte de patients que nous avons étudiée la mutation n’est jamais retrouvée à l’état homozygote ou hémizygote indiquant l’importance du dosage génique. Nos expériences de CRISPR-Cas9 sur la lignée U2940 dont le gène XPO1 est sauvage ont confirmé notre hypothèse. De manière intéressante, la présence de la mutation E571K modifie l’affinité de XPO1 pour le selinexor. En effet, le KO de l’allèle muté dans la lignée de cHL UH-01 rend les cellules moins sensibles au selinexor. Nous avons conclu que la balance entre l’allèle sauvage et l’allèle muté est un élément clé pour définir les propriétés oncogéniques de XPO1. Dans une étude préliminaire, une étude protéomique dans les lignées PMBL indique que XPO1-E571K est liée à l’importine-β1 ce qui pourrait modifier la localisation subcellulaire de XPO1.Nous avons montré que la localisation cytoplasmique de cycline D1 contrôle le mécanisme d’invasion et de migration dans cellules de lymphome à cellules du manteau (MCL). Cycline D1 étant une protéine cargo de XPO1 nous avons recherché d’éventuelles anomalies de XPO1 dans les lignées de MCL. Aucune anomalie d’expression de localisation ou de fonction de n’a été observée. Il pourrait être intéressant de déterminer la localisation subcellulaire de cycline D1 au moment du diagnostic afin de proposer une meilleure prise en charge pour les patients. De plus, bien que le selinexor soit toujours en essai clinique, son utilisation pour le traitement du MCL pourrait être envisagée dans les cas les plus agressif où cycline D1 est cytoplasmiqueExportin-1 (or XPO1) plays a key role in the nuclear export of several RNAs and more than 200 proteins. The XPO1-E571K "hot-spot" mutation is present in nearly 25% of patients with primary mediastinal B-cell lymphoma (PMBL), and the classical form of Hodgkin lymphoma (cHL) but at a lower frequency (3%) in chronic lymphocytic leukemia (CLL). Mutations affecting the JAK2/STAT6 pathway are common in PMBL and cHL. We first studied the role of XPO1 in the nuclear export of STAT6 in PMBL cell lines. Using immunofluorescence and proximity ligation assay (PLA) techniques, we showed that STAT6 is a cargo of XPO1. We also showed that a selinexor treatment induced a nuclear accumulation of wild-type and mutant STAT6 whatever the XPO1 status.In order to investigate the functional impact of the XPO1-E571K mutation, we compared several physiological parameters between the three PMBL cell lines bearing or not the mutation. No differences were observed despite the expression of the XPO1E571K allele. However, in the cell line harboring the XPO1 mutation (MedB1), the wild-type (wt) allele was amplified possibly masking the effects of the mutation. In addition, in the cohort of patients we studied, the mutation was never found in the homozygous or hemizygous state indicating the importance of the gene dosage. CRISPR-Cas9 experiments allowing the introduction of the mutation in the U2940 wt cell line confirmed our hypothesis. Interestingly, the presence of the E571K mutation changed the affinity of XPO1 for selinexor. Indeed, the knockout of the mutated allele in the cHL UH-01 line decreased its sensitivity to selinexor. We concluded that the balance between the wt and the mutated alleles is a key element in defining the oncogenic properties of XPO1. In a preliminary study, we conducted a proteomic analysis to identify XPO1E571K partners in the PMBL lines. Our results showed that XPO1E571K interacts with the importin-β1 which could modify the subcellular localization of XPO1.Mantle cell lymphoma (MCL) cells are characterized by the overexpression of cyclin D1. Cyclin D1 being an XPO1 cargo protein, we looked for possible XPO1 abnormalities in several MCL cell lines. No abnormalities in the expression, localization neither function of XPO1 were observed. But, we showed that the cytoplasmic portion of cyclin D1 controlled the invasion and migration of MCL cells. It might be interesting to determine the subcellular localization of cyclin D1 at the time of diagnosis in order to offer a better treatment management for MCL patients. In addition, although selinexor is still in clinical trials, its use for the treatment of MCL could be considered in the most aggressive cases where cyclin D1 is cytoplasmic
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Negatsch, Alexandra. « Vergleichende Analysen zur Replikation und zum intraaxonalen Transport des Pseudorabiesvirus und des Herpes Simplex Virus Typ 1 in primären Rattenneuronen ». Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-144375.
Texte intégralRésumé :
Nach dem Eintritt in den Wirtsorganismus und initialer Replikation infizieren Alphaherpesviren Neuronen zur weiteren Ausbreitung im Nervensystem und zur Etablierung einer Latenz. Dazu werden die Viruspartikel innerhalb der Axone retrograd von der Peripherie zum neuronalen Zellkörper transportiert. Die umgekehrte Richtung beschreibt den Weg des anterograden Transports vom Zellkörper zur Synapse für weitere Infektionen von Neuronen höherer Ordnung oder zurück zur Peripherie. Der retrograde intraaxonale Transport ist gut untersucht. Dagegen wird über den anterograden Transport kontrovers diskutiert. Zwei verschiedene Transportmodelle werden vermutet. Das „Married Model“ postuliert, dass umhüllte Virionen innerhalb von Vesikeln entlang des Axons transportiert werden. Die Freisetzung der Partikel erfolgt an der jeweiligen Synapse durch Endocytose. Das „Subassembly Model“ geht dagegen davon aus, dass einzelne Virusstrukurkomponenten (Nukleokapsid, Hülle) entlang des Axons transportiert werden. Der Zusammenbau und die Freisetzung erfolgt am Axonterminus bzw. an der Synapse (in vivo) oder am Wachstumskegel (in vitro) oder an speziellen Auftreibungen des Axons, den sogenannten Varicosities. Nach Infektion eines neuronalen Explantatsystems mit dem Pseudorabiesvirus (PrV) konnten ultrastrukturell umhüllte Virionen in Vesikeln detektiert werden und so der Nachweis der Gültigkeit des „Married Model“ als vorherrschendes Transportmodell geführt werden. Dagegen ist die Situation beim prototypischen Alphaherpesvirus, dem Herpes Simplex Virus Typ 1 (HSV-1), weiterhin ungeklärt. Aufgrund der zahlreichen unterschiedlichen Analysemethoden und -systeme war ein direkter Vergleich der beiden Viren bislang nicht möglich. Daher sollte in dieser Arbeit ein standardisiertes neuronales Kultursystem genutzt werden, um vier verschiedene HSV-1 Stämme im Vergleich zu PrV zu untersuchen. Für die Infektionen wurden sowohl Neuronen aus dem oberen Cervikalganglion als auch aus Spinalganglien genutzt. So konnte gezeigt werden, dass in Neuronen, welche mit den HSV-1 Stämmen HFEM, 17+ und SC16 infiziert waren ca. 75% als umhüllte Virionen in Vesikeln und ca. 25% als nackte Kapside vorlagen. Ingesamt war die Anzahl der Viruspartikel in HSV-1 infizierten Neuronen signifikant geringer als in PrV infizierten Kulturen. Überraschenderweise zeigten mit HSV-1 KOS infizierte Neuronen ein reverses Bild. Hier lagen nur 25% der Viruspartikel als umhüllte Virionen in Vesikeln vor, während 75% als nackte Kapside detektiert wurden. Dieser unerwartete Phänotyp sollte auf molekularbiologischer Ebene genauer untersucht werden. Dabei wurde auf die Genregion von US9 fokussiert. Das von US9 codierte Membranprotein spielt eine wichtige Rolle während des Zusammenbaus der Virionen und bei anschließenden axonalen anterograden Transportvorgängen. In dieser Arbeit konnte gezeigt werden, dass das HSV-1 KOS Genom durch verschiedene Basenaustausche an der vorhergesagten TATA-Box von US9 eine Mutation aufweist. Zusätzlich trägt das offene Leseraster durch eine weitere Mutation ein vorzeitiges Stopcodon auf und wird dadurch auf 58 Kodons reduziert, im Gegensatz zu anderen HSV-1 Stämmen, wo es 91 Kodons umfasst. Die Mutation an der TATA-Box verändert auch das ursprüngliche Stopcodon vom US8a Gen, was zur einer Verlängerung von ursprünglich 161 zu 191 Kodons führt. In Northern Blot Analysen konnte eine reduzierte Transkription von US9 in HSV-1 KOS infizierten Zellen detektiert werden. In HSV-1 KOS infizierten Zellen konnten mittels eines spezifischen Antiserums gegen US9 im Western Blot kein Genprodukt nachgewiesen werden. Auch Immunfluoreszenzanalysen zeigten, dass das abgeleitete verkürzte Protein offenbar nicht stabil exprimiert wird. Dagegen konnten Western Blot Analysen die Vergrößerung des pUS8a bestätigen. Der beobachtete auffällige intraaxonale Phänotyp könnte somit durch die Mutation des US9 Protein erklärt werden. Zusammenfassend wurde in dieser Arbeit gezeigt, dass auch bei HSV-1 vorwiegend das „Married Model“ für den anterograden intraaxonalen Transportweg bevorzugt wird und somit beide Alphaherpesviren, HSV-1 und PrV, denselben Transportweg nutzen.
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Joussain, Charles. « Construction and validation of HSV-1 vectors with selective and long-term expression in bladder afferent neurons for gene therapy of neurogenic detrusor overactivity. : A translational approach Botulinum Neurotoxin Light Chains Expressed by Defective Herpes Simplex Virus Type-1 Vectors Cleave SNARE Proteins and Inhibit CGRP Release in Rat Sensory Neurons Development and assessment of herpes simplex virus type 1 (HSV-1) amplicon vectors with expression from sensory neuron-selective promoters. Construction and properties of replication-incompetent HSV-1 recombinant vectors expressing transgenic botulinum toxins in primary cultures of human sensory neurons and displaying long-term expression in vivo. Therapeutic escalation for the neurogenic bladder in SCI patients : A bicentric study real life experience Long-term outcomes and risks factors for failure of intradetrusor onabotulinumtoxin A injections for the treatment of refractory neurogenic detrusor overactivity ». Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLV057.
Texte intégralRésumé :
Cinquante à 80% des patients atteints d'une lésion médullaire traumatique (LM) présente des d'épisodes d'incontinence urinaire liés à une hyperactivité détrusorienne neurogène (HDN). L’HDN est caractérisée par des contractions non inhibées du détrusor pendant la phase de remplissage vésical, qui conduit à une augmentation des pressions détrusoriennes, particulièrement lorsque l’HDN est associée à une dyssynergie vésico-sphinctérienne. L'objectif principal de la prise en charge de l’HDN est d'obtenir une vidange vésicale régulière, complète et à basse pression, ainsi que de maintenir la continence urinaire, afin d'améliorer la qualité de vie des patients et de prévenir les complications uro-néphrologiques dont l’insuffisance rénale. La prise en charge actuelle repose sur une pharmacothérapie agissant principalement au niveau de la branche efférente, motrice, du réflexe mictionnel, permettant ainsi un remplissage vésical à basse pression. Le traitement de première intention repose sur des antimuscariniques oraux, le plus souvent associés à la réalisation d’autosondages pluriquotidiens. En cas d’échec de cette thérapeutique, l’injection intradétrusorienne de toxine botulique A est proposée. Cependant, malgré leur efficacité, ces traitements induisent des effets secondaires et souffrent d’un échappement thérapeutique, conduisant à un traitement chirurgical de troisième ligne. La technique de Brindley, qui consiste en une désafférentation des racines postérieures sacrées innervant la vessie associée à une stimulation électrique, à la demande, des racines antérieures est une alternative prometteuse, mais reste peu proposée en raison de la complexité de la procédure chirurgicale requise. L'HDN résulte de l'émergence d'un réflexe spinal anormal médié par une plasticité des afférences vésicales de type-C dans les suites de la LM. Le projet de mon équipe est de réaliser une déafférentation vésicale par thérapie génique afin d'abolir le réflexe de miction spinale altérée. Dans un second temps, la miction sera déclenchée par une stimulation électrique à la demande, des neurones efférents de la vessie. Mon travail de thèse consistait à développer les outils nécessaires à une telle désafférentation moléculaire. En conséquence, j'ai construit des vecteurs défectifs HSV-1 délivrant comme transgène thérapeutique la chaine légère d’un toxine botulique (BoNT-LC), sous le contrôle du promoteur du gène codant pour la protéine liée au gène de la calcitonine (hCGRP), permettant une expression sélective au sein des neurones sensoriels. La cassette de transcription a été insérée dans le locus LAT du génome HSV-1, la seule région du génome du virus qui reste active sur le plan transcriptionnel pendant une infection latente. Ces vecteurs ont été évalués (i) in vitro, sur des lignées cellulaires d'origine neurale et sur des cultures primaires de neurones sensoriels embryonnaires et adultes de rats, ainsi que sur des cultures primaires de neurones sensoriels et sympathiques humains adultes, (ii) ex vivo, sur des cultures organotypiques de ganglions sensoriels, sympathiques et parasympathiques de rats adultes, et (iii) in vivo, post inoculation au niveau du coussinet plantaire de rats adultes. Nos résultats indiquent que (i) les vecteurs expriment des BoNT-LC fonctionnelles, clivant ainsi les protéines SNARE post infection de cultures primaires de neurones sensoriels de rats et d’être humain, et inhibant la libération du neuromédiateur CGRP dans les neurones sensoriels de rat, (ii) la sélectivité d’expression de ces vecteurs dans des neurones sensoriels humains, par rapport aux neurones sympathiques humains, et (iii) une expression transgénique prolongée in-vivo au sein de ganglions sensoriels (DRG), au moins pour trois mois, après injection. Par conséquent, ces vecteurs semblent présenter les trois principales spécifications requises pour le développement d’une future stratégie de thérapie génique visant à traiter l’HDNFifty to 80% of patients with traumatic spinal cord injury (SCI) undergo urinary incontinence episodes, mostly related to neurogenic detrusor overactivity (NDO). NDO is characterized by uninhibited detrusor contractions during the bladder-filling phase which could lead to a significant increase in bladder pressures, especially when associated to sphincter-destrusor-dyssynergia, leading to uro-nephrological complications. The main goal of NDO management following SCI is to achieve regular and complete bladder emptying, avoiding high intra-detrusor pressure and maintaining continence, in order to improve patients’ quality of life and to prevent renal failure. The current management is well characterized and relies on pharmacotherapy acting primarily at the level of efferent motor micturition reflex branch, thus allowing bladder filling at low pressure. First line treatment relies on oral antimuscarinics, often associated to clean intermittent bladder self-catheterization. When patients are refractory to antimuscarinics, injection of botulinum toxin A into the detrusor is proposed. However, despite their efficacy, these treatments fail to persist in the long term, leading to a third-line surgical treatment, which consists in cystoplasty augmentation or sacral neuromodulation. The Brindley technique, which consist in a sacral deafferentation of bladder posterior roots associated to an electrical stimulation, on demand, of anterior roots is a promising alternative, but remains seldom performed because of the complex surgical procedure required. NDO results from the emergence, secondary to neuronal plasticity following SCI, of an abnormal micturition reflex mediated by bladder afferent C-fibers, conveying aberrant sensory information to the spinal cord. The aim of the team where I developed my work is to silence these bladder afferent C-fibers in order to abolish the impaired spinal micturition reflex after SCI. In a second time, micturition would be fired, on demand, by electric stimulation of the bladder efferent neurons. My work consisted in developing the tools and methods required for such molecular deafferentation. Accordingly, I constructed replication-incompetent HSV-1 vectors conceived to deliver a therapeutic transcription cassette, consisting in the light chains of botulinum toxin (BoNT-LC) driven by the human version of the promoter of the gene encoding calcitonin gene-related protein (hCGRP), to achieve sensory neuron-selective transgenic expression. The transcription cassette was inserted into the LAT locus of the HSV-1 genome, the only region of the virus genome that remains transcriptionally active during latent infection. These vectors have been assessed (i) in vitro, on cell lines of neural origin and on primary cultures of rat embryonic and adult sensory neurons, and on primary cultures of adult human sensory and sympathetic neurons, (ii) ex vivo, on organotypic cultures of sensory, sympathetic and parasympathetic ganglia from adult rats, and (iii) in vivo, in sensory ganglia following infection at the hind footpad of adult rats.Our results indicate that (i) the vectors express functional BoNT-LC, thereby cleaving proteins of the SNARE complex in rat and human sensory neurons and inhibiting release of the neuromediator CGRP in rat sensory neurons, (ii) the transcription cassette delivered by the vectors display highly selectively expression towards human sensory neurons, as compared to human sympathetic neurons, and (iii) the vectors induced long-term transgenic expression in sensory (DRG) ganglia (at least for three months) following footpad injection. Therefore, the vectors seem to accomplish the three main specifications required for a future gene therapy strategy, allowing to restore urinary continence and micturition without catheterization and without any major surgery. This approach will represent a major breakthrough in the management of NDO in SCI patients with complete and incomplete lesion
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LORENZETTO, Antonio. « SHEDDING LIGHT ON THE MOLECULAR DEFECT OF TWOALANINE:GLYOXYLATE AMINOTRANSFERASE PATHOGENIC VARIANTS:A BIOCHEMICAL APPROACH ». Doctoral thesis, 2011. http://hdl.handle.net/11562/351830.
Texte intégralRésumé :
L’iperossaluria primaria di tipo 1 (PH1) è una malattia autosomica recessiva rara caratterizzata dal deposito di cristalli insolubili di ossalato di calcio prima nei reni e nel tratto urinario ed in seguito, in assenza di un appropriato trattamento, in tutto il resto del corpo. PH1 è causata da un deficit funzionale di alanina:gliossilato aminotransferasi umana (AGT), un enzima piridossal 5’-fosfato (PLP) dipendente che converte il gliossilato in glicina, prevenendo in tal modo la ossidazione del gliossilato ad ossalato e quindi la formazione di cristalli insolubili di ossalato di calcio. L’AGT normale è codificato dal gene AGXT che esiste nella popolazione umana in 2 varianti polimorfiche: l’allele maggiore (AGT-Ma) e l’allele minore (AGT-Mi), quest’ultimo caratterizzato da due mutazioni puntiformi, che portano alla sostituzione della prolina 11 con una leucina e della isoleucina 340 con una metionina, e da una duplicazione di 74 paia di basi nell’introne 1. Sebbene la presenza dell’allele minore non sia in sè patogenica, questa rende però l’enzima più suscettibile all’effetto di alcune mutazioni che non sarebbero patogeniche se associate all’allele minore. Perciò, c’è un grande interesse nel definire le proprietà dell’AGT-Mi, in quanto punto di partenza per capire il meccanismo molecolare alla base del sinergismo tra l’AGT-Mi e le mutazioni patogeniche che cosegregano con esso.
In questo lavoro, tramite un approccio “in vitro” su proteine purificate, sono stati studiati gli effetti delle 2 mutazioni combinate polimorfiche proprie dell’allele minore, sulle caratteristiche biochimiche dell’AGT, così come di 2 mutazioni che provacono PH1 se associate all’allele minore: F152I e G170R. I dati ottenuti hanno evidenziato che:
1) AGT-Mi mostra caratteristiche spettroscopiche, parametri cinetici, e affinità per il PLP simili a quelle di AGT-Ma. Tuttavia, la sua struttura dimerica è caratterizzata da una bassa resistenza allo stress sia chimico che termico. Questi effetti sembrano essere dovuti alla mutazione P11L dal momento che tale variante mostra un profilo di denaturazione comparabile con quello di AGT-Mi;
2) La mutazione patgenica F152I porta ad una diminuzione di ca. 200 volte nell’affinità dell’AGT per la piridossamina 5-fosfato (PMP) e quando associata all’allele minore, anche ad una inattivazione tempo dipendente ed ad una aggregazione a temperatura fisiologica.;
3) La mutazione patogenica G170R non incide né sulle proprietà spettroscopiche né su quelle cinetiche dell’AGT-Mi in condizioni native. D’altro canto, rende la struttura dimerica dell’apoG170R-Mi più sucettibile alla dissociazione rispetto al corrispondente apoAGT-Mi.
Riassumendo i dati ottenuti:
(i) rivelano le differenze tra AGT-Ma e AGT-Mi;
(ii) gettano luce sul difetto molecolare associato alle varianti F152I-Mi e G170R-Mi;
(iii) permettono di fare ipotesi sulla risposta alla terapia con piridossina osservata nei pazienti recanti queste 2 mutazioni.Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in the absence of appropriate treatments, in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and therefore the formation of calcium oxalate. Normal human AGT is encoded by the AGXT gene that exists in human populations in two polymorphic forms: the major allele (AGT-Ma) and the minor allele (AGT-Mi), which is characterized by two point mutations, leading to the Pro11Leu and Ile340Met substitutions, and a 74 bp-duplication in intron 1. Although the presence of the minor allele polymorphism is not pathogenic “per se”, it makes AGT more susceptible to the effect of some PH1-causing mutation that are expected to be not pathogenic when associated with the major allele. Thus, there is a great interest in defining the properties of AGT-Mi, as the base to unravel the molecular mechanism underlying the synergism between AGT-Mi and the pathogenic mutations that cosegregate with it. In this work, by an “in vitro” approach on purified proteins, we studied the effects on the biochemical features of AGT of the two combined polymorphic mutations typical of the minor allele as well as of two PH1-causing mutations associated with the minor allele, Phe152Ile and Gly170Arg. The data obtained have shown that: 1) AGT-Mi displays spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma. However, its dimeric structure is characterized by a low resistance to both chemical and thermal stress. This appears to be due to the P11L mutation since the P11L variant exhibits a denaturation pattern comparable to that of AGT-Mi; 2) The PH1-causing F152I mutation leads to a ~200 fold decrease in the affinity of AGT for pyridoxamine 5’-phosphate and, when associated with the minor allele polymorphism, to a time-dependent inactivation and aggregation at physiological temperature; 3) The pathogenic mutation G170R does not affect neither the spectroscopic nor the kinetic properties of AGT-Mi under native conditions. However, it makes the dimeric structure of apoG170R-Mi more susceptible to dissociation than the corresponding apoAGT-Mi. Overall, the obtained data: (i) reveal the biochemical differences between AGT-Ma and AGT-Mi; (ii) allow to shed light on the molecular defect associated with the F152-Mi and the G170R-Mi variants; (iii) permit to speculate on the responsiveness to pyridoxine therapy of the patients bearing these mutations.
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Domingues, Mara Sofia de Almeida. « 3D hiPSC to hepatocyte differentiation in bioreactor for Primary Hyperoxaluria type I disease model ». Master's thesis, 2018. http://hdl.handle.net/10362/52956.
Texte intégral
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DINDO, MIRCO. « Molecular analysis of the dimerization and aggregation processes of human alanine:glyoxylate aminotransferase and effect of mutations leading to Primary Hyperoxaluria Type I ». Doctoral thesis, 2017. http://hdl.handle.net/11562/960999.
Texte intégralRésumé :
Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT). AGT is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and calcium oxalate formation. Only two curative therapeutic approaches are currently available for PH1: the administration of pyridoxine (PN), a precursor of PLP, which is only effective in a minority of patients (25- 35%), and liver transplantation, a very invasive procedure. AGT is encoded by the AGXT gene, which is present in humans as two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). PH1 is a very heterogeneous disease with respect to the clinical manifestations, the response to treatment and the pathogenic mechanisms. In fact, more than 200 pathogenic mutations have been identified so far and the molecular mechanisms by which missense mutations cause AGT deficiency span from functional, to structural and to subcellular localization defects or to a combination of them. Several lines of evidence at both molecular and cellular level, indicate that many disease-causing missense mutations interfere with AGT dimer stability and/or aggregation propensity. However, neither the dimerization nor the aggregation process of AGT have been analyzed in detail. Therefore, we engineered a mutant form of AGT stable in solution in the monomeric form and studied its biochemical properties and dimerization kinetics. We found that monomeric AGT is able to bind PLP and that the coenzyme stabilizes the dimeric structure. Moreover, the identification of key dimerization hot-spots at the monomer-monomer interface allowed us to unravel the mechanisms at the basis of the aberrant mitochondrial mistargeting of two of the most common PH1-causing variants. We also elucidated the molecular and cellular consequences of the pathogenic mutations R36H, G42E, I56N, G63R and G216R, involving residues located at the dimer interface, and tested their in-vitro responsiveness to the treatment with PN. The latter results allowed us to suggest a possible correlation between the structural defect of a variant and its degree of responsiveness to PN. Finally, by combining bioinformatic and biochemical approaches, we analyzed in detail the tendency of AGT to undergo an electrostatically-driven aggregation. We found that the polymorphic changes typical of the minor allele have opposite effect on the aggregation propensity of the protein, and we predicted the possible effect/s of pathogenic mutations of residues located on the AGT surface. Overall, the results obtained allow not only to better understand PH1 pathogenesis, but also to predict the response of the patients to the available therapies as well as to pave the way for the development of new therapeutic strategies.
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Roncador, Alessandro. « THE DEFICIT OF ALANINE:GLYOXYLATE AMINOTRANSFERASE LEADS TO PRIMARY HYPEROXALURIA TYPE I : A BIOCHEMICAL STUDY TO UNDERSTAND THE ROLE OF INTERALLELIC COMPLEMENTATION IN COMPOUND HETEROZYGOUS PATIENTS AND TO PROJECT THE DEVELOPMENT OF AN ENZYME ADMINISTRATION THERAPY ». Doctoral thesis, 2014. http://hdl.handle.net/11562/723363.
Texte intégralRésumé :
Iperossaluria Primaria tipo I ( PH1 ) è una rara malattia autosomica recessiva caratterizzata da un elevato livello di ossalato nelle urine , che provoca la formazione di cristalli insolubili di ossalato di calcio dapprima nei reni e delle vie urinarie e , in assenza di un adeguato trattamento , in tutto il corpo . PH1 è causata da un deficit dell'enzima epatico Alanina: Gliossilato aminotransferasi ( AGT ). AGT è un enzima piridossal 5' - fosfato perossisomi ( PLP )-dipendente che converte il gliossilato in glicina, impedendo così l'ossidazione gliossilato di ossalato e la successiva formazione di ossalato di calcio.
AGT è codificata dal gene AGXT, che presenta nell'uomo, due forme polimorfe : l'allele maggiore (codifica AGT - Ma) e l'allele minore (codificante AGT - Mi). Finora sono state identificate più di 150 mutazioni associate a PH1. Diversi studi hanno consentito interessanti progressi nella comprensione dei meccanismi molecolari per cui ciascuna mutazione conduce alla carenza di AGT . Tuttavia, molto spesso i pazienti affetti da PH1 sono eterozigoti composti e il loro fenotipo enzimatico potrebbe dipendere da fenomeni di complementazione interallelic ( IC ). Fino ad ora , la patogenesi di PH1 è stata studiata solo tramite approcci che mimano la situazione cellulare di pazienti omozigoti , mentre il fenotipo clinico relazione genotipo- fenotipo enzimatico di pazienti eterozigoti composti è completamente sconosciuto . Durante il mio dottorato di ricerca , ho condotto studi volti a chiarire il fenotipo enzimatico legata alla mutazione S81L su AGT -Ma , concernente un residuo di interazione con il PLP , sia in omozigosi sia in eterozigosi composta con la mutazione G170R , la variante più comune in AGT – Mi. G170R è nota per determinare il mistargeting mitocondriale di AGT senza alterare le proprietà funzionali dell'enzima stesso.
Utilizzando un vettore di espressione eucariotico bicistronico abbiamo dimostrato che ( i) S81L -Ma ha una significativa ocalizzazione perossisomale, e ( ii ) l'interazione dei monomeri S81L e G170R si verifica nella cellula formando un eterodimero G170R-Mi/S81L-Ma, che viene importato in perossisomi e presenta una funzionalità migliorata rispetto agli enzimi parentali . Questi dati , integrati con i risultati della caratterizzazione biochimica dell' eterodimero purificato ottenuti da un vettore di espressione procariotico, sostengono l'ipotesi di un IC positiva tra i monomeri S81L e G170R. Questo studio rappresenta la prima indagine della patogenesi della PH1 in pazienti eterozigoti composti a livello molecolare.
PH1 è una malattia molto difficile da curare. Solo due approcci terapeutici sono attualmente a disposizione: la somministrazione di piridossina , un precursore di PLP, che è efficace solo in una minoranza di pazienti, e il trapianto di fegato, una procedura molto invasiva . Ne consegue che lo sviluppo di nuove strategie di trattamento , meno invasive ed efficaci per tutti i pazienti , sarebbe altamente desiderabile. Poiché PH1 è originata dal deficit di un singolo enzima , la possibilità di ripristinare la capacità catalitica degli epatociti somministrando enzima esogeno è una prospettiva intrigante. Uno dei problemi principali per lo sviluppo di una terapia somministrazione enzima è l'ingresso intracellulare della proteina esogena. Durante il mio dottorato di ricerca ho utilizzato un duplice approccio per ottenere una forma AGT in grado di attraversare il plasma membrana: ( i) la costruzione di una proteina di fusione tra AGT e la Tat peptide sfruttando le capacità di attraversamento di membrana del domino Tat , e ( ii ) la coniugazione di AGT con un nanocarrier polimerico in grado di trasportare l'enzima funzionale attraverso la membrana plasmatica. Entrambe le strategiehanno dimostrato di essere efficaci nella trasduzione di AGT in un modello cellulare permettendo di ripristinare la capacità di disintossicazione gliossilato senza alterare significativamente le proprietà strutturali e funzionali di AGT. Questi risultati possono essere considerati un incoraggiante punto di partenza per lo sviluppo di una terapia somministrazione enzima per PH1.Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive disorder characterized by a high level of oxalate in the urine, which in turn results in the formation of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in absence of an appropriate treatment, in the whole body. PH1 is caused by the deficiency of human liver alanine:glyoxylate aminotransferase (AGT), a peroxisomal pyridoxal 5'-phosphate (PLP)-dependent enzyme. AGT detoxifies glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and the subsequent calcium oxalate formation. AGT is encoded by the AGXT gene, which presents, in humans, two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). At the time of writing, more than 150 mutations associated with PH1 have been reported and several studies allowed for interesting progresses in the understanding of the molecular mechanisms by which each mutation leads to AGT deficiency. However, quite often patients affected by PH1 are compound heterozygous and their enzymatic phenotype could depend on interallelic complementation (IC) effects. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, while the genotype-enzymatic phenotype-clinical phenotype relationship of compound heterozygous patients is completely unknown. During my PhD, we elucidated the enzymatic phenotype linked to the S81L mutation on AGT-Ma, concerning a PLP binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functional properties, is present in the second allele. By using a bicistronic eukaryotic expression vector we demonstrated that (i) S81L-Ma has a significant peroxisomal localization, and (ii) the interaction of the S81L and G170R monomers occurs in the cell yielding the G170R-Mi/S81L-Ma heterodimer, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the recombinant purified heterodimer compared with those of the homodimeric counterparts obtained by a dual vector prokaryotic expression strategy, provided evidence for a positive IC between the S81L and G170R monomers. This study represents the first investigation of the pathogenesis of PH1 in compound heterozygous patients at molecular level. PH1 is a very difficult-to-treat disease. Only two curative therapeutic approaches are currently available: the administration of pyridoxine, a precursor of PLP that is only effective in a minority of patients, and liver transplantation, a very invasive procedure. It follows that the development of new treatment strategies, less invasive and effective for all the patients, would be highly desirable. In this regard, since PH1 originates from the deficit of a single enzyme, the opportunity to restore the catalytic pool of the hepatocytes by administering exogenous enzyme is an intriguing perspective. One of the major issues for the development of an enzyme administration therapy is the intracellular delivery of the exogenous protein. During my PhD, to obtain an AGT form able to cross the plasma membrane, a dual approach was used: (i) the construction of a fusion protein between AGT and the Tat peptide exploiting the membrane crossing capabilities of the Tat moiety, and (ii) the conjugation of AGT with a polymeric nanocarrier able to deliver the functional enzyme across the plasma membrane. Both strategies did not significantly alter the structural and functional properties of AGT and proved to be effective in transducing active AGT into a cellular disease model and in restoring their glyoxylate detoxification ability. These results can be considered an encouraging starting point for the development of an enzyme administration therapy for PH1.
30
Figueiredo, Rosa Mafalda Amorim. « GABA levels relate to BOLD signal in Neurofibromatosis Type 1 ». Master's thesis, 2019. http://hdl.handle.net/10316/89654.
Texte intégralRésumé :
Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de MedicinaA neurofibromatose Tipo 1 (NF1) é uma doença autossómica dominante na qual os níveis de ácido gama-aminobutírico (GABA) estão reduzidos em várias regiões cerebrais e cujas manifestações clínicas incluem alterações da motricidade. Este estudo investiga, pela primeira vez, a relação entre os níveis de GABA do córtex motor primário (M1) dominante e a atividade funcional de ambos os M1s e do cerebelo durante uma tarefa motora na NF1. Vinte e um participantes com NF1 e vinte controlos executaram movimentos síncronos e assíncronos com os dedos indicadores a ritmos crescentes (1 Hz, 3 Hz e 5 Hz). Os níveis de GABA foram medidos no M1 dominante por espetroscopia de ressonância magnética (MRS) e a atividade funcional de ambos os M1s e do cerebelo foi avaliada por ressonância magnética funcional (fMRI). Depois, investigámos a existência de uma correlação entre os níveis de GABA e a atividade fMRI em cada grupo. Este estudo mostrou que o sinal dependente do nível de oxigenação sanguínea (BOLD) é significativamente mais elevado no grupo NF1 do que no grupo controlo em ambos os M1s e no cerebelo. No movimento assíncrono, os níveis de GABA correlacionaram-se positivamente com a atividade fMRI em ambos os M1s dos doentes com NF1. Essa relação ocorreu sobretudo nos ritmos de tapping mais elevados e não foi observada no grupo controlo. Para além disso, os níveis BOLD do M1 não-dominante espelharam os do M1 dominante no grupo NF1. Em conclusão, alterações neuroquímicas e/ou funcionais no M1 e no cerebelo poderão ser a causa da diminuição das capacidades motoras observadas na NF1, devendo, por isso, ser objeto de estudos adicionais no futuro.
Neurofibromatosis Type 1 (NF1) is a common autosomal dominant disorder with reduced gamma-aminobutyric acid (GABA) levels in several brain regions and whose clinical manifestations include motor deficits. This study investigates for the first time the relation between GABA levels of the dominant primary motor cortex (M1) and the functional activity of both M1s and the cerebellum during a motor task in NF1. Twenty-one NF1 subjects and twenty controls executed a finger-tapping task with synchronous and asynchronous movements at increasing rhythms (1 Hz, 3 Hz, and 5 Hz). GABA levels were measured in the dominant M1 using magnetic resonance spectroscopy (MRS) and the functional activity of both M1s and cerebellum was evaluated using functional magnetic resonance imaging (fMRI). We then investigated the existence of a correlation between GABA levels and fMRI activity in each group. This study showed blood-oxygen-level-dependent (BOLD) signal to be significantly higher in the NF1 group compared to the control group in both M1s and the cerebellum. At asynchronous tapping, GABA levels of the dominant M1 positively correlated with the fMRI activity in both M1s of NF1 patients. That was mainly verified at the highest rhythms of tapping and it was not observed in the control group. In addition, the non-dominant M1 BOLD levels mirrored the dominant M1 in the NF1 group. In conclusion, neurochemical and activity changes in the M1 and the cerebellum may underlie the motor deficits observed in NF1 patients, which should be further addressed in future studies.
H2020
31
Liu, Ming-Tzen, et 劉明真. « Molecular pathology study of skin diseases : type 1 neurofibromatosis, hereditary epidermolytic palmoplantar keratoderma, and primary cutaneous amyloidosis ». Thesis, 2003. http://ndltd.ncl.edu.tw/handle/38866229842285666444.
Texte intégralRésumé :
博士國立陽明大學
臨床醫學研究所
91
Genetic factor plays an important role in determining the development of human skin disorders. Except for anomalies associated with chromosomal aberrations, single-gene and multiple gene disorders are known to cause skin phenotypes, easily detected clinically. In this thesis research, molecular genetic and biochemical approaches were taken to investigate the pathogenesis of three diseases: neurofibromatosis type 1 (NF1), epidermolytic palmoplantar keratoderma (EPPK), and primary cutaneous amyloidosis (PCA). The former two are inherited by autosomal dominant mode, while the latter is mostly sporadic in nature. Neurofibromatosis type 1 (NF1) is a common cancer predisposition syndrome affecting the nervous system. Mutations of the NF1 gene are different among patients, making NF1 molecular diagnosis difficult. Molecular genetic analysis of NF1 patients in Taiwan has not been reported before; therefore, a mutation screen protocol needs to be established to analyze NF1 mutations in our population. A RT-PCR based DNA diagnosis procedure was established to investigate the NF1 gene mutation in neurofibromatosis type I. Five overlapping segments covering approximately 8.6 Kb of the NF1 gene were amplified and sequenced to identify genetic alteration(s) in the coding region. Four new mutations in three patients were uncovered by this protocol. We further investigated what caused the cDNA deletion by PCR, using genomic DNA as a template. We found that a recombination between homologous intronic sequences caused the 7260-8167 deletion of the NF1 gene in the first patient. In the other two patients, we identified a single-base substitution in intron 13, designated IVS13+1G>A, in the second case, and an intron 3 mutation, IVS3+1G >T, in the third case. Both mutations affected the splicing donor signal and caused frame-shift and truncation of the NF1 protein. Epidermolytic palmoplantar keratoderma (EPPK) is a rare autosomal disorder of the skin. So far, only thirty-two kindreds of the keratin 9 gene have been documented in the English literature. We identified a four-generation family from Taiwan with typical clinical and histopathological features of EPPK. To identify the mutation of the new EPPK family and to determine whether the mutation in this Chinese family fall to the 1A region in the keratin 9 rod domain, we have analyzed the coding sequence of the keratin 9 gene in the family members and reviewed the mutation spectrum of familial EPPK in the literature. Whole blood sample was collected from affected and normal individuals of the family as well as 50 controls. Polymerase chain reaction was carried out to amplify the keratin 9 gene sequence. The PCR products were subjected to direct DNA sequencing for coding sequence analysis. A novel point mutation, designated 542T>G, (numbering from the first nucleotide of GenBank accession no. S69510) was identified. The mutation converts a leucine codon (CTC) to an arginine codon (CGC) at amino acid position 159 of keratin 9 protein in a conserved hydrophobic residue of the keratin heptad repeats. By literature review, we found that all familial EPPK mutations cluster within a 16-amino-acid region in the 1A rod domain of keratin 9 protein, and that the Taiwanese family adds a new base substitution type to the list of rare inherited mutations causing EPPK. Primary cutaneous amyloidosis (PCA) is a late-onset, slowly progressing skin disease prevalent in Southeast Asia and South America. The nature of cutaneous amyloid in PCA remains unknown. To understand the molecular basis of PCA pathogenesis, we investigated the nature of amyloid deposit by immunofluorescence and RNA in situ hybridization. RNA in situ hybridization revealed that melanocytes produced amyloid precursor protein in the epidermis, while immunofluorescence microscopy showed specific antibodies against A4 amyloid peptide reacted with the amorphous amyloid material in the papillary dermis. Together, the results indicate that amyloid deposit in PCA is probably originating from melanocytes. Additionally, we established a method for extracting amyloid proteins for biochemical characterization. We concluded that PCA can serve as a model for studying amyloid formation in neural crest-derived cells in the skin.
32
Costa, Adriana Emília Amaral. « Establishment of skin-derived fibroblast cell lines for the study of protein phosphorylation in Myotonic Dystrophy type 1 ». Master's thesis, 2021. http://hdl.handle.net/10773/31035.
Texte intégralRésumé :
Muscular dystrophies are a molecularly, genetically, and clinically
heterogeneous group of disorders characterized mainly by progressive muscle
weakness and degeneration. Within this group, the most common muscular
dystrophy in adults is Myotonic Dystrophy type 1 (DM1), an inherited autosomal
dominant disorder caused by an expansion of the (CTG)n trinucleotide repeats
on the 3’ untranslated region of the DMPK gene. Patients with DM1 not only
present muscular symptoms such as myotonia and muscle wasting, but also
extramuscular symptoms such as cataracts, cardiac conduction abnormalities
and insulin resistance. In DM1, the increased length of triplet expansions leads
to the accumulation of (CUG)n mRNA, that forms hairpin-like structures in the
nucleus, leading to a toxic “gain-of-function” that deregulates RNA binding
proteins such as MBNL1 and CUGBP1. This consequently affects alternative
splicing of different mRNAs, which damages the normal function of different
signalling pathways regulated through phosphorylation, an important regulatory
mechanism. To understand the different impaired phosphorylation signalling
pathways affected in DM1 we firstly conducted a systematic review about protein
phosphorylation in DM1. The results provided a compilation of the altered protein
phosphorylation events, namely the signalling pathways which regulate key
cellular events. Some main findings are the underactivation of signalling
pathways, such as AKT/mTOR and AMPK upon insulin signalling and starvation
conditions, respectively. Also, myoblast differentiation was impaired since,
during differentiation, there was an increase of activity of signalling pathways that
stimulate cell proliferation (e.g. MEK/ERK, PKR/PERK) and a decrease of
important proteins for muscle development, such as DMPK. In order to be able
to study the mechanisms underlying the impaired signalling pathways described
in the systematic review, it is necessary to establish DM1 cell models. For that
reason, fibroblasts have been widely used for the study of this disorder due to its
versatility and easy manipulation. We then successfully established skin-derived
human fibroblast cell lines from patients with DM1 through a skin punch biopsy
explant. These cell lines were subsequently characterized through indirect
immunocytochemistry using a fibroblast-specific marker TE-7. The intracellular
levels and localization of DMPK were also evaluated. It was possible to detect
differences, although not statistically significant, of a reduced expression of
DMPK in DM1-derived fibroblasts from late and juvenile onset with controls. To
conclude, these fibroblasts can be an important cell model for the study of
phosphorylation pathways and other mechanisms, such as nuclear envelope
alterations and being an important research tool for DM1. As future perspectives,
these cell models can be used to study protein phosphatases, such as PP1 and
PP2, since there is not enough evidence of how these are altered in DM1 and
could highly contribute to unravel new molecular mechanisms.As distrofias musculares são um grupo de patologias clínica e geneticamente heterogéneas, caracterizadas por fraqueza e degeneração muscular progressivas. Dentro deste grupo, a distrofia muscular mais comum em adultos é a distrofia miotónica tipo 1 (DM1), uma doença hereditária autossómica dominante causada por uma expansão das repetições de tripletos (CTG)n na região 3' não traduzida do gene DMPK. Os pacientes com DM1 apresentam não só sintomas musculares, como miotonia e perda de massa muscular, mas também extramusculares, como cataratas, problemas na condução cardíaca e resistência à insulina. Na DM1, o aumento das expansões CTG levam ao acúmulo de mRNA (CUG)n, que forma estruturas em hairpin no núcleo, levando a um "ganho de função" tóxico que desregula proteínas de ligação ao RNA, como a MBNL1 e CUGBP1. Isso, consequentemente, afeta o splicing alternativo de diferentes mRNAs, o que prejudica a função normal de diferentes vias de sinalização reguladas por fosforilação, um importante mecanismo regulatório. Para perceber as diferentes vias de sinalização de fosforilação afetadas na DM1, executamos uma revisão sistemática sobre a fosforilação de proteínas em DM1. Os resultados forneceram uma compilação das vias de sinalização alteradas e que regulam eventos celulares chave. Alguns dos principais resultados são a reduzida ativação das vias da AKT/mTOR e da AMPK quando estimuladas com insulina ou com condições de privação de nutrientes, respetivamente. Adicionalmente, a miogénese estava também alterada devido ao aumento de vias estimuladoras de proliferação celular (e.g. MEK/ERK, PKR/PERK) e uma diminuição de proteínas importantes para o desenvolvimento muscular, como a DMPK. Para poder estudar os mecanismos subjacentes às vias de sinalização prejudicadas descritas na revisão sistemática, é necessário estabelecer modelos de células DM1. Por esse motivo, os fibroblastos têm sido amplamente utilizados para o estudo dessa doença devido à sua versatilidade e fácil manipulação. Em seguida, estabelecemos com sucesso linhas de células de fibroblastos humanos derivadas da pele de pacientes com DM1 através de um explante de biópsia cutânea. Essas linhas celulares foram posteriormente caracterizadas por imunocitoquímica indireta usando um marcador específico para fibroblastos TE-7. Os níveis intracelulares e a localização de DMPK também foram avaliados. Foi possível detetar diferenças, embora não estatisticamente significativas, de uma expressão reduzida de DMPK em fibroblastos derivados de DM1 com fenótipos de início tardio e juvenil comparando com controlos. Concluindo, esses fibroblastos podem ser um importante modelo celular para o estudo das vias de fosforilação e outros mecanismos, como alterações do envelope nuclear, sendo uma importante ferramenta de pesquisa para DM1. Como perspetivas futuras, estas linhas celulares podem ser usadas para estudar as fosfatases, como a PP1 e PP2, uma vez que não há evidências suficientes de como estas se alteram no DM1 e podem contribuir fortemente para desvendar novos mecanismos moleculares.
Mestrado em Biomedicina Molecular
33
Chueh, Wei-Han, et 闕維涵. « Effects of berberine supplementation on immunomodulatory functions in type 1 non-obese diabetes mice and the protective mechanism in primary pancreatic islet cells ». Thesis, 2010. http://ndltd.ncl.edu.tw/handle/42233872090300676472.
Texte intégralRésumé :
碩士國立中興大學
食品暨應用生物科技學系所
98
Type 1 diabetes (T1D) is one of autoimmune diseases. T1D patients having Th1-skewed immune responses result in chronic inflammation. The chronic inflammation may further deteriorate diabetic complications. Berberine is an isoquinoline alkaloid. Recently, berberine is reported to have many pharmacological functions, including hypolipidemic and anti-inflammatory effects. However, the effect of berberine on T1D is still not clear. Therefore, this study first investigated the effects of berberine supplementation in vivo on immunomodulatory functions, especially anti-inflammation, using type 1 non-obese diabetes (NOD) mice. The NOD mice were randomly divided into four groups, including control (CO) group which was intragastric gavage with water, berberine low dose (BL), berberine medium dose (BM) and berberine high dose (BH) groups which were respectively administrated with 50, 150, and 500 mg berberine/kg bw through 14 weeks by consecutive tube feeding. ICR mice were also selected as a species control (SC) group to compare with NOD mice (CO). The results showed that secretion ratios of Th1/Th2 cytokines by splenocytes of NOD mice significantly decreased after berberine supplementation. Secretion ratios of anti-/pro-inflammatory cytokines by splenocytes of NOD mice significantly increased after high-dose berberine supplementation. Furthermore, berberine administration increased ratios of anti-/pro-inflammatory cytokines mRNA expression in the liver but decreased the ratios of pro-/anti-inflammatory cytokines mRNA expression in the kidney of NOD mice. Overall, the results suggest that berberine supplementation may improve T1D symptoms via its potent anti-inflammatory and Th2-inclination activities. We found that berberine supplementation increased islet cell numbers of NOD mice in a dose-dependent manner, possibly via an anti-apoptotic pathway. To unravel the protective mechanism of berberine against apoptosis, we established in vitro experimental models using primary pancreatic islet cells from ICR mice. Results showed that berberine administration before streptozotocin (STZ)-stimulation significantly down-regulated ratios of pro-/anti-apoptotic genes (Bax/Bcl-2) expression (mRNA levels) in islet cells compared to those in STZ-stimulation alone group. We concluded that the protective mechanism of berberine on primary islet cells may be via its anti-apoptotic effect in a preventive manner.
34
Negatsch, Alexandra. « Vergleichende Analysen zur Replikation und zum intraaxonalen Transport des Pseudorabiesvirus und des Herpes Simplex Virus Typ 1 in primären Rattenneuronen ». Doctoral thesis, 2013. https://ul.qucosa.de/id/qucosa%3A12491.
Texte intégralRésumé :
Nach dem Eintritt in den Wirtsorganismus und initialer Replikation infizieren Alphaherpesviren Neuronen zur weiteren Ausbreitung im Nervensystem und zur Etablierung einer Latenz. Dazu werden die Viruspartikel innerhalb der Axone retrograd von der Peripherie zum neuronalen Zellkörper transportiert. Die umgekehrte Richtung beschreibt den Weg des anterograden Transports vom Zellkörper zur Synapse für weitere Infektionen von Neuronen höherer Ordnung oder zurück zur Peripherie. Der retrograde intraaxonale Transport ist gut untersucht. Dagegen wird über den anterograden Transport kontrovers diskutiert. Zwei verschiedene Transportmodelle werden vermutet. Das „Married Model“ postuliert, dass umhüllte Virionen innerhalb von Vesikeln entlang des Axons transportiert werden. Die Freisetzung der Partikel erfolgt an der jeweiligen Synapse durch Endocytose. Das „Subassembly Model“ geht dagegen davon aus, dass einzelne Virusstrukurkomponenten (Nukleokapsid, Hülle) entlang des Axons transportiert werden. Der Zusammenbau und die Freisetzung erfolgt am Axonterminus bzw. an der Synapse (in vivo) oder am Wachstumskegel (in vitro) oder an speziellen Auftreibungen des Axons, den sogenannten Varicosities. Nach Infektion eines neuronalen Explantatsystems mit dem Pseudorabiesvirus (PrV) konnten ultrastrukturell umhüllte Virionen in Vesikeln detektiert werden und so der Nachweis der Gültigkeit des „Married Model“ als vorherrschendes Transportmodell geführt werden. Dagegen ist die Situation beim prototypischen Alphaherpesvirus, dem Herpes Simplex Virus Typ 1 (HSV-1), weiterhin ungeklärt. Aufgrund der zahlreichen unterschiedlichen Analysemethoden und -systeme war ein direkter Vergleich der beiden Viren bislang nicht möglich. Daher sollte in dieser Arbeit ein standardisiertes neuronales Kultursystem genutzt werden, um vier verschiedene HSV-1 Stämme im Vergleich zu PrV zu untersuchen. Für die Infektionen wurden sowohl Neuronen aus dem oberen Cervikalganglion als auch aus Spinalganglien genutzt. So konnte gezeigt werden, dass in Neuronen, welche mit den HSV-1 Stämmen HFEM, 17+ und SC16 infiziert waren ca. 75% als umhüllte Virionen in Vesikeln und ca. 25% als nackte Kapside vorlagen. Ingesamt war die Anzahl der Viruspartikel in HSV-1 infizierten Neuronen signifikant geringer als in PrV infizierten Kulturen. Überraschenderweise zeigten mit HSV-1 KOS infizierte Neuronen ein reverses Bild. Hier lagen nur 25% der Viruspartikel als umhüllte Virionen in Vesikeln vor, während 75% als nackte Kapside detektiert wurden. Dieser unerwartete Phänotyp sollte auf molekularbiologischer Ebene genauer untersucht werden. Dabei wurde auf die Genregion von US9 fokussiert. Das von US9 codierte Membranprotein spielt eine wichtige Rolle während des Zusammenbaus der Virionen und bei anschließenden axonalen anterograden Transportvorgängen. In dieser Arbeit konnte gezeigt werden, dass das HSV-1 KOS Genom durch verschiedene Basenaustausche an der vorhergesagten TATA-Box von US9 eine Mutation aufweist. Zusätzlich trägt das offene Leseraster durch eine weitere Mutation ein vorzeitiges Stopcodon auf und wird dadurch auf 58 Kodons reduziert, im Gegensatz zu anderen HSV-1 Stämmen, wo es 91 Kodons umfasst. Die Mutation an der TATA-Box verändert auch das ursprüngliche Stopcodon vom US8a Gen, was zur einer Verlängerung von ursprünglich 161 zu 191 Kodons führt. In Northern Blot Analysen konnte eine reduzierte Transkription von US9 in HSV-1 KOS infizierten Zellen detektiert werden. In HSV-1 KOS infizierten Zellen konnten mittels eines spezifischen Antiserums gegen US9 im Western Blot kein Genprodukt nachgewiesen werden. Auch Immunfluoreszenzanalysen zeigten, dass das abgeleitete verkürzte Protein offenbar nicht stabil exprimiert wird. Dagegen konnten Western Blot Analysen die Vergrößerung des pUS8a bestätigen. Der beobachtete auffällige intraaxonale Phänotyp könnte somit durch die Mutation des US9 Protein erklärt werden. Zusammenfassend wurde in dieser Arbeit gezeigt, dass auch bei HSV-1 vorwiegend das „Married Model“ für den anterograden intraaxonalen Transportweg bevorzugt wird und somit beide Alphaherpesviren, HSV-1 und PrV, denselben Transportweg nutzen.