53,287 research outputs found

    Cellular and molecular characterization of the corneal epithelium in Xenopus frogs

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    The vertebrate cornea is a transparent, avascular tissue that forms the front window of the eye. Specifically, the corneal epithelium is exposed to detrimental conditions including infections, solar (Ultraviolet, UV) irradiation, mechanical injuries, and undergoes constant self-renewal. Corneal epithelial stem cells (CESCs) and their progeny, the transit amplifying cells (TACs), play a prominent role in the maintenance of corneal homeostasis, transparency, and wound repair processes. Towards this end, my thesis focuses on understanding the molecular signature of the corneal epithelium using the frog, Xenopus laevis, with the goal to explore novel markers that will reliably identify populations of CESCs and TACs. In Chapter 2, I start by examining the expression of known corneal biomarkers (previously reported in literature from corneal studies of various vertebrates) in the vertebrate species, the African clawed frog. Here, I used antibody-based immunohistochemical staining to molecularly characterize the expression of nine proteins in the corneas of both Xenopus larvae and post-metamorphic adults. I found that localization of some markers changes between tadpole and juvenile adult stages. Markers such as p63, Keratin19, and beta1-integrin are restricted to basal corneal epithelial cells of the larvae. After metamorphosis their expression is found in basal and intermediate layer cells of the adult frog cornea epithelium. Another protein, Pax6 was expressed in the larval corneas, but surprisingly it was not detectable in the adult corneal epithelium. For the first time we report that Tcf7l2 can be used as a marker to differentiate cornea vs. skin in frogs. Tcf7l2 is present only in the frog skin, which differs from reports indicating that the protein is expressed in the human cornea. Furthermore, I identified the transition between the inner, and the outer surface of the adult frog eyelid as a key boundary in terms of marker expression. Although these markers are useful to identify different regions and cellular layers of the frog corneal epithelium, none was unique to CESCs or TACs. The results of this study substantiate findings from other studies in the field indicating that there may not be a single conserved, specific CESC marker in vertebrates. To overcome the limitation of candidate-based biomarker characterization, however, I undertook single-cell genomics approaches to understand the temporal cell atlas of the frog cornea (Chapter 3). Using ~22,000 corneal cells isolated from two distinct developmental time-points, this work provides key insights about the amphibian corneal transcriptome. The data also reveals several novel genes expressed in corneal cells and spatiotemporal changes in gene expression during corneal differentiation. In addition, the data helps in understanding the developmental trajectory of corneal cells during development and differentiation, and identifies key gene regulatory networks that are involved in corneal maturation. In conclusion, this work provides a detailed molecular characterization of the Xenopus corneal epithelium and establishes distinct and newly identified biomarkers of corneal cellular layers. Although a single biomarker for CESCs was not identified in this work (and may not even exist), it helps identify a range of proteins that could be tested for their functional role in regulating the population of corneal stem cells in vertebrates, including humans. Furthermore, this work will be valuable for future studies to understand the critical factors that regulate cornea epithelial cells and lens regeneration, the latter phenomenon being unique to the larval stages of Xenopus frogs.LimitedAuthor requested closed access (OA after 2yrs) in Vireo ETD syste

    Comparative histomorphometric and histochemical analysis of gastrointestinal tract of five rodent species

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    Prehrana Ňĺivotinja odraŇĺava se u grańĎi probavnog sustava, a razlike se mogu ońćitovati u debljini pojedinih slojeva te broju i tipu Ňĺljezdanih vrńćastih stanica koje izluńćuju mukozni sekret za probavu i zaŇ°titu. U ovom radu izabrala sam pet vrsta glodavaca koji Ňĺive u simpatriji na krŇ°kom podruńćju centralne Dalmacije, s ciljem utvrńĎivanja postoje li histomorfometrijske i histokemijske razlike u grańĎi probavnog sustava koje su rezultat razlika u prehrani ovih vrsta. Ukljuńćene vrste su Apodemus epimelas, Apodemus sylvaticus, Dinaromys bogdanovi, Eliomys quercinus i Glis glis. Histomorfometrijski rezultati su pokazali veńáu debljinu oroŇĺenog epitela jednjaka i manju debljinu vanjskog miŇ°ińánog sloja jednjaka kod karnivornih vrsta, dok vrste koje se hrane visoko kalorińćnom i lako probavljivom hranom imaju manju debljinu sluznice Ňĺlijezdanog dijela Ňĺeluca i deblji epitel debelog crijeva. Vrste kod kojih prehrana sadrŇĺi otrovne tvari imaju specifińćne prilagodbe debljine pojedinih slojeva. Histokemijska analiza pokazala je da karnivorne vrste imaju viŇ°e kiselih vrńćastih stanica u tankom crijevu, a granivorne viŇ°e mjeŇ°ovitih. Ovi rezultati predstavljaju nove podatke za ukljuńćene vrste i daju novi uvid u postojanje razlika izmeńĎu srodnih vrsta istraŇĺivanih glodavaca s razlińćitom prehranom, ali i izmeńĎu razlińćitih porodica, miŇ°eva iz roda Apodemus i puhova, sa slińćnom prehranom.Animal diet is reflected in the structure of the digestive tract. Some of the differences can be manifested in the thickness of individual layers or the number and type of glandular goblet cells that secrete mucose for digestion and protection. For this thesis, I selected five species of rodents that live in a sympatry in the karstic area of central Dalmatia, with the aim of determining if there are any histomorphometric and histochemical differences in the structure of the gastrointestinal tract, as a result of distinct diets of the species. Species that were included are Apodemus epimelas, Apodemus sylvaticus, Dinaromys bogdanovi, Eliomys quercinus i Glis glis. The histomorphometric results showed thicker esophageal keratenized epithelium and thinner esophageal outer muscular layer in carnovorous species, while species with highcalorie and easily digestible diets have thinner mucose layer of the glandular part of the stomach and thicker epithelium of the large intestine. Species whose diet contains toxic subtances showed specific adaptations in the thicknesses of certain layers. Histochemical analysis showed that carnivorous species have more acidic goblet cells in the small intestine, while granivorous species have more mixed ones. These results represent new data for included species and provide new insight into the existence of both differences between related species of rodents with distinct diets, and between species of different families but with similar diet, as between Apodemus genus and dormice species

    GABA receptors in the olfactory epithelium of the gilthead seabream (Sparus aurata)

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    Exposure to high PCO2/low pH seawater induces behavioural alterations in fish; a possible explanation for this is a reversal of Cl-/HCO3- currents through GABAA receptors (the GABAA receptor theory). However, the main evidence for this is that gabazine, a GABAA receptor antagonist, reverses these effects when applied to the water, assuming that exposure to systems other than the CNS would be without effect. Here, we show the expression of both metabotropic and ionotropic GABA receptors, and the presence of GABAA receptor protein, in the olfactory epithelium of gilthead seabream. Furthermore, exposure of the olfactory epithelium to muscimol (a specific GABAA receptor agonist) increases or decreases the apparent olfactory sensitivity to some odorants. Thus, although the exact function of GABAA receptors in the olfactory epithelium is not yet clear, this may complicate the interpretation of studies wherein water-borne gabazine is used to reverse the effects of high CO2 levels on olfactory-driven behaviour in fish.info:eu-repo/semantics/publishedVersio

    Papel de BAMBI en la respuesta inmune de las mucosas y en el desarrollo de patología inflamatoria intestinal y de neoplasias secundarias

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    RESUMEN: Previamente hemos demostrado que BAMBI (Bone Morphogenetic Protein and Activin Membrane Bound Inhibitor) un inhibidor de la se√Īalizaci√≥n de TGFő≤ a nivel de la membrana celular, inhibe la diferenciaci√≥n de los linfocitos T CD4+ hacia c√©lulas T reguladoras (Tregs) tolerog√©nicas y potencia la de las c√©lulas proinflamatorias Th17. A su vez, hemos observado que la ausencia de BAMBI incrementa la diferenciaci√≥n a c√©lulas Treg y que los ratones deficientes en BAMBI (BAMBI-KO) est√°n protegidos frente al desarrollo de distintas patolog√≠as autoinmunes como la artritis inducida por col√°geno o la psoriasis. En esta Tesis Doctoral se estudia el papel de BAMBI en la respuesta inmune de la mucosa intestinal (GALT) y en el desarrollo de patolog√≠a inflamatoria intestinal. Hemos visto que BAMBI regula la composici√≥n del sistema inmune adaptativo del GALT y que los ratones BAMBI-KO est√°n protegidos frente al desarrollo de colitis inducida por sulfato de dextrano s√≥dico (DSS). As√≠ mismo hemos observado que esta protecci√≥n est√° asociada al efecto de la ausencia de BAMBI en el epitelio intestinal, que es m√°s resistente en los ratones BAMBI-KO, y que la microbiota intestinal tiene un papel fundamental en esta protecci√≥n. La ausencia de BAMBI tambi√©n confiere protecci√≥n frente al desarrollo de c√°ncer de colon secundario a inflamaci√≥n cr√≥nica.ABSTRACT: We have previously shown that BAMBI (Bone Morphogenetic Protein and Activin Membrane Bound Inhibitor), an inhibitor of TGFő≤ signaling at the cell membrane level, inhibits the differentiation of CD4 + T lymphocytes into tolerogenic T-regulatory cells (Tregs) and enhances that of Th17 proinflammatory cells. In turn, we observed that the absence of BAMBI increases the differentiation of Treg and protects mutant mice against the development of different autoimmune pathologies such as collagen-induced arthritis or psoriasis. In this Doctoral Thesis we have studied the role of BAMBI in the immune response of the intestinal mucosa (GALT) and in the development of inflammatory bowel pathology. We demonstrate that BAMBI regulates the composition of the adaptive immune system of GALT and that BAMBI-KO mice are protected against the development of colitis induced by sodium dextran sulfate (DSS). We have also observed that this protection is secondary to the absence of BAMBI in the intestinal epithelium, which is more resistant in BAMBI-KO mice, and that the intestinal microbiota plays a fundamental role in this protection. The absence of BAMBI also confers protection against the development of colon cancer secondary to chronic inflammation

    Utiliza√ß√£o de plasma aut√≥logo no tratamento de √ļlceras da c√≥rnea no c√£o : estudo retrospetivo de 30 casos

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    Disserta√ß√£o de Mestrado Integrado em Medicina Veterin√°ria. √Ārea Cient√≠fica - Cl√≠nicaO plasma sangu√≠neo apresenta caracter√≠sticas semelhantes √†s da pel√≠cula lacrimal. Cont√©m componentes que, quando aplicados topicamente, promovem a regenera√ß√£o do tecido corneal e asseguram a sua integridade e viabilidade. Col√≠rios formulados a partir de sangue do paciente s√£o relativamente f√°ceis de obter e normalmente bem tolerados, sendo usados no tratamento de diversas doen√ßas oculares. Em √ļlceras de c√≥rnea complicadas, para al√©m de potenciar a cura, diminui ainda a atividade das colagenases envolvidas no processo. O presente estudo teve como objetivo realizar uma avalia√ß√£o retrospetiva sobre a utiliza√ß√£o de plasma aut√≥logo no tratamento de √ļlceras da c√≥rnea em c√£es. Para isso, foram avaliados pacientes diagnosticados com √ļlceras corneais no HRVM num per√≠odo de seis meses. Foi feita a caracteriza√ß√£o da amostra, da doen√ßa e do tratamento. Para determinar a efic√°cia da plasmaterapia, foram comparados os tempos medianos de cicatriza√ß√£o das les√Ķes superficiais tratadas com e sem plasma. Para defeitos graves (indolentes e profundos), estes tempos foram correlacionados de acordo com a frequ√™ncia da administra√ß√£o. Recorreu-se a uma an√°lise de sobreviv√™ncia para tratamento estat√≠stico destes dados. A amostra incluiu 30 c√£es de 14 ra√ßas distintas, maioritariamente braquic√©falas (70%). A idade m√©dia foi de 6.69 ¬Ī 4.03 anos (compreendida entre 0.17-14 anos). 43.33% (n=13) das les√Ķes eram estromais e 56.67% (n=17) afetaram apenas o epit√©lio. Destas, 40% (n=12) responderam ao tratamento e 16.67% (n=5) tiveram um comportamento indolente. A maioria das √ļlceras superficiais n√£o era extensa (n=6; 50%) e as profundas eram predominantemente vastas e mal√°cicas (n=4; 30.77%). O tratamento variou, tendo sido aplicado plasma aut√≥logo em 5 das 12 les√Ķes superficiais e em todas as graves (n=18). N√£o se encontraram diferen√ßas estatisticamente significativas entre os tempos medianos de recupera√ß√£o das les√Ķes superficiais tratadas com e sem plasma (Kaplan Meier; p=0.9), nem entre os tempos de cura dos defeitos graves de acordo com a frequ√™ncia de administra√ß√£o (Kaplan Meier; p=0.9). No presente estudo n√£o foram observados efeitos adversos decorrentes da utiliza√ß√£o de col√≠rios de plasma aut√≥logo, tendo sido demonstrado ser um produto bem tolerado pelos pacientes. N√£o foi poss√≠vel obter resultados estatisticamente significativos que comprovem a sua efic√°cia, devido √† heterogeneidade da amostra, diferen√ßas nos protocolos terap√™uticos adotados e aus√™ncia de grupos controlo. Estudos adicionais poder√£o contribuir para evidenciar os seus benef√≠cios terap√™uticos.ABSTRACT - Use of autologous plasma in the treatment of corneal ulcers in the dog: a retrospective study of 30 cases - Blood plasma presents characteristics similar to those of the tear film. It contains components that, when applied topically, promote corneal tissue regeneration and ensure its integrity and viability. Eye drops formulated from the patient's blood are relatively easy to obtain and usually well tolerated, and are used in the treatment of various ocular diseases. In complicated corneal ulcers, not only enhances healing but also decreases the activity of the collagenases involved in the process. The present study aimed to perform a retrospective evaluation on the use of autologous plasma in the treatment of corneal ulcers in dogs. Patients diagnosed with corneal ulcers at the HRVM were evaluated over a six-month period. Characterization of the sample, disease and treatment was performed. To determine the efficacy of plasmatherapy, median healing times of superficial lesions treated with and without autologous plasma were compared. For severe defects (indolent and deep) these times were correlated according to the administration frequency. Survival analysis was used for statistical treatment of these data. The sample included 30 dogs of 14 different breeds, mostly brachycephalic (70%). The mean age was 6.69¬Ī4.03 years (range 0.17-14 years). 43.33% (n=13) of the lesions were stromal and 56.67% (n=17) affected the epithelium only. From these, 40% (n=12) responded to treatment and 16.67% (n=5) had an indolent behavior. The majority of the superficial ulcers were not extense (n=6; 50%) and the deep ones were predominantly wide and melting (n=4; 30.77%). The treatment varied, with plasma being applied to 5 of the 12 superficial lesions and in all (n=18) severe ulcers. Median recovery times for superficial lesions treated with or without plasma were compared (Kaplan Meier; p=0.9) as well as healing times in severe defects according to the frequency of administration (Kaplan Meier; p=0.9). No significant statistical differences were found. In the present study, no adverse effects from the use of autologous plasma eye drops were observed, having been shown to be a product well tolerated by patients. It was not possible to obtain statistically significant results that prove its efficacy, due to the heterogeneity of the sample, differences in the therapeutic protocols adopted and the absence of control groups. Further studies may contribute to evidence its therapeutic benefits.N/

    Molecular diagnosis of Mycoplasma bovis

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    Disserta√ß√£o de Mestrado Integrado em Medicina Veterin√°riaMycoplasma bovis is a bacteria responsible for different disease presentations in cattle, such as pneumonia, mastitis, otitis, genital disorders, keratoconjuntivitis and arthritis, presently considered as one of the major emerging pathogens affecting cattle. Until this day, it is responsible for losses in animal production of over 150 million euros across Europe. The pathogenesis of Mycoplasma-associated diseases is multifactorial and the highly variable surface lipoproteins allows a fast and efficient dissemination of M. bovis within the host and the herd. Due to its high antigenic plasticity, its ability to survive within multiple host cells and the capacity to establish multiple synergistic interactions with other pathogens, makes M. bovis and associated infections are a major challenge in Veterinary Medicine, since the vaccine is not efficient and antibiotics are almost inefficient. This study aims at developing and validating a quantitative PCR protocol for the diagnosis of M. bovis. 93 milk samples, from 5 different Portuguese farms, were collected, processed and each one‚Äôs DNA extracted to be analyzed through a qPCR method targeting the uvrC and uvrC2024 genes. Given the percentage of positivity, which was high, the conclusion we can take from the study is that there is still work to do, in terms of establishing a uniformed practice to tackle the wide presence of M. bovis in farms.RESUMO - Diagn√≥stico Molecular de M. bovis - Mycoplasma bovis √© uma bact√©ria cuja infec√ß√£o pode ter diferentes apresenta√ß√Ķes tais como pneumonia, mastite, otite, afec√ß√Ķes genitais, queratoconjuntivite e artrites. √Č um dos agentes considerados emergentes e que afecta a produ√ß√£o agropecu√°ria, sendo respons√°vel por perdas na ordem dos 150 milh√Ķes de euros na Europa. Tem uma patog√©nese multifactorial e as prote√≠nas membranares √† sua superf√≠cie conferem uma variabilidade que permite uma r√°pida e eficiente dissemina√ß√£o no hospedeiro, e no rebanho. Esta variabilidade e capacidade de resistir √† imunidade do hospedeiro, assim como as suas interac√ß√Ķes sin√©rgicas com outros agentes patog√©nicos, tornam as infec√ß√Ķes por M. bovis um obst√°culo dif√≠cil de conter e ultrapassar na Medicina Veterin√°ria, isto porque quer a antibioterapia, quer a vacina, n√£o s√£o eficientes. Este estudo procura desenvolver e contribuir para o estabelecimento de um protocolo de diagn√≥stico para a detec√ß√£o de M. bovis. Vindas de 5 produ√ß√Ķes portuguesas diferentes, 93 amostras foram processadas e analisadas atrav√©s de um qPCR, com os genes uvrC e uvrC2024 como genes alvo. Dados os resultados, com uma positividade significativa pode-se considerar que ainda h√° trabalho pela frente em termos de estabelecer e uniformizar uma pr√°tica para combater a larga presen√ßa de M. bovis nas explora√ß√Ķes.N/

    Antisense locked nucleic acid gapmers to control Candida albicans filamentation

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    Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European RegionalDevelopment Fund under the scope of Norte2020-ProgramaOperacional Regional do Norte and Daniela Eira Ara√ļjo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge theproject funding by the‚Äú02/SAICT/2017‚ÄďProjetos de Investiga-√ß√£o Cient√≠fica e Desenvolvimento Tecnol√≥gico (IC&DT)‚ÄďPOCI-01-0145-FEDER-028893‚ÄĚ. VILLUM Fonden is acknowledgedfor funding the Biomolecular Nano-scale Engineering Center(BioNEC), a Villum center of excellence, grant numberVKR18333. Funding received by iBB-Institute for Bioengineer-ing and Biosciences from FCT (UID/BIO/04565/2020) andPrograma Operacional Regional de Lisboa 2020 (Project No.007317) is also acknowledged. We acknowledge Dr. Luc√≠liaGoreti Pinto, Life and Health Sciences Research Institute(ICVS), School of Medicine, University of Minho, forprocessing and sectioningG. mellonellatissue samples.The authors declare no conflict of interest.info:eu-repo/semantics/publishedVersio

    In vitro investigation of the effect of disulfiram on hypoxia induced NFőļB, epithelial to mesenchymal transition and cancer stem cells in glioblastoma cell lines

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    A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy.Glioblastoma multiforme (GBM) is one of the most aggressive and lethal cancers with a poor prognosis. Advances in the treatment of GBM are limited due to several resistance mechanisms and limited drug delivery into the central nervous system (CNS) compartment by the blood-brain barrier (BBB) and by actions of the normal brain to counteract tumour-targeting medications. Hypoxia is common in malignant brain tumours such as GBM and plays a significant role in tumour pathobiology. It is widely accepted that hypoxia is a major driver of GBM malignancy. Although it has been confirmed that hypoxia induces GBM stem-like-cells (GSCs), which are highly invasive and resistant to all chemotherapeutic agents, the detailed molecular pathways linking hypoxia, GSC traits and chemoresistance remain obscure. Evidence shows that hypoxia induces cancer stem cell phenotypes via epithelial-to-mesenchymal transition (EMT), promoting therapeutic resistance in most cancers, including GBM. This study demonstrated that spheroid cultured GBM cells consist of a large population of hypoxic cells with CSC and EMT characteristics. GSCs are chemo-resistant and displayed increased levels of HIFs and NFőļB activity. Similarly, the hypoxia cultured GBM cells manifested GSC traits, chemoresistance and invasiveness. These results suggest that hypoxia is responsible for GBM stemness, chemoresistance and invasiveness. GBM cells transfected with nuclear factor kappa B-p65 (NFőļB-p65) subunit exhibited CSC and EMT markers indicating the essential role of NFőļB in maintaining GSC phenotypes. The study also highlighted the significance of NFőļB in driving chemoresistance, invasiveness, and the potential role of NFőļB as the central regulator of hypoxia-induced stemness in GBM cells. GSC population has the ability of self-renewal, cancer initiation and development of secondary heterogeneous cancer. The very poor prognosis of GBM could largely be attributed to the existence of GSCs, which promote tumour propagation, maintenance, radio- and chemoresistance and local infiltration. In this study, we used Disulfiram (DS), a drug used for more than 65 years in alcoholism clinics, in combination with copper (Cu) to target the NFőļB pathway, reverse chemoresistance and block invasion in GSCs. The obtained results showed that DS/Cu is highly cytotoxic to GBM cells and completely eradicated the resistant CSC population at low dose levels in vitro. DS/Cu inhibited the migration and invasion of hypoxia-induced CSC and EMT like GBM cells at low nanomolar concentrations. DS is an FDA approved drug with low toxicity to normal tissues and can pass through the BBB. Further research may lead to the quick translation of DS into cancer clinics and provide new therapeutic options to improve treatment outcomes in GBM patients

    Mutational Profile of Human Papilloma Virus (HPV) Induced and Non-HPV Induced Head and Neck Squamous Cell Carcinoma

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    Head and Neck cancer accounts for approximately 900,000 cases and over 400,000 deaths annually worldwide. The primary risk factors associated with Head and Neck cancer include usage of tobacco, alcohol consumption, Human Papillomavirus (HPV) infection and Epstein-Barr virus (EBV) infection. Few subsites of Head and Neck Squamous Cell Carcinoma (HNSCC) are associated with Human Papilloma Virus (HPV) while others remain non-associated. The anatomical, physiological, genetic, protein profile and epigenetic changes that occur in both HPV-positive and HPV-negative HNSCC has been discussed in this chapter. The mutational profile plays a crucial role in the treatment of the HNSCC patients as the HPV-positive HNSCC patients have a better prognosis compared to the HPV-negative HNSCC patients. This chapter mainly focusses on the mutational profile of both HPV-associated and non-HPV associated HNSCC tumours
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