Els tumors de cervell humans (HBTs) són uns dels càncers més agressius i intractables. El sistema actual de diagnosi i prognosi dels HBTs es basa en l'examinació histològica d'un tall de biòpsia, el qual es considera el sistema de referència ("gold¬standard"). A més de ser invasiva, aquesta tècnica no és prou acurada per a diferenciar els graus de malignitat de determinats HBTs i la correlació amb la resposta del pacient a la teràpia sol ser variable. En aquest context, les signatures gèniques obtingudes a partir de microarrays de DNA poden millorar els resultats del "gold-standard". En aquesta tesi, vaig recollir 333 biòpsies de varis tipus de HBTs. Com un 38% de les mostres tenien l'RNA degradat, vam avaluar si el tipus de HBTs, el contingut aparent de sang de la biòpsia i el medi de recollida de la biòpsia hi afectaven. Com no vam determinar cap relació, hipotetitzo que un temps variable d'isquèmia a temperatura normal del cos abans de l'extracció de la biòpsia podria induir la degradació de l'RNA. Això va ser avaluat en un tumor glial pre-clínic desenvolupat en ratolí. Es va detectar que 30 minuts de temps d'isquèmia afecta la integritat del RNA en tumors no necròtics, però no en els necròtics. Una part crucial de la tesi va ser la demostració com una "prova de principis" de l'habilitat de les signatures gèniques per a predir objectivament els HBTs. Això es va mostrar mitjançant una predicció perfecta de glioblastoma multiforme (Gbm) i meningioma meningotelial (Mm) utilitzant microarrays de cDNA i microchips d'Affymetrix. Els histopatòlegs poden discriminar perfectament aquests dos tipus de tumors, però aquest treball demostra una predicció perfecta utilitzant una fórmula matemàtica objectiva. Un cop es va demostrar això, em vaig sentir confiat per a predir diferents graus de malignitat i possibles subtipus moleculars de tumors glials. En aquest sentit, es va descriure una signatura gènica basada en l'expressió de 59 transcrits, la qual va distingir dos grups de glioblastomes. Finalment, una anàlisi inicial de les dades clíniques associades suggereix que la signatura gènica podria correlacionar amb glioblastomes primaris i secundaris. / Human brain tumours (HBTs) are among the most aggressive and intractable cancers. The current system for diagnosis and prognosis of HBTs is based on the histological examination of a biopsy slice, which is considered the 'gold standard'. Apart from being invasive, this technique is not accurate enough to differentiate malignancy grades of some HBTs and it provides a variable correlation with response to therapy of the patient. In this context, gene signatures from DNA microarray experiments can improve the results of the 'gold standard'. In this thesis, I collected 333 biopsies from various types of HBTs. As 38% of samples displayed degraded RNA, I evaluated whether the HBT type, the apparent blood content and the collection medium of the biopsy could play a role in this. As no relationship was found, I hypothesized that the variable ischaemia time at normal body temperature prior to removal of the biopsy may induce degradation of RNA. This was tested in a preclinical glial tumour model in mice. It was detected that 30 minutes ischaemia time affects the integrity of the RNA in non-necrotic tumours, but not in the necrotic ones. A crucial part of this thesis was the demonstration of proof-of-principle of the ability of gene signatures for objective prediction of HBTs. This was shown by perfect prediction of glioblastoma multiforme (Gbm) and meningothelial meningioma (Mm) using cDNA and Affymetrix microarrays. Histopathologists perfectly discriminates both tumour types, but this work demonstrated perfect prediction using a simple mathematical formula. Once this was demonstrated, I felt confident to predict different malignancy grades and possible molecular subtypes of glial tumours. In this respect, a gene signature based on the expression of 59 transcripts, which distinguished two groups of glioblastomas, was described. Finally, a crude initial analysis of associated clinical data suggests that this gene signature may correlate to primary and secondary glioblastomas.

Gens Diana de les ProantocianidinesLa tesi es planteja demostrar la següent hipòtesis: les proantocianidines actuen modulant l'expressió diferencial de gens diana per condicionar una adaptació homeostàtica que previngui situacions d'estrès metabòlic, oxidatiu o inflamatori. S'han emprat diferents estratègies experimentals, unes utilitzant la metodologia de transcriptòmica no dirigida analitzant fetges de rates amb tractaments aguts i crònics dosi-resposta amb extractes de proantocianidines. Els resultats obtinguts a partir d'aquesta primera aproximació ens indiquen que no hi ha dosi resposta: a cada dosi el ventall de gens que es modifiquen es diferent i no d'una forma especialment significativa, possibilitant això no obstant adaptacions bioquímiques que van en el sentit postulat, modulant el metabolisme dels lípids i corregint potencials situacions d'estrès inflamatori. Destaca el paper proapoptòtic descobert. Una segona estratègia s'ha centrat en l'estudi de proantocianidines concretes: epicatequina, dímer i trímer de catequina fent servir cèl·lules hepàtiques en cultiu utilitzant també la transcriptòmica no dirigida. Destaquen com a resultats el fet que són molts pocs els gens que modifiquen l'expressió amb epicatequina, mentre que pel dímer i trímer la modificació de l'expressió és important i centrada en uns processos bioquímics alterats pel dímers, uns altres pel trímer i forces, especialment en el context d'actuar com inhibidors de la resposta immune, per ambdues molècules. En la tercera estratègia, centrada en la tècnica de la transcriptòmica dirigida s'han estudiat els canvis d'expressió gènica en cèl·lules hepàtiques i cèl·lules endotelials tractades amb extracte de proantocianidines de pinyol de raïm, extracte de proantocianidines ric en oligòmers, epicatequina gal·lat, dímer B2 i trímer C1, per tal de comprovar els canvis de la transcriptòmica no dirigida i ampliar l'estudi a cèl·lules endotelials. El conjunt de resultats obtinguts permeten avançar en la hipòtesi plantejada i avalen el potencial de les proantocianidines com a molècules reguladores i utilitzables en la indústria dels aliments funcionals. Gens Diana de les ProantocianidinesThe aim of this Thesis is to demonstrate the following hypothesis: proanthocyanidins act by modulating the differential expression of target genes for conditioning a homeostatic adaptation to prevent metabolic, oxidative or inflammatory stress.We have used different experimental strategies using transcriptomics tool for achieving the hypothesis.The first strategy is based on the methodology of untargeted transcriptomics analysis using the livers of rats that were administered an acute or a chronic treatment of a proanthocyanidin extract. The results obtained are firstly, that the effect is not dose-dependent. At each dose the range of genes that change is different and are not particularly significant. The conclusions derived from this first study showed a biochemical adaptation of animals, modulating lipid metabolism in addition to exert proapoptotic and anti-inflammatory action.The second strategy is focused on the study of pure molecules, using concrete proanthocyanidins such as the epicatechin monomer, a dimer of catechin and a trimer of catechin. These three treatments have been used in rat liver cells in culture and also using untargeted transcriptomics tool. The results derived from this secondstrategy postulated that the administration of epicatechinmodify the expression of a few genes. Moreover, administration of dimer of catechin and trimer of catechin exert a dual effect evident on these cells, inducing common changes mainly supressingthe immune response.The third strategy is focused on targeted transcriptomics. The models used have been liver cells and endothelial cells in culture treated with grape seed proanthocyanidins extract, an extract rich in oligomers purified from the generic extract, epigallocatechin gallate, dimer B2 and trimer C1 to visualize changes in gene expression of selected genes, all involved in the development of cardiovascular disease.The overall results obtained allow us to progress in the hypothesis and support the potential of proanthocyanidins as regulatory molecules and its use in the functional food industry.

Analysis of gene expression microarray data has traditionally been conducted using hierarchical clustering. However, such analysis has many known disadvantages and pattern discovery (PD) has been proposed as an alternative technique. In this work, three similar but different PD algorithms Teiresias, Splash and Genes@Work were benchmarked for time and memory efficiency on a small yeast cell-cycle data set. Teiresias was found to be the fastest, and best over-all program. However, Splash was more memory efficient. This work also investigated the performance of four methods of discretizing microarray data: sign-of-the-derivative, K-means, pre-set value, and Genes@Work stratification. The first three methods were evaluated on their predisposition to group together biologically related genes. On a yeast cell-cycle data set, sign-of-the-derivative method yielded the most biologically significant patterns, followed by the pre-set value and K-means methods. K-means, preset-value, and Genes@Work were also compared on their ability to classify tissue samples from diffuse large b-cell lymphoma (DLBCL) into two subtypes determined by standard techniques. The Genes@Work stratification method produced the best patterns for discriminating between the two subtypes of lymphoma. However, the results from the second-best method, K-means, call into question the accuracy of the classification by the standard technique. Finally, a number of recommendations for improvement of pattern discovery algorithms and discretization techniques are made.

The global gene expression profiles of industrial strains of <i>Saccharomyces cerevisiae</i> responding to nitrogen deficiency and very high sugar concentrations stresses were determined by oligonucleotide microarray analysis of ~ 6200 yeast open reading frames. Genomics analysis showed that 400 genes in S. cerevisiae was differentially expressed by more than 1.5-fold compared with controls at late-logarithmic phase of fermentation, as the yeast adapted to changing nutritional, environmental and physiological conditions. The genes of many pathways are regulated in a highly coordinated manner. The repressed expression of GDH1 and up-regulation of ARO10 within the contrast of Q270/Q10 indicated high energy demanding of yeast cells under high sugar stress. Activities of G3P shuttle indicated that under very high gravity environment, sufficient assimilatory nitrogen enhances yeasts ability of redox balancing, and therefore higher stress-tolerance and higher fermentation efficiency of yeast. Under contrast W270/Q270, the up-regulation of DUR1,2 responsible for urea degradation induces the glutamate biosynthesis and the consumption of -ketoglutarate. This may indicate that higher nitrogen level would enable higher activities in the TCA cycle, and therefore generate more energy for biosynthesis and yeast cell proliferation under very high gravity fermentation conditions. Nitrogen metabolism was also stimulated by high nitrogen level when yeast was grown in very high gravity environment.

The domestic dog, Canis lupus familiaris, is a unique model system for the dissection of hereditary diseases. Selective breeding practices have created more than 300 distinct breeds of dogs, born from a desire to create specific physical and behavioral characteristics. Breeds represent closed breeding populations and the extensive records maintained for members of each breed (e.g., multi-generational pedigrees, veterinary medical records) present an incredible tool for genetic research. Two closed populations were used in the work presented here: a colony of mixed-breed dogs segregating resistance and sensitivity to cholesterol feeding, and a purebred pet population of Havanese experiencing a high frequency of developmental abnormalities. Estimates of heritability were calculated for each disease to evaluate the degree of phenotypic variation attributable to genetics among dogs in the populations used. A heritability of 0.55 (± 0.16) was identified for cholesterol resistance and sensitivity in the mixed-breed colony. The small sample size prevented the use of complex segregation analyses to examine mode of transmission. A heritability of 0.36 (± 0.26) was calculated for the composite phenotype in the Havanese, encompassing the spectrum of abnormalities in the breed. Polygenic inheritance was identified for the composite phenotype, but the action of a major gene was identified by complex segregation analyses in the Havanese. Complex diseases preclude the use of a candidate gene approach, owing to the multitude of genes involved in the disease process. Whole genome screens provide a practical approach to the identification of chromosomal region(s) associated with a disease phenotype by narrowing the search for candidate gene(s). The Minimal Screening Set – 2 (MSS-2) was used in the present studies to evaluate the segregation of microsatellite markers in pedigrees for both the mixed-breed colony and the Havanese. No significant LOD scores were identified, though suggestive LOD scores were obtained in both analyses. A canine-specific oligonucleotide microarray was used to create gene expression profiles for developmental abnormalities in the Havanese and for cholesterol sensitivity in the mixed-breed colony dogs. Distinct expression profiles were generated for each group, and several genes of interest were identified as being both differentially expressed (>±2-fold change) and statistically significant (p-value<0.05).

Avian circadian clocks are composed of a distributed network of neural and peripheral oscillators. Three neural pacemakers, located in the pineal, the eyes, and the hypothalamus, control circadian rhythms of many biological processes through complex interactions with slave oscillators located throughout the body. This system, an astonishing reflection of the life history of this diverse class of vertebrates, allows birds to coordinate biochemical and physiological processes and harmonize them with a dynamic environment. Much work has been done to understand what roles these pacemakers have in avian biology, how they function, and how they interact to generate overt circadian rhythms. The experimental work presented in this dissertation uses the domestic chicken, Gallus domesticus, as a model to address these questions and carry forward current understanding about circadian biology in this species. To do so, we utilized a custom DNA microarray to investigate rhythmic transcription in cultured chick pineal cells. We then sought to identify genes which might be a component of the pineal clock by screening for rhythmic transcripts that are sensitive to a phase-shifting light stimulus. Finally, we surgically removed the eyes or pineal from chickens to examine the roles of these extra-SCN pacemakers in regulating central and peripheral rhythms in metabolism and clock gene expression. Using these methods, we show that the oscillating transcriptome is diminished in the chick pineal ex vivo, while the functional clustering of clock controlled genes is similar. This distribution reveals multiple conserved circadian regulated pathways, and supports an endogenous role for the pineal as an immune organ. Moreover, the robustness of rhythmic melatonin biosysnthesis is maintained in vitro, demonstrating that a functional circadian clock is preserved in the reduced subset of the rhythmic pineal transcriptome. In addition, our genomic screen has yielded a list of 28 genes that are candidates for functional screening. These should be evaluated to determine any potential role they may have as a component of the pineal circadian clock. Finally, we report that the eyes and pineal similarly function to reinforce rhythms in brain and peripheral tissue, but that metabolism and clock gene expression are differentially regulated in chick.

Equine laminitis is a debilitating disease that causes extreme sufferring in afflicted horses and often results in a lifetime of chronic pain. The exact sequence of pathophysiological events culminating in laminitis has not yet been characterized, and this is reflected in the lack of any consistently effective therapeutic strategy. For these reasons, we used a newly developed 21,000 element equine-specific whole-genome oligoarray to perform transcriptomic analysis on laminar tissue from horses with experimentally induced models of laminitis: carbohydrate overload (CHO), hyperinsulinaemia (HI), and oligofructose (OF). Samples were collected during the developmental (DEV) and Obel grade 1 (OG1) stages of laminitis for the CHO model. For the HI model, samples were collected at the Obel grade 2 (OG2) stage. For the OF model, samples were collected at the 12 h and 24 h time points. Appropriate control samples were obtained for all models. This is the first genome-wide transcriptome analysis of laminar tissue using an equine 21,000 70-mer long oligoarray approach in CHO, HI and OF induced laminitis. Overall, we identified the differential expression of genes encoding S100 calcium binding proteins, extracellular matrix proteins, glycoproteins, transporters, olfactory receptors, genes involved in signal transduction, body‟s homeostasis, apoptosis, and immune response. Between CHO and OF models of laminitis, there were more shared genes. We discovered several common differentially expressed genes (i.e., ADAMTS1, CYCS and CXCL14) among all three models that are likely important to the pathogenesis of equine laminitis. We also discovered what appear to be central roles of apoptosis, inflammatory response, and intracellular ion homeostasis molecular processes in CHO and OF models of laminitis. Pathway analysis detected the NOD-like receptor signaling pathway, which is involved in recognition of intracellular bacteria in both the CHO and OF models of laminitis. Genetic network analysis indicated convergent pathway core molecules present in equine acute laminitis: p38 MAPK and NF-κB. Most importantly, our results of overexpression of anti-microbial genes (i.e., DEFB4, PI3, and CXCL14) suggest the central involvement of these genes in the progression of early equine laminitis and will allow refinement of current hypotheses of disease pathogenesis.

RNA editing by adenosine deamination is catalyzed by members of an enzyme family known as adenosine deaminases that act on RNA (ADARs). ADARs can change the structure of RNA by changing an AU base-pair to an IU mismatch. This frequently modifies the function of the encoded protein, and an emerging theme associated with A-to-I mRNA editing is that tissues often regulate the ratio of proteins expressed from edited and unedited mRNAs to fine-tune cellular responses and functions. In mammals, pre-mRNA of receptor proteins involved in neurotransmission, including serotonin receptors and glutamate receptors, are edited. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. To identify RNAs containing inosine residues, this study used a multi step approach; including (1) inosine-specific base cleavage and RNase T1 digestion, (2) purification of polyA-tailed mRNA, (3) RT w/ T7-polydT primer, (4) probe synthesis and microarray analysis. Using this method it is possible to identify novel targets of A to I editing. Approximately 100 genes showed a significant decrease in two arrays. Future analysis of these targets should reveal the biomedical significance of A-to-I editing.

High-throughput technologies for rapid measurement of vast numbers of biolog- ical variables offer the potential for highly discriminatory diagnosis and prognosis; however, high dimensionality together with small samples creates the need for fea- ture selection, while at the same time making feature-selection algorithms less reliable. Feature selection is required to avoid overfitting, and the combinatorial nature of the problem demands a suboptimal feature-selection algorithm. In this dissertation, we have found that feature selection is problematic in small- sample settings via three different approaches. First we examined the feature-ranking performance of several kinds of error estimators for different classification rules, by considering all feature subsets and using 2 measures of performance. The results show that their ranking is strongly affected by inaccurate error estimation. Secondly, since enumerating all feature subsets is computationally impossible in practice, a suboptimal feature-selection algorithm is often employed to find from a large set of potential features a small subset with which to classify the samples. If error estimation is required for a feature-selection algorithm, then the impact of error estimation can be greater than the choice of algorithm. Lastly, we took a regression approach by comparing the classification errors for the optimal feature sets and the errors for the feature sets found by feature-selection algorithms. Our study shows that it is unlikely that feature selection will yield a feature set whose error is close to that of the optimal feature set, and the inability to find a good feature set should not lead to the conclusion that good feature sets do not exist.

Salmonella enterica serovar Enteritidis (SE) is one of the most common food-borne pathogens that cause human salmonellosis. Contamination of consumed poultry products continues to be a global threat to public health. Genetic resistance using genomic approach provides a promising solution to controlling SE infection in poultry. The mechanism of SE resistance in chickens remains elusive. Three different approaches, microarray techology, gene silencing, and computational gene analysis, have been utilized to study SE-induced transcriptional changes of host immune response in the chicken. A whole genome chicken 44K microarray was used to analyze the transcriptome of heterophils from SE-resistant (line A) and SE-susceptible chickens (line B) with/without in vitro SE stimulation. Many differentially expressed immune-related genes were found in the SE-infected to non-infected comparison, where more immune-related genes were down-regulated in line B than line A. These results suggested a similar Toll-like receptor (TLR) regulatory network might exist in heterophils of both lines, and provided strong candidates for further investigating SE resistance and susceptibility in chickens. In the gene silencing study, small interfering RNAs (siRNA) were used to specifically inhibit the expression of NFkB1 in the chicken HD11 macrophage cell line with SE challenge. Genes related to the NF-kB signaling pathway were selected to examine the effect of NFkB1 inhibition on TLR pathway. With 36% inhibition of NFkB1 expression, the results showed an increased expression of TLR4 and interleukin (IL)-6 following SE challenge and suggested a likely inhibitory regulation of NFkB1 on TLR signal pathway. Finally, two novel chicken C-type lectin-like receptors were identified and annotated to chicken CD69 and CD94/NKG2-like with multiple evidences generated by computational (in-silico) sequence analysis. Both genes located in a region on chicken chromosome 1 that is syntenic to mammalian Nature Killer Receptor Complex (NKC) region, which may have existed before the divergence between mammals and aves. While siRNA lays the foundation of using loss-of-function approach on testifying gene-gene interactions, in-silico analysis aids in gathering information of unknown genes of great interest. Both approaches provide great potential to use for down-stream analysis following microarray study.