Citrobacter rodentium induced liver changes in C57BL/6 mice : animal model of acute inflammatory stress and injury

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, 2011.Each page number preceded by chapter or appendix number. Cataloged from PDF version of thesis.Includes bibliographical references (p. F-150 - F-163).The activation of inflammatory responses, while critical for host defense, contributes to hepatic injury in numerous acute and chronic liver disease states as well as drug-induced liver injury (DILI). The interactions that mediate susceptibility to liver injury and disease, however, are still poorly understood, underscored by the complexity of immune interactions and the diverse cellular composition and functions of the liver. Using Citrobacter rodentium, a well characterized rodent-specific enteric pathogen as a source of extrahepatic inflammatory stress; host liver responses, metabolic dysregulation, and susceptibility to injury in C57BL/6 mice were investigated. For the first time, we show altered liver pathology during the early course of C. rodentium infection, characterized by periportal necrosis indicative of thrombic ischemic injury, correlating with distinct circulating and tissue specific cytokine/chemokine profiles. Using Acetaminophen (APAP), a widely used analgesic and well-characterized hepatotoxin, we evaluated liver responses in isolation and in the context of host inflammation to gain insight into the role of live bacterial infection in altering liver metabolism and susceptibility to DILI. We combined systemic and tissue-specific cytokine/chemokine levels, clinical serum chemistries, and histopathological assessments of hepatic and enteric inflammation and necrosis to measure molecularlevel responses to treatment and their physiological effect. Using principal components analysis (PCA), clustering, partial least squares regression (PLSR), and a combination mutual-information-correlation network, enabled detection and visualization of both linear and nonlinear dependencies between molecules and physiological states across tissues and timepoints. C. rodentium-induced inflammatory stress was finally investigated for its potential in altering drug pharmacokinetics (PK) of substrates varying in their metabolic biotransformation and clearance mechanisms. Infection resulted in increased systemic oral exposure (AUC) of clinically relevant xenobiotics such as verapamil, propranolol, and digoxin. Functionally, these changes were not found dependent on CYP-mediated biotransformation of parent compounds; rather, they appear driven more by proposed gut barrier compromise. In conclusion, gastrointestinal infection with C. rodentium alters systemic and hepatocytes specific responses, not previously appreciated from this enteric pathogen, making it a useful model for studying host-pathogen interactions under acute hepatic inflammatory stress and injury.by Arkadiusz R. Raczynski.Ph.D

Análise de agrupamento pelos métodos hierárquico aglomerativo e particional fuzzy utilizados para educational data mining em dados de educação a distância

Trabalho de Conclusão de Curso, apresentado para obtenção do grau de Bacharel no Curso de Ciência da Computação da Universidade do Extremo Sul Catarinense, UNESC.O crescimento da tecnologia, faz com que a quantidade de dados em repositórios aumente, impossibilitando a análise por métodos tradicionais, surgindo à mineração de dados, aplicada por meio da descoberta de conhecimento. A educação gera dados relacionados a alunos, principalmente a educação à distância em que os dados são provenientes de um ambiente virtual de aprendizagem, se tornando uma área de interesse dos pesquisadores educacionais. Com isso, surge o educational data mining, que utiliza métodos da mineração de dados. Mediante as técnicas e tarefas de mineração, tem o agrupamento, que é dividido em agrupamento hierárquico aglomerativo e agrupamento particional. De modo que nesta pesquisa é realizada a comparação entre o algoritmo de AGNES para o agrupamento hierárquico aglomerativo e o algoritmo fuzzy c-means para o agrupamento particional, com o objetivo de identificar qual dos métodos possui melhor desempenho em dados educacionais. Os dados são provenientes da disciplina ministrada a distância de Introdução a Engenharia de Segurança do Trabalho, na Universidade do Extremo Sul Catarinense. A ferramenta R foi usada, por ser um software livre, para implementação dos algoritmos e métodos de validação. Ao iniciar a mineração, é necessário definir a distância da matriz de similaridade, em que é aplicado as distâncias manhattan e euclidiana em AGNES e manhattan, euclidiana, correlattion e seuclidean no fuzzy c-means. O algoritmo AGNES, precisa da identificação do método de conexão, para gerar os resultados, sendo aplicado teste com os métodos de ward, distância média, maior distância e menor distância. A verificação dos resultados apresentados pelos algoritmos é realizada por meio das medidas de qualidade, aplicando índices de validação. O modelo final definido para fuzzy c-means, foi o que aplica a matriz de similaridade seuclidean e para o AGNES o que tem a matriz de similaridade de manhattan, pelo método de conexão distância média. Comparando o resultado gerado pelo índice de silhouette, o agrupamento particional, foi definido como modelo final de agrupamento sobre os dados educacionais

Addressing subjectivity in the classification of palaeoenvironmental remains with supervised deep learning convolutional neural networks

Archaeological object identifications have been traditionally undertaken through a comparative methodology where each artefact is identified through a subjective, interpretative act by a professional. Regarding palaeoenvironmental remains, this comparative methodology is given boundaries by using reference materials and codified sets of rules, but subjectivity is nevertheless present. The problem with this traditional archaeological methodology is that higher level of subjectivity in the identification of artefacts leads to inaccuracies, which then increases the potential for Type I and Type II errors in the testing of hypotheses. Reducing the subjectivity of archaeological identifications would improve the statistical power of archaeological analyses, which would subsequently lead to more impactful research. In this thesis, it is shown that the level of subjectivity in palaeoenvironmental research can be reduced by applying deep learning convolutional neural networks within an image recognition framework. The primary aim of the presented research is therefore to further the on-going paradigm shift in archaeology towards model-based object identifications, particularly within the realm of palaeoenvironmental remains. Although this thesis focuses on the identification of pollen grains and animal bones, with the latter being restricted to the astragalus of sheep and goats, there are wider implications for archaeology as these methods can easily be extended beyond pollen and animal remains. The previously published POLEN23E dataset is used as the pilot study of applying deep learning in pollen grain classification. In contrast, an image dataset of modern bones was compiled for the classification of sheep and goat astragali due to a complete lack of available bone image datasets and a double blind study with inexperienced and experienced zooarchaeologists was performed to have a benchmark to which image recognition models can be compared. In both classification tasks, the presented models outperform all previous formal modelling methods and only the best human analysts match the performance of the deep learning model in the sheep and goat astragalus separation task. Throughout the thesis, there is a specific focus on increasing trust in the models through the visualization of the models’ decision making and avenues of improvements to Grad-CAM are explored. This thesis makes an explicit case for the phasing out of the comparative methods in favour of a formal modelling framework within archaeology, especially in palaeoenvironmental object identification

ImMApp: An immersive database of sound art

The ImMApp (Immersive Mapping Application) thesis addresses contemporary and historical sound art from a position informed by, on one hand, post-structural critical theory, and on the other, a practice-based exploration of contemporary digital technologies (MySQL, XML, XSLT, X3D). It proposes a critical ontological schema derived from Michel Foucault's Archaeology of Knowledge (1972) and applies this to pre-existing information resources dealing with sound art. Firstly an analysis of print-based discourses (Sound by Artists. Lander and Lexier (1990), Noise, Water, Meat. Kahn (2001) and Background Noise: Perspectives on Sound Art. LaBelle (2006» is carried out according to Foucauldian notions of genealogy, subject positions, the statement, institutional affordances and the productive nature of discursive formation. The discursive field (the archive) presented by these major canonical texts is then contrasted with a formulation derived from Giles Deleuze and Felix Guattari: that of a 'minor' history of sound art practices. This is then extended by media theory (McLuhan, Kittler, Manovich) into a critique of two digital sound art resources (The Australian Sound Design Project (Bandt and Paine (2005) and soundtoys.net Stanza (1998). The divergences between the two forms of information technologies (print vs. digital) are discussed. The means by which such digitised methodologies may enhance Foucauldian discourse analysis points onwards towards the two practice-based elements of the thesis. Surface, the first iterative part, is a web-browser based database built on an Apache/MySQIlXML architecture. It is the most extensive mapping of sound art undertaken to date and extends the theoretical framework discussed above into the digital domain. Immersion, the second part, is a re-presentation of this material in an immersive digital environment, following the transformation of the source material via XSL-T into X3D. Immersion is a real-time, large format video, surround sound (5.ln.l) installation and the thesis concludes with a discussion of how this outcome has articulated Foucauldian archaeological method and unframed pre-existing notions of the nature of sound art

Monocyte and T cell plasticity in Crohn’s disease and ulcerative colitis

La maladie de Crohn (Crohn’s disease; CD) et la colite ulcéreuse (Ulcerative colitis ;CU) représentent deux formes distinctes de maladies inflammatoires chroniques de l’intestin (MICI), qui sont associées à une réponse immunitaire aberrante des tissus intestinaux à la flore intestinale. Les phagocytes mononucléés (MNPs) qui dialoguent, via les cytokines qu'ils produisent, avec les cellules immunitaires innées et adaptatives sont impliqués dans l’induction, la perpétuation et le maintien de la réponse inflammatoire des MICI. Chez la souris, les MNPs sont stratifiées en cellules dendritiques conventionnelles (cDCs), macrophages (M) et cellules dérivées de monocytes, une entité qui regroupe dans le tissu des cellules dendritiques dérivées de monocytes (Mo-DC), des M dérivés de monocytes et des « monocyte-like ». Toutefois, la diversité phénotypique, moléculaire et fonctionnelle des monocytes et des MNPs ainsi que la plasticité des monocytes restent à élucider dans les MICI. Les anticorps bloquant les cytokines IL12 et IL23 contrôlent la pathogénicité et la plasticité des cellules Th17, réduisant l'inflammation intestinale chez les patients atteints de MII. Cependant, il n’existe à ce jour aucun traitement curatif. L’étude approfondie de la plasticité des cellules T et du site tissulaire où elle pourrait se produire ne sont toujours pas clarifiés. Dans le premier chapitre, nous avons révélé l’existence de deux sous-populations distinctes de CD14+MNPs dans le colon de patients atteints de CU. Les cellules de type « inflammatory monocyte-like » CD14+CD163-CD64+ (P3) à l’opposé des CD14+CD163+CD64+ M (P4) s'accumulent dans le côlon inflammatoire. Nos résultats ont de plus établi un lien entre les P3 MNPs, l’IL12, l’IL1β et la détection de cellules Th17 mémoires produisant de l'IFN et de l'IL8, qui contribueraient collectivement à la pathogenèse de la CU. De plus, deux sous-populations CD14+ MNPs similaires sur le plan fonctionnel et moléculaire a ceux trouvés en CU, ont été détectées dans le côlon de patients atteints de CD. En revanche, dans le deuxième chapitre, nous fournissons des évidences que la sous-population monocytaire Slan+ pourrait contribuer à l’immunopathogenèse de la CD, mais pas à celle de la CU. La fréquence, le phénotype et la fonction des cellules Slan+ ont été examinés dans le sang, les ganglions mésentériques (MLN) et le côlon de patients atteints de MICI. Nous proposons que les cellules pro-inflammatoires CD14hiCD172α+Slan+ discriminent les tissus de CD et CU. En effet, elles ne s'accumulent que dans les MLNs et la muqueuse colique des patients atteints de CD. Dans le troisième chapitre, nous avons montré que les MLNs de CD et de CU, qui sont des tissus difficiles d'accès pour leur étude fonctionnelle en recherche, peuvent également être distingués par la distribution et le profil moléculaire des cellules T mémoire effectrices CXCR3-CCR6+ (Th17TEM). Nos données suggèrent également que la plasticité de Th17 se produit dans les MLNs avant leur migration vers l'intestin. Cette étude pourrait avoir des implications pour améliorer notre compréhension de la maladie. Enfin, il a été démontré qu’à l’homéostasie chez la souris, les monocytes sont continuellement recrutés dans la muqueuse intestinale où ils se différencient progressivement en M anti-inflammatoires. Ce processus de maturation est interrompu dans le contexte d'une inflammation. Les signaux environnementaux qui régulent la « cascade » de maturation d’un monocyte classique tissulaire demeurent inconnus chez l'homme. Dans le quatrième chapitre, nous avons récapitulé in vitro la cascade de différenciation des monocytes humains de «CD163- P3-like» en «CD163+P4-like» et avons montré leurs similitudes moléculaires avec les CD14+ MNP tissulaires. La manipulation de cette voie de différentiation pourrait ouvrir des pistes thérapeutiques pour restaurer l'homéostasie intestinale dans les MICI. En conclusion, une meilleure compréhension des sous-populations de MNPs, leurs fonction et plasticité dans la pathogenèse des MICI aidera à identifier des nouvelles cibles thérapeutiques et contribuera à augmenter les connaissances pour la mise au point de traitements personnalisés.Crohn’s disease (CD) and ulcerative colitis (UC), the two forms of inflammatory bowel diseases (IBD), are associated with dysregulated immune response in the intestinal tissue. It is mediated by mononuclear phagocytes (MNPs) that dialogue via the cytokine they produce with innate and adaptive immune cells. In mice, MNPs are stratified into conventional dendritic cells (DCs), macrophages (M) and monocyte-derived cells that regroup tissue monocyte-derived DCs, monocyte-derived M and monocytes-like cells. However, the phenotypic, molecular and functional diversity of MNPs and their plasticity remain to be elucidated in IBD patients. Therapies in IBD employ antibodies that block IL12 and IL23, thus control Th17 pathogenicity and plasticity and decrease intestinal inflammation. However, no cure exist nowadays for the treatment of IBD. In-depth study of T cell plasticity and the tissue where it occurs remain to be investigated. In the first chapter, we revealed the existence of two distinct CD14+ MNP subsets in colon of UC patients. Only, CD163-CD64+ inflammatory monocyte-like cells (P3) but not anti-inflammatory CD163+CD64+ M (P4) accumulate in inflamed UC colon. Our findings further established a link between monocyte-like CD14hiCD172α+ CD163- MNPs, IL12, IL1β and the detection of colonic memory Th17 cells that produce IFN and IL8, which might all contribute to UC pathogenesis. Two CD14+ MNP subsets, resembling their counterparts in UC mucosa at the functional and molecular level, were also detected in CD colon. In contrast, in the second chapter, we provide evidence that Slan+ monocyte subset may contribute to CD but not UC immunopathogenesis. Frequency, phenotype, and function of Slan+ cells were examined in blood, colon, and mesenteric lymph nodes (MLN) of patients with IBD. We showed that pro-inflammatory CD14hiCD172α+Slan+ cells are a distinguishing feature between CD and UC, as they only accumulate in MLNs and colonic mucosa of CD patients. In the third chapter, we showed that MLNs of CD and UC, tissues that were hard to access for research use, can also be distinguished by frequencies of CXCR3−CCR6+ Th17 effector memory T cells (TEM) and their molecular profile. Our data further suggested that Th17 plasticity is taking place in MLN, before T cell homing to gut tissues. This investigation has clear implications in furthering our understanding of the disease. Finally, it has been demonstrated that monocytes are continuously recruited into murine gut mucosa and progressively differentiate into macrophages under homeostatic conditions, a maturation process interrupted in the context of inflammation. However, the environmental cues that regulate tissue inflammatory monocyte “waterfall” remain to be investigated in humans. In the fourth chapter we recapitulated in vitro human monocyte differentiation cascade, from CD163- inflammatory monocyte-like cells (P3) towards anti-inflammatory CD163+ macrophages (P4) and showed their molecular similarities to tissue CD14+ MNPs. Manipulating this pathway might open therapeutic avenues to restore tissue homeostasis. In conclusion, a better understanding of MNP subsets, function and plasticity in IBD pathogenesis would help identify novel therapeutic targets and shed light for the development of personalized treatments

Intergeneric hybridisation in New Zealand Gnaphalieae (Compositae)

The occurrence of natural intergeneric hybridisation among the New Zealand Gnaphalieae was investigated using a case study approach. Putative hybrids between Anaphalioides bellidioides and Ewartia sinclairii were collected from beside the Yeo Stream, Inland Kaikoura Range, Marlborough and putative hybrids between Leucogenes grandiceps and Raoulia eximia from Mount Hutt, Mount Hutt Range, Canterbury. Cytology, pollen stainability and experimental crosses provided evidence for reduced fertility in the putative hybrids. Field evidence and the morphology and leaf anatomy of the putative hybrids supported the hybridity hypotheses for the majority of the putative hybrids. A range of isolating mechanisms may restrict the frequency of these hybrids in the field. In particular, environmental factors (the availability of suitable habitats and natural disturbance) and pre-zygotic and post-zygotic barriers (embryo and/or endosperm abortion, hybrid fitness and hybrid fertility) were suggested to be important. Cross-compatibility among indigenous Gnaphalieae and with related exotic Gnaphalieae was investigated through artificial crosses. Individual plants from six indigenous and five exotic species were preferentially selected as parents. The results provided evidence for the cross compatibility of many indigenous Gnaphalieae, including species of Anaphalioides, Euchiton, Ewartia, Helichrysum , Leucogenes and Raoulia. A plant of Euchiton audax was cross-compatible with individual plants of Ewartia planchonii and Gamochaeta spicata. The results indicate species groups among the indigenous Gnaphalieae are less genetically distinct than morphology suggests. The partial fertility of some natural intergeneric hybrids suggests intergeneric gene exchange has a potential role in the future evolution of the group

Pleiotropic effects of candidate genes on autism spectrum disorder and comorbidities: genetics, funcional studies and animal models

[eng] Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social communication and interaction, as well as repetitive and restricted patterns of behaviour. Although growing evidence supports a main contribution of genetic factors to its neurobiology and hundreds of candidate genes have been identified in recent years, the genetic architecture of the disorder is still not fully understood. Moreover, ASD frequently co-occurs with other developmental and psychiatric disorders, and shared genetic mechanisms are hypothesized to underlie these comorbidities. In this doctoral thesis, we aimed to study the contribution of several candidate genes to ASD and comorbidities. We have focused on the 14-3-3 gene family, RBFOX1 and the BEX/TCEAL gene family, performed genetic and functional studies and further characterized the neurobiological effects of their deficiency using animal models. First, our results suggest a role for the 14-3-3 genes in ASD and schizophrenia (SCZ). Ultra-rare variants in the 14-3-3 genes are enriched in ASD and common and rare variants in the YWHAE and YWHAZ genes, respectively, are associated with SCZ. We have also reported alterations in the expression of these genes in postmortem brains of ASD or SCZ patients. Furthermore, we have demonstrated a loss-of-function effect of a damaging variant in the YWHAZ gene present in two siblings with ASD and attention deficit/hyperactivity disorder (ADHD). In addition, we have characterized ywhaz expression in zebrafish across development and in adulthood and demonstrated that ywhaz depletion causes alterations in behaviour, in neuronal activity and connectivity and in monoamine signalling. The behavioural changes included freezing and were rescued with drug treatments that target monoamine neurotransmission. Second, we have demonstrated a relevant contribution of common variants in RBFOX1 to psychiatric disorders and traits. Also, we have shown that a high number of copy number variants (CNVs) spanning RBFOX1 are reported in patients with psychiatric conditions, the vast majority in patients with ASD or SCZ, and patients with these disorders also show a decreased expression of RBFOX1 in cortex. Finally, we have used knockout animal models to understand its role in psychiatric disorders, and demonstrated that both mice and zebrafish RBFOX1- deficient models present behavioural alterations that can be related to neurodevelopmental disorders such as ASD, ADHD and SCZ. Third, we found that all BEX/TCEAL genes are downregulated in postmortem brain regions of ASD and SCZ patients and that rare CNVs spanning several BEX/TCEAL genes have been reported in patients with severe neurodevelopmental problems. Furthermore, Bex3-deficient mice show anatomical and molecular alterations in brain, an excitatory/inhibitory imbalance and behavioural alterations that can be assimilated to ASD- and SCZ-like symptoms.[spa] El trastorno del espectro autista (TEA) es un trastorno del neurodesarrollo caracterizado por problemas en la comunicación e interacción social, así como patrones restrictivos y repetitivos de comportamiento. El peso de la genética en su etiología es cada vez más evidente, aunque la compleja arquitectura genética del trastorno sigue siendo una incógnita. Además, el diagnóstico de otros trastornos comórbidos es frecuente en pacientes con TEA, por lo que se hipotetiza una base genética común. El objetivo de esta tesis doctoral es elucidar la contribución de varios genes candidatos, concretamente la familia de genes 14-3-3, el gen RBFOX1 y la familia BEX/TCEAL, al TEA y otros trastornos comórbidos, realizando estudios genéticos y funcionales, así como caracterizando los efectos neurobiológicos de su deficiencia en modelos animales. En primer lugar, nuestros resultados sugieren que variantes ultra-raras en los genes 14-3-3 contribuyen al TEA y que variantes comunes y raras en los genes YWHAE y YWHAZ, respectivamente, están asociadas a esquizofrenia (SCZ). Además, la expresión de los genes 14- 3-3 está alterada en pacientes con TEA o SCZ. Hemos demostrado que una variante patogénica en el gen YWHAZ presente en dos hermanos con TEA y trastorno de déficit de atención e hiperactividad (TDAH) provoca una pérdida de función de la proteína. Asimismo, hemos demostrado que la deleción de ywhaz produce alteraciones en la actividad y conectividad neuronal, la señalización monoaminérgica y el comportamiento, pudiéndose este último recuperar mediante fármacos. En segundo lugar, hemos demostrado que variantes comunes en RBFOX1 están asociadas a diferentes trastornos psiquiátricos y que un número elevado de variantes del número de copias (CNV) afectan a RBFOX1 en pacientes con trastornos psiquiátricos, siendo especialmente frecuentes en pacientes con TEA o SCZ que, además, presentan una disminución en la expresión de RBFOX1 en corteza cerebral. Asimismo, hemos usado modelos animales genoanulados para estudiar la implicación de RBFOX1 en trastornos psiquiátricos, demostrando que tanto el modelo murino como los de pez cebra presentan alteraciones de comportamiento relacionadas con trastornos del neurodesarrollo, como ASD, TDAH y SCZ. Por último, la expresión de los genes BEX/TCEAL está disminuida en regiones cerebrales de pacientes con TEA o SCZ y, además, se han descrito CNVs que abarcan varios genes BEX/TCEAL en pacientes con trastornos severos del neurodesarrollo. Los ratones genoanulados para Bex3 muestran alteraciones anatómicas y moleculares en cerebro, un desequilibro excitación/inhibición y alteraciones de comportamiento asimilables a síntomas de TEA y SCZ

Molecular Techniques Reveal Wide Phyletic Diversity of Heterotrophic Microbes Associated with Discodermia spp. (Porifera: Demospongiae)

Sponges are well known to harbor large numbers of heterotrophic microbes within their mesohyl. Studies to determine the diversity of these associated microbes have been attempted for only a few shallow water species. We cultured various microorganisms from several species of Discodermia collected from deep water using the \u27Johnson-Sea-Link\u27 manned submersibles, and characterised them by standard microbiological identification methods. Characterisation of a small proportion (ca. 10%) of the total and potential eubacterial isolate collection with molecular systematics techniques revealed a wide diversity of microbes. Phylogenetic analyses of 32 small subunit (SSU) 16S-like rRNA gene sequences from different micorbes indicated high levels of taxonomic diversity assoiated with this genus of sponge. For example, bacteria from at least five cubacterial subdivisions - gamma, alpha, beta, Cytophaga and Gram positive - were isolated from the mesohyl of Discodermia. Several strains were unidentifiable from current sequence databases. No overlap was found between sequences of 24 isolates and 8 sequences obtained by PCR and cloning directly from sponge samples. The abundance and diversity of microbes associated with sponges such as Discodermia suggest that they may play important roles in marine microbial ecology, dispersal and evolution

Inflammatory response elements, glycan, and proteome profiles as salivary biomarkers for the early diagnosis of OSCC

El cáncer oral, sobre todo el, carcinoma oral de células escamosas (COCE), es la neoplasia maligna más común en la cavidad oral que se caracteriza por un mal pronóstico y una baja tasa de supervivencia, cuando se diagnostica en estadios avanzados. Puede ser tratado de forma efectiva si se detecta en estadios iniciales, mejorando el pronóstico. Por tanto, tras la prevención, el diagnóstico precoz se ha convertido en el principal objetivo en el manejo del cáncer oral, dado su impacto positivo en la supervivencia y calidad de vida de estos pacientes. A menudo, los escasos síntomas iniciales pasan desapercibidos y la enfermedad se descubre en fases avanzadas, por lo que se requiere mejorar el diagnóstico. Actualmente, no existen biomarcadores para COCE con la sensibilidad y especificidad suficiente para su uso clínico rutinario, lo que enfatiza la necesidad de una herramienta práctica y simple tanto para fines diagnósticos como de programas de detección precoz o screening. Los tumores de COCE aparecen a través de una serie de mutaciones moleculares que conducen a un crecimiento celular descontrolado, pasando por distintas fases: desde áreas de hiperplasia, lesiones displásicas, carcinoma in situ y, finalmente, carcinoma invasivo. Además, el desarrollo de muchos casos de COCE se ha correlacionado con la transformación cancerosa de lesiones orales potencialmente malignas, como la leucoplasia oral. Esta última presenta subtipos heterogéneos con diferentes potenciales de modificación, entre los que destaca la leucoplasia verrugosa proliferativa (LVP) con alto riesgo de conversión maligna. Los biomarcadores derivados de la saliva ofrecen un muestreo fácil con un mínimo riesgo para los pacientes, coste y tiempo de diagnóstico, y abarcan una variedad de parámetros detectables y medibles que permiten diferenciar la salud de la enfermedad. Objetivos El objetivo general es analizar un panel de citocinas inflamatorias salivares, así como perfiles proteicos y glicanos salivares con el fin de identificar potenciales biomarcadores para el diagnóstico precoz de COCE. Metodología Se utilizaron estrategias ómicas combinadas para perfilar diferentes tipos de moléculas con potencial utilidad diagnóstica en la saliva de pacientes con LVP, estadios tempranos y avanzados de COCE y personas sanas. Se utilizó un inmunoensayo multiplexado para examinar los niveles de ocho citocinas. Los perfiles proteicos y N-glicanos derivados de la saliva se analizaron mediante cromatografía líquida (CL) acoplada a espectrometría de masas (MS). Resultados y Conclusiónes Los niveles significativamente alterados de seis citocinas asociadas con los grupos de patología sugiere su posible participación en el proceso de carcinogénesis oral. El perfil comparativo de N-glicanos reveló varios glicanos bi- y tri-antenarios fucosilados expresados diferencialmente entre los grupos estudiados, proporcionando una plataforma razonable para investigar más a fondo la utilidad de la glicosilación salivar para el diagnóstico de COCE. Se cuantificaron más de 600 proteínas en la saliva de personas sanas y con lesiones pre/cancerosas mediante los perfiles proteicos generados por LC-MS cuantificadas. El análisis comparativo dio como resultado una lista de proteínas expresadas diferencialmente, caracterizadas en COCE, que indica mecanismos significativamente alterados como el sistema inmunológico, la inhibición de actividades enzimáticas y la adhesión celular. Entre los marcadores candidatos identificados, algunos se habían descrito previamente en la saliva. Además, se han anotado varias proteínas nuevas asociadas a COCE.Oral squamous cell carcinoma (OSCC) is the most common neoplasia in the oral cavity characterized by poor prognosis and a low survival rate when diagnosed at advanced phases. Many OSCC cases have been correlated to the cancerous transformation of oral potentially malignant disorders (OPMDs) such as oral leukoplakia. The latter exhibits heterogeneous subtypes, among which proliferative verrucous leukoplakia (PVL) stands out with a high risk of malignant conversion. Reliable biomarkers for OSCC are yet unavailable in the routine clinical setting, emphasizing the need for a practical and simple tool to be used for definitive diagnosis and screening programs. Saliva-derived biomarkers offer easy sampling with reduced risk for the patients, cost and diagnosis time, and a variety of measurable parameters that can discriminate health from disease. To identify novel salivary biomarkers of OSCC, we herein combined multi-omics analytical strategies to profile different types of molecules with potential diagnostic utility. As inflammation has previously been linked to oral pathologies, research so far indicates the possibility of using salivary cytokines for the screening of oral disorders. Altered cytokine responsiveness has been tightly associated with the development of OSCC, as well as detected in patients with OPMDs. To reveal changes related to oral carcinogenesis, a multiplex immune bead-based assay was used to survey the levels of 8 cytokines in the saliva of patients with PVL, at early and advanced OSCC stages and their healthy counterparts. Results indicated altered cytokine activity associated with the pathology groups, the predictive models for sensitivity and specificity of which showed high ROC AUC values. It is now known that defective glycosylation accompanies many chronic and infectious conditions and is a common feature of tumor cells that may affect N-glycans on glycoproteins. To compile a list of candidate biomarkers, another type of molecule has been studied. Saliva protein released N-glycans of healthy volunteers, PVL, and OSCC patients were analysed by the means of liquid chromatography (LC) coupled to mass spectrometry (MS). Comparative N-glycome profiling disclosed differentially expressed fucosylated bi- and tri-antennary glycans among the studied groups, providing a reasonable platform to further investigate the utility of salivary glycosylation for diagnosis of OSCC. Lastly, to get an insight into the complex molecular alterations associated with cancer, LC-MS generated proteomic profiles revealed more than 600 quantified proteins in the saliva of healthy and pre/cancerous lesions. The comparative analysis outlined a list of differentially expressed proteins, characterized in OSCC, indicating altered mechanisms implicating the immune system, inhibition of enzymatic activities, and cell adhesion. In this discovery phase of biomarkers identification, we provided a molecular panel of potential indicators of malignant transformation and/or early OSCC diagnosis