{"id":4799,"date":"2023-11-27T00:00:00","date_gmt":"2023-11-27T00:00:00","guid":{"rendered":"https:\/\/www.proefschriftmaken.nl\/portfolio\/samara-rosendo-machado\/"},"modified":"2026-03-18T08:27:54","modified_gmt":"2026-03-18T08:27:54","slug":"samara-rosendo-machado","status":"publish","type":"us_portfolio","link":"https:\/\/www.proefschriftmaken.nl\/en\/portfolio\/samara-rosendo-machado\/","title":{"rendered":"Samara Rosendo Machado"},"content":{"rendered":"","protected":false},"excerpt":{"rendered":"","protected":false},"author":7,"featured_media":4801,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_acf_changed":false,"footnotes":""},"us_portfolio_category":[45],"class_list":["post-4799","us_portfolio","type-us_portfolio","status-publish","has-post-thumbnail","hentry","us_portfolio_category-new-template"],"acf":{"naam_van_het_proefschift":"TRANSCRIPTIONAL REGULATION OF HOST RESPONSES TO VIRUS INFECTION IN AEDES AEGYPTI MOSQUITOES","samenvatting":"zien. Het mechanisme dat ten grondslag ligt aan de antivirale functie van \u00e9\u00e9n van deze RNA helicases, Dhx15, is vervolgens verder onderzocht. Dhx15 reguleert de expressie van genen die betrokken zijn bij glycolyse, een proces dat een centrale rol speelt in de energiehuishouding van de cel. Interessant was dat chikungunyavirus-infectie leidt tot een soortgelijke verandering in genexpressie, wat suggereert dat Dhx15 virusreplicatie be\u00efnvloedt door de modulatie van de glycolyse cascade.\n\nIn hoofdstuk 3 hebben we ons gericht op andere antivirale eiwitten die we hebben ge\u00efdentificeerd in de genetische screen uit hoofdstuk 2. Een interessant resultaat was dat vier van deze eiwitten, SPT4, SPT5, SPT6, en Brd4, allemaal een rol spelen in de regulatie van transcriptie, in een proces dat transcriptionele pauzering wordt genoemd. Remming van deze eiwitten zorgde voor een toename van de replicatie van verschillende arbovirussen, waaronder het chikungunyavirus. Gebaseerd op deze bevindingen hebben we de hypothese opgesteld dat virale replicatie een transcriptionele afweerreactie stimuleert, en dat transcriptionele pauzering deze reactie reguleert. Echter, chikungunyavirus-infectie in muggencellen gaf een lagere transcriptionele respons dan we hadden verwacht. Daarnaast behoorden de ge\u00efnduceerde genen niet tot de bekende afweermechanismen. In plaats daarvan zagen we een verhoging van de expressie van heat-shock eiwitten, een bekende stressreactie van de cel. Deze stressreactie was afhankelijk van specifieke eiwitten die betrokken zijn bij transcriptionele pauzering, wat suggereert dat dit mechanisme bijdraagt aan de regulatie van de stressrespons na virale infectie.\n\nIn hoofdstuk 4 hebben we het werkingsmechanisme van Brd4, \u00e9\u00e9n van de factoren die transcriptie reguleert, verder gekarakteriseerd. Dit eiwit is betrokken bij de zogenaamde epigenetische regulatie van genexpressie. Epigenetische regulatie vindt plaats door dynamische veranderingen van histonen, de structurele eiwitcomplexen waar het DNA omheen gebonden is. Hierin heeft Brd4 een belangrijke rol, met name tijdens actieve transcriptie. We hebben gebruik gemaakt van een sterke farmacologische remmer van Brd4, genaamd JQ1, om de rol van Brd4 in virusinfectie verder te ontrafelen. Het behandelen van muggencellen met JQ1 resulteerde in dramatische veranderingen in genexpressie. Opvallend hierin was dat vele differentieel gereguleerde genen afhankelijk zijn van de Forkhead box O (FOXO) transcriptie factor voor hun expressie. Het veranderen van FOXO genexpressie en activiteit had een vergelijkbaar effect op virusreplicatie als JQ1 behandeling. We concluderen daarom dat Brd4 de expressie be\u00efnvloedt van FOXO-afhankelijke genen en dat dit vervolgens de virusreplicatie be\u00efnvloedt.\n\nIn hoofdstuk 5 worden de bevindingen van dit proefschrift bediscussieerd in een bredere context. Verder reflecteer ik op de voordelen en uitdagingen van het muggenmodel dat in deze studies is gebruikt en bespreek ik wat er kan worden gedaan om deze studies voort te zetten. Samenvattend geeft dit proefschrift inzicht in nieuwe mechanismes die bijdragen aan de afweer van Aedes aegypti muggen tegen virussen.","summary":"Virus transmission from an infected host to a na\u00efve person is a crucial aspect of the viral life cycle and viruses have evolved multiple ways to achieve this challenge. A particularly interesting mode of transmission is by blood-feeding insects as it requires viruses to replicate in two evolutionary distinct hosts. Viruses that are transmitted between vertebrate host by insects or other arthropods are named arboviruses (arthropod-borne viruses), the majority of which are transmitted by mosquitoes. Most epidemic arboviruses, such as dengue and chikungunya viruses, were previously restricted to tropical and subtropical countries, but are nowadays also endemic in more template regions of the world. This development has largely been driven by the expansion of vector mosquitoes due to environmental change, rising global temperatures, or increased travel and trade.\n\nDengue and chikungunya virus transmission is mainly driven by Aedes aegypti, also known as the yellow fever mosquito, which is well-adapted to the urban habitat and can directly transmit disease from human to human. Importantly, the effectiveness of transmission depends on the ability of arboviruses to efficiently replicate in the insect vector. Mosquitoes acquire arboviruses during a blood meal on a viremic person, after which the viruses infect the midgut, the mosquito\u2019s equivalent of a stomach. Next, viruses need to replicate in secondary tissues to finally infect the mosquito salivary gland. In this organ, virus replication leads to shedding of new virus particles into the mosquito saliva which is essential for transmission to the next non-infected host. Besides physical tissue barriers, arboviruses have to overcome immune responses that are induced in the infected mosquito. Amongst the different immune pathways that exist in mosquitoes, RNA interference (RNAi) is the best characterized antiviral defense mechanism. This pathway is mediated by small RNAs derived from replicating viral RNA. Ultimately, the function of these small RNAs is to restrict virus replication by directly degrading the viral RNA. Besides the well-established RNAi pathway, other immune defense mechanisms such as transcriptionally-induced pathways (Toll, IMD, and JAK-STAT) have been suggested to act in antiviral defense and other currently unknown mechanisms likely exist. The aim of this doctoral thesis was to discover and characterize such unexplored pathways.\n\nIn chapter 2, we knocked down hundreds of so-called RNA binding proteins in mosquito cells to identify novel factors that act in mosquito antiviral defense. RNA binding proteins often play important roles in immune pathways, and indeed, we identified three RNA binding proteins belonging to the family of DEAD-box RNA helicases, that showed a broad antiviral phenotype against multiple arboviruses. From these, the mechanism underlying the antiviral function of the RNA helicase Dhx15 was further investigated. Dhx15 regulates the expression of genes that are involved in glycolysis, one of the central pathways that controls energy metabolism in the cell. Interestingly, also chikungunya virus infection leads to similar changes in glycolytic gene expression, suggesting that Dhx15 may affect virus replication in Ae. aegypti mosquito cells via modulation of the glycolysis pathway.\n\nIn chapter 3, we focused on the antiviral role of additional players that were picked up in our knockdown screen. Interestingly, four of the identified hits (SPT4, SPT5, SPT6, and Brd4) were proteins that act in the regulation of transcription in a process called transcriptional pausing. Knockdown of these transcriptional pausing factors caused increase replication of several arboviruses including chikungunya virus. Based on these findings, we hypothesized that virus replication triggers a transcriptional immune response and that transcriptional pausing controls this response. However, chikungunya virus infection only caused a surprisingly modest transcriptional response that lacked signatures of canonical immune pathway activation but instead resembled a general stress response involving the upregulation of heat-shock proteins. The heat-shock response was dependent on specific members of the transcriptional pausing machinery, suggesting that this mechanism contributes to the regulation of host responses to virus infection.\n\nIn chapter 4, we focused on the characterization of one of the factors that acts in transcriptional regulation: Brd4. This protein is involved in regulating gene expression at the epigenetic level. Epigenetic regulation of transcription involves, among other things, the dynamic deposition and interpretation of modifications of histones, the structural protein core that DNA is wrapped around. In this context, Brd4 is an important reader of histone modifications and is usually associated with active transcription. We made use of the fact that a potent pharmacological inhibitor (JQ1) is available for Brd4. Treating mosquito cells with JQ1 resulted in a fierce increase of virus replication that greatly exceeded the effect of Brd4 knockdown. The mechanism by which Brd4 inhibition affects antiviral defense was investigated. As expected for an inhibitor of a transcriptional regulator, JQ1 treatment resulted in a dramatic change in gene expression. Interestingly, amongst the differentially expressed genes target genes of the Forkhead box O (FOXO) transcription factors were enriched. FOXO had previously been associated with antiviral defense in insects, and indeed, modulating its expression or activity affected virus replication in a similar way as JQ1 treatment, suggesting that Brd4 controls virus replication at least partly by regulating FOXO target genes.\n\nIn chapter 5, the findings from this doctoral thesis are discussed in a broader context. Furthermore, I reflect on the advantages and challenges of the mosquito model used and I discuss suggestions for further research. Overall, this doctoral thesis provides insights into novel mechanisms that underlie antiviral defense in the Ae. aegypti mosquito.","auteur":"Samara Rosendo Machado","auteur_slug":"samara-rosendo-machado","publicatiedatum":"27 november 2023","taal":"EN","url_flipbook":"https:\/\/ebook.proefschriftmaken.nl\/ebook\/samararosendomachado?iframe=true","url_download_pdf":"https:\/\/ebook.proefschriftmaken.nl\/download\/e9f2f0c0-af2b-4101-add0-dddc4ce2149f\/optimized","url_epub":"","ordernummer":"FTP-202602261458","isbn":"978-94-6469-587-8","doi_nummer":"","naam_universiteit":"Radboud Universiteit","afbeeldingen":4801,"naam_student:":"","binnenwerk":"","universiteit":"Radboud Universiteit","cover":"","afwerking":"","cover_afwerking":"","design":""},"_links":{"self":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/4799","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio"}],"about":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/types\/us_portfolio"}],"author":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/users\/7"}],"replies":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/comments?post=4799"}],"version-history":[{"count":1,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/4799\/revisions"}],"predecessor-version":[{"id":4802,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio\/4799\/revisions\/4802"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media\/4801"}],"wp:attachment":[{"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/media?parent=4799"}],"wp:term":[{"taxonomy":"us_portfolio_category","embeddable":true,"href":"https:\/\/www.proefschriftmaken.nl\/en\/wp-json\/wp\/v2\/us_portfolio_category?post=4799"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}