1.Prostate Cancer (PC) 1.1.Epidemiology Prostate cancer (PC) is one of the major threats to men’s health worldwide (Siegel et al., 2016; Brawley, 2012; Jahn et al., 2015; Center et al., 2012). In the United States PC was estimated to make up roughly 20% of the new cancer cases in men in 2016. Deaths from PC are expected to account for 8% of cancer associated deaths (Siegel et al., 2016). Epidemiological data from China are still rare and incomplete but were recently supplemented by high-quality data provided by the National Central Cancer Registry of China (NCCR) (Chen et al., 2016). The incidence rate of prostate cancer in China increased from 1998 to 2008 by a factor of 3, from 35.2/100,000 to 110.0/100,000 and the average annual growth rate was as high as 12.07% reaching 60,300 cases in 2015 (Zhu et al., 2015; Coffey, 2001; Baade et al., 2013; Chen et al., 2016). While incidence rates in rural areas remained stable between 2006 and 2009, there was an increase in urban areas, especially documented in Hong Kong and Shanghai. The rapid rise of the incidence rate may be in part related to the aging of the population but there seems to be a strong link to Western-style diet (Lin et al., 2015). A comparison of the incidences of prostate cancer in 2015 showed that although the total number of patients in the United States has reached 3.66 times that of China, the estimated death tolls in the two countries are almost similar (Table 1) (Siegel et al., 2016; Ervik et al., 2016; Chen et al., 2016).Interestingly, the numbers in the European Union (EU, WHO region) are in between which might reflect more regional variations in living conditions and diet. However, further investigations are required to come to valid conclusions. Effectivity of PC treatment and cancer recurrence heavily depend on early detection and proper risk stratification (Moller et al., 2015; Schroder et al., 2012; Klotz et al., 2015; Moyer, 2012). In the US, the proportion of localized prostate cancer accounts for more than 80% of all cases, which is also one of the major reasons the mortality/morbidity rate in the US is much lower than that in Asian countries, and continues to decrease (Moller et al., 2015; Jemal et al., 2015; DeSantis et al., 2014). Therefore, early detection and diagnosis is the most effective way by which to improve the survival rate, and development of new biomarkers and/or reasonable combination of current diagnostic methods is a hot spot in the field of prostate cancer research (Felgueiras et al., 2014). Among countries that have implemented prostate cancer screening strategies, five-year survival rates have improved rapidly in Japan, with an average annual increase of about 11.7% and a 5-year survival rate of 93%, while in China, the annual increase was only 3.7% and the 5-year survival rate was 69.2%(Yao et al., 2021). In 2018, there were 1.3 million new cases of prostate cancer worldwide, and its morbidity and mortality ranked second and fifth among male malignancies, respectively.However, to date no serum or urine biomarker or biomarker panel meets the requirements for highly sensitive and specific detection of PC and differentiation between indolent and significant PC. We here explore the prospects of metabolomics to improve prostate cancer detection, patient stratification and treatment monitoring. 1.2. PC classification and grading The prostate gland is a walnut-sized gland located between the bladder neck and the external urethral sphincter. There are four main zones in the prostate gland: the peripheral zone (posteriorly), the fibromuscular zone (anteriorly), the central zone (centrally) and the transitional zone (surrounding the urethra). The anatomy of the prostate gland is shown in Fig. 1 (Adapted from: Bhavsar et al., 2014).Prostate cancer does not occur uniformly throughout the prostate. Although cancers of the prostate often are multifocal, from 80% to 85% arise from the peripheral zone, 10% to 15% arise from the transition zone, and 5% to 10% arise from the central zone (Buyyounouski et al., 2017). The biopsy Gleason grading system is the most important prognostic marker for prostate cancer. The higher the Gleason score, the higher the malignant degree of prostate cancer. The TNM staging system proposed by AJCC is a widely used independent index that can reflect the progression and prognosis of prostate cancer. Table 2 shows the definitions for clinical and pathological T, N, and M classifications (Buyyounouski et al., 2017). Radical prostatectomy (RP) has become the most effective method for the treatment of localized prostate cancer and some high-risk prostate cancer. RP is used when the cancer is believed to be confined to the prostate gland. During the procedure, the prostate gland and some tissue around the gland, including the seminal vesicles, are removed. Transurethral resection of the prostate, or TURP, which also involves removal of part of the prostate gland, is an approach performed through the penis with an endoscope (small, flexible tube with a light and a lens on the end). This procedure doesn't cure prostate cancer but can remove the obstruction while the doctors plan for definitive treatment. Laparoscopic surgery, done manually or by robot, is another method of removal of the prostate gland. Shortcomings in comprehensive medical check-ups in low- and middle-income countries lead to delayed detection of PC and are causative of high numbers of advanced PC cases at first diagnosis. The performance of available biomarkers is still insufficient and limited applicability, including logistical and financial burdens, impedes comprehensive implementation into health care systems. There is broad agreement on the need of new biomarkers to improve (i) early detection of PC, (ii) risk stratification, (iii) prognosis, and (iv) treatment monitoring. 2. PC Biomarkers Serum prostate specific antigen (PSA) level and digital rectal examination (DRE) constitute the major screening tests for prostate cancer (PC) diagnosis, while the transrectal ultrasound-guided prostate biopsy provides the final confirmation of cancer presence (Velonas et al., 2013). PSA level has been extensively used as a biomarker to detect PC. Nevertheless, due to prostate physiology, PSA testing results in a large frequency of false positives leading to numerous men each year undergoing unnecessary prostate biopsy procedures (Vickers et al., 2008; Link et al., 2004; McDunn et al., 2013; Roberts et al., 2011; Djavan et al., 2000). Hence, a non-invasive, cost-effective, efficient, and reasonably accurate test for early identification of PC is urgently needed. Compared with serum, urine is easier to obtain and handle, needs less sample preparation, and has higher amounts of metabolites and lower protein content (Rigau et al., 2013; Wilkosz et al., 2011; Zhang et al., 2013). Therefore, in attempt to solve this diagnostic dilemma, many previous studies have focused on urinary metabolomic profile, to identify the predictive biomarkers for PC (Chistiakov et al., 2018). Yang and colleagues conducted a study searching for urine metabolite biomarkers for the detection of PC. They found twenty differentially expressed urine metabolites in a cohort of 50 prostate cancer patients compared to non-cancerous individuals (Yang et al., 2021; Gordetsky et al., 2016; Nam et al., 2018; Di Meo et al., 2017). The combination of solely three metabolites, representing alterations in Glycine, Serine, and Threonine metabolism (KEGG database pathway), was able to identify PC patients with 77% accuracy at 80% sensitivity and 64% specificity. Furthermore, those metabolites could separate significant PC (Gleason score ≥ 7) from indolent PC (GS 6), which confirms urine metabolomics as a promising diagnostic tool in PC. However, to date, no single urine biomarker/biomarker panel meets the requirements for highly sensitive, and specific detection of PC. Therefore, the search for PC-specific biomarkers still is an active area of research. 3. PC prevalence is not equal in different populations There is a racial difference in incidence rate and interpatient heterogeneity of prostate cancer. By contrast, Asian men have lower disease prevalence compared with Asian-American or American PC cohorts. Despite lower PC incidence, the Asian populations have a higher prevalence of advanced disease, probably due to the lack of availability of more sensitive diagnostic tools (Ateeq et al., 2016). Therefore, it’s necessary to define the urine metabolome in an Asian population. 4. Aims of the study − Exploration of novel biomarkers for the detection of PC in an Asian cohort. − Are urinary metabolomics suitable to develop new PC biomarkers? − What are the advantages of urine biomarkers? − How to identify novel biomarkers in the urine and to investigate the possible functions and roles of potential biomarkers in PC?

This thesis is about protein profiling in serum and plasma using antibody suspension bead arrays for the analysis of biobanked samples and in the context of prostate cancer biomarker discovery. The influence of sample preparation methods on antibody based protein profiles were investigated (Papers I-III) and a prostate cancer candidate biomarker identified and verified (Papers III-V). Furthermore, a perspective on the research area affinity proteomics and its’ employment in biomarker discovery, for improved understanding and potentially improved disease diagnosis, is provided. Paper I presents the results of a comparative plasma and serum protein profiling study, with a targeted biomarker discovery approach in the context of metabolic syndrome. The study yielded a higher number of significant findings and a low experimental variability in blood samples prepared as plasma. Paper II investigated the effects from post-centrifugation delays at different temperatures prior sample storage of serum and plasma samples. Minor effects were found on the detected levels of more than 300 predicted or known plasma proteins. In Paper III, the detectability of proteins in plasma was explored by exposing samples to different pre-analytical heat treatments, prior target capture. Heat induced epitope retrieval was observed for approximately half of the targeted proteins, and resulted in the discovery of different candidate markers for prostate cancer. Several antibodies towards the prostate cancer candidate biomarker CNDP1 were generated, epitope mapped and evaluated in a bead based sandwich immunoassay, as presented in Papers IV and V. Furthermore, the developed sandwich immunoassay targeting multiple distinct CNDP1 epitopes in more than 1000 samples, confirmed the association of CNDP1 levels to aggres- sive prostate cancer and more specifically to prostate cancer patients with regional lymph node metastasis (Paper V). As an outcome of the present investigations and in parallel to studies within the Biobank profiling research group, valuable lessons from study design and multiplex antibody analysis of plasma within biomarker discovery to experimental, technical and biological verifications have been collected. / <p>QC 20130821</p>

Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...