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.

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. 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. 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. / QC 20101006

Resistance to broad spectrum of chemotherapeutic agents in cancer cell lines and tumors has been called multiple drug resistance (MDR). In this study, the molecular mechanisms of resistance to two anticancer agents (paclitaxel and vincristine) in mammary carcinoma cell line MCF-7 were investigated. MCF-7 cells were selected in the presence of paclitaxel and vincristine by stepwise dose increments. The cell viability and growth profiles of resistant sublines were examined. As the resistance indices increased, the growth rates of sublines were found to decrease. Gene and protein expression levels of the basic drug resistance proteins P-gp and MRP1 were studied in sensitive and drug resistant MCF-7 cells. It was shown that P-gp overexpression is significantly contributing to the developed drug resistance phenotype. Mutation analysis of beta tubulin gene which encodes the target of paclitaxel and vincristine was performed. Single histidine to proline mutation was identified near GTP binding site of beta tubulin in vincristine resistant subline which was not reported before. Apoptosis related BCL-2 and BAX were examined at both gene and protein expression levels and they were not found to be significantly related to the developed resistance in the sublines. The reversal of drug resistance by various inhibitory agents of P-gp and MRP1 was investigated by using flow cytometry. Synthetic silicon compounds were found to be the most effective MDR reversal agents. The effects of various combinations of anticancer drugs and reversal agents on cell proliferation were examined by checkerboard microplate method. ALIS409-paclitaxel and paclitaxel-doxorubicin pairs seem to have highest antiproliferative effects on resistant sublines. The microarray expression profiling of sensitive and resistant MCF-7 cells was performed for a much detailed and comprehensive analysis of drug resistance. The results indicated that the upregulation of MDR1 gene is the dominating mechanism of paclitaxel and vincristine drug resistance. Additionally up regulation of the genes encoding the detoxifying enzymes (i.e. GSTP1) was observed. Significant down regulation of apoptotic genes (i.e. PDCD2/4/6/8) and alterations in expression levels of genes related to invasion and metastasis (MMPs, ADAMs, COL4A2, LAMA etc.) were detected. Upregulation of some oncogenes (i.e. ETS, RAS) and cell cycle regulatory genes (CDKN2A, CCNA2 etc.) was seen which may be in close relation to MDR in breast cancer. Further studies will demonstrate the relationship between the components contributing to drug resistance phenotype in breast cancer cells.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Lic.-avh. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 2 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 5 uppsatser.

Thesis (Ph. D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Vita. Bibliography: 197-219.

Thesis (Ph. D.)--University of Tennessee, Memphis, 2006. / The electronic version of this thesis is available at http://d.utmem.edu/CAMPUS-ACCESS-ONLY/2006-001-chona.pdf Includes bibliographical references (leaves 128-136).

Thesis (Ph.D. in Human Medical Genetics) -- University of Colorado at Denver and Health Sciences Center, 2005. / Typescript. Includes bibliographical references (leaves 166-195).

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