Unravelling the role of mitochondrial TERT in a thyroid cancer cell line

Detalhes bibliográficos
Autor(a) principal: Chantre, Ana Sofia Moreira
Data de Publicação: 2023
Tipo de documento: Dissertação
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10400.22/24754
Resumo: Besides its established canonical function as the catalytic subunit of telomerase involved in telomere elongation, there is increasing evidence that suggest that Telomerase Reverse Transcriptase (TERT) plays several other (non-canonical) celular functions. In particular, i tis known that TERT can translocate into mitochondria. However, the precise mitochondrial function(s) of TERT remains to be fully unravelled. In the contexto of thyroid cancer TC), TERT reactivation is often associated with worse prognostic features. However, the canonical functions of TERT alone are not enough to account for these clinical associations. Therefore, the main objective of this project was to unravel non-canonical roles of TERT, in particular within the contexto of mitochondria, by comprehensively characterizing TPC-1 thyroid cancer cells which have been genetically modified to disrupt TERT’s-N-terminal Mitochondrial Target Sequence (MTS), thus preventing its translocation into mitochondria. For this, two clones were chosen, clone “noMTS1” and “noMTS2”, both preserving the in-frame TERT sequence but with only clone “noMTS1” retaining telomerase activity. Our results from phase contract microscopy and PrestoBlue viability assay revealed that the growth rate of the clones was diferente from that of their Mock control counterpart and also that it changed throughout the passege of the cells. Clone “noMTS1” cells seemed to be larger and their growth became increasingly higher over-time in culture. Clone “noMTS2” cells, allthough initially showing similar morphology and growth patterns to that of Mock cells, their size increased over-time and these cells lost thei initial growth capacity. Fluorescence and transmission electron microscopy (TEM) analysis revealed increased heterogeneity in cell size (which tended to be larger), and a noticeable increase in nucleus size, in lone “noMTS1” and “noMTS2” cells, compared to Mock cells. TEM analysis further reealed that clone “noMTS1” cells had higher mitochondria number, and that both clone “noMTS1” and “noMTS2” presented mitophagy- and autophagy- associated features. Seahorse metabolic assay showed that clone “noMTS1” had increased oxygen consumption rates in response to FCCP, and a slight tendency for higher spare respiratory capacity and maximal respiration levels. Overall, our study on the characterization of genetically manipulated TPc-1 cells which prevented TERT’s translocation into mitochondri revealed relevant diferences regarding cell growth and survival, and celular, nuclear, and mitochondrial features of these cells, further supporting that mitochondrial TERT has a fundamental role in thyroid cancer cells physiology.
id RCAP_30179ecc1d43b6bed12b4e3c8fe903fc
oai_identifier_str oai:recipp.ipp.pt:10400.22/24754
network_acronym_str RCAP
network_name_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository_id_str 7160
spelling Unravelling the role of mitochondrial TERT in a thyroid cancer cell lineTERTNon-canonical functionsMitochondriaTyroid cancerBesides its established canonical function as the catalytic subunit of telomerase involved in telomere elongation, there is increasing evidence that suggest that Telomerase Reverse Transcriptase (TERT) plays several other (non-canonical) celular functions. In particular, i tis known that TERT can translocate into mitochondria. However, the precise mitochondrial function(s) of TERT remains to be fully unravelled. In the contexto of thyroid cancer TC), TERT reactivation is often associated with worse prognostic features. However, the canonical functions of TERT alone are not enough to account for these clinical associations. Therefore, the main objective of this project was to unravel non-canonical roles of TERT, in particular within the contexto of mitochondria, by comprehensively characterizing TPC-1 thyroid cancer cells which have been genetically modified to disrupt TERT’s-N-terminal Mitochondrial Target Sequence (MTS), thus preventing its translocation into mitochondria. For this, two clones were chosen, clone “noMTS1” and “noMTS2”, both preserving the in-frame TERT sequence but with only clone “noMTS1” retaining telomerase activity. Our results from phase contract microscopy and PrestoBlue viability assay revealed that the growth rate of the clones was diferente from that of their Mock control counterpart and also that it changed throughout the passege of the cells. Clone “noMTS1” cells seemed to be larger and their growth became increasingly higher over-time in culture. Clone “noMTS2” cells, allthough initially showing similar morphology and growth patterns to that of Mock cells, their size increased over-time and these cells lost thei initial growth capacity. Fluorescence and transmission electron microscopy (TEM) analysis revealed increased heterogeneity in cell size (which tended to be larger), and a noticeable increase in nucleus size, in lone “noMTS1” and “noMTS2” cells, compared to Mock cells. TEM analysis further reealed that clone “noMTS1” cells had higher mitochondria number, and that both clone “noMTS1” and “noMTS2” presented mitophagy- and autophagy- associated features. Seahorse metabolic assay showed that clone “noMTS1” had increased oxygen consumption rates in response to FCCP, and a slight tendency for higher spare respiratory capacity and maximal respiration levels. Overall, our study on the characterization of genetically manipulated TPc-1 cells which prevented TERT’s translocation into mitochondri revealed relevant diferences regarding cell growth and survival, and celular, nuclear, and mitochondrial features of these cells, further supporting that mitochondrial TERT has a fundamental role in thyroid cancer cells physiology.Lima, Raquel T.Máximo, ValdemarVieira, Mónica Andreia AlmeidaRepositório Científico do Instituto Politécnico do PortoChantre, Ana Sofia Moreira2023-11-292026-11-29T00:00:00Z2023-11-29T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10400.22/24754TID:203472659enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-02-07T01:48:23Zoai:recipp.ipp.pt:10400.22/24754Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T01:59:07.753460Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse
dc.title.none.fl_str_mv Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
title Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
spellingShingle Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
Chantre, Ana Sofia Moreira
TERT
Non-canonical functions
Mitochondria
Tyroid cancer
title_short Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
title_full Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
title_fullStr Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
title_full_unstemmed Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
title_sort Unravelling the role of mitochondrial TERT in a thyroid cancer cell line
author Chantre, Ana Sofia Moreira
author_facet Chantre, Ana Sofia Moreira
author_role author
dc.contributor.none.fl_str_mv Lima, Raquel T.
Máximo, Valdemar
Vieira, Mónica Andreia Almeida
Repositório Científico do Instituto Politécnico do Porto
dc.contributor.author.fl_str_mv Chantre, Ana Sofia Moreira
dc.subject.por.fl_str_mv TERT
Non-canonical functions
Mitochondria
Tyroid cancer
topic TERT
Non-canonical functions
Mitochondria
Tyroid cancer
description Besides its established canonical function as the catalytic subunit of telomerase involved in telomere elongation, there is increasing evidence that suggest that Telomerase Reverse Transcriptase (TERT) plays several other (non-canonical) celular functions. In particular, i tis known that TERT can translocate into mitochondria. However, the precise mitochondrial function(s) of TERT remains to be fully unravelled. In the contexto of thyroid cancer TC), TERT reactivation is often associated with worse prognostic features. However, the canonical functions of TERT alone are not enough to account for these clinical associations. Therefore, the main objective of this project was to unravel non-canonical roles of TERT, in particular within the contexto of mitochondria, by comprehensively characterizing TPC-1 thyroid cancer cells which have been genetically modified to disrupt TERT’s-N-terminal Mitochondrial Target Sequence (MTS), thus preventing its translocation into mitochondria. For this, two clones were chosen, clone “noMTS1” and “noMTS2”, both preserving the in-frame TERT sequence but with only clone “noMTS1” retaining telomerase activity. Our results from phase contract microscopy and PrestoBlue viability assay revealed that the growth rate of the clones was diferente from that of their Mock control counterpart and also that it changed throughout the passege of the cells. Clone “noMTS1” cells seemed to be larger and their growth became increasingly higher over-time in culture. Clone “noMTS2” cells, allthough initially showing similar morphology and growth patterns to that of Mock cells, their size increased over-time and these cells lost thei initial growth capacity. Fluorescence and transmission electron microscopy (TEM) analysis revealed increased heterogeneity in cell size (which tended to be larger), and a noticeable increase in nucleus size, in lone “noMTS1” and “noMTS2” cells, compared to Mock cells. TEM analysis further reealed that clone “noMTS1” cells had higher mitochondria number, and that both clone “noMTS1” and “noMTS2” presented mitophagy- and autophagy- associated features. Seahorse metabolic assay showed that clone “noMTS1” had increased oxygen consumption rates in response to FCCP, and a slight tendency for higher spare respiratory capacity and maximal respiration levels. Overall, our study on the characterization of genetically manipulated TPc-1 cells which prevented TERT’s translocation into mitochondri revealed relevant diferences regarding cell growth and survival, and celular, nuclear, and mitochondrial features of these cells, further supporting that mitochondrial TERT has a fundamental role in thyroid cancer cells physiology.
publishDate 2023
dc.date.none.fl_str_mv 2023-11-29
2023-11-29T00:00:00Z
2026-11-29T00:00:00Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/masterThesis
format masterThesis
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10400.22/24754
TID:203472659
url http://hdl.handle.net/10400.22/24754
identifier_str_mv TID:203472659
dc.language.iso.fl_str_mv eng
language eng
dc.rights.driver.fl_str_mv info:eu-repo/semantics/embargoedAccess
eu_rights_str_mv embargoedAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron:RCAAP
instname_str Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
instacron_str RCAAP
institution RCAAP
reponame_str Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
collection Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
repository.mail.fl_str_mv
_version_ 1799137074922651648