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Characterization of the Nucleocytoplasmic Transport of the Cutaneous HPV8 E7 ProteinOnder, Zeynep January 2014 (has links)
Thesis advisor: Junona Moroianu / Some non melanoma skin cancers (NMSC) have been associated with human papillomavirus (HPV) pathogenesis, like epidermodysplasia verruciformis (EV) and squamous cell carcinoma (SCC). EV is a genetically inherited skin disease that develops when the individuals are infected with cutaneous HPV types belonging to the β-genus, especially types 5 and 8. Transgenic mouse lineages expressing all early genes of cutaneous HPV8 develop papillomas, dysplasias and SCC after UV irradiation and this correlates with enhanced HPV8 oncogenes expression. We have previously discovered that the nuclear localization of mucosal HPV16 E7 and HPV11 E7 proteins is mediated by their zinc-binding domain via a Ran-dependent pathway and independent of nuclear import receptors and that a patch of hydrophobic residues within the zinc-binding domain of HPV16 E7 and HPV11 E7 proteins is responsible for their nuclear import via hydrophobic interactions with FG nucleoporins. Here we investigated the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein using confocal microscopy to analyze the intracellular localization of EGFP-8E7, its subdomains and its mutants after transient transfections. We also investigated the nuclear import ability of GST-8E7, its subdomains and mutants using in vitro nuclear import assays in digitonin-permeabilized HeLa cells. In addition, we performed isolation assays to study the direct interaction between HPV8 E7 and two FG nucleoporins, Nup62 and Nup153 or the nuclear export receptor, CRM1. We found that the nuclear import of cutaneous HPV8 E7 is mediated by a nuclear localization signal (NLS) located within its zinc-binding domain. Furthermore, we determined that the hydrophobic residues within the 65LRLFV69 patch are responsible for the nuclear import and nuclear localization of HPV8 E7 via direct hydrophobic interactions with FG nucleoporins, Nup62 and Nup153, whereas the positively charged arginine 66 plays no significant role in the function of the NLS. In addition, we examined the nuclear export mechanism of cutaneous HPV8 E7 protein and showed that it has a leucine-rich nuclear export signal (NES) in its C-terminal domain that is recognized by the CRM1 nuclear export receptor. These studies are essential for understanding the nucleocytoplasmic traffic of cutaneous HPV8 E7 protein. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Studies on the Nucleocytoplasmic Transport of the E7 Oncoprotein of High-Risk HPV Type 16Eberhard, Jeremy January 2013 (has links)
Thesis advisor: Junona Moroianu / Human papillomaviruses (HPVs) have been estimated to be the most common sexually transmitted infection in the United States. In addition to condyloma accuminata, infection of the squamous basal epithelium by high-risk HPVs, notably type 16 (HPV16), has been shown to be the primary etiological agent in the majority of cervical carcinomas. The E7 major transforming protein of HPV16, along with E6, has been linked to tumorigenesis and malignancy. While the E7 protein itself possesses no enzymatic activity, its ability to bind a number of nuclear and cytoplasmic targets subverts a variety of cellular regulatory complexes and facilitates viral replication. Previous studies in the Moroianu Lab have shown the HPV16 E7 oncoprotein to translocate across the nuclear pore complex (NPC) in a facilitated manner dependent on a non-canonical, c-terminal, nuclear localization signal (cNLS) for import, and a consensus leucine-rich nuclear export sequence (NES) for export (28). While the leucine-rich NES has been characterized, a full examination of the cNLS has yet to be performed. Here we present evidence that the karyopherin independent nuclear import mediated by the cNLS of 16E7 is dependent on its c-terminal Zn binding domain. Furthermore, we demonstrate that nuclear import is mediated by the direct interaction of a small patch of hydrophobic residues, 65LRLCV69, with the FG domain of the central FG-nucleoporin Nup62. In addition, we examined a potential regulatory mechanism of 16E7 nucleocytoplasmic translocation. Previous work has shown that a serine conserved in the high-risk HPVs at position 71 is phosphorylated by an unknown kinase. Here we present evidence that while phosphorylation of S71 is not required for either 16E7 nuclear localization or nuclear export in HeLa cells, mimicking phosphorylation of the S71 residue results in a statistically significant shift in the distribution of localization phenotypes of the resultant cell population toward a larger percentage exhibiting more nuclear localization. These data suggest that nucleocytoplasmic transport of 16E7 is, at least in part, a regulated process. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.
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Avaliação da presença do Papilomavírus humano (HPV) em tumores de pulmãoAMARAL, Carolina Maria Medeiros Do 15 December 2015 (has links)
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Previous issue date: 2015-12-15 / FACEPE / Os Papilomavírus humano (HPV) infectam mucosas e epitélio contribuindo
para o desenvolvimento de tumores benignos como também malignos. São
amplamente conhecidos como causadores do câncer cervical, contudo,
atualmente, vem apresentando evidências de associação com diversos outros
tipos de canceres, como o câncer de pulmão. Sendo assim, o presente estudo
avaliou a presença do DNA do HPV em tumores de pulmão de pacientes do
Estado de Pernambuco bem como a expressão de suas oncoproteínas E6 e E7.
Para isto, a detecção foi feita em amostras de tecidos de tumores frescos e
parafinados de 63 pacientes. HPV estava presente em 52% das amostras, sendo
detectados os tipos 16 e 18 com frequências de 81 e 19%, respectivamente.
Quanto a presença do vírus nos diferentes tipos histológicos dos tumores, foi
detectado HPV em 40% dos carcinomas escamosos, 33% dos adenocarcinomas,
18% dos carcinomas de células pequenas e 9% em carcinoma de células
grandes. Através da técnica de imunohistoquimica detectou-se a presença das
oncoproteinas virais E6 (anticorpo anti-HPV 16 e anti-HPV 18) e E7 (anticorpo
anti-HPV 16 e anti-HPV 18) com frequências de 85 e 75%, respectivamente. Tal
resultado confirma os resultados obtidos molecularmente quanto à presença do
HPV e é sugestivo de que o vírus esteja em atividade nas células tumorais e
provavelmente esteja desempenhando um papel na carcinogênese de pulmão. No
entanto, mais estudos são necessários para se ter um maior esclarecimento sobre
interação de E6 e E7 com proteínas celulares na tumorigenese pulmonar. / Small DNA viruses - Human Papillomavirus (HPV) - infect oral mucosa and
the epthelium, which leads to the development of both benign and malign tumors.
They are widely known as the principal causes of cervical cancer although
currently there is evidence to show that they are associated with several other
types of cancer, such as lung cancer. In the light of this, this study evaluated the
presence of HPV in the tumors of lungs of patients from the State of Pernambuco,
as well as the E6 and E7 oncoproteins expression. This involved carrying out the
detection in tumor tissue samples that were fresh and paraffin-embedded and
taken from 63 patients. HPV was found to be present in 52% of the samples, and
types 16 and 18 were detected with frequencies of 81% and 19% respectively.
With regard to the presence of the vírus in different histological types of tumors,
HPV was detected in 40% of the squamous carcinomas, 33% of the
adenocarcinomas, 18% of the small cell carcinomas and 9% in large cell
carcinomas. The presence of the E6 (antibody anti-HPV 16 and anti-HPV 18) and
E7 (antibody anti-HPV 16 and anti-HPV 18) oncoproteins was detected by means
of the immunohistochemical technique and this confirmed the results obtained
from a molecular analysis with regard to the presence of the virus and it is
suggestive that the virus is active in tumor cells and is probably playing a role in
lung carcinogenesis. However, further studies are required to have a clearer
understanding of the interaction of E6 and E7 with the cell proteins in pulmonary
tumorigenesis.
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Vývoj experimentálních protinádorových DNA vakcín / Development of experimental antitumor DNA vaccinesKaštánková, Iva January 2017 (has links)
No description available.
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Biotechnologické využití rostlinných virů / Plant virus-based biotechnologyVaculík, Petr January 2015 (has links)
The latest model of tertiary structure of capsid protein of potato virus X (PVX CP) was used as a template to design new insertion sites suitable for the preparation of PVX-based antigen presentation system. Based on this model, seven insertion sites (A-G) located in putative surface loops were tested. As an antigen inserted into these sites was used 17 amino acids long epitope derived from human papillomavirus type 16 E7 oncoprotein (E7 epitope) fused with either 6xHis tag or StrepII tag in both possible orientations (6xHis-E7 and E7-6xHis, StrepII-E7 and E7-StrepII). Prior to plant expression, modified PVX CPs were expressed in Escherichia coli MC1061. The results showed that only PVX CP carrying StrepII-E7 or E7-StrepII in the insertion site A formed virus particles. The results from transient expression experiments with modified PVX CPs in Nicotiana benthamiana showed that only the insertion site A (located between 24th and 25th amino acid in the PVX CP) could tolerate all tested inserts. Importantly, viral particles were detected only in the presence of StrepII tag and their stability was affected by the insert orientation (StrepII-E7 vs. E7-StrepII) as only the viral particles presenting E7-StrepII could be purified. Besides the preparation of PVX-based antigen presentation system, an...
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Detecção e análise do Papilomavírus humano (HPV) em carcinomas mamários de mulheres do Nordeste do BrasilLIMA, Elyda Gonçalves de 11 March 2016 (has links)
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Previous issue date: 2016-03-11 / CAPES / O câncer da mama é o tipo de câncer que mais acomete mulheres em todo
o mundo. Diversos fatores estãoassociados ao desenvolvimento desta neoplasia,
dentre elas as infecções virais. Entre os três vírus mais estudados como causa de
carcinogênese mamária está oPapillomavirus humano (HPV). Assim, oobjetivo foi
detectar e analisar o HPV emcarcinomasmamáriosde mulheres do Nordeste do
Brasil. A detecção do DNA viral foi realizada PCR, as amostras positivasforam
tipificadas por sequenciamento. A quantificação da carga viral e a determinação
do status físico por qPCR, e a detecção as oncoproteínas de E6 e E7 de HPV por
imunohistoquímica. O DNA de HPV foi detectado em 46,7% dos carcinomas de
mama HPV-positivos. O HPV16foi omais prevalente, 92% dos casos. A carga viral
do HPV apresentou uma média de 14,2 cópias em 104 células, noscarcinomas de
mama. Além disso, em 57,2% dos carcinomas mamáriosHPV-positivas
apresentaram o DNA viral integrado ao genoma do hospedeiro. Altas taxas de
detecção das oncoproteínas E6(89,5%) e E7(90%) foram identificadas nos
carcinomas de mama HPV-positivos. Já as proteínas supressoras de tumor, p53 e
p16INK4A, apresentaram taxas menores 95,7% e 92,3% respectivamente. Os
resultados deste estudo sugerem que o vírus esteja em atividade nas células
tumorais e provavelmente desempenhem papel na carcinogênese mamária. / Breast cancer is the type of cancer that affects more women around
the world. Several factors are associated with the development of cancer, among
which viral infections. Of the three most-studied virus as a cause of mammary
carcinogenesis is the Human papillomavirus (HPV). The objective was to detect
and analyze HPV in breast carcinomas of women in northeastern Brazil. The
detection of viral DNA was performed PCR positive samples were typed by
sequencing. The quantification of viral load and to determine the physical status by
qPCR, and detection of the oncoproteins E6 and HPV E7 by
immunohistochemistry. HPV DNA was detected in 46.7% of HPV-positive breast
carcinomas. HPV16 was the most prevalent, 92% of cases. The HPV viral load
averaged 14.2 copies in 104 cells in breast carcinomas. Furthermore, 57.2% of
HPV-positive breast carcinomas showed the integrated viral DNA into the host
genome. High rates of detection of E6 (89.5%) and E7 (90%) were identified in
HPV-positive breast tumors. Already the tumor suppressor protein p53 and
p16INK4a, had lower rates 95.7% and 92.3% respectively. The results of this study
suggests that the virus is active in tumor cells and probably play role in breast
carcinogenesis.
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Využití rekombinantních virů vakcinie produkujících IGFBP3 pro terapii nádorů / IGFBP3 expressing rekombinant vaccinia virus used for tumor therapyMusil, Jan January 2010 (has links)
IGFBP-3 expressing rekombinant vaccinia viruses used for tumor therapy Insulin-like growth factor-binding protein-3 (IGFBP-3) is a major regulator of endocrine effects of IGF and is capable to suppress the growth of variety of cancer. Several studies have shown that IGFBP-3 can induce the apoptosis of cancer cells via IGF-dependent and IGF-independent mechanisms. In our study, we have constructed recombinant vaccinia viruses (VACV) expressing IGFBP-3 under the control of the early H5 and synthetic early/late (E/L) promoter to investigate the potential effect on cancer growth in our cervical cancer model. We have shown that the expression of IGFBP-3 alone had no effect on tumor growth. On the other hand, the co-expression of IGFBP-3 enhanced the anti-cancer effect of immunization with the fusion protein SigE7LAMP, which gave rise to the anti-cancer immunity directed against HPV16 induced tumors. We have shown that the double-recombinant P13-SigE7LAMP-H5-IGFBP-3 can enhance the protective immune responses against MK16/ABC induced tumors. Furthermore, we have show that both double-recombinant viruses P13-SigE7LAMP-H5- IGFBP-3 and P13-SigE7LAMP-E/L-IGFBP-3 can increase the anti-cancer effect of SigE7LAMP expression in the therapy of TC-1 induced tumors. Key words: IGFBP-3, IGF, VACV, HPV16, E7 oncoprotein,...
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Detecting uterine cervical cancer cells using molecular biomarkersMousa, Ahmed 11 1900 (has links)
Arrière-plan: les cellules tumorales circulantes (CTC) sont détectables dans de nombreux cancers et peuvent être utiles cliniquement pour le pronostic de la maladie, pour mesurer la récidive et pour prédire la sensibilité aux medicaments chimiothérapeutiques. Au cours des dernières années, l’études des CTC dans de nombreux cancers tels que le cancer du sein, du poumon, du côlon et de la prostate a grandement évolué. Alternativement, il y peu d'études à ce sujet concernant le cancer du col de l’utérus (CCU). Objectifs: Notre objectif est d’optimiser le processus d'enrichissement des CTC dans le CCU et la détection moléculaire des biomarqueurs E6 et E7. Matériel et Méthodes: Dans l’optique de mimer la présence de CTC dans le sang, nous avons dilué des cellules cancéreuses CaSki VPH16-positif provenant d’un CCU dans du sang humain prélevé sur des volontaires sains. Les CaSki ont été collectées suite à une centrifugation par densité avec le Ficoll, la lyse des globules rouges (RBC) et la lyse des RBC combinée avec un enrichissement positif et négatif à l’aide de marqueurs de surface cellulaire. Les CTC ont été détectées par la mesure d’expression des oncogènes E6 et E7 du virus du papillome humain (VPH), de la cytokératine 19 (CK19) et de la cycline p16INK4 en utilisant la technique quantitative en temps réel de Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR). Pour valider notre méthode de détection des CTC in vivo, nous avons recruté dix patientes atteintes d’un CCU VPH16 positif et six contrôles sains. Résultats: Dans le modèle de dilutions de cellules CaSki, la lyse des RBC seule ou combinée avec l'enrichissement négatif ou positif suggèrent des limites de détection de 1 CTC par mL de sang pour tous les biomarqueurs moléculaires utilisés. La sensibilité de détection est accrue lors de l'utilisation de l’enrichissement positif et négatif en réduisant le bruit de fond causé par les monocytes sanguins. Contrairement aux oncogènes E6 et E7, les marqueurs CK19 et p16INK4A ont été détectés chez des individus sains, les niveaux d'expression de base appropriés doivent donc être déterminés avec précision par rapport aux patientes CCU. Le gradient de densité par Ficoll a une limite de détection de seulement environ 1000 cellules par mL de sang. Enfin, les CTC ont été détectées dans 2/10 patientes en utilisant le marqueur CK19. Cependant, ces patientes étaient négatives pour les oncogènes E6/E7. Le marqueur p16INK4A était exprimé au même niveau dans tous les échantillons (CCU et normaux). Conclusion: Notre étude suggère que les oncogènes E6 et E7 du VPH16 sont les marqueurs biologiques les plus sensibles et spécifiques en qRT-PCR pour détecter les CTC dans le modèle de dilution de cellules de CCU dans le sang. Chez les patientes atteintes d’un CCU de stade précoce, seulement CK19 a révélé la présence potentielle de CTC, ce qui suggère que ces cellules sont rares à ce stade de la maladie.
Mots clés: cancer du col de l’utérus, cellules tumorales circulantes, RT-qPCR, E6 et E7, CK19, p16INK4A, enrichissement immunomagnétique, détection moléculaire. / Background: Circulating tumor cells (CTCs) have been detected in many cancers and are used in multiple clinical applications including disease prognosis, tumor recurrence prediction and prediction of tumor sensitivity to chemotherapeutic drugs. Studies in most major solid cancer(s) (breast, lung, colon and prostate) are progressing rapidly, but there has been very little progress concerning uterine cervical cancer (UCC).Objective: our aim is to optimize enrichment processes and the molecular biomarker-based detection of human circulating tumor cells (CTCs) in uterine cervical cancer (UCC). Material & Methods: To mimic CTCs in patients, we designed an experimental spiking model where the CaSki HPV16-positive UCC cell line was serially diluted and spiked into human blood collected from healthy volunteers. CaSki CTCs were enriched using either Ficoll density centrifugation, red blood cell (RBC) lysis or RBC lysis combined with cell surface markers negative or positive enrichment. CTCs were detected using real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure the gene expression of human papillomavirus (HPV) viral oncogenes (E6 and E7), cytokeratin 19 (CK19), or the cyclin dependent kinase inhibitor p16INK4A. Finally, ten HPV16- positive UCC patients and six healthy controls were recruited to validate CTCs detection in vivo. Result: In the spiking model, RBC lysis alone or combined with negative or positive enrichment suggests detection limits close to 1 CTC per mL of blood for all molecular biomarkers used. The sensitivity of detection increased when using positive and negative enrichment probably by reducing the peripheral blood mononuclear cell-derived RNA background. Unlike HPV oncogenes, CK19 and p16INK4A were detected in normal individuals, thus appropriate basal expression levels need to be accurately determined compared to cancer patients. Alternatively, Ficoll density gradient had a detection limit of only about 1000 cells per mL of blood. Finally CTCs were detected in 2/10 patients using CK19. None of the patients had E6/E7 transcripts and p16INK4A was expressed at similar level across all samples (cancer and healthy). Conclusion: qRT-PCR of HPV16 E6 and E7 is the most sensitive and specific biomarker used to detect CTCs in the spiking model. In early disease UCC patients, only CK19 revealed the presence of CTCs suggesting that these cells are rare at that stage of the disease.
Keywords: uterine cervical cancer, circulating tumor cells, qRT-PCR, E6 and E7 oncoprotein, CK19, p16INK4A, immune-magnetic enrichment, molecular detection.
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Produkce heterologních proteinů v rostlinách se zaměřením na antigeny odvozené od lidského papillomaviru (HPV 16) / Production of heterologous proteins in plants - human papillomavirus (HPV 16) derived antigensFolwarczna, Jitka January 2013 (has links)
5 Abstract Even though prophylactic vaccine against human papillomavirus (HPV) is currently licensed, infections by the virus continue to be the major health problem mainly in developing countries. Considerable effort is being devoted to preparation of therapeutic vaccine and to decrease of the production costs of current vaccine. Viral proteins such as the E7 oncoprotein and the L2 capsid protein from HPV type 16 are promising targets for the development of the experimental anti-HPV vaccine. The aim of our work was optimization of expression of mutagenized E7 oncoprotein (E7ggg) fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein (CP) or Potato virus X (PVX) CP in viral vectors derived from these plant viruses. The impact of linkers connecting CP and E7ggg fusion partners on expression and stability of fusion proteins was examined. The fusion proteins were first expressed in Escherichia coli (E. coli) MC1061 to assess the characteristics of the recombinant protein prior to their transient expression in both non-transgenic or transgenic Nicotiana benthamiana (N. benthamiana). We have obtained the high level expression in E. coli, but most of the expressed proteins based on TMV CP remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular...
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