Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of 'housekeeping' gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression 'signatures' that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of 'breaks' in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance.

To establish the gene expression profile and cascade subsequently on avian sex-determining candidate genes, seven avian sex-determining candidate genes including DMRT1, FET1, FOXL2, LHX9, HINT1, SMC2L1 and SOX9 were analyzed at early embryogenesis. Quantitative reverse transcription PCR (Quantitative RT-PCR) technology was used to establish the gene expression profiles among these genes at four, five, six and seven days of embryos. The results of quantitative RT-PCR reveal that the DMRT1 was expressed in chicken embryos of both sexes. DMRT1 gene expressions were up-regulated at four, five and six days of chicken embryos. DMRT1 expression increased at 5-Dpc. of male embryos, however, expression was not signification different in females embryos. Gene expression of FET1, FOXL2, LHX9 and HINT1 were higher in females than in males. The SMC2L1 and SOX9 were expressed in both sexes. Also, to identify the novel sex-determination genes in early chicken subtractive embryos, cDNA libraries from male-minus-female and female-minus-male 3.5 Dpc. embryos cDNA were established. Gene annotation was carried out by data-mining in public databases, GeneBank (NCBI, USA) and TIGR gene indices (The Institute for Genome Research, USA). A total 548 of colonies in male-minus-female library and 79 sequences were annotated. However, a total of 589 of colonies in female-minus-male library and 16 sequences were annotated. Sequences were homologous to the steroid 5£\-reductase protein (SRD5A1) using BLASTx in male-minus-female subtractive library. The SRD5A1 may play a sex-differentiation role in male chicken. We need more study to know function of steroid 5£\-reductase protein in future.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

The aim in this thesis was to gain an understanding of changes in gene expression in the hypothalamus of postpartum beef cows during the period of transition from suppressed ovarian follicular growth to increased follicular growth, and the resumption of ovulation. Beef cows tend to have an extended period of anoestrus after calving. This trait is particularly pronounced in tropically-adapted Zebu breeds. In addition to a genetic component, the postpartum anoestrous period can be influenced by age, body condition, the nutrient requirement of lactation, suckling stimulus, and maternal bonding. An extended postpartum anoestrous period is particularly evident in primiparous beef cows. This is understandable given that primiparous cows have yet to reach their mature body size which means there is a requirement to maintain maternal tissue growth whilst at the same time directing nutrients for milk production. Weaning removes maternal bonding, the suckling stimulus and nutrient requirement of milk production and, provided that nutrient supply and body condition are appropriate, primiparous cows show increased ovarian activity and resume ovulation after weaning. In the present thesis, groups of primiparous Zebu cows were weaned to promote increased ovarian follicular growth and hypothalamic gene expression was compared for weaned cows and contemporary cows that continued to lactate. Candidate genes were studied using quantitative real-time PCR (qRT-PCR) and a gene expression microarray was used to discover new genes and gene networks. Gene expression was examined in the anterior hypothalamic-preoptic area (sub-region H1) and posterior ventral hypothalamus (sub-region H2). The demarcation between H1 and H2 was a vertical line from the mid-point of the median eminence-pituitary stalk to the thalamus. Candidate genes studied by qRT-PCR included, gonadotrophin releasing hormone (GNRH1), kisspeptin (KISS1), neuropeptide Y (NPY), oestrogen receptor alpha (ESR1) and leptin receptor (LEPR). Marked regional expression was demonstrated for these genes. The expression of GNRH1 was greatest in the anterior hypothalamic region (sub-region H1) whilst the expression of KISS1 was greatest in the ventral posterior hypothalamic region (sub-region H2). Relative expression of LEPR, ESR1 and NPY was greater in H2 than H1. The regional gene expression patterns for GNRH1, KISS1, LEPR, ESR1 and NPY in the hypothalamus of cows were consistent with regional expression reported for other species. Weaning was associated with a decrease in the expression of LEPR, ESR1 and NPY. With regard to ovarian phenotype, there was a greater LEPR expression associated with ovarian phenotype 1 (OP1, follicles to 5mm) compared with ovarian phenotype 2 (OP2, follicles to 10mm) and ovarian phenotype 3 (OP3, recently ovulated) in sub-region H1. Relative expressions for ESR1, LEPR and NPY were highly correlated, particularly in sub-region H2. The evaluation of gene expression by microarray for cows with different ovarian phenotypes provided evidence of interactions between hormonal regulation and cell-cell signalling within the hypothalamus. Genes that were differentially expressed for different ovarian phenotypes were associated with reproduction, energy balance, the immune system and stress. Other genes that showed differential expression were involved with cell adhesion, synaptic transmission, ion signalling and neuronal development. The latter findings were interpreted to suggest that neuronal and glial cell plasticity is a feature of changes in reproductive functions of the hypothalamus. The evaluation of gene expression by microarray for weaned and suckled cows, irrespective of ovarian phenotype, identified differentially expressed genes associated with energy balance, fluid homeostasis, milk synthesis, stress, and oestrogen signalling. With regard the latter, thirty seven genes involved in oestrogen signalling through ESR1, or in other ways associated with oestrogen, were found to be differentially expressed between weaned and lactating cows. ESR1 occupied the central position of a primary gene network based on the present study. Six differentially expressed genes were shown by gene network analysis to be centred in nodes interacting closely with ESR1. Phospholipase-C-gamma (PLCG2), vitronectin (VTN) and endopin 1 (SERPINA3) are three genes associated with hypothalamic plasticity and neurotransmission that were differentially expressed between cows with OP1 and OP2, indicating a possible role in the shift to increased ovarian follicular growth and ovulation. The findings for ESR1 were consistent with the major role of oestrogen in female reproduction and in particular the known actions of oestrogen in regulating the hypothalamus during reproductive transition phases in females associated with puberty, seasonality and postpartum. Gonadotrophin inhibitory hormone (GnIH) is derived from Neuropeptide VF precursor (NPVF), which is encoded by NPVF gene transcripts. NPVF had reduced expression in cows that had ovulated (OP3) compared with OP1 and OP2. GnIH inhibits gonadotrophin secretion by directly acting on GnRH neurons as well as modulating the suppressive effects of oestrogen negative feedback. In addition, GnIH has been shown to play a role in seasonal regulation of reproduction in birds. The lesser expression of NPVF in cows that had resumed ovulation, particularly evident in sub-region H2, provides initial evidence that GnIH has an important role in maintaining the suppressive effects on reproduction during postpartum anoestrus in cattle. In summary, the studies in this thesis have identified hypothalamic genes and gene networks that potentially are important in the control of reproductive function in the postpartum cow. The thesis has also established that the postpartum cow can be used as an experimental model for fundamental studies that generate new knowledge on the reproductive biology of the postpartum period.

Growth of bacteria in beer leads to turbidity and off-flavors, resulting in a spoiled and unpalatable product and thus economic loss. The most common beer-spoilage organisms (BSOs) are lactic acid bacteria (LAB), with Lactobacillus and Pediococcus species being the most problematic. Because of the harsh environment (low nutrients, antimicrobial compounds ethanol and hops, anaerobic), only select isolates are able to sustain growth in and spoil beer. To begin understanding the phenomenon of LAB adapting to overcome stresses in beer, ethanol tolerance, hop resistance, and nutrient acquisition mechanisms were investigated. First, ethanol tolerance was analyzed in the context of beer-spoilage ability, and it was found that it is intrinsically high in LAB, thus leading to the conclusion that LAB ability to spoil beer is not dependent on ethanol resistance levels. This was then followed by genome sequencing of the BSO Pediococcus claussenii ATCC BAA-344T (Pc344) to elucidate mechanisms being used to resist hops and acquire low abundance or alternative nutrients. Subsequent analysis of Pc344 and Lactobacillus brevis BSO 464 via reverse transcription quantitative PCR demonstrated the variability found among BSOs in the presence of beer-spoilage-related genes and their use during growth in beer. Further analysis of Pc344 was performed via RNA-sequencing to get a global view of gene expression during mid-logarithmic growth in beer. It was found that several alternative nutrients were being used by Pc344 to sustain growth, and that hop resistance was enabled by a variety of mechanisms including oxidative stress response and pH control. Finally, genomic comparison of BSOs determined that conservation is only present for closely related organisms and that no specific genes/proteins are indicative of an isolate’s beer-spoilage potential. It is more likely that horizontal gene transfer plays a major role in LAB adaption for growth in beer, and that plasmids are very important for this evolution, as was demonstrated by plasmid-variants of Pc344. The main conclusions of this thesis are therefore that hop resistance is the main factor determining ability to grow in beer, and that transfer of genetic elements is the driving force behind LAB evolving into BSOs.

Background. Angiogenesis is essential for tumour growth. Vascular endothelial growth factor (VEGF) and its isoforms were investigated in relation to the clinical course in a large number of patients with renal cell carcinoma (RCC). Methods. RCC subtypes and behaviour were established by clinicopathological criteria and surveillance. VEGF expression was analysed in serum by enzyme-linked immuno-sorbent assay (ELISA) and in tumour tissue by reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC), and Western blot (WB). Results. Serum VEGF (S-VEGF) was increased in RCC compared to control group. S-VEGF correlated with tumour stage and grade and was associated with survival in men but not in women. S-VEGF correlated with blood platelet counts, which were inversely correlated to increasing age in women, and they were decreased in chronically medicated patients, particularly in men. In contrast to S-VEGF, platelet counts associated with survival only in patients free of medication and chronic diseases. RT-PCR showed a correlation between VEGF121/VEGF165 mRNA and between VEGF165/VEGF-R1 mRNA. There was no association between different VEGF mRNA isoforms and S-VEGF. Conventional renal cell carcinoma (CRCC) had higher VEGF165, VEGF121, and VEGF-R1 mRNA levels compared with papillary renal cell carcinoma (PRCC). IHC VEGF staining was strong in kidney cortex. Kidney tumour showed a considerable variation in cytoplasmatic VEGF expression, which correlated with tumour size. Although, there was no difference in VEGF expression between the RCC types, VEGF expression was associated with survival only in CRCC. WB showed a strong protein expression of both VEGF189 and VEGF165 in kidney cortex. In kidney tumour, expression of VEGF189 varied the most, VEGF165 less so, and VEGF121 was rarely detected. Both CRCC and PRCC expressed low levels of VEGF189 and VEGF165 compared with kidney cortex. Chromophobe renal cell carcinoma (ChRCC) expressed VEGF189 levels comparable to those from kidney cortex, while VEGF165 was lower. In PRCC and ChRCC, VEGF189 levels correlated inversely with advancing tumour stage, and in PRCC, VEGF165 levels correlated inversely with increasing tumour size. VEGF189 was an independent prognostic factor for survival in patients with PRCC. Conclusions. S-VEGF has a stronger association to progression in RCC than platelet count. CRCC showed high levels of VEGF mRNA isoforms and VEGF-R1 mRNA compared to PRCC. VEGF mRNA isoforms expression decreased with advancing stage. IHC demonstrated VEGF expression in cell cytoplasm related to tumour growth, particular in CRCC. Different VEGF isoform patterns were found in different RCC types. Protein VEGF189 expression was associated with tumour stage and was an independent prognostic factor in PRCC. Protein VEGF165 expression was generally low and had no prognostic value. The trend for decreasing levels of VEGF isoforms in advanced tumour stages may indicate that angiogenic activity is an early event in tumour growth induced by VEGF, but that during later tumour progression the role of VEGF is less clear.

To identify known genes or expressed sequence tags (ESTs) which are expressed specifically or preferentially in the chicken hypothalamus and pituitary gland related to highly reproductive performance, two reciprocal cDNA libraries were constructed using a subtractive hybridization strategy. Two different strains, L2 (dam line; n=12) and B (sire line; n=12) of Taiwan Country Chickens (TCCs), which were originated from one single strain and further subjected to 40-wk egg production and comb size, body weight, respectively since 1982, were used in our study. A total of 324 and 370 clones were identified from L2-subtract-B and B-subtract-L2 hypothalamus/pituitary cDNA libraries. 311 and 360 single inserted sequences from each cDNA library, 53 and 23 non-redundant candidate genes were identified. Quantitative reverse-transcription (RT)-PCR were used to validate the association of mRNA expression profiles of the identified candidate genes and high-egg yield trait in another 118 hypothalamuses and pituitary glands that were dissected from seven different chicken stocks, including B-, L2-, Black-, Red-feather TCCs, commercial Single-Comb White Leghorn (WL) layer at National Chung-Hsing University (NCHU) and Red-feather TCCs grouped into high eggs (Red-high) & low eggs (Red-low) to 40 wks of age at National Chiayi University (NCYU). Among identified genes including known genes and novel genes, involving 33 screened genes, Inhibitor-1 of protein phosphatase type 2A (ANP32A), 3-hydroxybutyrate dehydrogenase (BDH), Contactin (CNTN1), Deiodinase iodothyronine type II (DIO2), Inhibitor of growth family, member 3 (ING3), Lysosomal-associated transmembrane protein 4 beta (LAPTM4B), Neural cell adhesion molecule 1 (NCAM1), DJ-1 protein (PARK7), Prostaglandin D2 synthase (PGDS), Prolactin (PRL), Protocadherin 1 (PCDH1), Pleiomorphic adenoma gene 1 (PLAG1), GTP-binding protein SAR1a (SAR1A), Secretogranin II (SCG2), Stathmin 2 (STMN2), T-box protein 2 (TBX2) were up-regulated in B-subtract-L2 cDNA library. Among above-mentioned 16 identified genes, there were 9 genes related to high-egg yield in chickens., including BDH, NCAM1, PCDH1, PGDS, PLAG1, PRL, SAR1A, SCG2, STMN2.

名古屋大学博士学位論文 学位の種類:博士（医療技術学）（課程） 学位授与年月日:平成20年3月25日

L'anévrysme de l'aorte abdominale (AAA) est un problème de Santé Publique qui touche principalement les hommes de plus de 65 ans. L'AAA souvent asymptomatique évolue vers la rupture associée à un taux de mortalité élevé. Parmi les acides ribonucléiques (ARNs) non codants, les microARNs (miARNs), stables dans le tissu et les biofluides, sont des candidats intéressant dans la recherche de biomarqueurs. L'inflammation, la dégradation de la matrice extra-cellulaire (MEC) et la raréfaction de la média participent à l'AAA. De nombreuses cellules inflammatoires sont impliquées dans l'AAA. La raréfaction des cellules musculaires lisses (CML) est secondaire à l'anoïkis, apoptose par détachement cellulaire de la MEC. Une analyse protéomique différentielle de CML en culture, issues de patients porteurs d'AAA, réalisée au laboratoire a montré que la désintégrine et metalloprotéinase avec un motif de thrombospondine 5 (ADAMTS 5) est surexprimée dans les CML de patients présentant un AAA. L'isolement des cellules par la microdissection laser permet de conserver le phénotype des cellules isolées et de mettre en évidence des marqueurs potentiels de l'AAA masqués par l'analyse du tissu global. Mon travail de thèse a consisté à partir des cellules isolées de la paroi anévrysmale de l'aorte abdominale sous-rénale chez l'Homme: à effectuer une analyse globale des miARNS et une analyse ciblée de l'ADAMTS 5, métalloprotéase qui a une action enzymatique sur les protéines de la MEC. Les objectifs de ce travail sont une meilleure compréhension de l'AAA et l'identification de nouveaux biomarqueurs.La distribution des cellules dans la paroi anévrysmale est étudiée par immunohistochimie sur des biopsies anévrysmales et d'aortes saine obtenues chez l'Homme. Les cellules localisées sont isolées par microdissection laser. L'analyse par criblage de l'expression des miARNs des cellules isolées de l'AAA et des CML issues d'aorte saine est réalisée sur puce. L'expression différentielle de miARNs sélectionnés est analysée par PCR quantitative dans des cellules isolées de l'AAA et dans du tissu global. L'expression des miARNs sélectionnés est ensuite comparée dans le plasma des patients présentant un AAA et de patients athérosclérotiques sans AAA par PCR pour identifier de potentiels biomarqueurs. Dans l'AAA, les macrophages M1 proinflammatoires sont retrouvés dans l'adventice et les macrophages M2 anti inflammatoires dans le thrombus intraluminal, les lymphocytes de type B sont retrouvés organisé en organe lymphoïde tertiaire adventitielle ou ATLOs dans 11 échantillons sur 20 analysés. Les CML sont rares et strictement localises au niveau de la média. Sur 850 miARNs testés dans la puce, 205 miARNs sont exprimés dans les cellules isolées. Les miR-29b et let-7f sont augmentés dans le plasma de patients porteurs d'AAA et représentent de potentiel biomarqueurs.L'expression d'ADAMTS 5 dans les CML de la paroi anévrysmale est évaluée par immunohistochimie dans la paroi aortique saine et anévrysmale et quantifiée par Western-blot dans les CML isolées de la paroi aortique saine et anévrysmale.Deux morphotypes de CML anévrysmales ont été identifiés: un morphotype arrondi positif au marqueur de l'apoptose, caspase 3 et un morphotype allongé, similaire aux CML de l'aorte saine. Le profil d'expression des sous-unités d'ADAMTS 5 est diffèrent dans les CML arrondies et les CML allongées anévrysmales et saines. La mise en apoptose des CML a été mise au point in vitro pour étudier les mécanismes impliqués dans les modifications d’ expression d'ADAMTS 5 dans l'AAA et les conséquences sur son action enzymatique.L'approche systématique de l'expression transcriptomique des cellules anévrysmales isolées a identifié des marqueurs potentiels de l'AAA, les miR-29b et let-7f et l'analyse ciblée suggère l'implication d'ADAMTS 5 dans le profil évolutif des CML vers l'anoïkis dans l'AAA. Des études complémentaires permettront une meilleure compréhension de l'AAA. / Abdominal aortic aneurysm (AAA) is a public health problem, which mainly affects men older than 65 year. AAA are usually asymptomatic with a natural evolution towards rupture associated with a high mortality rate. Among non coding ribonucleic acids (RNAs), microRNAs are stable in tissue and biofluids and are interesting candidates for the search of biomarkers. Inflammation, extracellular matrix (ECM) degradation and media rarefaction are involved in AAA. Many inflammatory cells are involved in AAA. Anoikis is an apoptosis secondary to a cell detachment from ECM and is responsible for rarefaction of smooth muscle cells (SMC). Differential proteomic analysis of cultured SMC from AAA patients was performed in the laboratory and highlighted the overexpression of a disintegrin and metalloproteinase with thrombospondin motif of type 5 (ADAMTS 5) in SMC of AAA patients. Isolation of cells with laser microdissection allows to keep their phenotype and to find potential markers that may be masked by global tissue analysis.The aim of my PhD work was to perform a global miRNA screening of cells isolated from human aneurysmal wall and an analysis targeted on ADAMTS 5, a metalloprotease with an enzymatic activity on ECM proteins. The main objectives were a better understanding of AAA and the identification of new biomarkers.The distribution of cells in the aneurysmal wall was studied by immunohistochemistry in human aneurysmal and healthy aortic samples. Once located, the cells were isolated by laser microdissection and screened for miRNAs by microarrays. Differential expression of selected miRNAs was quantified by PCR in the cells isolated by laser microdissection and in whole aortas. They were then compared in plasma of AAA patients and atherosclerotic patients without AAA by quantitative PCR to identify potential biomarkers.In AAA, the M1 proinflammatory macrophages were located in the adventitia and the M2 antiinflammatory macrophages in the intraluminal thrombus; the type B lymphocytes were organized in tertiary lymphoid organs (ATLOs) in 11/20 of analysed samples. SMC were rare and restricted to the media. Among the 850 miRNAs tested on microarray, 205 miRNAs were detected in isolated cells. MiR-29b and let-7f were upregulated in plasma of AAA patients, and thus are potential biomarkers.The expression of ADAMTS 5 in aneurysmal SMC was evaluated by immunohistochemistry of healthy and aneurysmal aortic wall and quantified by Western blot in isolated SMC from healthy and aneurysmal wall.Two aneurysmal SMC morphotypes were identified: a rounded morphotype positive for caspase 3, an apoptotic marker, and a spindle-shaped morphotype similar to the healthy aortic SMC. The expression profile of ADAMTS 5 subunits was different in rounded SMC compared to aneurysmal and healthy spindle-shaped SMC. In vitro induction of apoptosis of SMC was established in order to study the mechanisms involved in ADAMTS 5 expression in AAA and their consequences on enzymatic actions.The global transcriptomic screening of aneurysmal cells isolated by laser microdissection has identified potential markers of AAA, miR-29b and let-7f. The targeted analysis suggested that ADAMTS 5 is involved in the evolution profile of SMC towards anoikis in AAA. Further investigations will allow a better understanding of AAA pathophysiology.