Molecular Background of Common Dyslipidemias

The leading cause of death in the Western world continues to be coronary heart disease (CHD). At the root of the disease process is dyslipidemia an aberration in the relevant amounts of circulating blood lipids. Cholesterol builds up in the arterial wall and following rupture of these plaques, myocardial infarction or stroke can occur. Heart disease runs in families and a number of hereditary forms are known. The leading cause of adult dyslipidemia presently however is overweight and obesity. This thesis work presents an investigation of the molecular genetics of common, hereditary dyslipidemia and the tightly related condition of obesity. Familial combined hyperlipidemia (FCHL) is the most common hereditary dyslipidemia in man with an estimated population prevalence of 1-6%. This complex disease is characterized by elevated levels of serum total cholesterol, triglycerides or both and is observed in about 20% of individuals with premature CHD. Our group identified the disease to be associated with genetic variation in the USF1 transcription factor gene. USF1 has a key role in regulating other genes that control lipid and glucose metabolism as well as the inflammatory response all central processes in the progression of atherosclerosis and CHD. The first two works of this thesis aimed at understanding how these USF1 variants result in increased disease risk. Among the many, non-coding single-nucleotide polymorphisms (SNPs) that associated with the disease, one was found to have a functional effect. The risk-enhancing allele of this SNP seems to eradicate the ability of the important hormone insulin to induce the expression of USF1 in peripheral tissues. The resultant changes in the expression of numerous USF1 target genes over time probably enhance and accelerate the atherogenic processes. Dyslipidemias often represent an outcome of obesity and in the final work of this thesis we wanted to address the metabolic pathways related to acquired obesity. It is recognized that active processes in adipose tissue play an important role in the development of dyslipidemia, insulin resistance and other pathological conditions associated with obesity. To minimize the confounding effects of genetic differences present in most human studies, we investigated a rare collection of identical twins that differed significantly in the amount of body fat. In the obese, but otherwise healthy young adults, several notable changes were observed. In addition to chronic inflammation, the adipose tissue of the obese co-twins was characterized by a marked (47%) decrease in amount of mitochondrial DNA (mtDNA) a change associated with mitochondrial dysfunction. The catabolism of branched chain amino acids (BCAAs) was identified as the most down-regulated process in the obese co-twins. A concordant increase in the serum level of these insulin secretagogues was identified. This hyperaminoacidemia may provide the feed-back signal from insulin resistant adipose tissue to the pancreas to ensure an appropriately augmented secretory response. The down regulation of BCAA catabolism correlated closely with liver fat accumulation and insulin. The single most up-regulated gene (5.9 fold) in the obese co-twins was osteopontin (SPP1) a cytokine involved in macrophage recruitment to adipose tissue. SPP1 is here implicated as an important player in the development of insulin resistance. These studies of exceptional study samples provide better understanding of the underlying pathology in common dyslipidemias and other obesity associated diseases important for future improvement of intervention strategies and treatments to combat atherosclerosis and coronary heart disease.

Molecular Pathways of Disturbed Sleep and Depression: Studies on Adenosine and Gene Expression Patterns

Background: Adenosine is a potent sleep-promoting substance, and one of its targets is the basal forebrain. Fairly little is known about its mechanism of action in the basal forebrain and about the receptor subtype mediating its regulating effects on sleep homeostasis. Homeostatic deficiency might be one of the causes of the profoundly disturbed sleep pattern in major depressive disorder, which could explain the reduced amounts of delta-activity-rich stages 3 and 4. Since major depression has a relatively high heritability, and on the other hand adenosine regulates sleep homeostasis and might also be involved in mood modulation, adenosine-related genes should be considered for their possible contribution to a predisposition for depression and disturbed sleep in humans. Depression is a complex disorder likely involving the abnormal functioning of several genes. Novel target genes which could serve as the possible common substrates for depression and comorbid disturbed sleep should be identified. In this way specific brain areas related to sleep regulation should be studied by using animal model of depression which represents more homogenous phenotype as compared to humans. It is also important to study these brain areas during the development of depressive-like features to understand how early changes could facilitate pathophysiological changes in depression. Aims and methods: We aimed to find out whether, in the basal forebrain, adenosine induces recovery non-rapid eye movement (NREM) sleep after prolonged waking through the A1 or/and A2A receptor subtype. A1 and A2A receptor antagonists were perfused into the rat basal forebrain during 3 h of sleep deprivation, and the amount of NREM sleep and delta power during recovery NREM sleep were analyzed. We then explored whether polymorphisms in genes related to the metabolism, transport and signaling of adenosine could predispose to depression accompanied by signs of disturbed sleep. DNA from 1423 individuals representative of the Finnish population and including controls and cases with depression, depression accompanied by early morning awakenings and depression accompanied by fatigue, was used in the study to investigate the possible association between polymorphisms from adenosine-related genes and cases. Finally to find common molecular substrates of depression and disturbed sleep, gene expression changes were investigated in specific brain areas in the rat clomipramine model of depression. We focused on the basal forebrain of 3-week old clomipramine-treated rats which develop depressive-like symptoms later in adulthood and on the hypothalamus of adult female clomipramine-treated rats. Results: Blocking of the A1 receptor during sleep deprivation resulted in a reduction of the recovery NREM sleep amount and delta power, whereas A2A receptor antagonism had no effect. Polymorphisms in adenosine-related genes SLC29A3 (equilibrative nucleoside transporter type 3) in women and SLC28A1 (concentrative nucleoside transporter type 1) in men associated with depression alone as well as when accompanied by early morning awakenings and fatigue. In Study III the basal forebrain of postnatal rats treated with clomipramine displayed disturbances in gamma-aminobutyric acid (GABA) receptor type A signaling, in synaptic transmission and possible epigenetic changes. CREB1 was identified as a common transcription denominator which also mediates epigenetic regulation. In the hypothalamus the major changes included the expression of genes in GABA-A receptor pathway, K+ channel-related, glutamatergic and mitochondrial genes, as well as an overexpression of genes related to RNA and mRNA processing. Conclusions: Adenosine plays an important role in sleep homeostasis by promoting recovery NREM sleep via the A1 receptor subtype in the basal forebrain. Also adenosine levels might contribute to the risk of depression with disturbed sleep, since the genes encoding nucleoside transporters showed the strongest associations with depression alone and when accompanied by signs of disturbed sleep in both women and men. Sleep and mood abnormalities in major depressive disorder could be a consequence of multiple changes at the transcriptional level, GABA-A receptor signaling and synaptic transmission in sleep-related basal forebrain and the hypothalamus.

Molecular Interactions Underlying Neuronal Ceroid Lipofuscinoses CLN1 and CLN5

Disorders resulting from degenerative changes in the nervous system are progressive and incurable. Both environmental and inherited factors affect neuron function, and neurodegenerative diseases are often the sum of both factors. The cellular events leading to neuronal death are still mostly unknown. Monogenic diseases can offer a model for studying the mechanisms of neurodegeneration. Neuronal ceroid lipofuscinoses, or NCLs, are a group of monogenic, recessively inherited diseases affecting mostly children. NCLs cause severe and specific loss of neurons in the central nervous system, resulting in the deterioration of motor and mental skills and leading to premature death. In this thesis, the focus has been on two forms of NCL, the infantile NCL (INCL, CLN1) and the Finnish variant of late infantile NCL (vLINCLFin, CLN5). INCL is caused by mutations in the CLN1 gene encoding for the PPT1 (palmitoyl protein thioesterase 1) enzyme. PPT1 removes a palmitate moiety from proteins in experimental conditions, but its substrates in vivo are not known. In the Finnish variant of late infantile NCL (vLINCLFin), the CLN5 gene is defective, but the function of the encoded CLN5 has remained unknown. The aim of this thesis was to elucidate the disease mechanisms of these two NCL diseases by focusing on the molecular interactions of the defective proteins. In this work, the first interaction partner for PPT1, the mitochondrial F1-ATP synthase, was described. This protein has been linked to HDL metabolism in addition to its well-known role in the mitochondrial energy production. The connection between PPT1 and the F1-ATP synthase was studied utilizing the INCL-disease model, the genetically modified Ppt1-deficient mice. The levels of F1-ATP synthase subunits were increased on the surface of Ppt1-deficient neurons when compared to controls. We also detected several changes in lipid metabolism both at the cellular and systemic levels in Ppt1-deficient mice when compared to controls. The interactions between different NCL proteins were also elucidated. We were able to detect novel interactions between CLN5 and other NCL proteins, and to replicate the previously reported interactions. Some of the novel interactions influenced the intracellular trafficking of the proteins. The multiple interactions between CLN5 and other NCL proteins suggest a connection between the NCL subtypes at the cellular level. The main results of this thesis elicit information about the neuronal function of PPT1. The connection between INCL and neuronal lipid metabolism introduces a new perspective to this rather poorly characterized subject. The evidence of the interactions between NCL proteins provides the basis for future research trying to untangle the NCL disease mechanisms and to develop strategies for therapies.

Nutrition and bone metabolism : In vivo effects of inorganic phosphate on rats and in vitro effects of bioactive tripeptides on human osteoblasts

Nutrition affects bone health throughout life. To optimize peak bone mass development and maintenance, it is important to pay attention to the dietary factors that enhance and impair bone metabolism. In this study, the in vivo effects of inorganic dietary phosphate and the in vitro effects of bioactive tripeptides, IPP, VPP and LKP were investigated. Dietary phosphate intake is increased through the use of convenience foods and soft drinks rich in phosphate-containing food additives. Our results show that increased dietary phosphate intake hinders mineral deposition in cortical bone and diminishes bone mineral density (BMD) in the aged skeleton in a rodent model (Study I). In the growing skeleton (Study II), increased phosphate intake was observed to reduce bone material and structural properties, leading to diminished bone strength. Studies I and II revealed that a low Ca:P ratio has negative effects on the mature and growing rat skeleton even when calcium intake is sufficient. High dietary protein intake is beneficial for bone health. Protein is essential for bone turnover and matrix formation. In addition, hydrolysis of proteins in the gastrointestinal tract produces short peptides that possess a biological function beyond that of being tissue building blocks. The effects of three bioactive tripeptides, IPP, VPP and LKP, were assessed in short- and long-term in vitro experiments. Short-term treatment (24 h) with tripeptide IPP, VPP or LKP influenced osteoblast gene expression (Study III). IPP in particular, regulates genes associated with cell differentiation, cell growth and cell signal transduction. The upregulation of these genes indicates that IPP enhances osteoblast proliferation and differentiation. Long-term treatment with IPP enhanced osteoblast gene expression in favour of bone formation and increased mineralization (Study IV). The in vivo effects of IPP on osteoblast differentiation might differ since eating frequency drives food consumption, and protein degradation products, such as bioactive peptides, are available periodically, not continuously as in this study. To sum up, Studies I and II raise concern about the appropriate amount of dietary phosphate to support bone health as excess is harmful. Studies III and IV in turn, support findings of the beneficial effects of dietary protein on bone and provide a mechanistic explanation since cell proliferation and osteoblast function were improved by treatment with bioactive tripeptide IPP.

Fatty acids, fibre, carotenoids and tocopherols in relation to glucose metabolism in subjects at high risk for type 2 diabetes : a cross-sectional analysis

Fatty acids, fibre, carotenoids and tocopherols in relation to glucose metabolism in subjects at high risk for type 2 diabetes a cross-sectional analysis Type 2 diabetes (T2D) is a heterogeneous disorder of carbohydrate, lipid and protein metabolism, resulting from genetics, environmental influences and interactions between these. The disease is characterized by insulin resistance, β-cell dysfunction, hepatic glucose overproduction and disordered fat mobilization and storage. The literature on associations between dietary factors and glucose metabolism is inconsistent. One factor behind the discrepant results may be genetic heterogeneity of study populations. Data on nutrient-gene interactions in relation to glucose metabolism are scarce. Thus, investigating high-risk populations and exploring nutrient-gene interactions are essential for improving the understanding of T2D aetiology. Ideally, this information could help to develop prevention programmes that take into account the genetic predisposition to the disease. In this study, associations between measures of glucose metabolism predicting T2D and fatty acids, antioxidative nutrients and fibre were examined in a high-risk population, i.e., in non-diabetic relatives of affected patients. Interactions between the PPARG Pro12Ala polymorphism and fatty acids on glucose metabolism were taken into consideration. This common polymorphism plays an important role in the regulation of glucose metabolism. The inverse associations observed between dietary fibre and insulin resistance are consistent with the prevailing recommendations urging increased intake of fibre to prevent T2D. Beneficial associations observed between the intake of carotenoids and glucose levels stress that a high consumption of vegetables, fruits and berries rich in carotenoids might also play a role in the prevention of T2D. Whether tocopherols have an independent association with glucose metabolism remains questionable. Observed interactions between fatty acids and glucose metabolism suggest that a high intake of palmitic acid is associated with high fasting glucose levels mainly in female Ala allele carriers. Furthermore, the PPARG Pro12Ala polymorphism may modify the metabolic response to dietary marine fat. The beneficial associations of high intake of marine n 3 fatty acids with insulin resistance and glucose levels may be restricted to carriers of the Ala allele. The findings pertain to subjects with a family history of T2D, and the cross-sectional nature of the study precludes inferences about causality. Results nevertheless show that associations of dietary factors with glucose metabolism may be modulated by the genetic makeup of an individual. Additional research is warranted to elucidate the role of probably numerous nutrient-gene interactions, some of which may be sex-specific, in the aetiology of T2D.

Effects of dietary phosphorus and calcium-to-phosphorus ratio on calcium and bone metabolism in healthy 20- to 43-year-old Finnish women

Dietary habits have changed during the past decades towards an increasing consumption of processed foods, which has notably increased not only total dietary phosphorus (P) intake, but also intake of P from phosphate additives. While the intake of calcium (Ca) in many Western countries remains below recommended levels (800 mg/d), the usual daily P intake in a typical Western diet exceeds by 2- to 3-fold the dietary guidelines (600 mg/d). The effects of high P intake in healthy humans have been investigated seldom. In this thesis healthy 20- to 43-year-old women were studied. In the first controlled study (n = 14), we examined the effects of P doses, and in a cross-sectional study (n = 147) the associations of habitual P intakes with Ca and bone metabolism. In this same cross-sectional study, we also investigated whether differences exist between dietary P originating from natural P sources and phosphate additives. The second controlled study (n = 12) investigated whether by increasing the Ca intake, the effects of a high P intake could be reduced. The associations of habitual dietary calcium-to-phosphorus ratios (Ca:P ratio) with Ca and bone metabolism were determined in a cross-sectional study design (n = 147). In the controlled study, the oral intake of P doses (495, 745, 1245 and 1995 mg/d) with a low Ca intake (250 mg/d) increased serum parathyroid hormone (S-PTH) concentration in a dose-dependent manner. In addition, the highest P dose decreased serum ionized calcium (S-iCa) concentration and bone formation and increased bone resorption. In the second controlled study with a dietary P intake of 1850 mg/d, by increasing the Ca intake from 480 mg/d to 1080 mg/d and then to 1680 mg/d, the S-PTH concentration decreased, the S-iCa concentration increased and bone resorption decreased dose-dependently. However, not even the highest Ca intake could counteract the effect of high dietary P on bone formation, as indicated by unchanged bone formation activity. In the cross-sectional studies, a higher habitual dietary P intake (>1650 mg/d) was associated with lower S-iCa and higher S-PTH concentrations. The consumption of phosphate additive-containing foods was associated with a higher S-PTH concentration. Moreover, habitual low dietary Ca:P ratios (≤0.50, molar ratio) were associated with higher S-PTH concentrations and 24-h urinary Ca excretions, suggesting that low dietary Ca:P ratios may interfere with homeostasis of Ca metabolism and increase bone resorption. In summary, excessive dietary P intake in healthy Finnish women seems to be detrimental to Ca and bone metabolism, especially when dietary Ca intake is low. The results indicate that by increasing dietary Ca intake to the recommended level, the negative effects of high P intake could be diminished, but not totally prevented. These findings imply that phosphate additives may be more harmful than natural P. Thus, reduction of an excessively high dietary P intake is also beneficial for healthy individuals.

Polymorphic low penetrance genes and breast cancer : The role of genes involved in metabolism of xenobiotics, estrogens and reactive oxygen species

Various endogenous and exogenous factors have been reported to increase the risk of breast cancer. Many of those are related to prolonged lifetime exposure to estrogens. Furthermore, a positive family history of breast cancer and certain benign breast diseases are known to increase the risk of breast cancer. The role of lifestyle factors, such as use of alcohol and smoking has been an area of intensive study. Alcohol has been found to increase the risk of breast cancer, whereas the role of smoking has remained obscure. A multitude of enzymes are involved in the metabolism of estrogens and xenobiotics including the carcinogens found in tobacco smoke. Many of the metabolic enzymes exhibit genetic polymorphisms that can lead to inter-individual differences in their abilities to modify hazardous substrates. Therefore, in presence of a given chemical exposure, one subgroup of women may be more susceptible to breast carcinogenesis, since they carry unfavourable forms of the polymorphic genes involved in the metabolism of the chemical. In this work, polymorphic genes encoding for cytochrome P450 (CYP) 1A1 and 1B1, N-acetyl transferase 2 (NAT2), sulfotransferase 1A1 (SULT1A1), manganese superoxide dismutase (MnSOD) and vitamin D receptor (VDR) were investigated in relation to breast cancer susceptibility in a Finnish population. CYP1A1, CYP1B1 and SULT1A1 are involved in the metabolism of both estrogens and xenobiotics, whereas NAT2 is involved only in the latter. MnSOD is an antioxidant enzyme protecting cells from oxidative damage. VDR, in turn, mediates the effects of the active form of vitamin D (1,25(OH)2D3, calcitriol) on maintenance of calcium homeostasis and it has anti-proliferative effects in many cancer cells. A 1.3-fold (95% CIs 1.01-1.73) increased risk of breast cancer was seen among women who carried the NAT2 slow acetylator genotype and a 1.5-fold (95% CI 1.1-2.0) risk was found in women with a MnSOD variant A allele containing genotypes compared to women with the NAT2 rapid acetylator genotype or to those with the MnSOD VV genotype, respectively. Instead, women with the VDR a allele containing genotypes were found to be at a decreased risk for breast cancer (OR 0.73; 95% CI 0.54-0.98) compared to women with the AA genotype. No significant overall associations were found between SULT1A1 or CYP genotypes and breast cancer risk, whereas a combination of the CYP1B1 432Val allele containing genotypes with the NAT2 slow acetylator genotypes posed a 1.5-fold (95% CI 1.03-2.24) increased risk. Moreover, NAT2 slow acetylator genotype was found to be confined to women with an advanced stage of breast cancer (stages III and IV). Further evidence for the association of xenobiotic metabolising genes with breast cancer risk was found when active smoking was taken into account. Women who smoked less than 10 cigarettes/day and carried at least one CYP1B1 432Val variant allele, were at 3.1-fold (95% CI 1.32-7.12) risk of breast cancer compared to women who smoked the same amount but did not carry the variant allele. Furthermore, the risk was significantly increased with increasing number of the CYP1B1 432Val alleles (p for trend 0.005). In addition, women who smoked less than 5 pack-years and carried the NAT2 slow acetylator genotype were at a 2.6-fold (95% CI 1.01-6.48) increased risk of breast cancer compared to women who smoked the same amount but carried the NAT2 rapid acetylator genotype. Furthermore, the combination of the CYP1B1 432Val allele and the NAT2 slow acetylator genotype increased the risk of breast cancer by 2.5-fold (95% CI 1.11-5.45) among ever smokers. Instead, the MnSOD A allele was found to be a risk factor among postmenopausal long-term smokers (>15 years of smoking) (OR 5.1; 95% CI 1.4-18.4) or among postmenopausal women who had smoked more than 10 cigarettes/day (OR 5.5; 95% CI 1.3-23.4) compared to women who had similar smoking habits but carried the MnSOD V/V genotype. Similarly, within subgroups of postmenopausal women who were using oral contraceptives, hormone replacement therapy or alcohol, women carrying the MnSOD A allele genotypes seemed to be at increased risk of breast cancer compared to women with the MnSOD V/V genotype. A positive family history of breast cancer and high parity were shown to be inversely associated with breast cancer risk among women carrying the VDR ApaI a allele or among premenopausal women carrying the SULT1A1*2 allele, respectively.

Thyroid Hormone Control of Cardiac Substrate Metabolism

Thyroid hormone (TH) plays an important role in maintaining a homeostasis in all the cells of our body. It also has significant cardiovascular effects, and abnormalities of its concentration can cause cardiovascular disease and even morbidity. Especially development of heart failure has been connected to low levels of thyroid hormone. A decrease in TH levels or TH-receptor binding adversely effects cardiac function. Although, this occurs in part through alterations in excitation-contraction and transport proteins, recent data from our laboratory indicate that TH also mediates changes in myocardial energy metabolism. Thyroid dysfunction may limit the heart s ability to shift substrate pathways and provide adequate energy supply during stress responses. Our goals of these studies were to determine substrate oxidation pattern in systemic and cardiac specific hypothyroidism at rest and at higher rates of oxygen demand. Additionally we investigated the TH mediated mechanisms in myocardial substrate selection and established the metabolic phenotype caused by a thyroid receptor dysfunction. We measured cardiac metabolism in an isolated heart model using 13Carbon isotopomer analyses with MR spectroscopy to determine function, oxygen consumption, fluxes and fractional contribution of acetyl-CoA to the citric acid cycle (CAC). Molecular pathways for changes in cardiac function and substrate shifts occurring during stress through thyroid receptor abnormalities were determined by protein analyses. Our results show that TH modifies substrate selection through nuclear-mediated and rapid posttranscriptional mechanisms. It modifies substrate selection differentially at rest and at higher rates of oxygen demand. Chronic TH deficiency depresses total CAC flux and selectively fatty acid flux, whereas acute TH supplementation decreases lactate oxidation. Insertion of a dominant negative thyroid receptor (Δ337T) alters metabolic phenotype and contractive efficiency in heart. The capability of the Δ337T heart to increase carbohydrate oxidation in response to stress seems to be limited. These studies provided a clearer understanding of the TH role in heart disease and shed light to identification of the molecular mechanisms that will facilitate in finding targets for heart failure prevention and treatment.

ORP2 is a sterol receptor that regulates cellular lipid metabolism

ORP2 is a member of mammalian oxysterol binding protein (OSBP)-related protein/gene family (ORPs), which is found in almost every eukaryotic organism. ORPs have been suggested to participate in the regulation of cellular lipid metabolism, vesicle trafficking and cellular signaling. ORP2 is a cytosolic protein that is ubiquitously expressed and most abundant in the brain. In previous studies employing stable cell lines with constitutive ORP2 overexpression ORP2 was shown to affect cellular cholesterol metabolism. The aim of this study was to characterize the properties and function of ORP2 further. ORP2 ligands were searched for among sterols and phosphoinositides using purified ORP2 and in vitro binding assays. As expected, ORP2 bound several oxysterols and cholesterol, the highest affinity ligand being 22(R)hydroxycholesterol. In addition, affinity for anionic membrane phospholipids, phosphoinositides was observed, which may assist in the membrane targeting of ORP2. Intracellular localization of ORP2 was also investigated. ORP2 was observed on the surface of cytoplasmic lipid droplets, which are storage organelles for neutral lipids. Lipid droplet targeting of ORP2 was inhibited when 22(R)hydroxycholesterol was added to the cells or when the N-terminal FFAT-motif of ORP2 was mutated, suggesting that oxysterols and the N-terminus of ORP2 regulate the localization and the function of ORP2. The role of ORP2 in cellular lipid metabolism was studied using HeLa cell lines that can be induced to overexpress ORP2. Overexpression of ORP2 was shown to enhance cholesterol efflux from the cells resulting in a decreased amount of cellular free cholesterol. ORP2 overexpressing cells responded to the loss of cholesterol by upregulating cholesterol synthesis and uptake. Intriguingly, also cholesterol esterification was increased in ORP2 overexpressing cells. These results may be explained by the ability of ORP2 to bind and thus transport cholesterol, which most likely leads to changes in cholesterol metabolism when ORP2 is overexpressed. ORP2 function was further investigated by silencing the endogenous ORP2 expression with short interfering RNAs (siRNA) in A431 cells. Silencing of ORP2 led to a delayed break-down of triglycerides under lipolytic conditions and an increased amount of cholesteryl esters in the presence of excess triglycerides. Together these results suggest that ORP2 is a sterol-regulated protein that functions on the surface of cytoplasmic lipid droplets to regulate the metabolism of triglycerides and cholesteryl esters. Although the exact mode of ORP2 action still remains unclear, this study serves as a good basis to investigate the molecular mechanisms and possible cell type specific functions of ORP2.

Comparative and functional genome analysis of fungi for development of the protein production host Trichoderma reesei

Filamentous fungi of the subphylum Pezizomycotina are well known as protein and secondary metabolite producers. Various industries take advantage of these capabilities. However, the molecular biology of yeasts, i.e. Saccharomycotina and especially that of Saccharomyces cerevisiae, the baker's yeast, is much better known. In an effort to explain fungal phenotypes through their genotypes we have compared protein coding gene contents of Pezizomycotina and Saccharomycotina. Only biomass degradation and secondary metabolism related protein families seem to have expanded recently in Pezizomycotina. Of the protein families clearly diverged between Pezizomycotina and Saccharomycotina, those related to mitochondrial functions emerge as the most prominent. However, the primary metabolism as described in S. cerevisiae is largely conserved in all fungi. Apart from the known secondary metabolism, Pezizomycotina have pathways that could link secondary metabolism to primary metabolism and a wealth of undescribed enzymes. Previous studies of individual Pezizomycotina genomes have shown that regardless of the difference in production efficiency and diversity of secreted proteins, the content of the known secretion machinery genes in Pezizomycotina and Saccharomycotina appears very similar. Genome wide analysis of gene products is therefore needed to better understand the efficient secretion of Pezizomycotina. We have developed methods applicable to transcriptome analysis of non-sequenced organisms. TRAC (Transcriptional profiling with the aid of affinity capture) has been previously developed at VTT for fast, focused transcription analysis. We introduce a version of TRAC that allows more powerful signal amplification and multiplexing. We also present computational optimisations of transcriptome analysis of non-sequenced organism and TRAC analysis in general. Trichoderma reesei is one of the most commonly used Pezizomycotina in the protein production industry. In order to understand its secretion system better and find clues for improvement of its industrial performance, we have analysed its transcriptomic response to protein secretion stress conditions. In comparison to S. cerevisiae, the response of T. reesei appears different, but still impacts on the same cellular functions. We also discovered in T. reesei interesting similarities to mammalian protein secretion stress response. Together these findings highlight targets for more detailed studies.