146 research outputs found

    Gene expression profiling of head and neck cancer

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    MDThe purpose of this study was to classify oral squamous cell carcinomas (OSCCs) based on their gene expression profiles, to identify differentially expressed genes in these cancers, and to correlate genetic deregulation with clinical-histopathological data and patient outcome. After conducting proof of principle experiments utilizing six head and neck squamous cell carcinomas (HNSCCs) cell lines, the gene expression profiles of 20 OSCCs and subsequently an additional 8 OSCCs were determined using cDNA microarrays containing 19,200 sequences and the Binary Tree-Structured Vector Quantization (BTSVQ) method of data analysis. Two sample clusters were identified in the group of 20 tumors that correlated with T3-T4 category of disease (P=0.035) and nodal metastasis( p=0.035). Samplec lustering of 28 OSCCsa nd the 6 cell lines revealed a correlation with disease free survival. BTSVQ analysis identified a subset of 23 differentially expressed genes with the lowest quantization error scores in the cluster containing more advanceds taget umors from the 20 OSCC dataset.T he expressiono f six of these differentially expressedg enesw as validated by quantitative real-time RT-PCR. Statistical analysis of quantitative real-time RT-PCR data was performed and, after Bonferroni correction, CLDNI (p = 0.007) over-expressionw as significantly correlated with the cluster containing more advanced stage tumors. Despite the clinical heterogeneity of OSCC, molecular subtyping by cDNA microarray analysis was able to identify distinct patternso f genee xpressiona ssociatedw ith relevant clinical parameters. The application of this methodology represents an advance in the classification of oral cavity tumors, and may ultimately aid in the development of more tailored therapies for oral carcinoma

    Transcriptional Alterations Related to Neuropathology and Clinical Manifestation of Alzheimer's Disease

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    Alzheimer's disease (AD) is the most common cause of dementia in the human population, characterized by a spectrum of neuropathological abnormalities that results in memory impairment and loss of other cognitive processes as well as the presence of non-cognitive symptoms. Transcriptomic analyses provide an important approach to elucidating the pathogenesis of complex diseases like AD, helping to figure out both pre-clinical markers to identify susceptible patients and the early pathogenic mechanisms to serve as therapeutic targets. This study provides the gene expression profile of postmortem brain tissue from subjects with clinic-pathological AD (Braak IV, V, or V and CERAD B or C; and CDR >= 1), preclinical AD (Braak IV, V, or VI and CERAD B or C; and CDR = 0), and healthy older individuals (Braak <= II and CERAD 0 or A; and CDR = 0) in order to establish genes related to both AD neuropathology and clinical emergence of dementia. Based on differential gene expression, hierarchical clustering and network analysis, genes involved in energy metabolism, oxidative stress, DNA damage/repair, senescence, and transcriptional regulation were implicated with the neuropathology of AD; a transcriptional profile related to clinical manifestation of AD could not be detected with reliability using differential gene expression analysis, although genes involved in synaptic plasticity, and cell cycle seems to have a role revealed by gene classifier. In conclusion, the present data suggest gene expression profile changes secondary to the development of AD-related pathology and some genes that appear to be related to the clinical manifestation of dementia in subjects with significant AD pathology, making necessary further investigations to better understand these transcriptional findings on the pathogenesis and clinical emergence of AD.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2005/04151-7]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

    Systems physiology and nutrition in dairy cattle: applications of omics and bioinformatics to better understand the hepatic metabolomics and transcriptomics adaptations in transition dairy cows

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    Application of systems concepts to better understand physiological and metabolic changes in dairy cows during the transition into lactation could enhance our understanding about the role of nutrients in helping to meet the animal’s requirements for optimal production and health. Four different analyses focused on the liver were conducted, and dealt with a metabolic disorder or thermal stress. The first three analyses dealt with supplementation of methionine to prevent clinical ketosis development in high-genetic merit dairy cows. Four groups of cows were formed retrospectively based on clinical health evaluated at 1 week postpartum: cows that remained healthy (OVE), cows that developed ketosis (K), and healthy cows supplemented with one of two commercial methionine products [Smartamine M (SM), and MetaSmart (MS)]. The liver tissue samples (n = 6/group) were harvested at -10 d before calving, and were used for metabolomics (GC-MS, LC-MS; Metabolon Inc.) and transcriptomics (44K-whole-transcriptome microarray; Agilent) analyses. Therefore, the main goals of the analyses were to 1) uncover metabolome and transcriptome patterns in the prepartum liver that were unique to those cows that became ketotic postpartum, and to 2) uncover unique patterns affected by supplemental methionine. The data were analyzed using the MIXED procedure of SAS. The metabolomics analysis (p ≤ 0.10) resulted in 13, 16, 26, 36, 13 and 43 biochemical compounds out of 313 identified for the comparisons K vs. OVE, SM vs. OVE, MS vs. OVE, SM vs. MS, K vs. SM and K vs. MS, respectively. The transcriptomics analysis (p ≤ 0.05 and fold change (FC) ≥ |1.5|) resulted in 3,065, 710, 786, 601, 1,021 and 771 number of differentially expressed genes (DEG) for the respective comparisons. The functional analysis of the data was performed using dynamic impact approach (DIA). The network reconstruction and data integration was performed with Ingenuity Pathway Analysis (IPA). In the first analysis of K vs. OVE, the results indicated inhibition of several carbohydrate- and lipid-related metabolic pathways, while activation of ‘Selenoamino acid metabolism’, ‘Ribosome’, and ‘Replication and repair’ was predominant. In the second analysis of SM vs. OVE, ‘Nitrogen metabolism’, ‘Glycosaminoglycan biocynthesis-chondroitin sulfate’, ‘Synthesis and degradation of ketone bodies’ and ‘Selenoamino acid metabolism’ were induced while the ‘Cyanoamino acid metabolism’, ‘Taurine and hypotaruine metabolism’ and ‘Inositol phosphate metabolism’ were inhibited. The analysis of MS vs. OVE revealed activation of ‘Riboflavin metabolism’, ‘Bile secretion’ and ‘Vitamin digestion and absorption’, while inhibition of ‘Base excision repair’, ‘Cyanoamino acid metabolism’, and ‘One carbon pool’. The analysis of SM vs. MS indicated activation of ‘Intestinal immune network for IgA production’, ‘Antigen processing and presentation’, and ‘Riboflavin metabolism’, while the inhibition ‘Glycosaminoglycan degradation’, ‘Other glycan degradation’ and ‘Bile secretion’. In the third analysis of K vs. SM, among the top 10 affected pathways, most were inhibited. Examples include ‘Cynoamino acid metabolism’, ‘Fructose and Mannose metabolism’, ‘Erb signaling’ and ‘Pentose phosphate pathway’. In contrast, the analysis of K vs. MS revealed an induction of ‘Nitrogen metabolism’ among the top 10 pathways, while pathways such as ‘Riboflavin metabolism’, ‘Pentose phosphate pathway’ and other carbohydrate and glycan biosynthesis related pathways were inhibited. The fourth analysis dealt with the effect of thermal stress on the liver transcriptome as it is related to health and productivity. During this study, we used gene network analysis on transcriptome data to uncover transcription regulators and their target genes in the liver tissue harvested at -30, +3, and +35 d relative to parturition during spring (SP, n = 6) and summer (SU, n = 6). Statistical analysis (FDR ≤ 0.10) of data from SU vs. SP revealed a total of 618, 1,030 and 894 DEG at -30, +3 and +35 d, respectively. IPA was used for gene network reconstructions. A total of 6, 7 and 7 transcription regulators were identified at -30, +3 and +35 d, respectively during SU vs. SP. The evaluation of these results suggests that calving during SU vs. SP is associated with the molecular phenotypes of the liver

    Identification of genes involved in macrophage activation and effector functions against intracellular pathogens

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    Includes bibliographical references.This dissertation addressed the hypothesis that macrophages have an alternative killing mechanism that is independent of superoxide and nitric oxide but dependent on IFN-γ, TNF and C/EBPβ. Since the mechanism and the genes involved in this alternative pathway are mostly unknown, the aim of this dissertation was to identify these macrophage effector genes and to functionally characterize their role during infection utilizing gene deficient mouse models. Since mice deficient for C/EBPβ (C/EBPβ-/-) expressed normal levels of IFN-y and TNF during Listeria monocytogenes infection, the macrophage effector genes involved in confinement and killing of L. monocytogenes were postulated to be downstream of C/EBPβ. Furthermore, C/EBPβ-/- mice are highly susceptible L. monocytogenes due to impaired listericidal activity. Comparison of the gene expression profiles of WT and C/EBPβ-/- macrophages infected with L. monocytogenes was postulated to increase the probability of identifying these effector genes, which would be differentially expressed between the two groups. Comparative gene expression profiling by DNA microarrays between L. monocytogenes in infected WT and C/EBPβ-/- macrophages, successfully identified 1268 genes to be differentially expressed between the two groups. A focussed functional clustering strategy reduced the number of candidate genes to 220. PKCδ was selected for further study since it was involved in humoral defense, immune signalling, production of superoxide, regulation of transcription and may be putatively transcriptionally regulated by C/EBPβ. Furthermore, PKCδ was indirectly shown to promote L. monocytogenes escape from the phagosome and to negatively regulate transcription activity of C/EBPβ. In addition, since PKCδ was un-regulated, as shown by microarray and confirmed by RT-PCR, in L. monocytogenes infected C/EBPβ-/- macrophages, it was therefore thought to play a detrimental role during L. monocytogenes. However, since this premise has never been investigated directly, the role PKCδ during innate immunity against L monocytogenes was examined using the PKCδ deficient (PKCδ-/-) mouse model. Data in this dissertation provides new insight into the role of PKCδ during innate immunity to L. monocytogenes. PKCδ-/- mice were highly susceptible to L. monocytogenes due to enhanced listerial escape and impaired listericidal activity. Despite full macrophage activation and production of nitric oxide, PKCδ-/- mice displayed uncontrolled bacterial growth and dissemination of L. monocytogenes, which led to early death of the mice. In contrast, PKCδ-/- mice were able to control Mycobacterium infection as well as WT mice, suggesting that the activity of PKCδ may be negatively regulated by L. monocytogenes. A systems biology approach generated the hypothesis that PKCδ may promote Rab5a activation, which together with localized release of superoxide into the phagosome and activation of C/EBPβ by PKCδ, resulted in the confinement of the L. monocytogenes within the phagosome. Alternatively, PKCδ may act in a separate pathway that confines L monocytogenes within the phagosome, by activating and/or synergizing with unidentified proteins to neutralize that activity of listerial LLO and PI-PLC. Data in this dissertation clearly demonstrates that PKCδ is critical for confinement of L monocytogenes within phagosomes and may be part of a listericidal mechanism that is independent or nitric oxide, superoxide and pro-inflammatory cytokines

    Análisis de la metástasis del cáncer colorectal mediante proteómica y microscopía

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    Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 08-11-2022Cancer is the second most common cause of death, only preceded by cardiovascular diseases. The main reason behind the high mortality rates of cancer is the appearance of metastasis, the dissemination and colonisation of secondary tissues by cells from the primary tumour. In particular, in the case of colorectal cancer (CRC) the survival rates of patients diagnosed, drop drastically if diagnosis happens at later stages when metastasis has occurred. A similar trend can be observed for most of the cancers commonly diagnosed. When metastasis occurs, treatment relies heavily on the use of chemotherapy, even if the original tumour mass is surgically removed, the metastases will still survive. It is thus paramount to find new tools that improve early diagnosis so that detection can occur before the tumour spreads. In this context, the main focus of this thesis has been the understanding and characterisation of CRC metastasis to find new diagnostic markers that can be used in the clinic. We have used isogenic cell lines, that share the same genetic background but have different metastatic capacities, to define the proteome of CRC metastasis in vitro...Tras las enfermedades cardiovasculares, la segunda causa de muerte en países desarrollados es el cáncer. El principal factor detrás de la alta mortalidad del cáncer es la aparición de metástasis, el proceso por el cual células provenientes de la masa tumoral original diseminan y son capaces de colonizar tejidos distantes. En el caso del cáncer colorrectal(CCR), las tasas de supervivencia de los pacientes caen de manera drástica si el diagnóstico de la enfermedad se produce en estadios tardíos en los que ya haya aparecido metástasis. Esta tendencia se puede observar también en la mayoría de los cánceres diagnosticados en la actualidad. Uno de los principales problemas de la metástasis es que una vez aparece, el tratamiento del cáncer se vuelve complejo, siendo la quimioterapia la principal forma de tratamiento ya que, aunque se elimine quirúrgicamente la masa tumoral original, los nichos metastáticos sobrevivirán a la operación. Es por tanto necesario el desarrollo de nuevas técnicas de diagnóstico que permitan mejorar la detección temprana, antes de que el tumor se extienda. En este contexto, el principal objetivo de esta tesis ha sido entender y caracterizar la metástasis del CCR para encontrar marcadores de diagnóstico que puedan trasladarse a la práctica clínica. Para definir el proteoma de la metástasis de CCR hemos empleado una serie de líneas celulares isogénicas, que comparten la misma carga genética, con diferente potencial metastático...Fac. de Ciencias QuímicasTRUEunpu

    Profiling B cell immune responses by microengraving

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    Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2008.Includes bibliographical references (leaves 83-87).The ability to monitor an immune response in the course of vaccination or disease progression is highly desirable. Currently, no technique is able to generate a comprehensive profile of the individual cells involved and the antibodies they produce at a particular point during the immune response. The ability to obtain such detailed "snapshots" describing the immune response with a high level of resolution would have implications for diagnostics and biological discovery. Improvement in vaccination schemes, specific tailoring of anti-viral administrations, large-scale monitoring of complex latent infections in a population are all possibilities that would stem from a better understanding of the dynamics of immune responses. currently available methods for profiling of B cells that produce antigen-specific antibodies helped clarify humoral responses, but it remains a challenge to generate measurements capable of detailing the phenotypic changes and secretion patterns of individual lymphocytes. To address this need a soft lithographic approach termed microengraving ([mu]En) - previously used for the isolation and rapid selection of monoclonal antibodies[31] - was further developed and adapted to measure the affinity and isotype of secreted antibodies. The objective of this thesis was to employ microengraving in conjunction with bioinformatics analysis to obtain routinely state-based comprehensive profiles detailing cellular and humoral immune responses to antigens to the level of clonal B cells. Here I show how bioinformatics methods were employed to generate multidimensional datasets for large numbers of individual primary B cells (10² - 10⁴). These data include three characteristics of the antibodies secreted by each cell: antigenic specificity, isotype, and affinity.(cont.) These data are sufficient to classify individual cells into distinct groups of related cells using algorithms for data clustering. In a series of mice immunizations designed to mimic a multipart vaccination, I apply this method to profile the resulting B cell response with single cell resolution.by Eliseo Papa.S.M

    Validation and functional characterization of a prognostic 4-miRNA signature in Glioblastoma

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    Regulation of gene expression by esrrb in embryonic stem cells

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    Ph.DDOCTOR OF PHILOSOPH

    Discovering pathways to autism spectrum disorder by using functional and integrative genomics approaches to assess monozygotic twin differences

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    Autism spectrum disorder (ASD) is a common developmental disorder typified by deficits in social communication and stereotyped behaviours. Despite evidence of a strong genetic basis to the disorder, molecular studies have thus far had little success in identifying risk variants or other biomarkers, and presently there is no unified pathomechanistic explanation. Monozygotic (MZ) twins show incomplete concordance in autistic traits, which suggests that alternative risk pathways involving non-shared environmental (NSE) factors could also have an important role to play in ASD. In this thesis, we describe microarray and RNA-seq studies characterising gene expression in a sample of 53 ASD MZ twin pairs from TEDS. The overall aims were to: 1) establish convergent evidence for genes and pathways involved in the etiology of ASD comparing affected and unaffected subjects across the sample 2) to identify those responsive to the environment by examining differences within the discordant pairs. We found a number of genes were differentially expressed including DEPDC1B - the most significant finding in cases vs controls, which also showed consistent down regulation within pairs. We further identified IGHG4, IGHG3, IGHV3-66, HSPA8P14, HSPA13, SLC15A2, and found that these results were enriched for transcriptional control, immune, and PI3K/AKT signalling pathways. We suggest that as these were found to be perturbed in the discordant twins, they could represent ASD risk pathways sensitive to the NSE. Next, we investigated integrative genomics methods for performing meta-dimensional analysis using the expression data along with methylation data on the same cohort. After applying regression-based joint analysis methods, and meta-analysis p-value combination methods to our datasets, a number of genes obtained nominal significance across the datasets, including potential genes of interest: NLGN2, UBE3A, OXTR. We suggest these represent genes with evidence for being functionally relevant to ASD
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