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Azzi-Nogueira, Deborah. "Os produtos dos genes Tsc1 e Tsc2 em processos neurodegenerativos." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-09122016-154805/.
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O complexo da esclerose tuberosa (TSC) é uma doença genética que pode afetar órgãos específicos de qualquer sistema do organismo humano. Em geral, as lesões surgem pela inativação bialélica de um dos genes supressores tumorais Tuberous Sclerosis Complex 1 (TSC1) ou 2 (TSC2). Por outro lado, nas regiões corticais e subcorticais do cérebro, as lesões decorrentes de falhas de migração neuronal e sua arborização podem ser explicadas pela haploinsuficiência de TSC1 ou TSC2. As lesões do córtex cerebral apresentam-se comumente com epilepsia refratária, a qual, por sua vez, pode se associar a deficiência intelectual e transtornos do comportamento. Estes quadros clínicos podem estar presentes em pacientes com TSC sem lesão anatômica detectável à ressonância nuclear magnética do crânio. As proteínas hamartina ou tuberina, conhecidas também como TSC1 e TSC2, são codificadas respectivamente pelos genes TSC1 e TSC2. Elas agem juntas em um complexo molecular citosólico que inativa a pequena GTPase Rheb, a qual tem ação ativadora da cinase alvo da rapamicina em mamíferos (mTOR), regulando diversos processos celulares, como proliferação, diferenciação, crescimento, migração e metabolismo. Com a hipótese de que a quantidade de TSC1 ou TSC2 no neurônio pode alterar suas funções de forma dependente do estado metabólico, tivemos, neste trabalho, o objetivo geral de caracterizar os padrões de expressão e atividade de TSC1 e TSC2 em dois modelos de neurodegeneração induzida no camundongo adulto e verificar se a redução de quantidade de TSC1 tem efeito sobre a extensão da lesão de neurônios dopaminérgicos em modelo de hemiparkinsonismo. No primeiro modelo empregado, cinco estruturas encefálicas de camundongos submetidos a dieta hiperlipídica mostraram alteração da quantidade de RNAm de Tsc1 e/ou Tsc2 ou sinais de estresse oxidativo. A redução de transcritos de Tsc1 e Tsc2 no córtex cerebral foi dependente de jejum realizado imediatamente antes da eutanásia. No córtex cingulado, houve evidência de estresse oxidativo. O aumento específico de RNAm foi observado no hipocampo (Tsc1 e Tsc2) e no estriado e hipotálamo (Tsc1), embora de forma independente do jejum, sugerindo se tratar de alterações relacionadas à dieta hiperlipídica. No modelo de hemiparkinsonismo, camundongos adultos submetidos a injeção intracerebral de 6-hidroxidopamina apresentaram redução da quantidade total de proteína S6 no lado encefálico tratado quando comparado ao segmento contralateral (p =0,004, r=0,8795; teste de Pearson, IC: 95%), sem alteração de TSC1 ou TSC2. Em análises de imunoperoxidase do encéfalo, descrevemos, de forma independente da lesão, a expressão de TSC1 no estriado, núcleos entopeduncular e arqueado e de TSC2 no tálamo e hipotálamo. Com o objetivo de obter um modelo de camundongo sem expressão pós-natal de Tsc1 em várias regiões encefálicas, de forma independente do tipo celular, realizamos cruzamentos entre uma linhagem de camundongo transgênico em que o gene Tsc1 contém sequências lox nos íntrons 16 e 18 e outra linhagem com Tsc1 tipo-selvagem (WT) em homozigose e o transgene para expressão da recombinase Cre em fusão ao domínio de ligação ao ligante do receptor de estrógeno humano (ESR1) sob o controle de expressão do promotor de ubiquitina C (UBC). Em F1, obtivemos camundongos portadores do transgene UBC-CreESR1 e heterozigotos para Tsc1 (Tsc1WT/Flox). Em F2, entre os animais homozigotos Tsc1Flox/Flox (N = 153) gerados por retrocruzamento, nenhum era portador do transgene (Nesperado = 85; Nobservado = 0; X2 = 348,185; p < 0,0001) É possível que o segmento genômico em que houve inserção do vetor lentiviral que carrega o transgene UBC-CreESR1 esteja ligado ao loco de Tsc1 no cromossomo 2 do camundongo, segregando juntos. O tratamento com 4-hidroxitamoxifeno de animais heterozigotos e portadores do transgene aumentou a quantidade de TSC1 no estriado (p < 0,05) e o cerebelo não apresentou alteração. É possível que mecanismos transcricionais ou traducionais, funcionais no estriado, tenham favorecido o aumento de TSC1 de forma dependente de 4-hidroxitamoxifenoTuberous sclerosis complex (TSC) is a genetic disorder that can affect any specific organs. In general, lesions are caused by biallelic inactivation of the tumor suppressor genes Tuberous Sclerosis Complex 1 (TSC1) or 2 (TSC2). On the other hand, in cortical and subcortical brain regions, lesions associated with neuronal migration and arborization failures can be explained by TSC1 or TSC2 haploinsufficiency. Brain cortical lesions commonly cause refractory epilepsy, which, in turn, may be associated with intellectual disabilities and behavioral disorders. These medical conditions may be present in TSC patients without detectable anatomic lesion on magnetic resonance images. TSC1 and TSC2 genes encode hamartin and tuberin, also known as TSC1 and TSC2, respectively. They act together in a cytosolic molecular complex that inactivates small GTPase Rheb, which is a mammalian target of rapamycin (mTOR) activator, regulating diverse cellular processes such as proliferation, differentiation, growth, migration and metabolism. With the hypothesis that the amount of TSC1 or TSC2 in the neuron can change its function depending on the metabolic state, the overall objective of this study was to characterize TSC1 and TSC2 expression patterns and activity in two mice models of induced neurodegeneration; and check whether TSC1 reduction changes dopaminergic neurons damage extent in a hemiparkinsonins model. For the first model, five brain structures from mice fed with high fat diet showed alterations in Tsc1 and/or Tsc2 mRNA, or oxidative stress signals. Reduction of Tsc1 and Tsc2 transcripts in the cerebral cortex was dependent on fasting performed immediately prior to euthanasiaThere was evidence of oxidative stress in the cingulate cortex. Increase in mRNA was observed in the hippocampus (Tsc1 and Tsc2) and striatum and hypothalamus (Tsc1), although independent of the fasting, suggesting that this effect is related to the high fat diet. In hemiparkinsonism model, adult mice subjected to intracerebral injection of 6-hydroxydopamine had decreased levels of S6 in the brain treated side compared to the contralateral segment (p = 0.004, r = 0.8795; Pearson test, CI: 95 %), without alterations in TSC1 nor TSC2. Using imunoperoxidase analysis, we described TSC1 expression in the striatum, entopeduncular and arcuate nuclei, and TSC2 in the thalamus and hypothalamus, independently from the 6-OHDA lesion. To obtain a mouse model without TSC1 postnatal expression in different brain regions, independently of the cell type, we performed crosses between transgenic mouse strain in which the Tsc1 gene contains lox sequences in introns 16 and 18 and strain with Tsc1 wild-type (WT) and the transgene for expression of Cre recombinase fused to the binding domain of the human estrogen receptor (ESR1) ligand, controlled by ubiquitin C (UBC) promoter expression. In F1, we obtained mice carrying the transgene UBC-CreESR1 and heterozygous for Tsc1 (Tsc1WT/flox). In F2, among animals homozygous Tsc1Flox/Flox (N=153) generated by backcrossing, none was carrying the transgene (Nexpected = 85; Nobserved = 0; X2= 348.185, p <0.0001) It is possible that the genomic segment containing the lentiviral vector insertion bearing UBC-CreESR1 transgene is linked to the TSC1 region on mouse chromosome 2, and they segregate together. Treatment with 4-hydroxytamoxifen in animals heterozygous and positive for the transgene showed increased TSC1 in the striatum (p <0.05), while there was no change in the cerebellum. It is possible that transcriptional or translational functional striatum mechanisms favored TSC1 increasing, in a 4-hydroxytamoxifen-dependent manner
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Khatri, Shikha. "FOXO3a Regulates Glycolysis via Transcriptional Control of Tumor Suppressor TSC1." University of Cincinnati / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1282570293.
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Marín, Alexandra Belén Saona. "Capacidade proliferativa in vitro de precursores neuro-gliais, telencefálicos e expressão dos genes 1 e 2 do Complexo da Esclerose Tuberosa (TSC1 e TSC2)." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-08032013-105224/.
Full textAbstract:
O complexo da esclerose tuberosa (TSC) é um transtorno clínico, com expressividade variável, caracterizado por hamartomas que podem ocorrer em diferentes órgãos. Tem herança autossômica dominante e é devido a mutações em um de dois genes supressores de tumor, TSC1 ou TSC2. Estes codificam para as proteínas hamartina e tuberina, respectivamente, que se associam formando um complexo macromolecular que regula funções como proliferação, diferenciação, crescimento e migração celular. As lesões cerebrais podem ser muito graves em pacientes com TSC e caracterizam-se por nódulos subependimários (SEN), astrocitomas subependimários de células gigantes (SEGA), tuberosidades corticais e heterotopias neuronais, podendo relacionar-se clinicamente à epilepsia refratária à terapia medicamentosa, deficiência intelectual, desordens do comportamento e hidrocefalia. O potencial de crescimento de SEGA até os 21 anos de idade dos pacientes exige acompanhamento periódico por exame de imagem e condutas clínicas ou cirúrgicas, conforme indicação médica. As lesões subependimárias têm sido explicadas por déficits de controle da proliferação, crescimento e diferenciação de precursores neuro-gliais na zona subventricular telencefálica. Embora a capacidade da tuberina em inibir a proliferação celular pela repressão do alvo da rapamicina em mamíferos (mTOR) esteja bem documentada, outros aspectos celulares do desenvolvimento de SEGA ainda não foram examinados. Assim, é importante estabelecer um sistema in vitro para o estudo de células da zona subventricular e testá-lo na análise das proteínas hamartina e tuberina. Neste sentido, o cultivo de neuroesferas em suspensão é muito apropriado. Neste estudo, buscamos relacionar a expressão e distribuição subcelular da hamartina e tuberina à capacidade proliferativa e de diferenciação das células de neuroesferas cultivadas in vitro a partir da dissociação da vesícula telencefálica de embriões de ratos normais. Analisamos a expressão e distribuição subcelular da hamartina e tuberina por imunofluorescência indireta em células entre a primeira e a quarta passagens das neuroesferas, sincronizadas nas fases G1 ou S do ciclo celular e após a reentrada no ciclo celular, através da incorporação de 5-bromo-2\'-desoxiuridina (BrdU) e imunofluorescência com anticorpo anti-BrdU. Em geral, células de neuroesferas apresentaram baixa colocalização entre hamartina e tuberina in vitro. A expressão da tuberina foi elevada em basicamente todas as células das esferas e fases do ciclo celular; ao contrário, a hamartina apresentou-se principalmente nas células da periferia das esferas. A colocalização entre hamartina e tuberina foi observada em células mais periféricas das esferas, sobretudo no citoplasma e, em G1, no núcleo celular. A proteína rheb, que conhecidamente interage diretamente com a tuberina, apresentou distribuição subcelular muito semelhante à desta. Ao carenciamento das células visando à parada do ciclo celular na transição G1/S, tuberina distribuiu-se ao núcleo celular em quase todas as células avaliadas e, de forma menos frequente, a hamartina também. À reentrada no ciclo celular pelo reacréscimo dos fatores de crescimento, avaliaram-se células com incorporação de BrdU ao seu núcleo celular, após 72 e 96 horas. Nestas, tuberina mostrou-se novamente no citoplasma de forma preponderante e hamartina manteve-se citoplasmática, em geral subjacente à membrana plasmática, em níveis mais baixos. Os grupos cujas células reciclaram por 72 ou 96 horas diferiram quanto ao aumento significativo da expressão da hamartina em células proliferativas no último. À diferenciação neuronal, aumentaram-se os níveis de expressão de hamartina observáveis à imunofluorescência indireta, tornando-se equivalentes àqueles da tuberina. Concluímos que as células de neuroesferas cultivadas em suspensão apresentam-se como um sistema apropriado ao estudo da distribuição das proteínas hamartina e tuberina e sua relação com o ciclo celularThe tuberous sclerosis complex (TSC) is a clinical disorder with variable expressivity, characterized by hamartomas that can occur in different organs. It has autosomal dominant inheritance and is due to mutations in one of two tumor suppressor genes, TSC1 or TSC2. These encode for the proteins hamartin and tuberin, respectively, which are associated in a macromolecular complex which functions as a regulator of cell proliferation, differentiation, growth and migration. TSC brain lesions may be severe and are characterized by subependymal nodules (SEN), subependymal giant cell astrocytomas (SEGA), neuronal heterotopias and cortical tubers, and may be clinically related to refractory epilepsy, intellectual disability, behavioral disorders and hydrocephaly. The growth potential of SEGA up to 21 years of age in TSC patients requires regular monitoring by imaging. Clinical and surgical interventions may be medically indicated. Subependymal lesions have been explained by deficient control of proliferation, growth and differentiation of neuro-glial progenitors from the telencephalic subventricular zone. While tuberin ability to inhibit cell proliferation by repressing the mammalian target of rapamycin (mTOR) has been well documented, other cell aspects of SEGA development have not been thoroughly examined. Therefore, it is important to establish conditions for an in vitro system to study the cells from the subventricular zone and to test its suitability for the study of the TSC proteins. In this regard, the neurosphere suspension culture is very appropriate. We evaluated the expression and subcellular distribution of hamartin and tuberin in relation to the proliferation and differentiation capability of neurosphere cells derived in vitro from the dissociation of the telencephalic vesicle of normal E14 rat embryos. These analyses were performed by indirect immunofluorescence in cells from first through fourth passages of neurospheres, synchronized in G1 or S phases of the cell cycle, and after reentry into the cell cycle by the addition of 5-brome-2\'-desoxyuridine (BrdU) and immunolabeling with anti-BrdU antibody. In general, neurosphere cells presented low colocalization between hamartin and tuberin in vitro. Tuberin expression was relatively high in basically all neurosphere cells and cell cycle phases, whereas hamartin distributed mainly to cells from the periphery of the spheres. In these cells, hamartin and tuberin colocalization was evident mostly in the cytoplasm and, in G1, also in the cell nucleus. Rheb, which is known to interact directly with tuberin, had subcellular distribution very similar to tuberin. Cell starvation indicating cell cycle arrest at G1/S redistributed tuberin to the cell nucleus in virtually all cells examined, what was accompanied by nuclear location of hamartin in a small subset of cells. When cells were allowed to reenter cell cycle by adding growth factors, we evaluated BrdU-labeled nuclei 72 and 96 hours later. In the two groups, tuberin was shown to move back to the cytoplasm as well as hamartin, which apparently maintained its lower expression levels distribution underneath the plasma membrane. Group of cells that recycled for 96 hours had significantly more expression of hamartin than those cells that cycled for only 72 hours. After neuronal differentiation, hamartin expression levels observed by immunofluorescence were similar to those of tuberin. We conclude that neurosphere cells cultured in suspension showed to be an appropriate cell system to study hamartin and tuberin distribution in respect to the cell cycle
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Schmid, Maria [Verfasser], and Joachim-Ulrich [Akademischer Betreuer] Walther. "Genotyp-Phänotyp-Korrelation beim Tuberöse-Sklerose-Komplex : Auswertung der Mutationsanalyse von TSC1 und TSC2 aus der Diagnostik von TSC-Patienten und Vergleich unterschiedlicher Techniken / Maria Schmid ; Betreuer: Joachim-Ulrich Walther." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1127527851/34.
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Schmid, Maria Anna Katharina [Verfasser], and Joachim-Ulrich [Akademischer Betreuer] Walther. "Genotyp-Phänotyp-Korrelation beim Tuberöse-Sklerose-Komplex : Auswertung der Mutationsanalyse von TSC1 und TSC2 aus der Diagnostik von TSC-Patienten und Vergleich unterschiedlicher Techniken / Maria Schmid ; Betreuer: Joachim-Ulrich Walther." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2017. http://d-nb.info/1127527851/34.
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Alzhrani, Jasser Ali S. "Na+/K+ Pump and Cl--coupled Na+ and K+ co-transporters in Mouse Embryonic Fibroblasts lacking the Tuberous Sclerosis Complex TSC1 and TSC2 genes." Wright State University / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=wright1440683830.
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Zügge, Karin Louise. "Molecular genetic investigation of the variability of the GTPase activating protein- (GAP-) related domain of the tuberous sclerosis-2 (TSC2) gene in TSC patients and healthy subjects." [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972115366.
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Wilson, Catherine. "Molecular analysis of a Tsc1-deficient mouse." Thesis, Cardiff University, 2006. http://orca.cf.ac.uk/54274/.
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Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutation in either the TSC1 or TSC2 genes and characterised by the development of benign hamartomatous growths in multiple organ systems. We have inactivated Tsd in the mouse germ line by gene targeting in ES cells and confirmed that the mutant allele (Tsc1) has a recessive embryonic lethal phenotype. Tsc1+, mice developed macroscopically visible renal lesions as early as 3-6 months. Renal lesions progressed from cysts through cystadenomas to solid carcinomas. Eighty percent of Tsc1+/ mice on a Balb/c background exhibited solid renal cell carcinomas (RCC) by 15-18 months and in 41%, RCCs were _5mm, resulting in grossly deformed kidneys. Some RCCs had a sarcomatoid morphology of spindle cells in whorled patterns and metastasised to the lungs. This new murine model of hamartin deficiency exhibits a more severe phenotype than existing models. A Bloom's deficient mouse model (Blmm3/m3) has been shown to induce colorectal tumourigenesis when crossed with Apc+/M,n mice. Here, we investigate whether the Blmm3/m3 genotype could induce tumourigenesis in extra-colonic tissues in Tsc1+/ mice that are predisposed to renal cystadenomas and carcinomas. Tsc1+/ B mm3,m3 mice had significantly more macroscopic and microscopic renal lesions at 3-6 months compared to Tsc1+' Blm+/m3 mice. Tsc1+, Blrrf3*"3 mice tumours showed significantly increased levels of somatic LOH of the wild type Tsd (Tsrf *) allele, as compared to those from Tsc1+, Blm+/+ mice. This work demonstrates the utility of the Blmm3/m3 mice for inducing renal tumourigenesis and the high levels ( 87%) of LOH in the resultant tumours will help facilitate mapping of loci involved in tumour progression. TSC1 and TSC2 are generally considered to act as tumour suppressors that fulfil Knudson's '2-hit hypothesis'. Here, we identified somatic Tsd mutations (2nd hits) in -80% of CAs and RCCs, but only 31.6% of cysts from Tsc1+/- mice, raising the possibility that haploinsufficiency for Tsd plays a role in cyst formation. Consistent with this proposal, many cysts showed little or no staining for phosphorylated mTOR and phosphorylated S6 ribosomal protein, whereas >90% of CAs and RCCs showed strong staining for both markers. We also sought somatic mutations in renal lesions from Tsc1+/ Blm'A mice that have a high frequency of somatic LOH, thereby facilitating the detection of 2nd hits. We also found significantly less somatic mutations in cysts, as compared to CAs and RCCs from these mice. Our data indicate that although activation of the mTOR pathway is an important step in Tsc-associated renal tumourigenesis, it may not be the key initiating event in this process.
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Hien, Annie. "Regulation of Translation and Synaptic Plasticity by TSC2." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1097.
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Mutations in TSC2 cause the disorder tuberous sclerosis (TSC), which has a high incidence of autism and intellectual disability. TSC2 regulates mRNA translation required for group 1 metabotropic glutamate receptor-dependent synaptic long-term depression (mGluR-LTD), but the identity of mRNAs responsive to mGluR-LTD signaling in the normal and TSC brain is largely unknown. We generated Tsc2+/- mice to model TSC autism and performed ribosome profiling to identify differentially expressed genes following mGluR-LTD in the normal and Tsc2+/- hippocampus. Ribosome profiling reveals that in Tsc2+/-mice, RNA-binding targets of Fragile X Mental Retardation Protein (FMRP) are increased. In wild-type hippocampus, induction of mGluR-LTD caused rapid changes in the steady state levels of hundreds of mRNAs, many of which are FMRP targets. Moreover, mGluR-LTD signaling failed to promote phosphorylation of eukaryotic elongation factor 2 (eEF2) in Tsc2+/- mice, and chemically mimicking phospho-eEF2 with low cycloheximide enhances mGluR-LTD in the Tsc2+/- brain. These results suggest a molecular basis for bidirectional regulation of synaptic plasticity by TSC2 and FMRP. Furthermore, deficient mGluR-regulated translation elongation contributes to impaired synaptic plasticity in Tsc2+/- mice.
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Virdi, Simerjot Kaur. "Roles of the TSN1 and TSC2 Genes in Conferring Susceptibility of Durum Wheat to Tan Spot and Septoria Nodorum Blotch." Thesis, North Dakota State University, 2015. https://hdl.handle.net/10365/27628.
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Tan spot is an important disease caused by the necrotrophic fungus Pyrenophora triticirepentis.
Two common necrotrophic effectors produced by this fungus are Ptr ToxA and Ptr
ToxB, which recognize host sensitivity genes Tsn1 and Tsc2, respectively. In this research, a
tetraploid recombinant inbred line population was evaluated for reaction to the Ptr ToxA and Ptr
ToxB-producing isolates 86-124 (race 2) and DW5 (race 5). The results indicated that a
compatible Tsc2-Ptr ToxB interaction accounted for 26% of the disease variation, which states
that this interaction plays a significant role in the development of tan spot. On the contrary, the
Tsn1-Ptr ToxA interaction was not associated with tan spot caused by 86-124. However,
evaluation of a ToxA-producing isolate of Parastagonospora nodorum, indicated that the Tsn1-
ToxA interaction accounted for 38% of the variation. Therefore, the Tsn1-ToxA interaction
played a significant role in the development of septoria nodorum blotch, but not tan spot.
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Moosa, Alym Platinum. "Characterization of cellular abnormalities due to loss of TSC2." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/46520.
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Tuberous sclerosis complex (TSC) is a disorder characterized by multiple benign tumours in all major organs that can result in neurological manifestations including mental retardation and autism. TSC results from mutation in TSC1 or TSC2, which together form a complex that serves as a negative regulator of protein kinase mTORC1, a key regulator of cell growth and metabolism. Thus, cells with diminished TSC1:TSC2 function display elevated mTORC1 signaling, leading to the formation of benign tumours with very large cells. Rapamycin is a potent mTORC1 inhibitor, and rapamycin derivative everolimus has been approved for treatment of TSC patients with inoperable subependymal giant cell astrocytomas. However, these drugs can have serious side effects and should not be used for extended periods of time. In a screen of approved human drugs, amiodarone, dronedarone, perhexiline, and niclosamide were found to inhibit the elevated mTORC1 signaling seen in mouse embryo fibroblast (MEF) cells lacking TSC2. The goal of this project was to determine whether the many abnormal cell phenotypes associated with loss of TSC2 are directly related to elevated mTORC1 levels, and whether these drugs can ameliorate these abnormal phenotypes.
Unlike wild type MEFs, TSC2-null MEFs showed an epithelial-like morphology with an increase in localization of actin to the cell periphery, focal adhesions, localization of β-catenin to cell-cell junctions, and localization of N-cadherin to cell-cell junctions. Exposure of TSC2-null MEFS to the mTORC1 inhibitors for seven days caused a transition from an epithelial-like to a fibroblast-like morphology in all of the aforementioned phenotypes, resembling that of wild type MEFs.
TSC2-null MEFs were shown to express E-cadherin, a cell adhesion protein not normally found in MEFs. Knocking down levels of TSC2 in wild-type MEFs did not induce expression of E-cadherin, but restoring TSC2 expression in TSC2-null MEFs slightly reduced E-cadherin expression. A tentative model was proposed to explain how TSC2 can control E-cadherin expression, which has not yet been described in literature.
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Jeganathan, Dharini. "Identification and analysis of mutations in the TSC1 gene." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326099.
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Ali, Johari Bin Mohd. "Comparative mapping and mutation analysis of the human TSC1 gene." Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621738.
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Spångberg, Christian. "En studie om regleringen av nanomaterial : - i The Toxic Substances Control Act (TSCA) och Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)." Thesis, Uppsala universitet, Juridiska institutionen, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-327381.
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Aguirre, Egaña Itziar de. "LKB1/ AMPK / TSC2 signaling pathway alterations in non-small-cell-lung-carcinoma." Doctoral thesis, Universitat Autònoma de Barcelona, 2014. http://hdl.handle.net/10803/284322.
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El gen supresor tumoral LKB1/STK11b, codifica una serina/treonina quinasa. Las mutaciones de línea germinal en LKB1 están asociadas al síndrome de Peutz-Jeghers (PJS), trastorno dominante autosómico caracterizado por el crecimiento de pólipos en el tracto gastrointestinal, y depósitos de melanina mucocutánea, aumentando el riesgo de ciertos cánceres, incluido el de pulmón. El locus de PJS se encuentra situado en el cromosoma 19p13.3 y el gen responsable fue identificado como LKB1. Las mutaciones del gen LKB1 también están asociadas a los adenocarcinomas de pulmón, ocurren aproximadamente en un tercio de los tumores primarios y en la mitad de las líneas celulares de adenocarcinoma de pulmón, implicando a LKB1 en la patogénesis del cáncer de pulmón. Estudios recientes sugieren que la vía de señalización de LKB1 juega un papel en la protección de las células frente a la apoptosis, en respuesta a niveles de energía o nutrientes bajos, lográndose en parte a través de la supresión de la actividad de mTOR. Hay evidencias en la bibliografía que demuestran que LKB1 activa al complejo heterotrimerico AMPK, formado por tres subunidades, una catalítica (α) y dos reguladoras (β y γ). AMPK es un sensor de energía celular que contribuye a regular el balance energético y la ingesta calórica en la célula. Posteriormente, AMPK fosforila a TSC2. El heterodímero TSC1-TSC2 actúa para inhibir la actividad de mTOR, que es esencial para el control del crecimiento celular y la proliferación. Así, LKB1 funciona como un regulador negativo de mTOR. Está demostrado que LKB1 también regula 11 de las 12 quinasas de la subfamilia de quinasas AMPK in vitro, sugiriendo que una de las funciones del gen supresor LKB1 podría ser la regulación de la vía de señalización de AMPK. Por tanto, LKB1 podría ser un blanco potencial para el tratamiento de cáncer y trastornos metabólicos.
Nuestros principales objetivos fueron: ▪ Determinar la frecuencia de alteraciones genéticas y epigenéticas en la vía de señalización LKB1/AMPK/TSC2 en CPCNP, en un panel de 23 líneas celulares (4 líneas celulares epiteliales normales bronquiales inmortalizadas con SV40 y 19 líneas celulares de CPCNP). ▪ Estudiar el estado de metilación de 4 quinasas de la subfamilia de quinasas AMPK: BRSK1, BRSK2, MARK1 MARK4 en 23 líneas celulares. ▪ Examinar la vía LKB1/BRSK2. ▪Analizar el estado de metilación de BRSK2 en 58 FFIP de pacientes con CPCNP. ▪ Determinar el impacto de la pérdida de LKB1 en líneas celulares de CPCNP.
Las principales conclusiones fueron: ▪ Las mutaciones de LKB1 no se limitan a los adenocarcinomas, también ocurren en otro subtipo de CPCNP, los carcinomas de células grandes. No se encontró metilación en el promotor de LKB1 en ninguna de las líneas celulares de cáncer pulmón analizadas en este estudio. ▪ No se detectaron mutaciones ni ningún grado de metilación en los distintos componentes estudiados de AMPK y TSC en las 23 líneas celulares analizadas. ▪ De la subfamilia de quinasas de AMPK analizadas, el estado de metilación del promotor BRSK2 representa el de mayor porcentaje: ~ 26% de las líneas celulares y 25,8% del grupo de 58 FFIP de pacientes con CPCNP. ▪ El estado de metilación de BRSK2 se correlaciona significativamente con cuatro parámetros clinicopatológicos: estadio tumoral (P = 0, 025), histología (P = 0, 001), tamaño tumoral (P = 0, 073) y nódulos afectados (P = 0, 04), sugiriendo que el estado de metilación del promotor BRSK2 podría utilizarse como un nuevo biomarcador en la progresión del cáncer de pulmón. ▪ La interrupción de la vía LKB1/BRSK2 podría ser un importante mecanismo molecular, durante el desarrollo del cáncer de pulmón.LKB1, also known as STK11b, is a serine/threonine kinase that functions as a suppressor of tumor growth. Germ line mutations in LKB1 are associated with the Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by benign hemartomas of the gastrointestinal tract and mucocutaneous melanin deposits and an increased risk of certain cancers, including lung. The main PJS locus has been mapped to chromosome 19p13.3, and the gene responsible was identified as LKB1. Mutations in LKB1 gene are also associated with sporadic lung adenocarcinomas and occurs in approximately one-third of primary tumors and half of lung adenocarcinoma cells lines, implicating LKB1 in the pathogenesis of lung cancer. Recent evidence suggests that LKB1 signaling plays a role in protecting cells from apoptosis in response to energy or nutrient deprivation, which is achieved in part through the suppression of mTOR activity. Evidence from the literature supports a protein kinase cascade model in which LKB1 phosphorylates AMPK. AMPK is a heterotrimeric complex comprising a catalytic subunit (α) and two regulatory subunits (β and γ) that act as an intracellular energy sensor maintaining the energy balance within the cell. AMPK subsequently phosphorylates TSC2. Together the TSC1-TSC2 heterodimer acts to suppress the activity of mTOR, which is essential for the control of cell growth and proliferation. Thus, LKB1 functions as a negative regulator of mTOR. Additionally, LKB1 has been shown to regulate 11 of the 12 AMPK family members in vitro, suggesting that one of the tumour suppressor functions of LKB1 may be regulation of AMPK signaling. Therefore, LKB1 could be a potential target for treatment of both cancer and metabolic disorders. In this work we sought to study the signal transduction LKB1/AMPK/TSC2 pathway alterations that contribute to the pathogenesis NSCLC. Our major objectives were: ▪To determine the frequency of genetic and epigenetic alterations in the signal traduction LKB1/AMPK/TSC2 pathway in NSCLC in a panel of 23 cell lines (4 normal bronchial epithelial cell lines immortalized with SV40 and 19 NSCLC cell lines). ▪ To study some components of AMPK-related kinase family that could be activated downstream of LKB1, in particular we analyzed the methylation status of: BRSK1, BRSK2, MARK1, MARK4 in our panel of 23 cell lines. ▪ To examine LKB1/BRSK2 signaling.▪ To make a clinical validation of BRSK2 methylation status, in 58 FFPE of patients with NSCLC. ▪ To determinate the impact of LKB1 depletion on NSCLC cell lines. The major conclusions of this work were: ▪ LKB1 mutations are not confined to adenocarcinomas, they also occur in other NSCLC subtype such as large cell carcinomas. But we found no evidence of LKB1 methylation in any NSCLC cell lines used in this study. ▪ There are not sequence alterations or/and promoter methylation of AMPK and TSC components studied in our 23 panel cell lines. ▪ Of the four AMPK related kinases -BRSK1, BRSK2, MARK1 and MARK4- studied, BRSK2 represents the highest percentage of methylation; ~26 % of NSCLC cell lines and 25.8% in the 58 NSCLC samples. ▪ BRSK2 methylation is significantly correlated with four clinicopatological parameters: tumor stage (P=0,025), histology (P=0,001), tumor size (P=0,073) and nodules affected (P=0,04), suggesting that methylation of BRSK2 could provide a novel biomarker for disease progression in lung cancer. ▪ The disruption of LKB1/BRSK2 signaling could be important in the carcinogenesis of lung, as a molecular mechanism for the development of lung cancer.
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Obayashi, Yoko. "Novel physiological function of proline and mTOR regulator tuberin." Kyoto University, 2018. http://hdl.handle.net/2433/232154.
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Woodward, Karen Jane. "Characterisation of the TSCI candidate region on human chromosome 9q34." Thesis, University College London (University of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283661.
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Anderson, John A. "Designing and modeling a torque and speed control transmission (TSCT)." Morgantown, W. Va. : [West Virginia University Libraries], 1999. http://etd.wvu.edu/templates/showETD.cfm?recnum=1194.
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Thesis (M.S.)--West Virginia University, 1999.Title from document title page. Document formatted into pages; contains viii, 69 p. : ill. Includes abstract. Includes bibliographical references (p. 68-69).
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Kariyawasam, Gayan Kanishka. "Molecular Genetic Characterization of Ptr ToxC-Tsc1 Interaction and Comparative Genomics of Pyrenophora tritici-repentis." Diss., North Dakota State University, 2018. https://hdl.handle.net/10365/28964.
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Tan spot of wheat, caused by Pyrenophora tritici-repentis, is an economically important disease worldwide. The disease system is known to involve three pairs of interactions between fungal-produced necrotrophic effectors (NEs) and the wheat sensitivity genes, namely Ptr ToxA-Tsn1, Ptr ToxB-Tsc2 and Ptr ToxC-Tsc1, all of which result in susceptibility. Many lines of evidence also suggested the involvement of additional fungal virulence and host resistance factors. Due to the non-proteinaceous nature, Ptr ToxC, has not been purified and the fungal gene (s) controlling Ptr ToxC production is unknown. The objective for the first part of research is to map the fungal gene (s) controlling Ptr ToxC production. Therefore, A bi-parental fungal population segregating for Ptr ToxC production was first developed from genetically modified heterothallic strains of AR CrossB10 (Ptr ToxC producer) and 86-124 (Ptr ToxC non-producer), and then was genotyped and phenotyped. Using the data, the gene (s) was mapped to the distal end of chromosome 2 in the reference genome of Pt-1c-BFP. The objective for the second part of my research is to develop genomic and genetic resources for the fungal pathogen. A high quality of genome sequence for AR CrossB10 and the first P. tritici-repentis genetic linkage map was generated. The AR CrossB10 genome and genetic linkage map is highly comparable to newly published reference genome except some noticeable chromosomal structural variations (SVs). Comparison of the genome sequences between parental isolates and twenty progeny isolates also revealed some SVs including deletion, insertion and inversion were detected that likely occurred during the fungal sexual reproduction. The objective for the third of my research is to characterize genetic resistance in Nebraskan winter wheat cultivar ?Wesley? using QTL mapping in a recombinant inbred line population. The results showed that resistance in Wesley is largely due to the lack of susceptibility genes Tsc1 and Tsn1. My Ph.D. research provides a further understanding of the genetics of host-pathogen interaction in wheat tan spot and contributes knowledge and tools for breeding tan spot resistant cultivars.USDA-NIFA-AFRI
North Dakota Wheat Commission
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Momose, Shuji. "Identification of the coding sequences responsible for Tsc2-mediated tumor suppression using a transgenic rat system." Kyoto University, 2003. http://hdl.handle.net/2433/148668.
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Pymar, Louis Spencer. "Functional analysis of the TSCI gene product hamartin in urothelial cells." Thesis, University of Leeds, 2007. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.485306.
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Bruno, Odemir Martinez. "Sistema automatizado de medidas TSC em plataforma GUI." Universidade de São Paulo, 1995. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-03062014-151648/.
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Neste trabalho desenvolvemos um método de controle e aquisição de dados, particularmente aplicado a medidas de correntes termoestimuladas (TSC), mas que podem ser estendidas a sistemas semelhantes. O sistema automatizado de medidas TSC em plataforma GUI, foi implementado de modo a realizar o controle da temperatura de uma estufa, de forma que seja possível gerar rampas lineares com taxas programáveis, e também a leitura de sinais analógicos referentes ao sistema, como corrente e temperatura, e processar os sinais obtendo como resultado curvas gráficas (sinais processados em função do tempo). Os resultados são armazenados e podem ser exibidos, ou impressos e transferidos para o padrão de arquivo de entrada de outros aplicativos. Os dados são obtidos em tempo real e o sistema utiliza conceitos da tecnologia GUI, onde provemos um estudo desta, gerando janelas com interação orientada a eventos e uma interface gráfica com o usuário bastante interativa. O sistema faz uso de microcomputador padrão IBM PC AT e utiliza o MS-Windows 3.1 como plataforma GUI (\"GraphicsUser Interface\").A method for control and data acquisition using GUI environment, applied to thermal and electrical measurements was developed. The method was specifically applied to thermal stimulated current (TSC), but it can be extended to similar systems. The GUI - based automated TSC system maked possible the accurate control of the oven temperature, at different heating rates. This system performs the data acquisition of analogic signals (current and temperature, for instance), and those signals are processed generating functions of time and/or temperature (TSC curves, for example). Stored results can be displayed, printed, as well as transferred to files, which are compatible to standard applicative for data treatment. Data are obtained in real time and the system uses GUI concepts, which are extensively studied in this work, generating events oriented windows and a user-friendly graphical interface. The system configuration consists of an IBM PC AT microcomputer and the MS-Windows 3.1 is used as the GUI environment.
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Almeida, Luiz Gustavo Dufner de. "Estudo mutacional em pacientes com o complexo da esclerose tuberosa." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-09122014-085619/.
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O complexo da esclerose tuberosa (TSC) é um transtorno genético, sistêmico, com expressividade variável e herança autossômica dominante. Clinicamente manifesta-se devido ao desenvolvimento de hamartomas e hamártias em diferentes tecidos, principalmente no cérebro, rins, coração, pele e pulmões, podendo causar disfunção do órgão. Mutação em um de dois genes supressores tumorais, TSC1 ou TSC2, são responsáveis pelo TSC. Os genes TSC1 e TSC2 codificam para hamartina e tuberina, respectivamente. Ambas as proteínas interagem fisicamente formando um complexo citosólico que inibe mTOR (mammalian target of rapamycin). Testes moleculares para TSC1 e TSC2 são úteis para auxiliar no diagnóstico de casos clínicos difíceis, em aconselhamento genético e estudos de associação genótipo-fenotípica, além de permitirem a caracterização molecular de mecanismos patogenéticos da formação dos hamartomas e análises funcionais de seus produtos gênicos. Apesar de o diagnóstico de TSC ser basicamente clínico, a partir da revisão de seus critérios em 2012 por um grupo de especialistas, o achado de uma mutação em TSC1 ou TSC2 passou a ser considerado suficiente para o diagnóstico definitivo da doença. O estudo apresentado aqui é parte de um projeto em andamento para estabelecer condições ao desenvolvimento de análise de mutações causadoras de TSC nos genes TSC1 e TSC2. Nosso objetivo neste trabalho foi avaliar por sequenciamento de Sanger o DNA genômico de 28 pacientes brasileiros com diagnóstico definitivo de TSC, procedentes dos estados de São Paulo ou Paraná, tendo como alvo a sequência codificadora do gene TSC1, parte de seus segmentos intrônicos, o promotor basal, bem como o segmento imediatamente a 5\' deste. Sete pacientes (25%) apresentaram mutações de sentido trocado (nonsense) ou com deslocamento da leitura à tradução (frameshift) no gene TSC1. Entre 31 outras variantes de DNA encontradas, 23 são polimorfismos conhecidos e oito apresentaram frequência inferior a 1%, como verificado in silico entre mais de mil sequências de genomas humanos. Entre essas oito variantes de DNA novas ou raras, quatro foram detectadas em pacientes para os quais uma mutação patogênica havia sido identificada e, por isso, foram reclassificadas como polimorfismos. Duas e uma variantes de DNA do mesmo paciente flanqueavam um sítio de ligação em potencial para um fator de transcrição específico, a 5\' do promotor basal de TSC1. Por fim, uma nova variante de DNA na região não codificadora do éxon 2 do gene TSC1 foi predita com potencial para alterar um elemento candidato a acentuador de splicing. Em resumo, como observado em estudos anteriores, descrevemos aqui 25% dos pacientes com TSC apresentando mutações patogênicas na sequência codificadora do gene TSC1. Nossos dados mostraram quatro novas variantes de DNA em regiões potencialmente reguladoras da expressão do gene TSC1, que podem revelar-se como mutações patogênicas e, portanto, necessitam ser testadas experimentalmenteTuberous sclerosis complex (TSC) is a multisystem disorder, with variable expression and autosomal dominant inheritance. Clinically it is due to hamartia and hamartoma development in different tissues, notably in the brain, kidneys, heart, skin and lungs, causing organ dysfunction. Mutations in either tumor suppressor gene, TSC1 or TSC2, are responsible for TSC. TSC1 and TSC2 genes code for hamartin and tuberin, respectively. Both proteins physically interact forming a cytosolic complex that inhibits the mammalian target of rapamycin (mTOR). TSC1 and TSC2 molecular testing has been useful in diagnosing clinically challenging cases, in genetic counseling and genotype/phenotype association studies, besides evaluation of the molecular basis of hamartoma formation and functional analyses of both gene products. Although TSC diagnosis is basically clinical, since the 2012 specialist panel review the finding of a TSC1 or TSC2 mutation has been considered sufficient for the definite diagnosis of the disease. The results presented here are part of an ongoing project to establish conditions for TSC1 and TSC2 mutation studies. Our first aim is to evaluate by Sanger sequencing TSC1 coding sequence, and an average of 132 base pairs of intronic regions next to exon boundaries from TSC patients, in addition to the gene core promoter. We present preliminary results of a sample of 28 patients with definite TSC diagnosis, from São Paulo and Paraná states. Seven patients (25%) displayed TSC1 nonsense or frameshift mutations. Among 31 other DNA variants identified, 23 were known polymorphisms, and eight had frequencies below 1% as verified in silico among more than a thousand human genomes. Out of eight novel or rare DNA variants, four were detected in patients for whom a pathogenic mutation had been found. Two and one additional DNA point variants from the same patient flanked a putative transcription factor binding site. Finally, a novel DNA variant residing in the TSC1 noncoding exon 2 was predicted to change the sequence potential to behave as a splicing enhancer. In summary, similar to previous studies, we describe 25% of TSC patients with mutations in the TSC1 coding sequence. Differently from other reports, our data disclose four novel DNA variants in TSC1 potentially regulatory regions that are likely to unravel novel pathogenic mutations, and thus need to be experimentally tested
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Young, Janet Mary. "Identification of the TSCI gene and neighbouring transcripts on human chromosome 9Q34." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.299865.
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Liang, Ning. "Regulation of YAP by mTOR and autophagy reveals a therapeutic target of Tuberous Sclerosis Complex." Thesis, Paris 5, 2014. http://www.theses.fr/2014PA05T055/document.
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La sclérose tubéreuse complexe (TSC) est une maladie génétique caractérisée par une croissance des hamartomes dans différents organes y compris le cerveau, les reins, les poumons, la peau et le cœur. Ces lésions sont des sources de morbidité et de mortalité chez les patients TSC, car ils peuvent provoquerl’ épilepsie, l’autisme, le retard de développement et l’insuffisance rénale et pulmonaire. Les causes connues de TSC sont la perte de la fonction et les mutations des gènes TSC1 et TSC2. La majorité des lésions TSC contient plusieurs types cellulaires de la lignée mésenchymateuse, comme dans le cas des angiomyolipomes, l’lymphangioleiomyomatose et les angiofibromes. Un type unique de cellules épithélioïdes périvasculaires nommé (PEC) est constamment présent dans les lésions de TSC mésenchymateuses, comme angiomyolipomes et lymphangioleiomyomatose, basant sur les caractérisations morphologiques et l'expression des marqueurs communs mélanocytaires et myogéniques. Par conséquent, ces lésions sont officiellement classées, ainsi que d'autres tumeurs, comme PEComes. Leur origine cellulaire et les mécanismes moléculaires impliqués dans la pathologie restent à élucider. Ici, nous avons généré un modèle souris mosaïque TSC1 knockout qui développe des lésions rénales mésenchymateuses récapitulant périvasculaire épithélioïde cellules tumorales humaines (Pecoma) observés chez les patients TSC. Nous avons identifié YAP, le co-activateur transcriptionnel de la voie Hippo, a été régulée d'une manière mTOR-dépendante dans les lésions rénales de notre TSC1 knockout souris et les échantillons de l’angiomyolipome humaines. L'inhibition de YAP avec des outils génétiques ou pharmacologiques atténue considérablement la prolifération et la survie des cellules nulles TSC1 in vivo et in vitro. En outre, l’accumulation de YAP dans les cellules déficientes TSC1 / TSC2 pourra être dû à la dégradation de la protéine altéré par le système de l’autophagosome / lysosome. Ainsi, la régulation de YAP par mTOR et l'autophagie est un nouveau mécanisme de contrôle de la croissance, l'activité de YAP correspondant à la disponibilité des éléments nutritifs dans les conditions de croissance permissives. Il pourra servir comme une cible thérapeutique potentielle pour TSC et d'autres maladies avec une activité de mTOR déréguléeThe Tuberous Sclerosis Complex (TSC) is a genetic disease characterized by growth of hamartomas in different organs including brain, kidney, lung, skin, and heart. These lesions are sources of morbidity and mortality in patients with TSC, as they may cause intractable epilepsy, autism, developmental delay, renal and pulmonary failure. Known causes of TSC are loss of function mutations in TSC1 and TSC2 genes. The majority of TSC lesions contain multiple cell types of the mesenchymal lineage, as in the case of angiomyolipomas, lymphangioleiomyomatosis and angiofibromas. A unique cell type named perivascular epithelioid cell (PEC) is constantly present in mesenchymal TSC lesions, such as angiomyolipomas and lymphangioleiomyomatosis, basing on morphological features and the common expression of melanocytic and myogenic markers. Therefore, these lesions are officially classified, along with other tumors, as PEComas. Their cell of origin and the molecular mechanisms underlying their pathogenesis remain poorly defined. Here we generated a novel mosaic Tsc1 knockout mouse model which develop renal mesenchymal lesions recapitulating human Perivascular Epithelioid Cell tumor (PEComa) observed in TSC patients. We identified YAP, the transcriptional coactivator of Hippo pathway, was upregulated in both renal lesions of TSC mouse model and human angiomyolipoma samples in a mTOR-dependent manner. Inhibition of YAP with genetic or pharmacological tools greatly attenuates the proliferation and survival of Tsc1 null cells in vivo and in vitro. Futhermore, we found YAP accumulation in TSC1/TSC2 deficient cells is due to impaired degradation of the protein through the autophagosome/lysosome system. Thus the regulation of YAP by mTOR and autophagy is a novel mechanism of growth control, matching YAP activity with nutrient availability under growth permissive conditions. It may serve as a potential therapeutical target for TSC and other diseases with dysregulated mTOR activity
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Aldred, Mark. "Investigating the mechanism of cystogenesis in TSC and ADPKD." Thesis, Cardiff University, 2011. http://orca.cf.ac.uk/55109/.
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Tuberous sclerosis complex (TSC) is characterised by the development of benign growths across the body and is caused by mutations in TSC1 or TSC2. The TSC gene products have an established role in the regulation of mammalian target of Rapamycin (mTOR) signalling. Clinical trials are underway for the treatment of TSC- associated tumours using mTOR inhibitors. Here, we show that many of the earliest renal lesions from Tsc1+, and Tsc2+/' mice (cysts) do not exhibit mTOR activation, suggesting alternative pathways should be targeted to prevent tumour formation. Patients with TSC often develop renal cysts (derived from dilated tubules) and those with inherited co-deletions of the autosomal dominant polycystic kidney disease (ADPKD) gene 1 (PKD1) develop severe, early onset polycystic kidneys. Using mouse models, we have shown that the Tsc and Pkd1 gene products are required for correct cell polarisation during renal tubule and bile duct elongation. When polarity is disrupted in Tsc1+I', Tsc2+, and Pkd1+' mice, we found significant alterations in the length of primary cilia projecting from pre-cystic tubule and duct cells (consistent with the highly polar nature of this organelle). The primary cilium is proposed to facilitate many signalling events and provides a mechanosensory input into renal tubule cells. Despite widespread defects in cell polarity and primary cilia in the developing kidney of a Tsc1+I , Tsc2+/ or Pkd1+I' mouse, we found no evidence of tubule dilation, occlusion or cyst formation until around 3-6 months of age. On the basis of this delay period, combined with our data showing significantly higher levels of cleaved caspase-3 in pre-cystic renal tubules from these mice, we suggest that apoptosis destroys these misaligned cells to protect against cyst formation. We found that almost all cysts without mTOR-activation failed to stain for cleaved caspase-3, and therefore sought activation of a pro-survival pathway. There was strong upregulation of Bcl2 in mTOR inactive cysts that were not undergoing apoptosis, suggesting this was the mediator of survival in our cysts. In cysts without activation of mTOR or apoptosis, we found significant activation of Jak2 and its downstream target Stat3. We finally sought gain-of-function mutations in this pathway, and found several somatic Jak2 mutations with likely oncogenic potential in Tsc-associated cysts. These data suggest that defective cell polarity in the context of abnormal Jak2 signalling can drive Tsc-associated cystogenesis in the absence of mTOR dysregulation and targeting of this pathway may be of key therapeutic benefit.
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Han, Juliette. "The Role of TSC in Oligodendrocyte Differentiation and Myelination." Thesis, Harvard University, 2012. http://dissertations.umi.com/gsas.harvard:10194.
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Tuberous Sclerosis Complex (TSC) is an autosomal dominant syndrome characterized by epilepsy, intellectual disability, and autism. Recent studies have suggested that white matter abnormalities, including hypomyelination, contribute to the cognitive deficits in TSC patients, but the mechanism has remained elusive. I used the neuron-specific Tsc1 knockout mice that display a marked decrease in myelin and show that oligodendrocytes are arrested at immature stages of development in vivo resulting in a reduction in the number of myelinating cells. I established an oligodendrocyte culture system and examined the effect of neuron-conditioned media and found that the Tsc1 mutant phenotype was replicable in vitro using medium collected from Tsc1 knockdown (TSC-KD) neurons, confirming that a secreted signal is responsible for inhibiting differentiation of the oligodendrocytes. I took an unbiased genome-wide approach and identified Connective Tissue Growth Factor (CTGF) as a putative candidate for the secreted signal. I confirmed that CTGF was upregulated in Tsc1 mutant neurons and characterized its spatial and developmental expression pattern in our mouse model. In vitro, CTGF was sufficient to inhibit differentiation of oligodendrocytes. The addition of CTGF neutralizing antibody to the TSC-KD neuronal media was able to reverse the suppression of oligodendrocyte maturation, strongly suggesting that CTGF is a major component of the oligodendrocyte inhibitory signal derived from Tsc mutant neurons. Since TSC mutation affects all cells, I investigated the role of TSC in oligodendrocytes. In response to TSC knockdown, oligodendrocytes demonstrate an upregulation of cellular stress marker. I also found a decrease in myelin protein genes, a finding that offers interesting implications for the role of TSC in hypomyelination. Furthermore, I expanded my research into Zellweger disease, a syndrome that involves TSC in its neuropathological manifestations including white matter deficits, and found that localization of TSC to the peroxisome is a critical factor in neuron development. Together, this body of work developed new approaches in Tuberous Sclerosis research in the brain to investigate a previously under-appreciated aspect of TSC pathology - myelination. I have demonstrated that the TSC pathway has important roles in neuron-oligodendrocyte communication and emphasize the critical importance of neuron-derived signals in the establishment of myelination.
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Leclezio, Loren. "Identification of natural TSC-Associated Neuropsychiatric Disorders (TAND) clusters." Doctoral thesis, University of Cape Town, 2017. http://hdl.handle.net/11427/27084.
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Tuberous Sclerosis Complex (TSC) is associated with many learning, behavioural, neurodevelopmental and psychiatric difficulties. Over 90% of individuals with TSC will have some of these concerns yet no more than 20% receive support and treatment, even though these issues may cause the greatest burden of disease in TSC. The Neuropsychiatry Panel at the 2012 TSC Consensus Conference coined the term TAND (TSC-Associated Neuropsychiatric Disorders) to capture the multidimensional concerns seen in TSC, and recommended that each person with TSC should be screened for TAND every year. To facilitate the process, a TAND Checklist was designed. Many professionals and families feel overwhelmed by the complexity of TAND and say that they do not know where to start and how to access relevant information, tips or 'next step' approaches. This may in part be due to the multi-dimensionality of TAND, and in part due to lack of access to clear, useful and evidence-based resources for TAND. This project aimed to address the complexity of TAND. The hypothesis was that, even though each individual will typically have their own unique TAND profile, there will be key natural TAND Clusters - combinations of behaviours across multi-dimensional levels - that will simplify further evaluations and treatment. The study was performed over 36 months, in two phases using a mixed-methods approach. Phase I was a pilot phase. TAND Checklist data were collected from 56 individuals with TSC in South Africa (n=20) and in Australia (n= 36). Using R, these data were explored with various multivariate data analysis techniques to identify suitable analysis methods for the identification of potential natural TAND clusters. WARD's cluster analysis method rendered six TAND clusters with good face validity, and convergence with a six-factor exploratory factor analysis solution. Pilot results suggested that a combination of cluster analysis and exploratory factor analysis methods may be able to identify clinically-meaningful natural TAND clusters. Phase II set out to replicate and expand on pilot results. TAND checklist data were collected from n=453 across six international TSC sites, and the multivariate analysis techniques identified in phase I were applied. WARD's method rendered seven natural TAND clusters with good clinical face validity. This data-driven strategy identified a 'Scholastic' cluster of TAND manifestations, a 'Neuropsychological' cluster, a 'Mood/Anxiety' cluster, an 'ASD-like' cluster, a 'Behaviours that Challenge' cluster, a 'Hyperactive/Impulsive' cluster, and an 'Eating/Sleeping' cluster. Results showed significant convergence with an exploratory factor analysis solution. The larger-scale study findings were remarkably consistent with pilot findings, supporting the robustness of these naturally occurring clusters. We propose that the seven natural TAND clusters identified can in future be used to generate clinical toolkits for use in real-life setting. In addition, findings suggest that the aetiology and molecular treatments of TAND may also show differential clustering across human and animal models, pointing towards novel hypotheses regarding neuropsychiatric phenomena in TSC to be explored in future studies.
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Elling, Eva. "Effects of MIFID II on Stock Trade Volumes of Nasdaq Stockholm." Thesis, KTH, Matematisk statistik, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-257510.
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Introducing new financial legislation to financial markets require caution to achieve the intended outcome. This thesis aims to investigate whether or not the newly installed revised Markets in Financial Instruments Directive- the MIFID II regulation - temporally influenced the trading stock volume levels of Nasdaq Stockholm during its introduction to the Swedish stock market. A first approach of a generalized Negative Binomial model is carried out on aggregated data, followed by an individual Fixed Effects model in an attempt to eliminate omitted variable bias caused by missing unobserved variables for the individual stocks. The aggregated data is attained by taking the equally weighted average of the trading volume and adjusting for seasonality through Seasonal and Trend decomposition using Loess in combination with a regression model with ARIMA errors to mitigate calendar effects. Due to robustness of the aggregated data, the Negative Binomial model manage to capture significant effects of the regulation on the Small Cap. segment, even though clusters of the data show signs of divergent reactions to MIFID II. Since the Fixed Effects model operate on non-aggregated TSCS data and because of the varying effects on each stock the Fixed Effect model fails in its attempt to do the same.Implementation av nya finansiella regelverk på finansmarknaden kräver aktsamhet för att uppnå de tilltänka målen. Det här arbetet undersöker huruvida MIFID II regleringen orsakade en temporär medelvärdesskiftning av de handlade aktievolymerna på Nasdaq Stockholm under regelverkets introduktion på den svenska marknaden. Först testas en generaliserad Negative Binomial regression applicerat på aggregerad data, därefter en individuell Fixed Effects modell för att försöka eliminera fel på grund av saknade, okända variabler. Det aggrigerade datasettet erhålls genom att ta genomsnittet av handelsvolymerna och justera dessa för sässongsmässiga mönster med metoden STL i kombination med regression med ARIMA residualer för att även ta hänsyn till kalender relaterade effekter. Eftersom den aggrigerade datan är robust lyckas the Negative Binomial regressionen fånga signifikanta effekter av regleringen för Small Cap. segmentet trots att datat uppvisar tecken på att subgrupper inom segmentet reagerat väldigt olika på den nya regleringen. Eftersom Fixed Effects modellen är applicerad på icke-aggrigerad TSCS data och pågrund av den varierande effekten på de individuella aktierna lyckas inte denna modell med detta.
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Gómez, Baldó Laia. "The link between the centrosome, TSC disease and epithelial differentiation." Doctoral thesis, Universitat de Barcelona, 2010. http://hdl.handle.net/10803/1113.
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Studies of the role of tuberous sclerosis complex (TSC) proteins (TSC1/TSC2) in pathology have focused mainly on their capacity to regulate translation and cell growth, but their relation with alterations of cellular structures and the cell cycle is not yet fully understood. The transforming acidic coiled-coil (TACC) domain-containing proteins are central players in structures and processes involving the microtubule network. Here, TACC3 interactome mapping identified TSC2 and 15 other proteins, including TACC homo and heterodimers, and two evolutionary conserved interactors (ch-TOG/CKAP5 and FAM161B). TACC3 and TSC2 co-localize and co-purify with components of the nuclear envelope, and their deficiency causes morphological alterations of this structure. During cell division, TACC3 is necessary for the proper localization of phospho-Ser939 TSC2 at the spindle poles and cytokinetic bridges. Consistently, abscission alterations and increased frequency of binucleated cells were observed in Tacc3- and Tsc2-deficent cells relative to controls. In regulating cell division, TSC2 acts epistatic to TACC3 and, in addition to canonical TSC/mTOR signaling and cytokinetic associations, converges to the early mitotic checkpoint mediated by CHFR. Our findings link TACC3 to novel structural and cell division functions of TSC2, which may provide additional explanations of the clinical and pathological manifestations of lymphangioleiomyomatosis disease and TSC syndrome, including the greater clinical severity associated with TSC2 germline mutations relative to TSC1."ESTUDI DE LA CONNEXIÓ ENTRE EL CENTROSOMA, L'ESCLEROSI TUBEROSA I LA DIFERENCIACIÓ EPITELIAL"
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La major part dels estudis realitzats per tal d'esbrinar les funcions de les proteïnes TSC1 i TSC2 causants de l'Esclerosi Tuberosa (TSC) s'han centrat en la seva capacitat de regular la transcripció i el creixement cel·lular, però la seva relació amb alteracions d'estructures cel·lulars i el cicle cel·lular no estan ben determinades. Les proteïnes TACC tenen un paper primordial en estructures i processos relacionats amb les xarxes de microtúbuls (MTs). En aquest estudi hem realitzat un mapatge d'interaccions centrat en TACC3 i hem identificat TSC2 i 15 proteïnes més, com són els homo- i heterodímers de TACC, i dues proteïnes (ch-TOG/CKAP5 i FAM161B), la interacció de les quals està conservada entre espècies. TACC3 i TSC2 co-localitzen i co-purifiquen amb components de l'embolcall nuclear i la seva deficiència dóna lloc a alteracions d'aquesta estructura. Al llarg de la divisió cel·lular, TACC3 és necessària per a la localització de phospho-Ser939-TSC2 als pols del fus mitòtic i al punt de divisió de citocinesi. En concordança, cèl·lules deficients en Tacc3 i Tsc2 presenten un retràs en la citocinesi i una elevada freqüència de cèl·lules binucleades. Pel que fa a la regulació del cicle cel·lular, TSC2 actua de forma epistàtica sobre TACC3 i, a més de la seva funció de senyalització en la via TSC/mTOR i les associacions amb la citocinesi, convergeix amb el punt de regulació temprà de mitosi mediat per CHFR. Els nostres resultats relacionen TACC3 amb noves funcions estructurals i de divisió cel·lular per part de TSC2, la qual cosa podria proporcionar noves explicacions a les manifestacions clíniques i patològiques de la limfangioleiomiomatosi (LAM) i TSC.
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Jonsson, Oskar, and Filip Papic. "Studies of Squamous Cell Carcinoma of the Tongue(TSCC), with Focus on Histological Factors." Thesis, Umeå universitet, Institutionen för odontologi, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-144078.
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Squamous cell carcinomas of the head and neck (SCCHN) is a diverse group of tumours includingtumours of the mouth, of which most are manifested in the tongue. Previous studies have shown that assessment of histological risk factors including worstpattern of invasion (WPOI) and lymphocytic response (LR) is of clinical relevance in treating SCCHN. It hasalsobeen suggested that evaluation of the inflammatory infiltrate could be of prognostic importance.The purpose of this study was to further investigate, analyze and map histological factors associated with TSCC.A total of 58biopsies fromtwo different ethnic groups,Swedish and Italian,were evaluated regarding factors like WPOI andLR. Results were thencorrelated to clinical data.Approximately halfof the patients, 53%, displayed patches ofdense lymphocytic infiltrate at the tumour interface. There was,however,no statistically relevant correlation seen between LR, recurrence of disease, survival rate or ethnicity. Considering WPOI, 83% of patients showed a tumour growth pattern withsmall invasive islands <15 cells (WPOI 4). No correlationbetween WPOI, recurrence of disease, survival rate, ethnicity or lymphocyticresponse was found.Our findings confirm that SSC of the tongue hasa very split patternof invasion. No conclusive result was found concerning inflammatory response and prognostic factors. Data collected showed that ethnic differences could potentiallybe of interest for further study of the prevalence of tongue SCC.
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Hale, Amber N. "ANALYSIS OF THE ROLE OF TWO AUTOPHAGY PATHWAY RELATED GENES, BECN1 AND TSC1, IN MURINE MAMMARY GLAND DEVELOPMENT AND DIFFERENTIATION." UKnowledge, 2014. http://uknowledge.uky.edu/biology_etds/18.
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The mammary gland is a dynamic organ that undergoes the majority of its development in the postnatal period in four stages; mature virgin, pregnancy, lactation, and involution. Every stage relies on tightly regulated cellular proliferation, programmed cell death, and tissue remodeling mechanisms. Misregulation of autophagy, an intracellular catabolic process to maintain energy stores, has long been associated with mammary tumorigenesis and other pathologies. We hypothesize that appropriate regulation and execution of autophagy are necessary for proper development of the mammary ductal tree and maintenance of the secretory epithelia during late pregnancy and lactation. To test this hypothesis we examined the role of two genes during development of the mammary gland.
Beclin1 (Becn1) is an essential autophagy gene. Since the Becn1 knockout model is embryonic lethal, we have generated a Becn1 conditional knockout (cKO). We used two discrete mammary gland-specific Cre transgenic lines to interrogate the role of BECN1 during development. We report that MMTV-CreD; Becn1fl/fl mice have a hyper-branching phenotype and WAP-Cre; Becn1fl/- mice are unable to sustain a lactation phase. Becn1 mutants exhibit abnormal glandular morphology during pregnancy and after parturition. Moreover, when autophagy is chemically inhibited in vitro, mammary epithelial cells have an increased mean number of lipid droplets per cell.
MTOR inhibits autophagy upstream of BECN1; we looked higher in the regulatory pathway for regulatory candidates. It has been well characterized that Tuberous sclerosis complex 1 (TSC1), in a heterodimer with its primary binding partner TSC2, inhibits MTOR signaling via inhibition of RHEB. Using the Tsc1 floxed model we generated a mammary gland specific Tsc1 cKO and found that these mice phenocopy the Becn1 cKO mice, including a gross lactation failure. Tsc1 cKO glands have altered morphology, retained lipid droplets in secretory epithelia, and an overall increase in MTOR signaling. We show that TSC1 and BECN1 are interacting partners, and that the interaction is nutrient responsive.
These results suggest that Becn1 and Tsc1 are necessary for proper mammary gland development and differentiation. Furthermore, we have demonstrated a novel murine protein-protein interaction and an important link between regulation of MTOR pathway and regulation of autophagy in a developmental context.
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Joly, Fanny. "Impacts d’une perturbation de la voie TSC2/mTOR dans l’amygdale dès l’adolescence sur le comportement de peur et la fonctionnalité du cortex préfrontal chez le rat adulte Disruption of Amygdala Tsc2 in Adolescence Leads to Changed Prelimbic Cellular Activity and Generalized Fear Responses at Adulthood in Rats." Thesis, université Paris-Saclay, 2021. http://www.theses.fr/2021UPASL016.
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L’adolescence est une période développementale vulnérable marquée par d’intenses modifications structurelles et fonctionnelles des réseaux qui assurent la régulation des comportements émotionnels et cognitifs. Les processus de maturation de ces réseaux sont influencés par les facteurs environnementaux et génétiques. Le syndrome de stress post-traumatique (PTSD) est un trouble psychiatrique caractérisé par des peurs exagérées, des généralisations de la peur et des déficits d’extinction de peur. Les facteurs de prédisposition au PTSD sont peu connus, mais nous savons qu’un stress intense subi au cours de l’adolescence favorise son apparition à l’âge adulte lorsqu’un individu est confronté à un nouvel événement traumatique.Au cours de cette thèse, nous avons étudié la fonctionnalité de deux structures du circuit de la peur, l’amygdale et le cortex préfrontal, qui suivent une maturation asynchrone. L’amygdale étant fonctionnellement mature à l’âge juvénile, elle est en position de modifier la maturation tardive du cortex préfrontal (mPFC). Nous avons donc voulu tester l’impact d’une dérégulation de la voie Tsc2/mTOR dans les cellules excitatrices du noyau basolatéral de l’amygdale (BLA) du rat au début (25 jours post-natal, PN25), ou à la fin (PN50) de l’adolescence. Le comportement émotionnel à l’âge adulte a été évalué à l’aide d’un conditionnement Pavlovien et l’activité basale du mPFC par la mesure de l’expression du gène immédiat précoce c-FOS. Nous montrons que seuls les rats altérés à l’adolescence (PN25) présentent à l’âge adulte des symptômes typiques du PTSD (déficit d’extinction et généralisation de la peur), associés à une augmentation de l’activité basale du mPFC, en particulier dans les couches corticales impliquées dans le maintien de la mémoire de peur. Ainsi, une modification de la fonctionnalité de l’amygdale au début de l’adolescence pourrait être un facteur de prédisposition à l’apparition d’un PTSD à l’âge adulteAdolescence is a highly sensitive developmental period characterized by massive structural and functional changes in networks regulating emotional and cognitive behaviors, with maturational processes influenced by environmental and genetical factors. Post-traumatic stress disorder (PTSD) is a psychiatric disorder characterized by an exaggerated fear, overgeneralization, and deficits in fear extinction. Nowadays, genetical and/or environmental predisposal factors for PTSD are not fully understood, but we know that an intense stress or a trauma endured during adolescence promotes the appearance of PTSD at adulthood following a novel trauma exposure.In this thesis, we particularly studied two structures that belong to the fear-network, the amygdala and prefrontal cortex, which follow an asynchronous maturation. While the amygdala is functionally mature at a juvenile age, its activity could impact the late maturation of the medial prefrontal cortex (mPFC). We aimed to study the impact of a disruption of Tsc2/mTOR pathway in the excitatory cells of the basolateral nucleus of the amygdala (BLA) in rats at young adolescence (post-natal day 25, PN25) or at the end of adolescence (PN50). When animals had reached adulthood, we assessed emotional behavior through a Pavlovian fear conditioning protocol, and the basal mPFC activity through the measure of expression of immediate early gene c-FOS. We show that only animals altered during young adolescence presented at the adult age typical symptoms of PTSD (fear extinction deficits, overgeneralization of fear), associated with an increase of mPFC basal activity, especially in cortical layers known to be involved in the maintenance of fear memory and expression. Thus, we suggest that a developmental dysfunction of the amygdala early in adolescence could be a predisposal factor to PTSD appearance at adulthood
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Dettenmaier, Erik. "Measuring and Modeling of Plant Root Uptake of Organic Chemicals." DigitalCommons@USU, 2008. https://digitalcommons.usu.edu/etd/18.
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Determining the root uptake of xenobiotic organic chemicals into plants is critical for assessing the human and ecological health risks associated with the consumption of plants growing in contaminated environments. Root uptake of xenobiotic organics occurs passively in conjunction with transpiration and the transport from root to shoot is ultimately controlled by passage through one or more lipid root membranes. The transpiration stream concentration factor (TSCF), the ratio between the concentration of a chemical in the xylem to that in the solution used by the roots, is used to describe the relative ability of an organic chemical to be passively transported from root to shoot. However, relatively few experimental TSCF values exist due to the cost and the lack of regulatory requirements for generating such data. Where literature data exist for chemicals having more than one TSCF, the variability is often large due to the lack of standardized methods and difficulty in accounting for metabolism and volatilization losses occurring during the uptake experiments. Because of the scarcity of experimental values, estimated TSCFs are often used. Widely cited estimation approaches relating TSCF and the logarithm octanol/water partition coefficient (log KOW) suggest that only compounds that are in the intermediate lipophilicity range (log KOW = 2) will be taken up and translocated by plants. However, recent data for highly water soluble compounds such as 1,4-dioxane, MTBE, and sulfolane suggest that these estimation techniques should be critically reviewed. To re-evaluate the relationship between TSCF and log Kow, TSCFs were measured for 25 organic chemicals ranging in log KOW from -0.8 to 5 using an improved pressure chamber technique. The technique provides an approach for efficiently generating consistent plant uptake data. By using this data, a new mass transfer model relating TSCF and log KOW was developed that indicates that neutral, polar organic compounds are most likely taken up by plant roots and translocated to shoot tissue. An extensive review of literature TSCF studies supports the updated model.
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Delaney, Sean Phillip. "Modeling and Therapeutic Development for the Tuberous Sclerosis Related Neoplasm Lymphangioleiomyomatosis." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39810.
Full textAbstract:
The multisystemic tumors characteristic of the monogenic neoplastic diseases, tuberous sclerosis complex (TSC) and lymphangioleiomyomatosis (LAM), share common signaling aberrations upon the loss of heterozygosity in either the TSC1 or TSC2 genes. However, their physical manifestations are vastly different and can generally be classified as being either neurological (TSC) or mesenchymal (TSC & LAM; referred to herein as LAM for simplicity) in origin. In this study, I present a comprehensive stem cell model of LAM utilizing multiple TSC2 knockout (TSC2-/-) pluripotent stem cell lines differentiated to the putative cell of origin for mesenchymal tumors, neural crest cells (NCCs). TSC2-/- NCCs faithfully recapitulate LAM phenotypes and temporal RNA-seq analysis of neural and neural crest differentiation was performed to model disease pathogenesis. Analysis revealed immediate activation of stress response signaling resulting in protein aggregation and lysosome and autophagosome accumulation upon neuralization in TSC2-/- cells. This resulted in acute and lasting effects specific to neural progenitor cells (NPCs), that are transient and ameliorated in NCCs. These lineage-specific effects resulted in selective sensitization of NPCs to cell death via proteasome inhibition, suggesting a potential therapeutic avenue for neurological TSC, but not LAM. Thus, a genome-wide CRISPR knockout screen was performed in TSC2-/- NCCs. Analysis of synthetic lethal genes reveals pathways previously targeted for LAM, but provides gene-level resolution to the vulnerable nodes within these pathways. Importantly, 18 novel gene targets were identified that display synthetic lethality to TSC2-/- cells with high specificity. 3 genes within this list were targetable using commercially available small molecule inhibitors, one of which, FGFR1, shows highly selective lethal targeting of TSC2-/- NCCs. Importantly, this model system, paired with the expansive resource of transcriptomic and synthetic lethal data, serves as a foundation for the development of next generation treatment strategies for LAM, and potentially the entire spectrum of TSC manifestations.
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Fields, Justin R. "Implementing the Transforming School Counseling Initiative into practice the experience of TSCI-trained professional school counselors /." Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1196284456.
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Hsieh, Ting-Chiu. "Tuberous sclerosis complex 1 (Tsc1) regulates dE2F1 protein expression during development and cooperates with Rbf1 to control proliferation and survival in «Drosophila melanogaster»." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95246.
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Retinoblastoma tumour suppressor Rb is a cell cycle regulator that is active during early G1 preventing the transition from G1 to S-phase. This is achieved by Rb inhibiting E2F transcription factors from activating expression of genes required for G1 to S-phase progression and DNA synthesis. In our initial genetic test searching for genes that interact with mutations of rbf1, the homologue of rb in Drosophila melanogaster, one of the genes identified was tsc1, which is also a tumour suppressor gene that regulates translation and cell growth. We found that in Drosophila eye imaginal disc cells, tsc1 and rbf1 mutations have a synergistic effect on increasing the level of cell death and promoting ectopic S-phase entry. In addition, I found that dE2F1 protein level increased in tsc1 mutant eye disc cells, which implies that Tsc1 is a negative regulator of dE2F1 expression. The goal of my thesis study was to characterize the synergistic relation between Rbf1 and Tsc1 as well as the regulation of dE2F1 expression by Tsc1. In cells triple-mutant for rbf1, tsc1, and de2f1, I found that the observed elevation in cell death in rbf1 and tsc1double-mutant cells was suppressed, which suggests that the cooperation between Rbf1 and Tsc1 is dE2F1-dependent. Moreover, by using a reporter construct for dE2F1 activity, PCNA-GFP, and performing in situ hybridization with anti-sense RNA probes of dE2F1 target genes, rnrS, Cyclin E, and PCNA, I showed that activities of de2f1 downstream target genes were activated by tsc1 mutations, suggesting that Tsc1 also regulates dE2F1 target gene expression. Through clonal analysis of loss-of-function mutant alleles of the canonical Tsc pathway genes, I found that Tsc1 regulates dE2F1 via the Tsc pathway, specifically tsc/rheb/Tor/s6k. Finally, my RTq-PCR result showed that the regulation of dE2F1 protein expression by Tsc1 is at post-transcriptional level. To address whether the regulation is at the level of translation, I cloned the 5' untranLe suppresseur de tumeur du Rétinoblastome, Rb, est un régulateur du cycle cellulaire qui est actif dans la phase précoce G1, prévenant le passage en phase S. Pour ce faire Rb inhibe le facteur de transcription E2F, l'empêchant d'activer l'expression de gènes requis pour le passage de la phase G1 à la phase S et pour la synthèse d'ADN. Dans nos tests génétiques initiaux, cherchant des gènes interagissant avec la mutation rbf1, l'homologue de rb chez Drosophila Melanogaster, un des gènes identifié fut tsc1, qui est également un gène suppresseur de tumeur qui régule la traduction et la croissance cellulaire. Nous avons découvert que dans les cellules des disques imaginaux des yeux, les mutations tsc1 et rbf1 ont un effet synergique sur l'augmentation du taux de mort cellulaire et promeuvent l'entrée en phase ectopique S. Il fut également découvert que le taux de la protéine dE2f1 augmente dans les cellules mutantes du disque des yeux, ce qui implique que Tsc1 est un régulateur négatif de l'expression de dE2F1. Le but de ma thèse était de caractériser la régulation de l'expression de dE2F1 par Tsc1 et la relation de synergie entre Rbf1 et Tsc1. Dans les cellules triples mutantes pour rbf1, tsc1, et de2f1, j'ai trouvé que l'augmentation du taux de mort cellulaire observé disparaissait dans les cellules doubles mutantes rbf1 et tsc1, ce qui suggère que la coopération entre Rbf1 et Tsc1 est dE2F1-dependante. Egalement, en utilisant un gène rapporteur de l'activité de de2f1, PCNA-GFP, et en réalisant des hybridations in situ avec des sondes ARN anti sens, rnrS, Cyclin E, and PCNA, j'ai montré que l'activité de la région en aval des gènes cibles de de2f1 était activé par la mutation tsc1, suggérant que Tsc1 régule également l'expression des gènes cible de dE2F1. Par l'analyse de clones possédant des allèles mutants perte de fonction pour les gènes de la cascade canonique Tsc, j'ai trouvé que Tsc1 régule dE2F1 par le biais$
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Breyer, Carlos Alexandre. "Investigação dos determinantes moleculares envolvidos na interação com os substratos de Tsa1 e Tsa2 de Saccharomyces cerevisiae." Universidade Federal de São Carlos, 2015. https://repositorio.ufscar.br/handle/ufscar/7323.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Peroxiredoxins (Prx) are antioxidant proteins capable of decomposing a variety of hydroperoxide are very abundant in the cell and in eukaryotes are distributed in many cell compartments. The Prx are able to reduce their substrates using a highly reactive cysteine residue, named peroxidatic cysteine (CP-S-), which is maintained in the thiolate form as a consequence of the active site environment. In hydroperoxide decomposition process the CP is oxidized to cysteine sulfenic acid (CP-SOH). These vast majorities of these proteins are obligate dimers and has a second cysteine involved in the catalytic cycle, named resolution cysteine (CR), which forms a disulfide with CP and is often reduced by the enzyme thioredoxin (Trx). In addition to the reduction of Prx, the Trx are involved in several other biological processes such as cell growth, inhibition of apoptosis, transcriptional activation and DNA synthesis. When cell are exposed to oxidative stress, the CP can suffer overoxidation, forming species such as cysteine sulfinic acid (CP-SO2H) and cysteine sulfonic acid (CP-SO2H), which are not reduced by Trx. However, the CP overoxidation results in a change of the of Prx quaternary structure, resulting in the formation of high molecular weight structures (HMW) with molecular chaperone property and related to signal transduction triggered by hydroperoxides. Saccharomyces cerevisiae, have two isoforms of cytosolic Prx (Tsa1 and Tsa2), which shares high similarity (86% identity and 96% similarity), but differ in cellular abundance and expression levels. However, these enzymes are often considered redundant proteins. This study aimed the comparative analysis searching for functional and structural differences between Tsa1 and Tsa2. We evaluated Tsa1 and Tsa2 relationships with its oxidizing (hydroperoxide) and reducing substrates (Trx1 and Trx2). We also performed structural and kinetic analysis showed a significant relationship between maintaining the decameric structure the activity of enzymes which occurs by a series polar interactions but not equal to the active site amino acids. Highlighting the Thr44 of Tsa1 (Ser in Tsa2) which is involved in decamer structure stabilization by a CH-π interaction type with the Tyr77 and through the Oγ atom (from Thr) polar interactions with Phe45 and Leu41. Overoxidation susceptibility assays were performed using Tsa1 and Tsa2, and our results revealed that organic hydroperoxides were able to promote the CP overoxidation more efficiently when compared to H2O2. In turn, Tsa2 was very more resistant to CP overoxidation than Tsa1. This difference was also investigated concerning the catalytic triad Thr (Tsa1) to Ser (Tsa2). We generated mutants carrying reciprocal substitutions Tsa1T44S and Tsa2S44T and our results revealed that Tsa1T44S become more resistant to CP overoxidation and Tsa2S44T become more sensitive. The formation of high molecular weight structures (HMW) of Tsa1, Tsa2 and mutants were also investigated by size exclusion chromatography (SEC) and transmission electron microscopy (TEM). The Tsa1, Tsa2 and mutants HMW formation were analyzed by size exclusion chromatograph (SEC) and transmission electron microscopy (TEM). The results showed an increase of HMW formation after Trx system reaction using high concentrations of CHP. Were observed the stacking of the ring-shaped structures besides spherical species. Our results demonstrate that Tsa1 and Tsa2 proteins differ significantly in overoxidation susceptibility and HMW complex formation, indicating that these proteins have not redundant biological roles and small changes in active site promote high functional and structural changes in these enzymes.
Peroxirredoxinas (Prx) são proteínas antioxidantes capazes de decompor uma grande variedade de hidroperóxidos, são muito abundantes na célula e em eucariotos estão distribuídas nos diversos compartimentos celulares. As Prx são capazes de reduzir seus substratos utilizando um resíduo de cisteína altamente reativa, denominada de cisteína peroxidásica (CP-S-), que se apresenta na forma de tiolato. No processo de decomposição de hidroperóxidos é oxidada a cisteína ácido sulfênico (CP-SOH). A grande maioria destas proteínas se apresenta como dímeros obrigatórios e apesar de algumas Prx apresentarem somente uma cisteína, grande parcela possui uma segunda cisteína envolvida no ciclo catalítico que recebe o nome de cisteína de resolução (CR), a qual forma um dissulfeto com CP, que frequentemente é reduzido pela enzima tiorredoxina (Trx). Adicionalmente à redução das Prx, as Trx estão envolvidas em diversos outros processos biológicos como crescimento celular, inibição de apoptose, ativação de transcrição e síntese de DNA. Quando a célula é exposta a elevado estresse oxidativo, pode ocorrer a superoxidação de CP formando espécies superoxidadas como a cisteína ácido sulfínico (CP-SO2H) e sulfônico (CPSO3H) que não podem ser reduzidas por Trx. Entretanto, também resulta na alteração da estrutura quaternária das Prx, levando à formação de estruturas de alto peso molecular (HMW) que possuem propriedade de chaperona molecular e estão relacionadas a transdução de sinal desencadeada por hidroperóxidos. Em Saccharomyces cerevisiae, há duas isoformas citosólicas de Prx (Tsa1 e Tsa2), que possuem grande semelhança (86% de identidade e 96% de similaridade), mas apresentam diferenças na concentração celular e nível de expressão. Entretanto, muitas vezes são consideradas proteínas redundantes. Este trabalho teve como objetivos uma análise comparativa aprofundada buscando um maior entendimento das diferenças funcionais e estruturais de Tsa1 e Tsa2. Foram avaliados a relação de Tsa1 e Tsa2 com seus substratos oxidantes (hidroperóxidos) e substratos redutores (Trx1 e Trx2). Análises estruturais e cinéticas demonstraram uma importante relação entre a manutenção da estrutura decamérica a atividade das enzimas a qual ocorre por uma serie interações polares similares mas não iguais entre aminoácidos do sítio ativo. Com destaque para a Thr44 de Tsa1 (Ser em Tsa2) que possui importância na estabilização da estrutura decamérica através de uma interação do tipo C-H-π com Tyr77 e através do átomo de O que possui interações polares com Phe45 e Leu41. Também foram realizadas análises de susceptibilidade a superoxidação de Tsa1 e Tsa2, e demonstrou-se que peróxidos orgânicos são capazes de promover mais eficientemente a superoxidação quando comparados a H2O2, sendo que Tsa1 apresenta uma maior sensibilidade a superoxidação. Essa diferença foi também relacionada à substituição Thr/Ser e análises de susceptibilidade a superoxidação dos mutantes Tsa1T44S e Tsa2S44T demonstraram que a substituição reciproca tornou Tsa1T44S mais resistente a superoxidação e Tsa2S44T mais sensível. A formação de estruturas de alto peso molecular (HMW) de Tsa1, Tsa2 e mutantes foram investigadas através de cromatografia de exclusão molecular (SEC) e microscopia eletrônica de transmissão (TEM). Os resultados demonstraram um aumento da formação de HMW após reações de superoxidação utilizando o sistema Trx em altas concentrações de CHP e foi verificada a presença de empilhamentos de decâmeros, além de esferas, descritas na literatura. Os resultados obtidos neste trabalho demonstram de forma clara que as proteínas Tsa1 e Tsa2 diferem de forma significativa tanto na suscetibilidade a superoxidação quanto na formação de complexos HMW distintos, indicando fortemente que estas proteínas possuem papeis biológicos não redundantes e que alterações sutis de aminoácidos no sitio ativo promovem grandes alterações funcionais e estruturais nas enzimas.
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Brown, Sarah Denyse. "An Investigation of Trauma Symptom Reduction in a Clinical Sample of Sexually Abused Children Using the Trauma Symptom Checklist for Children." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/cps_diss/10.
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School counselors have a duty to formulate strategies that aid in the detection and prevention of child sexual abuse (ASCA, 2003). This may be accomplished in a number of ways, such as designing programs, providing training to teachers regarding recognizing and reporting abuse indicators, and collaborating with child protection and other mental health professionals to provide additional aftercare for sexually abused children in the school setting. Much can be learned about trauma symptomology from a clinical sample of sexually abused children. The Trauma Symptom Checklist for Children (TSCC; Briere, 1996) is a 54-item self-report instrument for children and adolescents 8-16 years of age which assesses the frequency of thoughts, feelings, and behaviors related to traumatic events they have experienced. To understand better the trauma symptomology of children and adolescents, the author analyzed an existing data set of TSCC protocols from children who received treatment for sexual abuse from a children’s advocacy center in a metropolitan area near a large city in the southeastern United States. Although a large number of potential participants were lost to follow up (N = 54), T2 analyses revealed significant differences between the groups only on the length of time in therapy. A repeated measures analysis of variance was performed on data from children and adolescents who completed therapy (N = 31) to test whether differences on Depression and Posttraumatic Stress scale scores would exist across the course of therapy. Although no statistically significant findings emerged, implications for clinical practice and research became apparent. Specifically, differences in cutoff T-scores on TSCC scales may be more useful to clinicians for treatment and termination planning purposes than statistically significant differences. In addition, assessing clients at intervals measured by session number, rather than by length of time, may provide more generalizable results for within- and between-participants clinical and research comparisons. These implications may aid clinical and school counselors and researchers to recognize and serve the specific needs of sexually abused children in their respective settings.
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Puterbaugh, Rebekah Lee. "The Effect of Dissolved Air on the Cooling Performance of a Partially-Confined FC-72 Spray." Wright State University / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=wright1219841299.
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Hambrock, Harald. "Struktur-Funktions-Beziehungen der extrazellulären Calcium-bindenden Proteine SC1, Hevin und TSC-36, FRP." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=964169975.
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Pepin, Aurelie. "Régulation de l'apoptose des lymphocytes T par les protéines de la famille TSC-22D." Phd thesis, Université Paris Sud - Paris XI, 2011. http://tel.archives-ouvertes.fr/tel-00713526.
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Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l'apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l'apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l'activation des caspases ainsi qu'une régulation positive de l'expression de BIM. Nous avons en outre montré que l'expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l'expression de l'ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n'était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l'entrée en apoptose des lymphocytes T en régulant négativement l'expression de la protéine anti-apoptotique GILZ.
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Pépin, Aurélie. "Régulation de l'apoptose des lymphocytes T par les protéines de la famille TSC-22D." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA114814/document.
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Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l’apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l’apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l’activation des caspases ainsi qu’une régulation positive de l’expression de BIM. Nous avons en outre montré que l’expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l’expression de l’ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n’était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l’entrée en apoptose des lymphocytes T en régulant négativement l’expression de la protéine anti-apoptotique GILZGILZ (Glucocorticoid-Induced Leucine Zipper) and TSC-22 (Transforming growth factor-beta Stimulated Clone-22) belong to the TSC-22D (TSC-22 Domain) family of proteins. GILZ has been previously shown to be induced upon interleukin-2 (IL-2) deprivation in the T-cell line CTLL-2, allowing cells to delay apoptosis. The aim of our study was to elucidate the respective roles of GILZ and TSC-22 during IL-2 deprivation-induced T-lymphocytes apoptosis.Our results demonstrated that TSC-22 increased CTLL-2 cells apoptosis induced upon IL-2 deprivation. We highlighted in TSC-22 expressing cells both an increase in caspases activation and BIM expression up-regulation. We also demonstrated that GILZ expression, an anti-apoptotic protein, known to be induced after IL-2 withdrawal, was down-regulated in the presence of TSC-22. Moreover, we showed that gilz mRNA expression was also significantly repressed, but gilz mRNA half-life was not modified.Altogether, these results suggest that, in T-cells, TSC-22 could behave as a repressor of GILZ expression, accelerating IL-2 deprivation-induced apoptosis
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Karlsson, Ki. "Projektframgång : och vilka faktorer påverkar denna?" Thesis, Växjö University, School of Technology and Design, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:vxu:diva-1304.
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Definitionen av ett projekt är att det måste gå att mäta om ett projekt nått sitt mål eller inte. Ett projekt skall också ha en klar och tydligt fastställd slutpunkt, där projektgruppen upplöses och projektet avslutas.
De metoder som valts att användas för datainsamling är intervjuer med projektledarna och kunderna. Även litteraturstudier har använts för att kartlägga redan existerande kunskap inom området samt för att bygga upp en teoretisk referensram.
I denna studie beskrivs arbetsgången hos två projekt. Vid jämförande och analys av arbetsgången vid de båda projekten visade det sig att det fanns både likheter och skillnader. I resultatet redovisas de båda projektens arbetsgång, samt vad projektledarna och kunderna har för åsikter om respektive projekts genomförande och slutresultat. Slutsatsen som man kan dra av jämförandet av Projekt A och B med den teoretiska projektgången är att i många fall oberoende på storleken på ett projekt så genomförs kundorderprojekt på samma sätt.
The definition of a project is that the goal must be able to be measured after the project is finished. A project must have a clear ending point where the project group is dissolved. The methods that have been chosen for collecting material are interviews with the project managers and the customers. Litterature has also been studied for a theoretical view.
In this study two projects are analyzed. At comparison between the projects, it shows that there are both similarities and differences between the way to work in a project. In the result it is described how the two projects where carried out and also what the project managers and the customers thought of the realization and end result. The conclusion that you can draw is that in many cases the realization are not depending of size of the project. The projects are carried out the same way independent of the projects size.
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Gonzalo, Peces Carlos. "Energy-Efficient Communication with Lightweight M2M in IoT Networks." Thesis, KTH, Skolan för elektroteknik och datavetenskap (EECS), 2018. http://urn.kb.se/resolve?urn=urn:nbn:se:kth:diva-249943.
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OMA’s Lightweight Machine to Machine (LwM2M) is an application protocol for device management in the Internet of Things (IoT) that has been recently published and widely adopted in a lot of projects. The protocol is designed to operate in sensor networks and machine-to-machine environments, where one of the main constraints is the energy consumption since the nodes are usually battery powered. Different strategies to achieve high energy efficiency in IoT networks have been developed, but there is no deep knowledge about the performance of LwM2M operating with them. Moreover, the specification of this protocol includes one strategy, called the Queue Mode, which could be more efficient than the usual ones because it has been specified for this particular protocol. This project aims to implement this Queue Mode at both sides of the communication, and then evaluate its performance by comparing it with TSCH, which is the standard MAC protocol used in IEEE 802.15.4 that defines a way of radio duty cycling. It has been proven to achieve a high energy efficiency, and that is the main reason why it is selected. The comparison is performed according to several metrics to have a comprehensive evaluation, and in different kind of scenarios, with different numbers of IoT devices and different parameters in the communication. The implementation was done inside the Contiki-NG OS for the client side, which is an operating systems designed for constrained devices. For the server side it has been carried out inside the Eclipse Leshan code, which is a LwM2M implementation in Java made by the Eclipse Foundation. As a result of the evaluation, it shown that both implementations operate correctly. This thesis contributes as a guideline for making decisions about which low power strategy is better to use depending on the IoT scenario and the type of application. It shows that for many use cases Queue Mode is a better option than TSCH because it achieves a higher energy efficiency and the rest of the metrics used in the evaluation have also improved values. TSCH has a better performance only in demanding scenarios or in cases where the communication is not produced at fixed time instants. The thesis was developed in cooperation with RISE SICS AB, Networked Embedded Systems Group.OMA:s Lightweight Machine to Machine (LwM2M) är ett applikationsprotokoll för enhetshantering i Sakernas Internet (IoT) som nyligen har publicerats och börjat användas i många projekt. Protokollet är utformat för att fungera i sensornätverk och maskin-till-maskin miljöer, där en av de viktigaste begränsningarna är energiförbrukningen eftersom noderna vanligtvis är batteridrivna. Olika strategier för att uppnå hög energieffektivitet i sensornätverk har utvecklats, men det finns ingen djup kunskap om hur LwM2M fungerar med dem. Dessutom innehåller specifikationen av LwM2M en strategi kallad Queue Mode (köläge) som kan vara effektivare än de vanliga strategierna eftersom den har utvecklats direkt för det här protokollet.Detta examensarbete syftar till att implementera detta köläge på båda sidor av kommunikationen och sedan utvärdera prestandan genom att jämföra det med TSCH, vilket är ett MAC-protokoll specificerat i IEEE 802.15.4-standarden. Tidigare arbeten har visat att TSCH kan uppnå en låg energiförbrukning, vilket är den främsta anledningen till att detta protokoll väljs ut för att jämföra mot LwM2M:s köläge. Jämförelsen inkluderar flera olika typer av mätvärden och scenarier för att få en omfattande utvärdering, samt med flera olika antal sensor noder och parametrar.Implementationen gjordes för Contiki-NG OS på klientsidan, vilket är ett operativsystem för resursbegränsade IoT-enheter. På serversidan har implementationen gjorts för Eclipse Leshan, vilken är en LwM2M-implementation skriven i Java och publicerad av Eclipse Foundation. Som en följd av utvärderingen har det visat sig att båda implementationerna fungerar korrekt.Detta examensarbete bidrar med riktlinjer för att fatta beslut om vilken energibesparingsstrategi som är bättre att använda beroende på IoT-scenariot och typen av applikation. Utvärderingen visar hur Queue Mode i många användningsfall är ett bättre alternativ än TSCH eftersom det uppnår en högre energieffektivitet utan att de andra typerna av mätvärden påverkas av det. I vissa fall uppnås dessutom förbättrade resultat även i de andra typerna av mätvärden. TSCH har endast bättre prestanda i krävande scenarier eller i fall där kommunikationen inte genereras vid bestämda tillfällen.Examensarbetet har genomförts hos Networked Embedded Systems-gruppen på RISE SICS AB.
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Bell, Catherine M. "RHEB DYNAMICS ON LYSOSOMAL MEMBRANES DETERMINES MTORC1 ACTIVITY AFTER LOSS OF P53 OR ACTIVATION OF AMPK." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4036.
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The tumor suppressor TP53 is the most frequently altered gene in human cancers. The growth-promoting complex, mTORC1 plays a part of the oncogenic profile caused by dysfunctional p53. mTORC1 sits downstream of AMPK and other crucial tumor suppressors/oncogenes, PTEN, LKB1, and Akt. The antifolate pemetrexed was found by this laboratory to activate AMPK via the inhibition of the enzyme AICART in de novo purine synthesis. This work presents a mechanism of mTORC1 activation with p53 loss, as well as of mTORC1 inhibition by pemetrexed-induced AMPK. We have found that mTORC1 activity was substantially upregulated by the loss or mutation of p53. This activation involves the loss of TSC2 from lysosomal membranes, the site of mTORC1 activation by Rheb. We demonstrate that loss of lysosomal TSC2 increased the levels of lysosomal Rheb. Control of mTORC1 was restored by overexpression of TSC2, which correlated with decreased lysosomal Rheb. Surprisingly, pemetrexed-activated AMPK did not phosphorylate TSC2 because of an accumulation of nonfunctional p53, and a subsequent decrease in TSC2 mRNA. Accordingly, lysosomal TSC2 decreased, however, the levels of lysosomal Rheb decreased. Future studies will question whether the robust Raptor phosphorylation by pemetrexed is involved in this decrease in lysosomal Rheb. AMPK activation by pemetrexed also significantly increased the translocation of AMPK to the nucleus, and we will explore the function of this nuclear AMPK. Overall, these findings present a mechanism involved in the oncogenic signaling of mTORC1 with loss of p53 and offer insight into how pemetrexed reinstates control.
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Tomasoni, Romana. "TSC/mTOR signalling controles thymocyte development and profiliferation/differentiation of T lymphocytes and nerve cells." Thesis, Open University, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.542455.
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Agarwal, Stuti. "Pemetrexed, A Modulator of AMP-activated Kinase Signaling and an Inhibitor of Wild type and Mutant p53." VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/4003.
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New drug discoveries and new approaches towards diagnosis and treatment have improved cancer therapeutics remarkably. One of the most influential and effective discoveries in the field of cancer therapeutics was antimetabolites, such as the antifolates. The interest in antifolates increased as some of the antifolates showed responses in cancers, such as mesothelioma, leukemia, and breast cancers. When pemetrexed (PTX) was discovered, our laboratory had established that the primary mechanism of action of pemetrexed is to inhibit thymidylate 22 synthase (TS) (E. Taylor et al., 1992). Preclinical studies have shown that PTX has a broad range of antitumor activity in human and murine models of cancer (Adjei, 2000; Adjei, 2004; S. Chattopadhyay, Moran, & Goldman, 2007; Miller et al., 2000). Accordingly, in February 2004, the FDA issued first-line treatment approval for pemetrexed in malignant pleural mesothelioma and in 2008 for first line treatment for locally advanced or metastatic NSCLC (reviewed in (Rollins & Lindley, 2005). As an antifolate this level of therapeutic activity of PTX against lung cancers was surprising and atypical (Hazarika, White, Johnson, & Pazdur, 2004). This led us to the question whether the effects of pemetrexed on other folate-dependent targets could explain the clinical activity of the drug. Our lab showed that, in addition to inhibiting thymidylate synthase, PTX also inhibits aminoimidazolecarboxamide ribonucleotide formyltransferase (AICART), the second folate-dependent enzyme of de novo purine synthesis. Inhibition of AICART leads to massive accumulation of its substrate 5-amino-4-imidazolecarboxamide ribonucleotide (ZMP), causing activation of AMP-dependent kinase (AMPK), which ultimately leads to suppression of mTORC1 signaling, a central regulator of cell growth and proliferation. This secondary mechanism could explain the unusual activity of PTX against mesothelioma and lung cancers. The large proportion of lung cancers are either null or mutant for p53 function. Therefore, this thesis focused on defining what the role of p53 is in the PTX-mediated AMPK activation and mTORC1 inhibition and how the loss of p53 affects mTORC1 signaling. These two questions proved to be interlinked. Chapter 2 investigates this relationship in detail. We found that, upon loss of p53, mTORC1 signaling is enhanced to a significant degree in colon carcinoma and lung cancer cell lines. Clearly, this observation required explanation. We found that the major factors responsible for these differences in mTORC1 activity upon loss of p53 23 were lower levels of two p53 target genes Tuberin (TSC2) and sestrin2. Immunoprecipitation studies of mTORC1 complexes from p53 wt and p53 null cells revealed quite interesting differences in the components of the mTORC1 complex. Immunoprecipitates from p53 null cells had higher levels of mTOR and lower levels of TSC2 and PRAS40 bound to raptor. This suggested that, in comparision to p53 competent cells, p53 null cells have more mTORC1 complex with enhanced activity due to decreased interaction of TSC2 and PRAS40, both of which are inhibitors of mTORC1. These observations explained the higher mTORC1 in p53 null cells and laid the foundation for determining the role of p53 in PTX-activated AMPK and mTORC1 inhibition. In the experiments described in Chapter 3, we found that PTX-mediated AMPK activation inhibited mTORC1 regardless of the p53 status in colon carcinoma cells. This suggested that mTORC1 inhibition by PTX was either independent of p53 mediated negative regulation of mTORC1 or was somewhere bypassing it. Therefore, we compared the effects of PTX with the classic AMPK activator aminoimidazolecarboxamide ribonucleoside (AICAR). In spite of a common mechanism of AMPK activation, namely, expansion of cellular ZMP levels, signaling from AMPK activated by PTX or AICAR were quite different. PTX-activated AMPK phosphorylated the mTORC1 component Raptor but not tuberin (TSC2), whereas AICARactivated AMPK phosphorylated both the targets. This differential behavior of two AMPK activators was due to differential behavior of p53 under these two treatments. Both, AICAR and PTX treatment led to increase in p53 levels but the p53 that accumulated after AICAR treatment was transcriptionally active while the p53 that accumulated after PTX treatment was not. Transcription of p53 targets, including TSC2 and sestrin2, was activated in AICAR- but not in PTX-treated cells. In the absence of p53 function, TSC2 was deficient and mTORC1 activity 24 enhanced, but Raptor phosphorylation by AMPK following PTX was robust and independent of both p53 and TSC2. Therefore we concluded that p53 deficiency suppresses TSC2 and upregulates mTORC1, but AMPK-phosphorylation of Raptor after pemetrexed treatment was sufficient to suppress mTORC1, even in TSC2 deficiency. This suggested pemetrexed as a drug for treatment of Tuberous Sclerosis, a genetic disease caused by functional inactivity of TSC1 or TSC2 due to point mutations in these genes. Mutation of p53 is one of the most common genetic alterations in human cancers and tumors. Cancers that express mutant p53 tend to be more aggressive, resistant to chemotherapy and show worse prognosis then p53-null tumors (Elledge et al., 1993; Olivier et al., 2006). This tumor-promoting activity of mutant p53 has been correlated with acquired and novel transcriptional activities of mutant p53. It has been shown that mutp53 can activate the transcription of cell growth promoting genes, such as, NFκB2, PCNA, MDR1, Axl, EGFR, hTERT, and HSP70, which are not usually transcriptional targets of wt p53. Interestingly, we found that whereas DNA damaging drugs enhance the acquired oncogenic transcriptional activities of mutp53, PTX interferes with this transcription activation. We also found in Chapter 4 that PTX can limit or block the DNA damaging drug-mediated increment of transcriptional activation of mutp53. This suggests that blockade of transcriptional activation of mutp53 by pemetrexed may provide an additional therapeutic benefit in mutp53 bearing cancers. As discussed in Chapter Three, although pemetrexed (with TdR) increases the levels of p53 and its binding to the promoter of its target gene, p21, this p53 is transcriptionally inactive. In order to understand the mechanism of the pemetrexed-mediated transcriptional defect of wt p53, we studied the PTX-mediated signaling towards ATM and ATR and their effects on their substrates Chk2 and Chk1, respectively. These studies suggested that the difference between 25 signaling under AICAR treatment and PTX treatment was that, unlike PTX, AICAR treatment was leading to DNA damage, followed by Chk2 phosphorylation at Thr68. We found there were three major differences between AICAR and pemetrexed (+ TdR) mediated signaling: AICAR caused DNA damage, followed by ATM mediated phosphorylation of Chk2 at Thr68 and phosphorylation of p53 at Ser15 all of which lead to activation of p53 transcriptional activity, events which do not take place under PTX treatment. Studies aimed at understanding the effects of PTX on wt and mutp53 transcriptional activities are discussed in detail in Chapters Three and Four of this dissertation. Overall, we concluded that PTX interferes with the transcription activity of wild type as well as gain-of-function mutant p53. The blockade of DNA damaging agent-mediated enhancement of mutp53 transcription activity by PTX, suggests the clinical relevance of PTX in carcinomas with mutp53. We suggest that this could be one of the contributing factors in the effects of PTX against human lung cancers.
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Orita, Naho. "Root uptake of organic contaminants into plants: Species differences." DigitalCommons@USU, 2012. https://digitalcommons.usu.edu/etd/1287.
Full textAbstract:
Trace amounts of xenobiotic organic contaminants have been frequently identified in the environment, including surface water and wastewater streams, and some are even in drinking water. The concern of unintended ingestion by humans or wildlife of such compounds resulting from the uptake by plants has risen in recent years. Although the uptake of a variety of xenobiotic organic contaminants by plants has been reported and the contaminants are found in the fruits in some cases, the differences between plant species are not fully understood. The emphasis of this research is to investigate the unique uptake ability of zucchini that has been reported repeatedly in recent years. Xylem saps, collected using a pressure chamber technique, were used to determine the values of Transpiration Stream Concentration Factor (TSCF), the ratio of the contaminant concentration in the xylem to that in the solution. Soybean "hoyt," squash "zephyr," and zucchini "gold rush" were used to compare the uptake ability of each plant. The root tissue was analyzed for total carbon and lipid content. Xylem sap was analyzed for total organic carbon and protein contents. The solubilities of the compounds in the xylem sap and deionized water were also determined using a modified shake flask method. From the measurement of TSCF, the uptake of hydrophobic contaminants in zucchini "gold rush" was found to be three-to tenfold of the other two plant species. The lipid content of the root tissue from zucchini "gold rush" was twice as much of that in soybean and squash "zephyr," indicating enhanced adsorption of the hydrophobic compounds. The solubility of triclocarban in the xylem sap of zucchini "gold rush" was also twice the amount of that in soybean xylem sap. The enhanced solubility could be a result of high protein content measured in zucchini "gold rush" xylem sap, which may be increasing the facilitated transport of the hydrophobic compounds. The data generated in this study will be used to better understand the mechanistic differences associated with the plant uptake of organic contaminants by different species. This information can also be used in the selection of the plant species used in risk assessment studies and phytoremediation studies.
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Lafeillade, Bruno. "CISP/thrombospondine-2, une protéine synthétisée par les cellules corticosurrénaliennes en réponse à l'ACTH : étude comparative de la régulation de l'expression de CISP/TSP2 avec TSP1 et distribution tissulaire de TSP2." Université Joseph Fourier (Grenoble ; 1971-2015), 1997. http://www.theses.fr/1997GRE10084.
Full textAbstract:
L'acth induit la synthese par les cellules corticosurrenaliennes bovines (bac) d'une proteine trimerique de 600 kda, appelee cisp pour corticotropin induced secreted protein. Des anticorps polyclonalux anti-cisp ont ete produits chez le lapin et un fragment d'adnc de 386 pb a ete amplifie par pcr et sequence. Ce fragment code 128 acides amines n-terminaux et presente 87 % d'identite avec la sequence de thrombospondine-2 (tsp2) humaine. Cet amplicon utilise comme sonde nucleique en northern blot detecte un arn messager de 6. 0 kb, dont la synthese est induite par l'acth. Une sonde nucleique specifique de tsp1 bovine a egalement ete developpe. L'etude de la regulation de l'expression de cisp/tsp2 et de tsp1 par les cellules bac a montre un effet oppose de l'acth avec (1) une augmentation de l'expression de cisp et une diminution de celle de tsp1, (2) une induction precoce par le serum de la synthese de tsp1 (3) une induction de l'expression de tsp1 et de tsp2 par tgf-b1. Dans les cellules bac traitees par l'acth, cisp est localisee au niveau de l'appareil de golgi comme le montrent les experiences d'immunofluorescence. L'emploi de l'anticorps anti-cisp a permis de montrer que cisp est necessaire au maintien de l'arrondissement cellulaire induit par l'acth. Dans la lignee de cellules d'osteosarcome ros, une inhibition de l'expression de tsp1 par l'ampc et la parathormone a ete observee. En revanche, l'induction de cisp par l'ampc n'est pas retrouvee dans ce modele, suggerant une regulation specifique aux cellules bac. In vivo, cisp peut etre extraite de differents tissus bovins, dont la surrenale. Plus precisement, en immunofluorescence, cisp est localisee au niveau du cortex surrenalien, dans la zone glomerulee et la zone fasciculo-reticulee. La presence de cisp a egalement ete retrouvee dans les tumeurs de la surrenale, sans difference d'expression detectee selon la malignite de la tumeur. L'existence d'une regulation differentielle de l'expression de tsp1 et de cisp/tsp2 est en faveur de roles distincts de ces deux thrombospondines et suggere que les cellules corticosurrenaliennes ainsi que les cellules ros representent de bons modeles d'etude des fonctions de tsp1 et de tsp2.