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1
Miller, Paula. "Oxygen sensing by hTREK1, a twin-pore-domain potassium channel". Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.403031.
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FANTONE, SONIA. "Role of HtrA1 in pregnancy: a possible early marker of Preeclampsia". Doctoral thesis, Università Politecnica delle Marche, 2021. http://hdl.handle.net/11566/289654.
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L’ High temperature requirement A 1 (HtrA1), un membro della famiglia HtrA, è una proteina di secrezione multidominio con attività serin-proteasica. Alcuni studi suggeriscono che questa proteina sia coinvolta nello sviluppo fisiologico di alcuni organi, compresa la placenta. Inoltre, è stata dimostrata un'alterazione dell'espressione di HtrA1 in vari carcinomi e in alcune malattie placentari come la preeclampsia (PE).
La PE è una sindrome gestazionale che colpisce il 3-5% della gravidanza nel mondo, caratterizzata dalla nuova insorgenza di ipertensione e proteinuria materna. Sebbene i sintomi della PE compaiano dopo la 20° settimana di gestazione, la patologia inizia a svilupparsi prima di questo periodo. Una corretta identificazione prima delle 12 settimane di gestazione delle donne che svilupperanno la PE durante la gravidanza, potrebbe consentire di trattare le gestanti ad alto rischio di PE prima della comparsa dei sintomi al fine di prevenire danni alla placenta e di conseguenza al feto.
È interessante notare che l'espressione dell’HtrA1 era alterata sia nel tessuto placentare che nel plasma materno delle gravidanze complicate da PE. Pertanto, l’HtrA1 potrebbe essere una molecola chiave nello sviluppo di questa malattia, nonché un marker utile per la sua diagnosi e cura.
Lo scopo di questo studio è valutare se l’HtrA1 possa essere considerato un utile marker precoce dell'insorgenza di PE e come l'espressione dell’HtrA1 possa essere modificata attraverso la somministrazione di sostanze di uso comune e innovative per il trattamento di PE. L'identificazione di un marker predittivo di PE e di possibili nuovi approcci terapeutici per la gestione della PE è attualmente una delle questioni ginecologiche più rilevanti, al fine di ridurre il rischio di mortalità e morbilità materna e fetale.The High temperature requirement A 1 (HtrA1), a member of the HtrA family, is a multidomain secretory protein with serine-protease activity. Some studies suggest that this protein is involved in physiological development of some organs including the placenta. Furthermore, it has been proved an alteration in HtrA1 expression in various carcinomas and in some placental disease such as preeclampsia (PE). PE is a gestational syndrome that affect the 3-5% of the pregnancy worldwide, characterized by new onset of maternal hypertension and proteinuria. Although PE symptoms appear after 20th week of gestation, the pathology begins to develop before this period. A correct identification of pregnant woman that will develop PE, before 12 weeks of gestation, makes it possible to manage women at high risk of PE before the appearance of symptoms in order to prevent damage to the placenta and consequently to the fetus. It is interesting to note that HtrA1 expression was altered in both placental tissue and maternal plasma of pregnancies complicated by PE. Therefore, HtrA1 could be considered a key molecule in the development of this disease. The aim of this study is to evaluate: i) whether the HtrA1 protein could be considered a useful early marker of PE onset and ii) how common and innovative compounds for treatment of PE modify the HtrA1 expression. In conclusion, the identification of a predictive markers of PE and possible new therapeutic approaches for the management of PE is currently one of the relevant gynecological issues in order to reduce the risk of maternal and fetal mortality and morbidity.
3
Scharrer, Eva. "Consequences of HtrA1 deficiency on TGF-Beta signaling". Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-185742.
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Verdura, Edgard. "Familial Cerebral Small Vessel Diseases of unknown etiology : a high throughput approach towards a better understanding of pathophysiological mechanisms". Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC264.
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Les maladies des petites artères cérébrales sont un groupe hétérogène de maladies qui affectent les petites artères, artérioles, veines et/ou capillaires du cerveau. La plupart des patients sont des cas sporadiques, mais plusieurs formes héréditaires ont été identifiées.Toutefois, 15 % seulement des patients atteints d’une cSVD familiale sont porteurs d’une mutation dans l’un de ces gènes, suggérant l’implication d’autres gènes. Dans cette thèse, nous avons montré que des mutations hétérozygotes du gène HTRA1 étaient responsables d’environ 5 % des cSVD familiales. L’analyse fonctionnelle de ces mutations a montré un effet perte de fonction. L’âge de début chez les sujets hétérozygotes était beaucoup plus tardif que chez les patients CARASIL, où les deux allèles d’HTRA1 sont mutés. Ensuite, nous avons identifié 2 familles (incluant la famille rapportée sous l’acronyme PADMAL / Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy) portant deux mutations distinctes dans un site d’accrochage du microRNA miR-29, dans la partie 3’UTR du gène COL4A1.Quatre autres patients index porteurs du même type de mutations ont été identifiés dans notre cohorte de cas cSVD. L’analyse fonctionnelle de ces mutations a mis en évidence une up-régulation de l’expression du gène COL4A1. Le phénotype observé était très stéréotypé, caractérisé par la survenue d’infarctus pontiques dans la 3ème décade. L’identification des bases moléculaires de ces deux nouvelles formes de cSVD héréditaire a des applications diagnostiques immédiates. Elle renforce par ailleurs l’hypothèse du rôle essentiel d’une altération du matrisome dans les mécanismes physiopathologiques des cSVDCerebral small vessel diseases (cSVD) are a heterogeneous group of disorders affecting small arteries, arterioles, veins, and/or capillaries of the brain. In most cases cSVD are sporadic, but several hereditary monogenic forms have been identified. Nevertheless, only 15% of familial cSVD patients sent for genetic screening are carriers of mutations in one of these genes, suggesting the implication of other genes. In this thesis work, we showed that heterozygous mutations in HTRA1 are found in 5% of familial cSVD cases. Functional analysis of these mutations showed that most of them behave as loss-of-function mutations. Disease onset was much later (>25 years) than in CARASIL patients, in which both2 HTRA1 alleles are mutated. Afterwards, we identified 2 informative families (including the original family reported to be affected by PADMAL / Pontine Autosomal Dominant Microangiopathy and Leukoencephalopathy) harboring two different mutations in the binding site of miR-29 microRNA within the 3’UTR of COL4A1 gene. Four other index patients carrying the same type of mutations were identified in our patient cohort. Functional analysis of these mutations showed an up-regulation of COL4A1 gene expression. The observed phenotype was highly stereotyped in all patients, characterized by pontine infarcts appearing in the 3rd decade. Identification of the molecular defects underlying these two novel hereditary cSVD forms provides tools to improve the molecular diagnosis of cSVD. Besides, it reinforces the hypothesis of an essential role of matrisome alteration in cSVD pathophysiological mechanisms
5
Yamawaki, Satoko. "HtrA1 Is Specifically Up-Regulated in Active Keloid Lesions and Stimulates Keloid Development". Kyoto University, 2019. http://hdl.handle.net/2433/245295.
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Stuqui, Bruna [UNESP]. "Caracterização funcional de HTRA1 em linhagens celulares HPV positiva e HPV negativa". Universidade Estadual Paulista (UNESP), 2014. http://hdl.handle.net/11449/111013.
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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
O Papilomavirus humano (HPV) é um dos vírus mais prevalentes entre as infecções sexualmente transmissíveis e está associado com doenças malignas. Os HPVs de alto risco possuem proteínas, denominadas de E6 e E7, caracterizadas como oncoproteínas devido aos seus papéis na transformação celular e na inativação de supressores de tumor. Um dos mecanismos usados na transformação celular pela proteína E6 do HPV de alto risco é a interação do seu domínio carboxi-terminal, PDZ, com domínios PDZs presentes em algumas proteínas celulares, destinando-as à degradação. Uma proteína que está associada com várias condições patológicas e tem domínio PDZ é a protease HtrA1. Esta proteína é pouco expressa em alguns cânceres, sugerindo um papel supressor de tumor. O objetivo deste estudo foi avaliar o efeito da superexpressão de HTRA1 em linhagem celular HPV16 positiva (HF698) e HPV negativa (C33). As linhagens celulares foram transfectadas com vetor contendo a ORF de HTRA1 ou vetor vazio. A superexpressão do mRNA e proteína foi confirmada por qPCR e imuno-histoquímica, respectivamente. As linhagens celulares transfectadas foram submetidas a ensaio de formação de colônia, de viabilidade celular, de apoptose e ciclo celular. As células C33 superexpressando HtrA1 formaram significantemente menos colônias e apresentaram redução de viabilidade celular comparadas as células sem expressão de HtrA1. Diferentemente, na linhagem HPV positiva ocorreu aumento no número de colônias nas células superexpressando HtrA1 e não houve diferença no ensaio de viabilidade celular. Esses resultados sugerem que os diferentes padrões observados nas duas linhagens celulares são decorrentes da presença do HPV na HF698 e da ausência na C33. A fim de confirmar se o aumento do número de colônias nas células HF698 superexpressando HtrA1 é decorrente da interação dessa proteína com E6, foi produzida linhagem estável de C33 com ...
The Human Papillomavirus (HPV) is one of the most prevalent virus among sexually transmitted infections and it is associated with some malignancies. High risk HPVs contain proteins, E6 and E7, characterized by oncoproteins due to their roles in cellular transformation and suppressor tumor inactivation. One of the mechanisms used in cell transformation by E6 protein from high-risk HPVs is the interaction of its carboxy-terminal domain, known as PDZ, with PDZs domains present in some cellular proteins, triggering them to degradation. A protein that is associated with various pathological conditions and has PDZ domain is the protease HtrA1. This protein is poorly expressed in some cancers, suggesting its tumor suppressor role. The aim of this study was to evaluate the effect of the HtrA1 overexpression in HPV 16 positive (HF698) and HPV negative (C33) cell lines. The cell lines were transfected with vector containing the HTRA1 ORF or empty vector. The mRNA and protein overexpression were confirmed by qPCR and immunohistochemical, respectively. The cell lines transfected were subjected to cell proliferation, viability, apoptosis and cell cycle assays. C33 cells expressing HtrA1 presented significantly fewer colonies and showed reduced viability than cells without HtrA1 expression. On the other hand, in HPV-positive cell line there was an increase in the number of colonies in cells expressing HtrA1 and there was no difference in the cell viability assay. These results suggest that the different patterns observed between the two cell lines studied may be due to the HPV presence in HF698 and its absence in C33 cells. To confirm if the increase in the number of colonies in HPV positive cells (HF698) overexpressing HtrA1 arises from the interaction of this protein with E6, stable lines of C33 containing gene E6 were produced and subsequently performed cell proliferation assay. C33 cells overexpressing E6 and HTRA1 showed an increased number of ...
7
Stuqui, Bruna. "Caracterização funcional de HTRA1 em linhagens celulares HPV positiva e HPV negativa /". São José do Rio Preto, 2014. http://hdl.handle.net/11449/111013.
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Orientador: Marilia de Freitas CalmonCoorientador: Paula Rahal
Banca: Carolina Colombelli Pacca
Banca: Sebastião Roberto Taboga
Resumo: O Papilomavirus humano (HPV) é um dos vírus mais prevalentes entre as infecções sexualmente transmissíveis e está associado com doenças malignas. Os HPVs de alto risco possuem proteínas, denominadas de E6 e E7, caracterizadas como oncoproteínas devido aos seus papéis na transformação celular e na inativação de supressores de tumor. Um dos mecanismos usados na transformação celular pela proteína E6 do HPV de alto risco é a interação do seu domínio carboxi-terminal, PDZ, com domínios PDZs presentes em algumas proteínas celulares, destinando-as à degradação. Uma proteína que está associada com várias condições patológicas e tem domínio PDZ é a protease HtrA1. Esta proteína é pouco expressa em alguns cânceres, sugerindo um papel supressor de tumor. O objetivo deste estudo foi avaliar o efeito da superexpressão de HTRA1 em linhagem celular HPV16 positiva (HF698) e HPV negativa (C33). As linhagens celulares foram transfectadas com vetor contendo a ORF de HTRA1 ou vetor vazio. A superexpressão do mRNA e proteína foi confirmada por qPCR e imuno-histoquímica, respectivamente. As linhagens celulares transfectadas foram submetidas a ensaio de formação de colônia, de viabilidade celular, de apoptose e ciclo celular. As células C33 superexpressando HtrA1 formaram significantemente menos colônias e apresentaram redução de viabilidade celular comparadas as células sem expressão de HtrA1. Diferentemente, na linhagem HPV positiva ocorreu aumento no número de colônias nas células superexpressando HtrA1 e não houve diferença no ensaio de viabilidade celular. Esses resultados sugerem que os diferentes padrões observados nas duas linhagens celulares são decorrentes da presença do HPV na HF698 e da ausência na C33. A fim de confirmar se o aumento do número de colônias nas células HF698 superexpressando HtrA1 é decorrente da interação dessa proteína com E6, foi produzida linhagem estável de C33 com ...
Abstract: The Human Papillomavirus (HPV) is one of the most prevalent virus among sexually transmitted infections and it is associated with some malignancies. High risk HPVs contain proteins, E6 and E7, characterized by oncoproteins due to their roles in cellular transformation and suppressor tumor inactivation. One of the mechanisms used in cell transformation by E6 protein from high-risk HPVs is the interaction of its carboxy-terminal domain, known as PDZ, with PDZs domains present in some cellular proteins, triggering them to degradation. A protein that is associated with various pathological conditions and has PDZ domain is the protease HtrA1. This protein is poorly expressed in some cancers, suggesting its tumor suppressor role. The aim of this study was to evaluate the effect of the HtrA1 overexpression in HPV 16 positive (HF698) and HPV negative (C33) cell lines. The cell lines were transfected with vector containing the HTRA1 ORF or empty vector. The mRNA and protein overexpression were confirmed by qPCR and immunohistochemical, respectively. The cell lines transfected were subjected to cell proliferation, viability, apoptosis and cell cycle assays. C33 cells expressing HtrA1 presented significantly fewer colonies and showed reduced viability than cells without HtrA1 expression. On the other hand, in HPV-positive cell line there was an increase in the number of colonies in cells expressing HtrA1 and there was no difference in the cell viability assay. These results suggest that the different patterns observed between the two cell lines studied may be due to the HPV presence in HF698 and its absence in C33 cells. To confirm if the increase in the number of colonies in HPV positive cells (HF698) overexpressing HtrA1 arises from the interaction of this protein with E6, stable lines of C33 containing gene E6 were produced and subsequently performed cell proliferation assay. C33 cells overexpressing E6 and HTRA1 showed an increased number of ...
Mestre
8
Tahmaseb, Kambiz. "Biochemical Characterization of hTRF1 and hTEP1, Two Proteins Involved in Telomere Maintenance". Wright State University / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=wright1182306717.
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Stanton, Chloe May. "Investigating the genetic and molecular basis of age-related macular degeneration". Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9608.
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Age-related macular degeneration (AMD) is the leading cause of blindness worldwide, affecting an estimated 50 million individuals aged over 65 years. Environmental and genetic risk-factors contribute to the development of AMD. An AMD-risk locus on chromosome 10q26 spans two genes, ARMS2 and HTRA1, and controversy exists as to which variants are responsible for increased risk of disease. Recent work suggests that HTRA1 expression levels are significantly increased in carriers of the risk haplotype associated with AMD. However, relatively little is known about the interactions, substrate specificity and roles in disease played by this secreted serine protease. This thesis aims to elucidate the potential role played by HTRA1 in AMD pathogenesis. A combination of tandem affinity purification (TAP) and yeast two-hybrid techniques was used to identify interacting partners of HTRA1. A number of proteins, with diverse roles in the alternative complement pathway, cell signaling, cell-matrix interactions, inflammation, angiogenesis and fibrosis, were identified. These are attractive candidates for further study as such processes are disturbed in AMD, implicating HTRA1 and its binding partners in disease development. One interacting partner, Complement Factor D (CFD), is a key activator in the alternative complement pathway. CFD, a 24 kDa serine protease, is expressed as an inactive zymogen, from which a signal peptide and activation peptide are cleaved before release of the mature, active protein into the circulation. In vitro studies show that CFD interacts with, and can be a substrate for, HTRA1. The interacting domain between the two proteins is localised to a region of 30 amino acids at the N-terminal end of proCFD. The 5 amino acid pro-peptide of CFD appears to be both necessary and sufficient for proteolysis of CFD by HTRA1. Investigation of the functional relevance of the interaction between HTRA1 and CFD shows that proCFD is cleaved by HTRA1, whilst mature CFD is not subjected to proteolysis. HTRA1-mediated cleavage of CFD forms an active protease, leading to activation of factor B in the alternative complement pathway in in vitro assays. Furthermore, a normal complement response is restored to CFD-depleted serum by addition of proCFD activated by HTRA1. Thus, an HTRA1- mediated increase in alternative complement pathway activity may explain a proportion of the AMD-risk attributed to the chr10q26 locus. Genetic and protein-based approaches were used to study the potential role of CFD in AMD pathogenesis, independent of an interaction with HTRA1. An intronic SNP, rs3826945, was significantly associated with increased risk of AMD in two British case-control cohorts, and in a combined meta-analysis with 4 additional cohorts from North America and Europe (p-value = 0.032, Odds Ratio = 1.112 in 4765 cases and 2693 controls). Assessment of copy number variation and sequencing of CFD did not identify any functional variants which may explain the association with disease. However, plasma levels of CFD were measured by ELISA in 751 AMD cases and 474 controls, and were found to be significantly elevated in AMD cases compared to controls (p-value = 0.00025). This further implicates complement activation in AMD pathogenesis, and makes CFD an attractive candidate for therapeutic intervention. An alteration in the level of activated CFD, possibly mediated via an interaction with HTRA1, either at the systemic or local tissue level, may play a role in disease development and progression.
10
Scharrer, Eva [Verfasser], i Christian [Akademischer Betreuer] Wahl-Schott. "Consequences of HtrA1 deficiency on TGF-Beta signaling / Eva Scharrer. Betreuer: Christian Wahl-Schott". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1075457033/34.
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Vierkotten, Sarah [Verfasser], Mats [Akademischer Betreuer] Paulsson i Sigrun [Akademischer Betreuer] Korsching. "HTRA1: Ein Kandidatengen für die Altersbedingte Makuladegeneration? / Sarah Vierkotten. Gutachter: Mats Paulsson ; Sigrun Korsching". Köln : Universitäts- und Stadtbibliothek Köln, 2011. http://d-nb.info/1038168481/34.
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Ripkens, Kamilla [Verfasser], i Michael [Akademischer Betreuer] Ehrmann. "Untersuchungen zur Regulation und Bedeutung der Serinprotease HTRA1 in Tumorzellen / Kamilla Ripkens. Betreuer: Michael Ehrmann". Duisburg, 2016. http://d-nb.info/1081899603/34.
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Akagi, Yumiko. "MMP20 and ARMS2/HTRA1 are Associated with Neovascular Lesion Size in Age-Related Macular Degeneration". Kyoto University, 2016. http://hdl.handle.net/2433/204581.
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Beguier, Fanny. "La sérine protéase HTRA1 et l'inflammation sous-rétinienne dans le contexte de la dégénérescence maculaire liée à l'âge". Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS008/document.
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Localisé entre l'Epithélium Pigmentaire Rétinien (EPR) et les segments externes des photorécepteurs, l'espace sous-rétinien est une zone immunosuppressive ; régulée par des signaux comme la thrombospondine-1 (TSP-1) ou Fas Ligand (FasL), qui empêchent l'accumulation des phagocytes mononucléés (PMs), en particulier des monocytes inflammatoires. La Dégénérescence Maculaire Liée à l'Age (DMLA) est associée à une rupture de l'immunosuppression de cet espace, et s'accompagne d'une accumulation de PMs ; causant la mort des photorécepteurs, la dédifférenciation de l'EPR et une néovascularisation pathologique. Des études d'associations génétiques ont établi un lien entre la DMLA et un haplotype qui affecte le locus 10q26, qui contient trois gènes : PLEKHA1, ARMS2 et HTRA1. L'haplotype est associé à une augmentation de la transcription de HTRA1 dans les lymphocytes ou les cellules de l'EPR. HTRA1 code pour une sérine protéase qui a une multitude de substrats ; mais le mécanisme par lequel elle pourrait être impliquée dans la pathogenèse de la DMLA reste inconnu. TSP-1 est une glycoprotéine exprimée par l'EPR, les macrophages résidents et inflammatoires. Le domaine C-terminal de TSP-1 contient deux séquences VVM qui peuvent chacune interagir avec un récepteur CD47. Dans cette étude, nous montrons que HTRA1 clive TSP-1 et inhibe l'élimination des PMs régulée par l'interaction entre TSP-1 et CD47 à l'état physiologique, in vitro et in vivo. L'activation pharmacologique de CD47 nous a permis d'annuler les effets pro-inflammatoires de HTRA1 et pourrait représenter un espoir thérapeutique pour le contrôle de la progression de la DMLA chez les patients porteurs de l'haplotype à risqueLocalized between the Retinal Pigment Epithelium (RPE) and the photoreceptors outer segments, the subretinal space is an immunosuppressive zone, mediated by signals such as Thrombospondin-1 (TSP-1), Fas Ligand (FasL) that prevent the accumulation of Mononuclear Phagocytes (MPs) and in particular pathogenic inflammatory monocytes. Age related Macular Degeneration (AMD) is associated with a breakdown of this immunosuppressivity and an accumulation of MPs, which causes photoreceptor degeneration, RPE dedifferentiation and pathological neovascularization. Genome association studies showed a strong link between AMD and a relatively common haplotype of 10q26 locus that contains the PLEKHA1, ARMS2 and HTRA1 genes. The disease haplotype is associated with increased HTRA1 transcription in cell types such as lymphocytes and RPE cells. HTRA1 is a serine protease with a number of substrates, but the mechanism by which it might be involved in AMD pathogenesis is unknown. TSP-1 is a glycoprotein expressed by RPE, resident macrophages and inflammatory macrophages. The C-terminal domain of TSP-1 contains two VVM sequences that can each interact with a CD47 receptor. We show that HTRA1 induced subretinal MP accumulation is dependent on TSP-1 deactivation in an RPE/Mo co-culture model and in a laser induced inflammation model in vivo. This pathogenic effect of HTRA1 was reversible by synthetic CD47 agonists. Our study reveals a comprehensive mechanism how the risk-allele 10q26 participates in the pathogenesis of AMD and opens new therapeutic avenues to restore subretinal immunosuppressivity and inhibit the inflammation-dependent neurodegeneration
15
Breiden, Maike [Verfasser], Michael [Akademischer Betreuer] Ehrmann i Markus [Akademischer Betreuer] Kaiser. "Charakterisierung der Interaktion von HTRA1 und Calpain 2 / Maike Breiden. Gutachter: Markus Kaiser. Betreuer: Michael Ehrmann". Duisburg, 2014. http://d-nb.info/1058323385/34.
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Paolinelli, Francesca. "La serin proteasi HtrA1: studio del suo potenziale ruolo di "biomarker" tissutale, urinario e plasmatico del cancro uroteliale vescicale umano e del suo possibile coinvolgimento nello sviluppo della malattia neoplastica". Doctoral thesis, Università Politecnica delle Marche, 2013. http://hdl.handle.net/11566/242683.
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Il carcinoma della vescica è uno dei tumori di più frequente riscontro da parte dell’urologo,
rappresentando la seconda principale causa di decesso fra tutte le neoplasie del tratto genitourinario.
Più del 90% delle neoplasie maligne della vescica è rappresentato dai carcinomi delle
cellule uroteliali.
La scarsità di procedure non invasive attendibili per la diagnosi precoce, cui va aggiunta la
complessa eterogeneità biologica che questo tumore riscontra nella pratica clinica, sono alla base
dei decennali tentativi della comunità scientifica nell’individuazione di biomarcatori tumorali,
configurabili come indicatori precoci dell’esistenza del processo neoplastico, da utilizzare anche
per la sua sorveglianza a lungo termine. Alla luce di tali considerazioni, la necessità di
identificare nel tumore vescicale alcuni indicatori biochimici e genetici altamente specifici,
sensibili e valutabili, oltre che su frammenti di tessuto, anche su liquidi biologici (urine e
plasma), è tutt’oggi al centro di numerosi studi scientifici.
Lo scopo di questo lavoro è stato quello di analizzare l’espressione della serina proteasi HtrA1,
che è nota fungere da soppressore tumorale in diversi tumori solidi, nel tessuto vescicale
uroteliale umano in condizioni fisiologiche e tumorali, al fine di valutarne una eventuale
alterazione dei livelli in presenza di neoplasia. Inoltre, abbiamo voluto estendere lo studio
all’analisi dei fluidi biologici e alla valutazione di un possibile coinvolgimento dell’HtrA1 nella
progressione della malattia. Infatti, studi più o meno recenti hanno dimostrato come l’HtrA1 sia
una molecola in grado di esercitare una azione di controllo sulla crescita e proliferazione
cellulare e di indurre la morte cellulare stimolando l’apoptosi.
Sono stati reclutati per lo studio pazienti affetti da neoplasia vescicale uroteliale a diverso grado
e stadio, soggetti sani e con cistite. Di ciascun individuo, sono stati raccolti campioni bioptici
tissutali assieme ad urine e plasma.
Gli studi di immunoistochimica da noi condotti hanno dimostrato che l’HtrA1 è una molecola
espressa dall’urotelio della vescica in condizioni fisiologiche ed in patologie infiammatorie come
la cistite batterica. Al contrario, la proteina è risultata assente in tutti i casi esaminati di
carcinoma uroteliale con diverso grado di malignità e a differenti stadi di infiltrazione, fin dalle
fasi più precoci di comparsa visibile della neoplasia.
Una diversa espressione dell’HtrA1 tra i tessuti patologici ed i tessuti sani, nonostante simili
livelli del trascritto, è stata evidenziata dall’analisi western-blotting, dalla quale è emersa la
presenza di due forme dell’HtrA1, una nativa di peso molecolare di ~50 kDa ed una, che si
origina per autoproteolisi della prima, di ~38 kDa. Solo la forma a più basso peso molecolare ha
mostrato una diminuzione significativa in tutti i tessuti patologici analizzati rispetto ai sani,
dimostrandosi idonea ad essere considerata un buon biomarcatore tumorale.
Poiché questa proteina fu originariamente descritta come una proteasi di secrezione, abbiamo
ipotizzato che essa potesse essere secreta da parte dell’urotelio nella cavità vescicale o dal
tessuto nel sangue. Abbiamo quindi esaminato la presenza dell’HtrA1 anche nelle urine e nel
plasma di tutti i soggetti reclutati, dimostrando un incremento significativo della proteina
nell’urina e nel plasma dei pazienti con carcinoma rispetto ai soggetti sani.
Il presente lavoro ha quindi evidenziato l’HtrA1 quale possibile marker tissutale e
urinario/plasmatico utile nella diagnosi del carcinoma uroteliale della vescica.
Inoltre, dati di biologia molecolare supportati dai risultati ottenuti in vivo ci hanno suggerito che,
anche nella vescica umana, l’HtrA1 può assumere il ruolo di soppressore tumorale e che
probabilmente sia l’urotelio normale adiacente a quello tumorale a determinare un aumento
dell’HtrA1 nelle urine dei soggetti con carcinoma piuttosto che l’urotelio interessato dalla
neoplasia, forse come risposta di protezione alla progressione della malattia.Bladder cancer is one of the cancers most commonly encountered by the urologist, making it the second leading cause of death among all cancers of the genito-urinary tract. More than 90% of malignant neoplasms of the bladder is represented by the carcinomas of the urothelial cells. The lack of reliable non-invasive procedures for early diagnosis, together with the complex biological heterogeneity that this tumor has in clinical practice, are the basis of decades of efforts of the scientific community in identifying cancer biomarkers, configurable as early indicators of the existence of the neoplastic process, to be used also for its long-term surveillance. In the light of these considerations, the need to identify some highly specific and sensitive biochemical and genetic markers in bladder cancer, to be valued, as well as in tissue fragments, even in biological fluids (urine and plasma), is still the focus of numerous scientific studies. The purpose of this work was to analyze the expression of serine protease HtrA1, which is known to act as a tumor suppressor in various solid tumors, in human urothelial bladder tissue under physiological and neoplastic conditions, in order to assess a possible alteration of its levels in presence of cancer. In addition, we wanted to extend the study to the analysis of biological fluids and evaluation of possible involvement of HtrA1 in the progression of the disease. In fact, more or less recent studies, showed how the HtrA1 is a molecule capable of exerting a control action on cell growth and proliferation and to induce cell death by stimulating apoptosis. We recruited for the study patients with urothelial bladder cancer at different grade and stage, healthy subjects and with cystitis. Of each individual, tissue biopsy samples were collected along with urine and plasma. The immunohistochemical studies carried out showed that HtrA1 is a molecule expressed in bladder urothelium under physiological conditions and in inflammatory diseases, such as bacterial cystitis. On the contrary, the protein was absent in urothelial carcinoma with different degree of malignancy and at different stages of infiltration, right from the earliest stages of visible appearance of the neoplasm. A different expression of HtrA1 between the pathological and normal tissues, despite similar levels of the transcript, was detected by Western blotting, which revealed the presence of two forms of HtrA1, a native form with the molecular weight of ~ 50 kDa and another, which originates by autoproteolysis from the native one, of ~ 38 kDa. Only the HtrA1 form with lower molecular weight showed a significant decrease in all analyzed pathological tissues compared to the healthy counterparts, proving to be suitable to be considered a good cancer biomarker. Since this protein was originally described as a secreted protease, we hypothesized that it might be secreted by the urothelium in the bladder cavity or by tissue into blood. Thus, we examined the presence of HtrA1 also in the urine and plasma of all patients enrolled, demonstrating a significant increase of the protein in the urine and plasma of cancer patients compared to healthy subjects. The present work has therefore shown that HtrA1 may be considered a possible tissue and urinary/plasma biomarker, useful in the diagnosis of urothelial carcinoma of the bladder. In addition, data of molecular biology supported by the results obtained in vivo have suggested that, even in the human bladder, the HtrA1 can assume the role of tumor suppressor and that, probably, the normal urothelium adjacent to the tumor is responsible of the increase of HtrA1 in the urine of patients with carcinoma rather than the urothelium affected by cancer, perhaps as a protective response to disease progression.
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Gagné, Andréanne, i Andréanne Gagné. "Expression de la protéase tissulaire HtrA1 et le pronostic des femmes atteintes de cancer épithélial de l'ovaire". Master's thesis, Université Laval, 2015. http://hdl.handle.net/20.500.11794/27482.
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Introduction : La protéase High Temperature Requirement Factor A1 (HtrA1) pourrait être associée au pronostic du cancer de l’ovaire (CO). Objectif : Évaluer l’effet de l’expression de HtrA1 dans des tissus tumoraux sur le pronostic des femmes avec un CO séreux. Méthodes : Une étude de cohorte a été menée chez 122 femmes ayant un CO séreux traitées au CHU de Québec entre 1993-2006. Par immunohistochimie, l’expression de HtrA1 a été mesurée de façon visuelle et informatisée (% noyaux positifs). Des risques relatifs (HR) de progression et de décès ont été estimés avec le modèle de Cox multivarié. Résultats: Un faible pourcentage de noyaux marqués par HtrA1 était associé à une diminution des risques de progression (visuel HR=0,66, p=0,04, informatique HR=0,63, p=0,03) et de décès (visuel HR=0,69, p=0,09, informatique HR=0,56, p=0,01). Conclusion : La sous-expression de HtrA1 était associée à un meilleur pronostic des femmes avec un CO séreux.Introduction : La protéase High Temperature Requirement Factor A1 (HtrA1) pourrait être associée au pronostic du cancer de l’ovaire (CO). Objectif : Évaluer l’effet de l’expression de HtrA1 dans des tissus tumoraux sur le pronostic des femmes avec un CO séreux. Méthodes : Une étude de cohorte a été menée chez 122 femmes ayant un CO séreux traitées au CHU de Québec entre 1993-2006. Par immunohistochimie, l’expression de HtrA1 a été mesurée de façon visuelle et informatisée (% noyaux positifs). Des risques relatifs (HR) de progression et de décès ont été estimés avec le modèle de Cox multivarié. Résultats: Un faible pourcentage de noyaux marqués par HtrA1 était associé à une diminution des risques de progression (visuel HR=0,66, p=0,04, informatique HR=0,63, p=0,03) et de décès (visuel HR=0,69, p=0,09, informatique HR=0,56, p=0,01). Conclusion : La sous-expression de HtrA1 était associée à un meilleur pronostic des femmes avec un CO séreux.
Background: The protease High Temperature Requirement Factor A1 (HtrA1) might be associated with prognosis in ovarian cancer (OC). Objective: To evaluate the effect of HtrA1 expression in tumoral tissues on prognosis of women with serous OC. Methods: A cohort study was conducted among 122 women with a serous OC treated at the CHU de Québec between 1993-2006. Tissue microarrays were immunostained for HtrA1. HtrA1 expression was assessed visually and by digital image analysis (% of positive nuclei). Cox regression multivariate models taking into account standard prognostic factors were used to estimate adjusted hazard ratios (HR) of progression and death. Results: Low percentage of HtrA1 marked nuclei was associated with lower risks of progression (visual HR=0.66, p=0.04, digital HR=0.63, p=0.03) and death (visual HR=0.69, p=0.09, digital HR=0.56, p=0.01). Conclusion: Nuclear downregulation of HtrA1 was associated with a better prognosis in women with serous OC.
Background: The protease High Temperature Requirement Factor A1 (HtrA1) might be associated with prognosis in ovarian cancer (OC). Objective: To evaluate the effect of HtrA1 expression in tumoral tissues on prognosis of women with serous OC. Methods: A cohort study was conducted among 122 women with a serous OC treated at the CHU de Québec between 1993-2006. Tissue microarrays were immunostained for HtrA1. HtrA1 expression was assessed visually and by digital image analysis (% of positive nuclei). Cox regression multivariate models taking into account standard prognostic factors were used to estimate adjusted hazard ratios (HR) of progression and death. Results: Low percentage of HtrA1 marked nuclei was associated with lower risks of progression (visual HR=0.66, p=0.04, digital HR=0.63, p=0.03) and death (visual HR=0.69, p=0.09, digital HR=0.56, p=0.01). Conclusion: Nuclear downregulation of HtrA1 was associated with a better prognosis in women with serous OC.
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Schillinger, Jasmin [Verfasser], i Michael [Akademischer Betreuer] Ehrmann. "Die Rolle der humanen Serinprotease HTRA1 in der Regulation von Zellzyklus und Apoptose / Jasmin Schillinger ; Betreuer: Michael Ehrmann". Duisburg, 2018. http://d-nb.info/1150654481/34.
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ANCELIN, KATIA. "Chromatine et telomeres chez les mammiferes : le role central des proteines htrf1 et htrf2 dans les fonctions telomeriques". Lyon, École normale supérieure (sciences), 2001. http://www.theses.fr/2001ENSL0189.
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Les extremites d'un chromosome eucaryote sont constituees par des edifices nucleoproteiques particuliers : les telomeres. Les telomeres protegent les chromosomes contre la perte de materiel genetique lors de la replication et l'instabilite genetique. Leur integrite est donc vitale pour une bonne transmission de l'information genetique. Leurs fonctions sont correlees aux differentes proteines qui se fixent sur l'adn telomerique. Nous avons mis en evidence deux proteines humaines nouvelles : htrf1 et htrf2. Elles possedent un motif proteique de fixation a l'adn telomerique conserve parmi les proteines telomeriques affines de la partie double brin des telomeres. Ce motif a ete appele telobox. Dans certaines cellules eucaryotes (cellules germinales, souches somatiques, cancereuses ou micro-organismes), la perte d'adn telomerique due a la replication est compensee par une enzyme de type transcriptase inverse, la telomerase. Dans tous ces cas, l'action de la telomerase permet de maintenir une taille moyenne des telomeres relativement constante. Je me suis interessee aux roles de htrf1 et htrf2 dans les mecanismes de regulation de la taille des telomeres dans des cellules possedant une activite telomerase. Nous avons mis en place un nouveau systeme de ciblage a facon de proteines telomeriques humaines sur un telomere. L'influence de la presence en cis des proteines telomeriques sur l'elongation ou la degradation des sequences telomeriques. A ensuite ete etudiee. Ainsi, le role de cis -regulateur negatif de la taille de htrf1 et htrf2 a pu etre montre. Enfin, nous avons egalement pu obtenir quelques evidences concernant le mode d'action des deux proteines htrf1 et htrf2 dans la regulation de la taille des telomeres. Tandis que htrf1 parait agir dans une voie inhibant la telomerase, htrf2 au contraire semble avoir un role independant, et une hypothese serait que htrf2 participe a des voies d'activation de degradation des sequences telomeriques.
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Trübestein, Linda [Verfasser], Michael [Akademischer Betreuer] Ehrmann i Peter [Akademischer Betreuer] Bayer. "Strukturelle und Biochemische Charakterisierung der Humanen Serin Protease HtrA1 / Linda Trübestein. Gutachter: Michael Ehrmann ; Peter Bayer. Betreuer: Michael Ehrmann". Duisburg, 2011. http://d-nb.info/1015268242/34.
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Weber, Niklas [Verfasser], Harald [Akademischer Betreuer] Kolmar i Heribert [Akademischer Betreuer] Warzecha. "Structure-Based Monomerization of Human Serine Protease HTRA1 towards Evolutive Engineering of Activity Modulators / Niklas Weber ; Harald Kolmar, Heribert Warzecha". Darmstadt : Universitäts- und Landesbibliothek Darmstadt, 2017. http://d-nb.info/1147968349/34.
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Lehner, Anna Veronika [Verfasser], Marion B. [Akademischer Betreuer] [Gutachter] Kiechle i Barbara [Gutachter] Schmalfeldt. "Expressionsanalyse und Epigenetik der Serinprotease HTRA1 im Mammakarzinom / Anna Veronika Lehner. Betreuer: Marion B. Kiechle. Gutachter: Marion B. Kiechle ; Barbara Schmalfeldt". München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1103658484/34.
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Akhtar-Schäfer, Isha [Verfasser], Elena [Gutachter] Rugarli i Thorsten [Gutachter] Hoppe. "Role of the complement system and HtrA1 in microglia and age-related macular degeneration / Isha Akhtar-Schäfer ; Gutachter: Elena Rugarli, Thorsten Hoppe". Köln : Universitäts- und Stadtbibliothek Köln, 2019. http://d-nb.info/118060153X/34.
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Pöpsel, Simon [Verfasser], Michael [Akademischer Betreuer] Ehrmann i Hemmo [Akademischer Betreuer] Meyer. "Proteolysis and ATP-independent disaggregation of Tau aggregates by the human serine protease HTRA1 / Simon Pöpsel. Betreuer: Michael Ehrmann. Gutachter: Hemmo Meyer". Duisburg, 2015. http://d-nb.info/1079793550/34.
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Datta, Shyamtanu [Verfasser], i Bernhard [Akademischer Betreuer] Weber. "Functional analysis of genetic variants associated with age-related macular degeneration (AMD) - The HtrA serine peptidase 1 (HTRA1) / Shyamtanu Datta. Betreuer: Bernhard Weber". Regensburg : Universitätsbibliothek Regensburg, 2016. http://d-nb.info/1104480506/34.
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Dias, Sandra Martha Gomes. "Estudos estruturais dos receptores nucleares humanos para os hormônios tireoidianos Isoforma ß1 (hTRß1) e para o ácido retinóico 9-cis Isoforma a (hRXRa)". Universidade de São Paulo, 2004. http://www.teses.usp.br/teses/disponiveis/76/76132/tde-21092007-141432/.
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Os receptores nucleares são de suma importância para os processos de sinalização intercelular nos eucariotos, uma vez que possuem a capacidade de convergir diferentes sinais internos e externos na regulação de programas genéticos. Estas proteínas funcionam, na sua maioria, como fatores de transcrição ativados por ligantes, sendo a via de comunicação direta entre as moléculas de sinalização e a resposta transcricional eliciada pelas mesmas. A programação genética, estabilizada ou modificada pelos receptores, afeta virtualmente todos os aspectos da vida dos organismos multicelulares, tais como a embriogênese, a homeostase, a reprodução, o crescimento e a morte celular. A regulação transcricional e a seletividade promovida por estas proteínas têm fomentado intensas pesquisas, as quais estão decifrando a complexa rede de eventos moleculares que relatam sua forma de ação. Será um desafio para o futuro o conhecimento completo das regras moleculares que definem sua maneira de promover o controle espacial e temporal da expressão gênica. Estas informações prometem trazer detalhes cruciais para o desenvolvimento de drogas mais eficientes e de grande valor terapêutico. Neste contexto, o principal objetivo dos estudos aqui apresentados foi o de aumentar o conhecimento sobre o comportamento e estrutura do receptor nuclear humano dos hormônios tireoidianos, isoforma β1 (hTRβ1), e do receptor nuclear humano do ácido retinóico 9-cis, isoforma ? (hRXRα). Para tal, aplicou-se a técnica de espalhamento de raios X a baixos ângulos para determinar-se, em solução, o envelope destes receptores contendo os domínios de ligação ao DNA e ao ligante. Paralelamente, investiu-se em diversas tentativas de cristalização dos mesmos. Os resultados obtidos permitiram a determinação da localização espacial dos diferentes domínios e as organizações quaternárias dos homodímeros e homotetrâmeros. Conseqüentemente, foram propostos os primeiros modelos estruturais de receptores nucleares contendo os domínios de ligação ao DNA e ao ligante. O comportamento oligomérico, em solução, do hTRβ1 também foi analisado qualitativamente. Verificou-se que a formação do homodímero e do homotetrâmero é influenciada pela presença do hormônio T3, pela concentração protéica, pelos domínios presentes e por mutações específicas. Estes estudos geraram a hipótese de que o receptor nuclear hTRβ1 é capaz de se autoreprimir. Até então, dentro da superfamília dos receptores nucleares, esta capacidade de autorepressão somente havia sido descrita para o receptor hRXRα. Por fim, cristalizou-se o domínio LBD do receptor hTRβ1 com os ligantes T3, Triac e GC-1. O objetivo foi o de determinar estruturas cristalográficas importantes para o futuro desenvolvimento de tiromiméticos de ação isoforma-seletiva.In eukaryotes, nuclear receptors are of major importance for intercellular signaling because they join different intra and extracellular signals during regulation of genetic programs. The great majority of these proteins function as ligand activated transcription factors providing a direct link between signaling molecules and the transcriptional responses elicited by them. The genetic programs that these receptors establish or modify affect virtually all aspects of the multicellular organisms? life, such as embryogenesis, homeostasis, reproduction, cell growth, and death. Their gene-regulatory power and selectivity has prompted intense research which is now starting to decipher the complex network of molecular events involved in transcription regulation. The future challenge will be to uncover the molecular rules that define spatial and temporal control of gene expression. Such knowledge would be essential to the development of more efficient drugs with better therapeutic values. Therefore, the main purpose in this study was to extend the understanding on the behavior and the structure of human thyroid receptor, isoform ?1 (hTRβ1), and human retinoic acid X receptor, isoform ? (hRXRα). It was applied the small angle X-ray scattering technique to determine, in solution, the envelop of both receptors containing DNA and ligand binding domains. Beside this, several crystallization conditions were tried for both receptors. The results made possible to define the spatial localization of the domains and the quaternary structure of the homodimers and homotetramers. Consequently, we were able to propose the first structural models for nuclear receptors containing the DNA and ligand binding domains. The oligomeric behavior of the hTRβ1, in solution, was also analyzed qualitatively. We verified that it was influenced by the presence of T3 hormone, the protein concentration, the presence of both DNA and ligand binding domains, and by specific mutations. Based on these results, we were able to hypothesize that the hTRβ1 has the capacity of autorepression. Up to now, only the hRXRα, in the whole nuclear receptor superfamily, had been described to behave similarly. Finally, we crystallized the ligand binding domain of the hTRβ1 in the presence of the ligands T3, Triac, and GC-1. The objective was to solve crystallographic structures essential for the future development of tiromimetics with isoform-selective action.
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Simmons, Michael. "Identifying Genetic Pleiotropy through a Literature-wide Association Study (LitWAS) and a Phenotype Association Study (PheWAS) in the Age-related Eye Disease Study 2 (AREDS2)". Thesis, The University of Arizona, 2017. http://hdl.handle.net/10150/623630.
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A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine.Genetic association studies simplify genotype‐phenotype relationship investigation by considering only the presence of a given polymorphism and the presence or absence of a given downstream phenotype. Although such associations do not indicate causation, collections of phenotypes sharing association with a single genetic polymorphism may provide valuable mechanistic insights. In this thesis we explore such genetic pleiotropy with Deep Phenotype Association Studies (DeePAS) using data from the Age‐Related Eye Study 2 (AREDS2). We also employ a novel text mining approach to extract pleiotropic associations from the published literature as a hypothesis generation mechanism. Is it possible to identify pleiotropic genetic associations across multiple published abstracts and validate these in data from AREDS2? Data from the AREDS2 trial includes 123 phenotypes including AMD features, other ocular conditions, cognitive function and cardiovascular, neurological, gastrointestinal and endocrine disease. A previously validated relationship extraction algorithm was used to isolate descriptions of genetic associations with these phenotypes in MEDLINE abstracts. Results were filtered to exclude negated findings and normalize variant mentions. Genotype data was available for 1826 AREDS2 participants. A DeePAS was performed by evaluating the association between selected SNPs and all available phenotypes. Associations that remained significant after Bonferroni‐correction were replicated in AREDS. LitWAS analysis identified 9372 SNPs with literature support for at least two distinct phenotypes, with an average of 3.1 phenotypes/SNP. PheWAS analyses revealed that two variants of the ARMS2‐HTRA1 locus at 10q26, rs10490924 and rs3750846, were significantly associated with sub‐retinal hemorrhage in AMD (rs3750846 OR 1.79 (1.41‐2.27), p=1.17*10‐7). This associated remained significant even in populations of participants with neovascular AMD. Furthermore, odds ratios for the development of sub‐retinal hemorrhage in the presence of the rs3750846 SNP were similar between incident and prevalent AREDS2 sub‐populations (OR: 1.94 vs 1.75). This association was also replicated in data from the AREDS trial. No literature‐defined pleiotropic associations tested remained significant after multiple‐testing correction. The rs3750846 variant of the ARMS2‐HTRA1 locus is associated with sub‐retinal hemorrhage. Automatic literature mining, when paired with clinical data, is a promising method for exploring genotype‐phenotype relationships.
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Leveziel, Nicolas. "Génétique de la dégénérescence maculaire liée à l'âge variants majeurs de prédisposition à la forme exsudative". Paris 6, 2008. http://www.theses.fr/2008PA066183.
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Pazdera, Radek. "Efektivní metoda čtení adresářových položek v souborovém systému Ext4". Master's thesis, Vysoké učení technické v Brně. Fakulta informačních technologií, 2013. http://www.nusl.cz/ntk/nusl-236169.
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Cílem této práce je zvýšit výkon sekvenčního procházení adresářů v souborovém systému ext4. Datová struktura HTree, jenž je v současné době použita k implementaci adresářu v ext4 zvládá velmi dobře náhodné přístupy do adresáře, avšak není optimalizována pro sekvenční procházení. Tato práce přináší analýzu tohoto problému. Nejprve studuje implementaci souborového systému ext4 a dalších subsystému Linuxového jádra, které s ním souvisí. Pro vyhodnocení výkonu současné implementace adresářového indexu byla vytvořena sada testů. Na základě výsledků těchto testů bylo navrženo řešení, které bylo následně implementováno do Linuxového jádra. V závěru této práce naleznete vyhodnocení přínosu a porovnání výkonu nové implementace s dalšími souborovými systémy v Linuxu.
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Mukherjee, Sourajit. "Single-channel studies on human TREK-1 (hTREK-1) channels to intracellular ischemia related factors". Thesis, 2020. https://etd.iisc.ac.in/handle/2005/5023.
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TREK-1, a member of the two-pore domain family of potassium channels, majorly contributes
to the maintenance of resting membrane potential of a cell and has been reported to respond to
ischemic levels of intracellular lactate and acidic pH to provide neuroprotection. There are two
N-terminal variants that arise due to Alternative splicing: the shorter variant having a shorter
N-terminus than the full-length human TREK-1 (hTREK-1) which is widely expressed in the
acute hypoxia sensitive regions of the adult brain like the cerebellum and hippocampus and is
upregulated under ischemia. Previous whole-cell patch-clamp experiments on the shorter
variant of hTREK1 have shown contradictory results to hypoxia- a condition attributed to
ischemia, which has put the neuroprotective role of the hTREK-1 channel into question.
Although these experiments were performed on the shorter hTREK-1, there have been no
studies on the effect of hypoxia and other ischemia-related factors like lactate, pH, and hemin
on the full-length hTREK-1 channel.
In the present study, using single-channel inside-out patch-clamp electrophysiology on the
full-length hTREK-1 expressed in HEK293 cells, we show that the extended N-terminus of the
full-length hTREK-1 channel is required to sense hypoxia. The probability of opening of the
full-length hTREK-1 channel reversibly increased on exposure to hypoxia. However, there was
a decrease in the open probability of the shorter hTREK-1 channel under similar conditions
suggesting that the N-terminus might be responsive to hypoxia or it might interact with the Cterminus
of the protein or a sensor situated elsewhere in the channel.
Due to the shift in glucose metabolism from aerobic to anaerobic mode upon ischemia, there
is an increase in intracellular lactate that has been shown to be a potent modulator of the fulllength
hTREK-1 channel. However, the modulatory effect of lactate and hypoxia on the fulllength
hTREK-1 has not been reported. We observed a significant increase in the open
probability when the channel was first exposed to hypoxia, followed by hypoxia and 20mM
lactate together. Interestingly, the extent of increase was reproducible when the channel was
exposed to 20mM lactate first, followed by 20mM lactate and hypoxia together. This
observation suggests that the sites of action of hypoxia and lactate are independent of each
other and additive when presented together.
It is known from previous literature that a decrease in intracellular pH due to ischemia leads to
activation of the TREK-1 channel. However, the effect of hypoxia and lactate under ischemic
pH conditions on the full-length hTREK1 remained elusive. We showed that the channel’s
open probability increases with hypoxia under acidic pH 6, which gets further elevated with
the addition of 20mM lactate in the medium.
Cerebral ischemia is associated with several pathological microenvironmental changes like
membrane distortion and changes in intracellular pH. A critical amino acid residue, E306
(E321 in the full-length hTREK-1) in the intracellular C-terminus of the channel has been
shown to be involved in mechano-gating of the channel by associating with the inner leaflet of
the plasma membrane during intracellular acidosis. Since TREK-1 is mechano-gated and
sensitive to intracellular acidosis, we hypothesized that the intactness of the physiological state
of the channel by the C-terminus is essential for the hypoxic response in ischemic conditions.
Single-channel inside-out recordings from the E321A mutant with a disrupted interaction of
the C-terminus with the lipid membrane that traps it in a constitutively open state showed a
significant decrease in activity to hypoxia. The importance of the C-terminus that purportedly
hangs in the cytosol and the extended N-terminus of the full-length hTREK1 in eliciting the
response to hypoxia indicated possible N-C terminus interactions that have been shown in other
ion channels. While the non-heme-based oxygen sensing was clear from these isolated insideout
single ion channel recordings, it must be pointed out that hypoxia has been previously
shown to dimerize HIF (hypoxia-inducible factor) alpha and beta chains through proline
residues that are lost under normoxic conditions.
Since heme-based oxygen sensing is the common biological mechanism to sense oxygen, I
explored the plausible existence of such a mechanism in the full-length hTREK-1 channel.
Single-channel recordings in inside-out patches in the presence of bath perfused hemin- a
known oxygen sensor and a channel modulator were studied. Bath perfusion of 500nM hemin
followed by 500nM hemin and hypoxia together failed to significantly increase the channel
activity, although the same channel responded to hypoxia alone by increasing the channel
activity. Although beyond the scope of the present thesis work, the non-heme-based hypoxia
sensing through N-C terminus interaction in the full-length as a plausible mechanism needs
further investigation. A bioinformatics search for a hemin-binding motif that has been indicated
in other ion channels was not seen in the full-length hTREK1, indicating that a heme-based
oxygen sensing mechanism might be absent in the hTREK1.
Finally, the key point that emerges from the study is the polymodal regulation of the hTREK1
where the channel appears to integrate the responses to the ischemia-related factors hypoxia,
intracellular lactate, and pH.
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Metri, Vishal. "Stochastic Chemical Kinetics : A Study on hTREK1 Potassium Channel". Thesis, 2013. http://etd.iisc.ac.in/handle/2005/3329.
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Chemical reactions involving small number of reacting molecules are noisy processes. They are simulated using stochastic simulation algorithms like the Gillespie SSA, which are valid when the reaction environment is well-mixed. This is not the case in reactions occuring on biological media like cell membranes, where alternative simulation methods have to be used to account for the crowded nature of the reacting environment. Ion channels, which are membrane proteins controlling the flow of ions into and out of the cell, offer excellent single molecule conditions to test stochastic simulation schemes in crowded biological media.
Single molecule reactions are of great importance in determining the functions of biological molecules. Access to their experimental data have increased the scope of com-putational modeling of biological processes. Recently, single molecule experiments have revealed the non-Markovian nature of chemical reactions, due to a phenomenon called `dynamic disorder', which makes the rate constants a deterministic function of time or a random process. This happens when there are additional slow scale conformational transitions, giving the molecule a memory of its previous states. In a previous work, the hTREK1 two pore domain potassium channel was revealed to have long term memory in its kinetics, prompting alternate non-Markovian schemes to analyze its gating.
Traditionally, ion channel gating is modeled as Markovian transitions between fixed states. In this work, we have used single channel data from hTREK1 ion channel and have provided a simple diffusion model for its gating. The main assumption of this model
is that the ion channel diffuses through a continuum of states on its potential energy landscape, which is derived from the steady state probability distribution of ionic current recorded from patch clamp experiments. A stochastic differential equation (SDE) driven by Gaussian white noise is proposed to model this motion in an asymmetric double well potential. The method is computationally very simple and efficient and reproduces the amplitude histogram very well.
For the case when ligands are added, leading to incorporation of long term memory in the kinetics, the SDE is modified to run on coloured noise. This has been done by introducing an auxiliary variable into the equation. It has been shown that increasing the noise correlation with ligand concentration improves the fits to the experimental data. This has been validated for several datasets. These methods are more advantageous for simulation than the Markovian models as they are true to the physical picture of gating and also computationally very efficient. Reproducing the whole raw data trace takes no more than a few seconds with our scheme, with the only input being the amplitude histogram and four parameters.
Finally a quantitative model based on a modified version of the Chemical Langevin equation is given, which works on random rate parameters. This model is computationally simple to implement and reproduces the catalytic activity of the channel as a function of time.
From the computational analysis undertaken in this work, we can infer that ion channel activity can be modeled using the framework of non-Markovian processes, lending credence to the recent understanding that single molecule reactions are basically processes with long-term memory. Since the ion channel is basically a protein, we can also hypothesize that the some of the properties that make proteins so vital to living organ-isms could be attributed to long-term memory in their folding kinetics, giving them the ability to sample specific regions of their conformation space, which are of interest to biological functions.
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Metri, Vishal. "Stochastic Chemical Kinetics : A Study on hTREK1 Potassium Channel". Thesis, 2013. http://etd.iisc.ernet.in/2005/3329.
Pełny tekst źródłaStreszczenie:
Chemical reactions involving small number of reacting molecules are noisy processes. They are simulated using stochastic simulation algorithms like the Gillespie SSA, which are valid when the reaction environment is well-mixed. This is not the case in reactions occuring on biological media like cell membranes, where alternative simulation methods have to be used to account for the crowded nature of the reacting environment. Ion channels, which are membrane proteins controlling the flow of ions into and out of the cell, offer excellent single molecule conditions to test stochastic simulation schemes in crowded biological media.
Single molecule reactions are of great importance in determining the functions of biological molecules. Access to their experimental data have increased the scope of com-putational modeling of biological processes. Recently, single molecule experiments have revealed the non-Markovian nature of chemical reactions, due to a phenomenon called `dynamic disorder', which makes the rate constants a deterministic function of time or a random process. This happens when there are additional slow scale conformational transitions, giving the molecule a memory of its previous states. In a previous work, the hTREK1 two pore domain potassium channel was revealed to have long term memory in its kinetics, prompting alternate non-Markovian schemes to analyze its gating.
Traditionally, ion channel gating is modeled as Markovian transitions between fixed states. In this work, we have used single channel data from hTREK1 ion channel and have provided a simple diffusion model for its gating. The main assumption of this model
is that the ion channel diffuses through a continuum of states on its potential energy landscape, which is derived from the steady state probability distribution of ionic current recorded from patch clamp experiments. A stochastic differential equation (SDE) driven by Gaussian white noise is proposed to model this motion in an asymmetric double well potential. The method is computationally very simple and efficient and reproduces the amplitude histogram very well.
For the case when ligands are added, leading to incorporation of long term memory in the kinetics, the SDE is modified to run on coloured noise. This has been done by introducing an auxiliary variable into the equation. It has been shown that increasing the noise correlation with ligand concentration improves the fits to the experimental data. This has been validated for several datasets. These methods are more advantageous for simulation than the Markovian models as they are true to the physical picture of gating and also computationally very efficient. Reproducing the whole raw data trace takes no more than a few seconds with our scheme, with the only input being the amplitude histogram and four parameters.
Finally a quantitative model based on a modified version of the Chemical Langevin equation is given, which works on random rate parameters. This model is computationally simple to implement and reproduces the catalytic activity of the channel as a function of time.
From the computational analysis undertaken in this work, we can infer that ion channel activity can be modeled using the framework of non-Markovian processes, lending credence to the recent understanding that single molecule reactions are basically processes with long-term memory. Since the ion channel is basically a protein, we can also hypothesize that the some of the properties that make proteins so vital to living organ-isms could be attributed to long-term memory in their folding kinetics, giving them the ability to sample specific regions of their conformation space, which are of interest to biological functions.
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FASANO, ALESSANDRO. "HTRA1 expression and functionality in HTRA1 mutation carriers CARRIERS". Doctoral thesis, 2019. http://hdl.handle.net/2158/1166650.
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High temperature requirement A1 (HTRA1) belongs to heat shock-induced serine proteases and is ubiquitously expressed in normal human adult tissues. HTRA1 plays a modulatory role in various cell processes, particularly regulates the transforming growth factor-β (TGF-ß) signalling. Biallelic mutations in HTRA1 lead to cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), a rare cerebral small vessel disease (CSVD).
Nowadays, fifteen HTRA1 mutations have been identified. Recent data reported that heterozygous HTRA1 mutations seem to be linked to
familial CSVD of unknown aetiology, which is characterized by a later age at onset.
These data suggest that HTRA1 mutation could behave as autosomal recessive or dominant mutation.
Our aim is to obtain further data about the pathogenic effect of various heterozygous HTRA1 mutations. We compared expression profiles of HTRA1 and intermediaries of TGF-β signalling proteins both in heterozygous carriers, with missense and stop codon HTRA1 mutations, and in heterozygous and homozygous mouse embryonic fibroblasts harbouring HTRA1-R274Q mutation. Moreover, we used heterozygous and homozygous murine models harbouring HTRA1-R274Q in order to evaluate in vivo the effects of mutant HTRA1.
Further, we performed supplementary studies to evaluate the possibility of a dominant negative effect on HTRA1-WT by HTRA1-mutants, and the possibility torescue HTRA1-protease activity in homozygous HTRA1-R274Q carriers.
The cell lysates and culture medium of cultured cells were used to analyse the expression pattern of both HTRA1 and intermediaries of TGF-β signalling proteins by western blot and immunofluorescence analysis. RNAs extracted from cultured cells and from mice tissues were used to analyse HTRA1-RNA and CTGF-RNA expression level by RT-qPCR.
We found a ∼50% reduction in HTRA1 expression in human fibroblasts carrying heterozygous HTRA1-mutations compared to control. RT-qPCR analysis confirmed these data for two of the analysed subjects, whilst showed no significant reduction in the remaining carriers compared to control. Analysis of the murine models showed that there is no alteration of HTRA1-RNA expression nor in heterozygous nor in homozygous HTRA1-R274Q mice. No significant alteration of Smad2/3 phosphorylation and CTGF expression, down- and up-stream intermediaries of
TGF-β signalling pathway, respectively, were found, suggesting that dysfunction of TGF-β signalling in fibroblasts might not contribute to the pathogenesis of CARASIL and CSVD linked to heterozygous HTRA1 mutations reported in this study.
Heterozygous and homozygous HTRA1-R274Q murine cells displayed an increased fibronectin accumulation of 10-fold and 40-fold, respectively, than HTRA1-WT cells, suggesting that even the heterozygous HTRA1-mutations could be enough to cause deleterious phenotypic alterations in brain small vessels.
HTRA1-WT protease activity did not display any remarkable alteration in presence of HTRA1-mutants.These findings seem to rule out a dominant negative effect in HTRA1 mutations we investigated. Finally, MEFs transfected with the rescue protein give an outcome similar to that obtained with MEFs transfected with HTRA1-WT, opening actual possibility to rescue the functionality of HTRA1-mutants.
In conclusion, our results seem to suggest that CSVD, linked to heterozygous HTRA1 mutations, may occur in the presence of ~50% expression of the protein.
Progressive tissue damage accumulation in small vessels leading to delayed and milder clinical expression with later onset with respect to classical CARASIL phenotype may be hypothesized.
Moreover, data collection on heterozygous HTRA1 mutants is still limited, so investigation on the HTRA1 expression and activity, in cells from a wider number of subjects harboring different heterozygous HTRA1 missense mutations, could be helpful to verify a possible correlation between specific aminoacidic variations and particular protein alterations.
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Choudhury, Nasreen. "G-Protein Coupled Estrogen Receptor (hGPER)- Mediated Action of 17β-Estradiol on hTREK-1 Potassium Channel". Thesis, 2017. http://etd.iisc.ac.in/handle/2005/4159.
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TREK-1 is a two-pore domain potassium channel that contributes to maintenance of the resting membrane potential of a cell. TREK-1 is involved in several physiological and pathophysiological conditions like nociception, anaesthesia, epilepsy, ischemia and depression. Activity of TREK-1 is modulated by a number of physical and chemical stimuli including the activation of G-protein coupled receptors by several neurotransmitters and hormones. An important modulator of neuronal activity and function is 17β-estradiol, which by acting through its classical receptors ERα and ERβ, can bring about genomic changes in the cell. 17β-Estradiol can also act through membrane receptors like the G-protein coupled estrogen receptor (GPER) and activate intracellular signaling pathways. Several neuroprotective effects of 17β-estradiol in epilepsy, ischemia and diseases like Alzheimer‟s and Parkinson‟s is mediated through activation of GPER. 17β-Estradiol is also known to modulate the activity of several ion channels in a non-genomic manner, thus, regulating the neuronal function. Of the different membrane ionic channels, the leak potassium channel TREK-1 is implicated in neuroprotection since their activation hyperpolarizes the membrane of neurons and astrocytes and reduces neuronal excitability. However, it is not known whether 17β-estradiol can physiologically modulate the activity of TREK-1 channels and use this as an additional mechanism to mediate neuroprotection.
In the present study, using single-channel cell-attached patch-clamp electrophysiology in HEK293 cells, we show that 17β-estradiol increases the activity of hTREK-1 by an hGPER-dependent mechanism. The probability of opening of the hTREK-1 channel increased rapidly and irreversibly on application of 17β-estradiol, not directly but only in the presence of hGPER. The potentiation of hTREK-1 activity by 17β-estradiol was mimicked by hGPER agonist and inhibited by hGPER antagonist, supporting the hGPER-dependence of 17β-estradiol action.
Pharmacological studies demonstrated that the hGPER-mediated potentiation of hTREK-1 by 17β-estradiol occurred in a pertussis toxin-sensitive manner, mediated by the Gβγ subunits. Raising the intracellular cAMP levels reversed the potentiation of hTREK-1 induced by 17β-estradiol suggesting the inhibition of cAMP production in the hGPER-mediated increase of hTREK-1 activity. The hGPER-dependent rise in hTREK-1 activity induced by 17β-estradiol was occluded by inhibition of PKA which indicated that 17β-estradiol action involves inhibition of PKA.
The serines at position 315 and 348 in the C-terminal domain of hTREK-1 are involved in phosphorylation-mediated inhibition of channel activity as known from earlier studies. Mutational studies with S315 and S348 suggested that S348 was the target site for dephosphorylation and potentiation of hTREK-1 by hGPER-mediated action of 17β-estradiol. 17β-Estradiol-induced potentiation of hTREK-1 was abolished on inhibition of serine/threonine phosphatases suggesting the requirement of serine/threonine phosphatases for the action of 17β-estradiol. Thus, the inhibition of PKA acts jointly with the activation of serine/threonine phosphatases to dephosphorylate S348 in the C-terminal domain of hTREK-1 leading to an increase in its activity.
It was known from previous literature that TREK-1 and 17β-estradiol play important roles in neuroprotection. However, the effect of 17β-estradiol on the TREK-1 channel was not explored. The study undertaken as part of this thesis provides the link between 17β-estradiol and TREK-1 activity, giving an insight into a plausible mechanism underlying the several neuroprotective roles of 17β-estradiol.
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Irle, Inga C. [Verfasser]. "Epigenetische Regulation der konservierten Serinprotease HtrA1 / vorgelegt von Inga Irle". 2010. http://d-nb.info/100041812X/34.
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Tirniceriu, Anca Laura [Verfasser]. "Die genetische und funktionelle Bedeutung des High-Temperature-Requirement A1-Proteins (HtrA1) und eines HtrA1-Single Nucleotide Polymorphismus für Morbus Alzheimer / vorgelegt von Anca Laura Tirniceriu". 2008. http://d-nb.info/997241497/34.
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"Mechanism of age-related macular degeneration: the role of HtrA1 and related molecules". Thesis, 2010. http://library.cuhk.edu.hk/record=b6075056.
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Ng, Tsz Kin.Thesis (Ph.D.)--Chinese University of Hong Kong, 2010.
Includes bibliographical references (leaves 151-185).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
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Weber, Niklas. "Structure-Based Monomerization of Human Serine Protease HTRA1 towards Evolutive Engineering of Activity Modulators". Phd thesis, 2017. http://tuprints.ulb.tu-darmstadt.de/6908/1/171025_Diss_Weber.pdf.
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Human serine protease HTRA1 plays an important role in a plethora of physiological processes by recognition and conversion of numerous different substrates. Various studies suggested that HTRA1 is involved in several diseases, such as osteoarthritis, cancer, age-related macular degeneration or Alzheimer disease. The aim of this work was the generation of molecules that modulate HTRA1 activity, which could act as tools for validation of HTRA1 as a potential therapeutic target.
An initial approach was the monomerization of multimeric HTRA1 that is composed of trimers as well as higher oligomers of unknown composition to get an easier to handle and more controllable target for evolutionary design of interacting molecules. Accordingly, structural data of the trimer was applied for the design of amino acid replacements that are supposedly important for trimerization, resulting in stable monomeric fractions of HTRA1 catalytic domain.
Three different scaffold libraries were screened by yeast surface display towards binding of monomeric HTRA1. The miniprotein McoTI II-based library, which is a trypsin inhibitor from squash plant, delivered a single molecule that bound monomeric HTRA1 with nanomolar affinity, but not native trimeric HTRA1. Similarly, the two molecules isolated from an immunized VHH library, which is the variable domain of a single domain camelid immunoglobulin, also bind only monomeric HTRA1 with nanomolar affinities, but not native trimeric HTRA1.
The third library was a vNAR library, which is the variable domain of a single domain shark immunoglobulin that was synthetically randomized in CDR3. Six independent molecules were isolated against monomeric HTRA1 and five of them were shown to bind native trimeric HTRA1 with micromolar affinities. By stepwise affinity maturation of CDR1 and HV2, affinities were improved to double digit nanomolar affinities.
Finally, promising vNAR molecules were soluble expressed and evaluated for modulation of HTRA1 activity by activity assays, resulting in three molecules that enhance HTRA1 activity in a range from 150 % to 400 %. Therefore, we successfully generated activators that enhance HTRA1 activity with different strengths that can be used in in vitro and in vivo assays for validation of human HTRA1 as novel therapeutic target.
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Hou, Shirui. "The secreted serine protease xHtrA1 is a positive feedback regulator of long-range FGF signaling". Doctoral thesis, 2007. http://hdl.handle.net/11858/00-1735-0000-0006-B36F-7.
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Tennstädt, Annette [Verfasser]. "Die protektive Rolle der konservierten Serinprotease HtrA1 in der Alzheimerschen Krankheit / vorgelegt von Annette Tennstädt". 2009. http://d-nb.info/1001399102/34.
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Nappo, Francesco. "Screening of the CTSA gene in a population of NOTH3 and HTRA1 negative patients with Small Vessel Disease". Doctoral thesis, 2020. http://hdl.handle.net/2158/1198725.
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Screening genetico di pazienti affetti da small vessel disease che sono risultati negativi a mutazioni nei geni NOTCH3 e HTRA1, causativi di CADASIL e CARASIL, le forme di SVD più comuni. Mai prima d'ora il gene CTSA è stato associato a questo tipo di malattia.
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Boyne, J. R., K. J. Colgan i A. Whitehouse. "Recruitment of the complete hTREX complex is required for Kaposi's sarcoma-associated herpesvirus intronless mRNA nuclear export and virus replication". 2008. http://hdl.handle.net/10454/5869.
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A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.
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Schmidt, Nina [Verfasser]. "Die Serin-Protease HtrA1 ist ein neuer Regulator der Zellteilung und spielt eine wichtige Rolle in der malignen Transformation / vorgelegt von Nina Schmidt". 2010. http://d-nb.info/1004791321/34.
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