Previous studies have demonstrated specific biochemical abnormalities in the noradrenergic locus coeruleus (LC) that are strongly associated with major depressive disorder (MDD). Here, we studied the LC of 4 pairs of MDD and matched control subjects by gene expression microarray analysis in an effort to accelerate the discovery of pathobiological abnormalities of these cells in MDD. Among matching criteria, pH values of control (6.71±0.06) and MDD (6.66±0.12) subjects were closely matched. Gene expression profiling using whole human genome microarrays (Agilent) revealed statistically significant changes in approximately 50 transcripts in the LC of depressive subjects. Quantitative real-time PCR (qPCR) was used to analyze transcripts identified by microarray anlayses. In initial studies of 11 of these transcripts that demonstrated a >2-fold change in microarrays, only 3 transcripts were confirmed by qQPCR in a larger sample of 11-12 pairs of MDD and matched control subjects. Amounts of bone morphogenetic factor-7 (BMP7; p=0.001) and potassium channel subfamily K, member 7 (KCNK7; p=0.049) mRNAs were significantly lower in MDD subjects compared to control subjects (~2-fold difference). In contrast, neurolysin mRNA levels were significantly higher (~3-fold; p=0.03) in MDD than in control subjects. BMP7 is a member of the TGF-β superfamily and has neuroprotective and neurotrophic effects on catecholaminergic neurons. The KCNK family of potassium channels contribute to the excitability of neurons. Neurolysin is a zinc-dependent metallopeptidase involved in neuropeptide metabolism. The present study is the first report of these novel gene expression abnormalities in the LC of MDD subjects. These findings enhance our understanding of the pathobiology of MDD and may represent novel targets for pharmacological management of depression.

Natural microbial communities are the ultimate drivers of change in any ecosystem. Through chemical contamination of natural environments, these communities are exposed to many different types of chemical stressors; however, research on whole genome responses to this contaminant stress is limited. This research examined the stress response of a common soil bacterium, <i>Pseudomonas aeruginosa</i>, to a common environmental pollutant, pentachlorophenol (PCP). In the first part of the research, it was revealed that nutrient-limited <i>P. aeruginosa</i> is able to respond to PCP with minimal physiological damage due to the upregulation of multidrug efflux pumps. Further study of this PCP-mediated induction of efflux pumps revealed a simultaneous increase in antibiotic resistance. It was discovered that the resistance nodulation-cell division (RND) efflux pump, MexAB-OprM, in particular is responsible for the PCP-induced increase in antibiotic resistance. Both whole cell physiological indicators and whole genome analysis were used to examine the stress response of <i>P. aeruginosa</i> to PCP. Cells were grown in a chemostat at a low growth rate to simulate nutrient-limiting growth in the natural environment. Whole cell acetate uptake rates (WAUR) and viable cell counts as colony forming units (CFU) were determined as cells were exposed to increasing concentration of PCP. At the same time, changes in gene expression were examined by Affymetrix microarray technology. Results showed little change in whole-cell physiology, with no difference in WAUR and only a slight reduction in CFU. However, the microarrays revealed that over 100 genes either increased or decreased expression greater than two-fold due to the PCP exposure. In particular, multiple multidrug efflux genes were upregulated in response to the PCP. The results were validated by real time reverse transcription polymerase chain reaction (RT-PCR) for one of these genes. Further analysis of the effects of MexAB-OprM showed that this particular efflux pump is essential for the response of <i>P. aeruginosa</i> to the toxin PCP. Induction of multidrug efflux pumps is responsible for the development of antibiotic resistance in strains of <i>P. aeruginosa</i>. Therefore, it was investigated whether PCP might induce resistance to a variety of antibiotics. The research was further extended to examine the effect of a variety of organic contaminants on MexAB-OprM efflux and antibiotic resistance development. PCP, 2,4-dinitrophenol, benzoate and Roundup® all induced antibiotic resistance. However, although MexAB-OprM is required for optimal growth in the presence of all chemicals, this particular efflux pump is only involved in increased resistance with PCP. This was confirmed using RT-PCR as <i>mexB</i> expression was induced by PCP, but not by the other three chemicals. A long term generational study on the effects of PCP did not result in a stable antibiotic-resistant phenotype; however, RT-PCR showed that <i>mexB</i> induction is a direct result of PCP exposure and can be reversed by removal of PCP. Together, these results demonstrate the necessity to understand functional responses to contaminant stress. Discovery of direct induction of multidrug efflux pumps and the resulting increase in antibiotic resistance has significant implications for environmental microbiology and public health. This research suggests that organic contamination may result in antibiotic resistance and that antibiotic resistant strains may have a survival advantage in contaminated environments. / Ph. D.

Indiana University-Purdue University Indianapolis (IUPUI) / The heart is the first organ to form during development in vertebrates and Nkx2-5 is the first marker of cardiac specification. In Xenopus laevis, Nkx2-5 is essential for heart formation, but early targets of this homeodomain transcription factor have not been fully characterized. In order to discover potential early targets of Nkx2-5, synthetic Nkx2-5 mRNA was injected into eight-cell Xenopus laevis embryos and changes in gene expression measured using microarray analysis. While Xenopus laevis is a commonly used model organism for developmental studies, its genome remains poorly annotated. To compensate for this, a cross-species annotation database called CrossGene was constructed. CrossGene was created by exhaustively comparing UniGene transcripts from Homo sapiens, Mus musculus, Rattus norvegicus, Gallus gallus, Xenopus laevis, Danio rerio, Drosophila melanogaster, and Caenorhabditis elegans using the BLAST family of algorithms. Networks were then assembled by recursively combining reciprocal best matches into groups of orthologous genes. Gene ontology annotation from all organisms could then be applied to all members of the reciprocal group. In this way, the CrossGene database was used to augment the existing genomic annotation of Xenopus laevis. Combining cross-species annotation with differential gene expression analysis of Nkx2-5 overexpression led to the discovery of 99 potential targets of Nkx2-5.

<p> DNA microarrays can measure the gene expression of thousands of genes at a time to identify differentially expressed genes. The Affymetrix GeneChip system is a platform for the high-density oligonucleotide microarray to measure gene expression using hundreds of thousands of 25-mer oligonucleotide probes.</p> <p> To deal with Affymetrix microarray data, there are three stages of preprocessing to produce gene expression measurements/values. These are background correction, normalization and summarization. At each stage, numerous methods have been developed.</p> <p> Our study is based on Affymetrix MG_U74Av2 chip with 12488 probe sets. Two strains of mice called NOR and NOR.NOD_Idd4/11 mouse are hybridized for the experiment. We apply a number of commonly used and state-of-art normalization methods to the data set, thus compute the expression measurements for different methods. The major methods we discuss include Robust Multi-chip Average (RMA), MAS 5.0, GCRMA, PLIER and dChip.</p> <p> Comparisons in terms of correlation coefficient, pairwise expression measures plot, fold change and Significance Analysis of Microarray (SAM) are conducted.</p> / Thesis / Master of Science (MSc)

This thesis presents a new ant-optimized biclustering technique known as SNAP biclustering, which runs faster and produces results of superior quality to previous techniques. Biclustering techniques have been designed to compensate for the weaknesses of classical clustering algorithms by allowing cluster overlap, and allowing vectors to be grouped for a subset of their defined features. These techniques have performed well in many problem domains, particularly DNA microarray analysis and collaborative filtering. A motivation for this work has been the biclustering technique known as bicACO, which was the first to use ant colony optimization. As bicACO is time intensive, much emphasis was placed on decreasing SNAP's runtime. The superior speed and biclustering results of SNAP are due to its improved initialization and solution construction procedures. In experimental studies involving the Yeast Cell Cycle DNA microarray dataset and the MovieLens collaborative filtering dataset, SNAP has run at least 22 times faster than bicACO while generating superior results. Thus, SNAP is an effective choice of technique for microarray analysis and collaborative filtering applications. / Master of Science

No / Genome-wide microarray technology has facilitated the systematic discovery of diagnostic biomarkers of cancers and other pathologies. However, meta-analyses of published arrays often uncover significant inconsistencies that hinder advances in clinical practice. Here we present an integrated microarray analysis framework, based on a genome-wide relative significance (GWRS) and genome-wide global significance (GWGS) model. When applied to five microarray datasets on melanoma published between 2000 and 2011, this method revealed a new signature of 200 genes. When these were linked to so-called ‘melanoma driver’ genes involved in MAPK, Ca2+, and WNT signaling pathways we were able to produce a new 12-gene diagnostic biomarker signature for melanoma (i.e., EGFR, FGFR2, FGFR3, IL8, PTPRF, TNC, CXCL13, COL11A1, CHP2, SHC4, PPP2R2C, and WNT4). We have begun to experimentally validate a subset of these genes involved in MAPK signaling at the protein level, including CXCL13, COL11A1, PTPRF and SHC4 and found these to be over-expressed in metastatic and primary melanoma cells in vitro and in situ compared to melanocytes cultured from healthy skin epidermis and normal healthy human skin. While SHC4 has been reported previously to be associated to melanoma, this is the first time CXCL13, COL11A1, and PTPRF have been associated with melanoma on experimental validation. Our computational evaluation indicates that this 12-gene biomarker signature achieves excellent diagnostic power in distinguishing metastatic melanoma from normal skin and benign nevus. Further experimental validation of the role of these 12 genes in a new signaling network may provide new insights into the underlying biological mechanisms driving the progression of melanoma.

<p>Through a number of biological, technological and computational achievements during the 20th century and the devoted work of hundreds of researchers the sequence of the human and other genomes are now available in public databases. The current challenge is to begin to understand the information encoded by the DNA sequence, to elucidate the functions of the proteins and RNA molecules encoded by the genes as well as how they are regulated. For this purpose new technologies within the area of functional genomics are being developed. Among those are powerful tools for gene expression analysis, such as microarrays, providing means to investigate when and where certain genes are used.</p><p>This thesis describes a method that was developed to enable gene expression analysis, on the transcriptome level, in small tissue samples. It relies on PCR amplification of the 3’-ends of cDNA (denoted 3’-end signature tags). PCR is a powerful technology for amplification of nucleic acids, but has not been used much for transcript profiling since it is generally considered to introduce biases, distorting the original relative transcript levels. The described method addresses this issue by generating uniformly sized representatives of the transcripts/cDNAs prior to amplification. This is achieved through sonication which, unlike restriction enzymes, does not require a specific recognition sequence and fragments each transcript randomly. The method was evaluated using cDNA microarrays, Affymetrix™ oligonucleotide arrays and real-time quantitative PCR. It was shown to perform well, yielding transcript profiles that correlate well to the original, unamplified material, as well as being highly reproducible.</p><p>The developed method was applied to stem cell biology. The variability in gene expression between different populations of cultured neural stem cells (neurospheres) was investigated. It was shown that neurospheres isolated from different animals or passaged to different degrees show large fluctuations in gene expression, while neurospheres isolated and cultured under identical conditions are more similar and suitable for gene expression analysis. A second study showed that withdrawing epidermal growth factor (EGF) from the culture medium when treating the cells with an agent of interest has profound effects on gene expression, something which should be taken into consideration in future neurosphere studies.</p>

Adenomas que se desenvolvem a partir da linhagem corticotrófica (corticotropinomas) secretam ACTH (hormônio adrenocorticotrófico) de modo autônomo. Esta secreção induz a produção crônica e excessiva de cortisol, pelo córtex das glândulas suprarrenais, caracterizando a doença de Cushing (DC). A grande maioria dos adenomas visível à ressonância magnética é microadenoma ( < 10 mm) e apenas 10-30 % dos indivíduos com DC possuem macroadenomas ( > 10 mm), enquanto macroadenomas invasivos são considerados raros. Para investigar os diferentes fenótipos destes tumores, estudamos o padrão de expressão gênica entre microadenomas e macroadenomas, incluindo como critério de classificação sua capacidade de invasão. Utilizando a metodologia de microarray, estudamos 12 amostras de corticotropinomas de indivíduos com diagnóstico clínico, laboratorial e histopatológico de DC (microadenomas não-invasivos n = 4, macroadenomas não-invasivos n = 5 e macroadenomas invasivos n = 3). Além disso, foi investigada a presença de mutações do gene USP8. Observamos que micro e macrocorticotropinomas não-invasivos possuem uma assinatura gênica semelhante, com apenas 48 genes diferencialmente expressos entre si. Por outro lado, macroadenomas invasivos apresentaram um perfil de expressão diferencial mais acentuado, com 168 genes diferencialmente expressos em relação aos não-invasivos (ANOVA p-valor < 0,05; fold change cut off = 2; FDR = 0,05). Nenhum dos pacientes apresentou variantes do USP8. Baseado em sua significância de expressão e funcionalidade, destacamos os genes CCND2, ZNF67 (hiper-expressos, DAPK1 e TIMP2 9 (hipo-expressos). A expressão desses transcritos foi validada por QPCR em 15 corticotropinomas não-invasivos e 3 invasivos, onde 28% destes tumores apresentou mutações somáticas para o gene da USP8. Dentre as vias biológicas comprometidas com pelo menos dois genes hipo ou hiperexpressos estão: via do receptor de Vitamina D, TGF-beta, sinalização por proteína G, resposta ao dano no DNA e controle do ciclo celular. Nossos resultados podem ser úteis para identificar novos marcadores envolvidos no fenótipo invasivo dos corticotropinomas clinicamente ativos. Apesar das funções específicas destes potenciais marcadores ainda precisarem ser elucidadas nos corticotropinomas, nossos resultados podem apresentar um impacto positivo na escolha e eficácia terapêutica, no prognóstico e na previsão de recorrência destes tumores / Adenomas that develop from the corticotrophic lineage (corticotrophinomas) secrete ACTH (adrenocorticotropic hormone) autonomously. This secretion leads to chronic and excessive production of cortisol, by the cortex of the adrenal glands, featuring Cushing\'s disease (CD). Most of the adenomas visible to the MRI are microadenomas (< 10 mm) and macroadenomas (> 10 mm) occur in only 10-30 % of individuals with CD, while invasive macroadenomas, although rare, have great clinical relevance. To investigate the different phenotypes of these tumors, we studied the pattern of differential gene expression between microadenomas and macroadenomas, including their invasiveness as classification a criterion. Using DNA microarray methodology, we studied 12 samples of corticotrophinomas of patients with clinical, laboratory and histopathologic diagnosis of CD (non-invasive microadenomas n = 4, non-invasive macroadenomas n = 5 and invasive macroadenomas n=3). In addition, we investigated the presence of USP8 mutations. We observed that non-invasive corticotrophinomas have a similar genic signature with each other, with only 48 genes differentially expressed between them. Moreover, invasive macroadenomas showed a more pronounced differential expression profile, with 168 differentially expressed genes compared to sellar corticotrophinomas (ANOVA p value < 0.05; fold change cut-off = 2; FDR = 0.05). None of them exhibited USP8 variants. Based on expression significance and functionality, we highlighted CCND2, ZNF676 (overexpressed), DAPK1 and TIMP2 (underexpressed). These results were validated through alfaRT-PCR in another cohort of 15 sellar and 3 invasive corticotrophinomas, in which 28% of these tumors harbored USP8 somatic mutations. Among the biological pathways committed with at least two under or overexpressed genes are: Vitamin D receptor pathway, TGF-beta, G-protein signaling, response to DNA damage and control of the cell cycle. Our results can be useful to identify new markers involved in the invasive phenotype of clinically active corticotrophinomas. Although the specific functions of these potential markers still need to be elucidated in corticotropinomas, our results may have a positive impact on choice and therapeutic efficacy, prognosis and prediction of recurrence of these tumors

目前廣泛應用于胎兒醫學的唐氏綜合症篩查法，即結合早孕期胎兒頸項透明層的超聲檢查，及母體血清生化指標的綜合篩查法。頸項透明層是指在早孕期利用超聲檢測到的胎兒頸后的皮下積水，其作為預測胎兒異常的一項重要“軟指標，其臨床意義，尤其是與胎兒染色體異常及器官結構異常之間的關係，逐漸得到深入的認識，但其形成機制尚未明確。現在已知有一百餘種畸形及遺傳綜合征與胎兒頸項透明層增厚相關，但其染色體異常譜系，尤其是亞顯微的染色體異常仍有待明確。大部分頸項透明層增厚但核型正常的胎兒預後良好，但約3-10%的這部分胎兒會伴有畸形或出生后的神經智力發育缺陷。而傳統核型分析無法檢測到亞顯微的染色體異常，從而無法判斷這部分核型正常卻伴有缺陷的胎兒是否因為這類染色體異常而致病。 / 微陣列比較基因組雜交芯片作為檢測兒童發育遲緩者及器官結構異常原因的重要手段已廣泛應用于臨床。在染色體核型正常的胎兒中，若伴有器官結構異常的胎兒，5-12%被檢出與該畸形相關的微缺失及微重複；若僅伴有孕婦高齡或唐氏篩查高危，則微缺失及微重複檢出率約1%。 / 該課題旨在研究頸項透明層增厚但核型正常的胎兒中，染色體拷貝數變異發生的頻率及頻譜；評估微陣列比較基因組雜交芯片在協助臨床判斷胎兒預後中的作用。因此，我們開展該多中心隊列研究，通過納入449例頸項透明層厚度≧3.5 mm但正常核型胎兒的，檢測其染色體拷貝數變異，監測并記錄其圍產、產後及新生兒期情況。微陣列比較基因組雜交芯片總共檢出2.8%的異常拷貝數變異，其大小範圍為0.1 kb至18Mb。在伴有器官結構異常的胎兒組中，異常拷貝數變異檢出率達7.8%。對於頸項透明層厚度≧4.0 mm的胎兒，異常拷貝數變異檢出率可達7.3%。 / 對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜。但，該研究中發現重複的致病拷貝數變異，如22號染色體長臂1區1帶的微重複或微缺失，2號染色體長臂2區2帶的微缺失。未在3號、7號、12號、13號、18號、20號、21號或Y染色體上發現與胎兒頸項透明層增厚相關的致病拷貝數變異。 / 頸項透明層增厚的胎兒79.3%預後良好；若經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則81.2%預後良好。如果僅頸項透明層增厚不伴有結構異常的胎兒，經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，則93.5%預後良好。 / 綜上所述，微陣列比較基因組雜交芯片顯著提高了致病拷貝數變異的檢出率。可考慮將微陣列比較基因組雜交芯片作為頸項透明層厚度≧4.0 mm的胎兒染色體異常檢查的首要方法。對於僅頸項透明層增厚不伴有結構異常的胎兒，且經微陣列比較基因組雜交芯片未檢出致病拷貝數變異，絶大部分預後良好。對於頸項透明層增厚的胎兒，致病拷貝數變異暫未發現特定的頻譜，但發現重複出現的致病拷貝數變異。通過初步的基因本體分析及基因通路分析，神經嵴細胞的分化遷徙功能異常可作為今後研究頸項透明層增厚的病理生理機制的方向。 / Measurement of nuchal translucency (NT) has been recognized as a sensitive marker for fetal chromosomal disorders for more than a decade, and is presently used as a routine first-trimester screening test. Although over 100 abnormalities and genetic syndromes have been reported to be associated with increased NT, these associations have not been fully explored and the relevant spectrum of associated submicroscopic chromosomal abnormalities has not been sufficiently investigated. The majority of euploid fetuses with increased NT have a good outcome, but around 3-10% of fetuses present with structural or neurodevelopmental abnormalities postnatally. A range of genetic syndromes has been reported, many of which are linked to submicroscopic chromosomal abnormalities that are typically missed by conventional karyotyping. / Microarray-based comparative genomic hybridization (arrayCGH) has been applied as the first-tier diagnostic tool for the evaluation of developmental delay and structural malformations in children. In fetuses with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 5-12% with a structural anomaly and in about 1% of those whose indications were advanced maternal age or positive screening results. / The objectives of this study were to delineate the frequency and spectrum of pathogenic chromosome copy number variants (CNVs) among fetuses with increased NT and normal karyotype; to evaluate the role of arrayCGH to predict the prognosis of the high NT fetuses; to explore the genotype-phenotype correlations of increased NT. Therefore, a multi-centre cohort of 449 fetuses with NT ≧3.5 mm and normal karyotype were further investigated by arrayCGH. Antenatal surveillance, pregnancy outcome and paediatric follow up were documented. ArrayCGH detected abnormal CNVs in 2.8% (14 of 449) of the fetuses with high NT; the size of CNVs ranged from 0.1 kb to 18Mb. Among fetuses with major congenital abnormalities the incidence of abnormal CNV reached 7.8% (4 of 51). By adjusting the NT to ≧4.0 mm as the referral indication, 7.3% (14 of 192) of the fetuses would have abnormal arrayCGH results. The spectrum of pathogenic CNVs found associated with increased NT was diverse. However, there were recurrent ones such as the deletions or duplications at chromosomal region 22q11, and deletions in ZEB2. There was no pathogenic CNV related with increased NT found in chromosomes 3, 7, 12, 13, 18, 20, 21, or Y. The total normal outcome rate of euploid fetuses with an increased NT was 79.3%; for fetuses with normal arrayCGH results 81.2% had a normal outcome. In fetuses with isolated increased NT, normal arrayCGH results predict a favorable prognosis of 93.5%. / In conclusion, arrayCGH significantly increased the diagnostic yield of pathogenic CNVs. In clinical practice arrayCGH may be considered as the first tier investigation in fetuses with an increased NT more than 4.0 mm. In cases with an isolated increased NT with normal arrayCGH results the pregnancy outcome is likely to be favorable. The spectrum of abnormal CNVs found by arrayCGH is diverse but there are recurrent cases such as del/dup 22q11 and del ZEB2. Our preliminary gene ontology and pathway analysis showed that gene pathways related to neural crest cells may be considered as a future study for physiopathologic mechanisms of NT. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Huang, Jin. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 106-120). / Abstracts also in Chinese.

<p>The genetic basis for behavioural traits is largely unknown. The overall aim of this thesis was to find genes with importance for behavioural traits related to fear and anxiety. Microarray analysis was used to screen expression profiles of brain regions important for emotional behaviour in dogs, wolves, foxes and mice. In a first experiment, dogs and their wild ancestors the wolves were compared. Our results suggested that directed selection for behaviour might have resulted in expression changes in few genes acting on several brain functions, possibly affecting behaviour. However, the observed expressional differences were confounded with environmental effects. This was addressed in a second study on domesticated silver foxes. By correlating behaviour and brain gene expression in foxes selected for tameness to non-selected foxes raised in the same environment, we found large behavioural differences but only few genes with differential expression in the brain. Fifteen of the 40 genes showing evidence of expression difference were related to haem or haemoglobins. Further studies showed an additive genetic effect on brain gene expression, similar to the additive genetic inheritance of behaviour, indicating an involvement in domestication. Transcriptional profiling was also used for finding genes involved with the sleep disorder narcolepsy. Narcoleptic Doberman pinschers homozygous for the canarc-1 mutation were compared to their unaffected heterozygots revealing reduced expression of three genes, TAC1, PENK and SOCS2, with relevance to the narcoleptic phenotype. Finally gene expression was investigated in relation to anxiety-related traits in a mouse model. Surprisingly, as in the fox study, genes coding for haemoglobins indicated differential expression in the brain between animals with different anxiety levels. Our combined results suggest that genes like haemoglobins, best known for their function in oxygen transport in blood, may also participate in brain functions related to decreased anxiety in domestic animals. </p>