There is currently no clinically useful screening test available to identify nulliparous women at high risk of developing pre-eclampsia. This study aimed to identify novel biomarkers using hypothesis generating proteomic methods applied to plasma samples obtained prior to clinical diagnosis of pre-eclampsia. Plasma samples taken at 15 weeks gestation from women who subsequently developed late pre-eclampsia (> 34 weeks), early pre-eclampsia (< 34 weeks) and two distinct groups of women with uncomplicated pregnancies (each n=12) were pooled. Pooled plasma was immunodepleted, labelled using iTRAQ-8 plex reagent and separated into fractions using high pH reverse phase chromatography. Fractions were analysed by LC-MS/MS and data interrogated using ProteinPilot 3.0. The merits of two immunodepletion systems were compared; the Seppro® IgY 14 -SuperMix LC column system removes up to 100 highly abundant plasma proteins and the Multiple Affinity Removal LC column depletes 14 highly abundant plasma proteins. Removal of more high abundance proteins allowed identification of more, potentially interesting, low abundance proteins, but was less reproducible than removing fewer proteins. Two methods of LC-MS/MS analysis were assessed; the QStar XL qTOF and 5800 MALDI-TOF-TOF. The protein identifications and the quantification data acquired by each method was comparable and complementary and increased the total number of proteins identified. A total of 502 proteins were identified. A stringent two stage analysis was developed to identify candidate proteins which changed in abundance in plasma from women who later developed pre-eclampsia compared to women with uncomplicated pregnancies. Analysis identified a total of 113 proteins which were both reproducibly quantified and changed by more than the expected range of biological variation. Six candidate proteins changed in abundance in the plasma taken from women who subsequently developed early pre-eclampsia were selected for further validation. A high throughput, low cost, method of multiple reaction monitoring which allows relative quantitation without the use of costly isotopically labelled peptides was developed to validate candidate proteins. Candidate proteins were also assessed by western blot and ELISA. Only one candidate protein; platelet basic protein, was validated by all three methods and demonstrated similar increases in the abundance. This investigation suggests that measurement of platelet basic protein at 15 weeks gestation is a novel candidate predictive marker for pre-eclampsia. Validation of platelet basic protein in a large, independent, sample set is required to confirm changes in protein expression and to evaluate potential, alongside other factors, to identify nulliparous women at high risk of developing pre-eclampsia later in pregnancy.

ude.dravrah.egelloc@gnufynnej / This research was supported by awards from the National Institutes of Health (NIH), the Eunice Kennedy Shriver Institute of Child Health and Human Development (R01-HD-059835) and the National Institute for Minority Health and Health Disparities (T37-MD000149). The NIH had no further role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the paper for publication. The authors wish to thank the dedicated staff members of Asociacion Civil Proyectos en Salud (PROESA), Peru and Instituto Especializado Materno Perinatal, Peru for their expert technical assistance with this research. / Revisión por pares

Prostate carcinoma is one of the most frequent cancer diseases of maturated men. Diagnose of this cancer disease is based on level of prostatic specific antigen and digital exam per rectum. However, presence of tumor, grade and stage must be examined by biopsy. This method may not to be accurate. This is the reason why are new diagnostic methods investigated, to increase the accuracy of diagnostic prostate cancer. Amino acid sarcosine is currently on of the most widely discussed biomarker, that can serve as a diagnostic biomarker for early stage detection in prostate cancer. The present work deals with the study of sarcosine metabolism in prostate cancer cell lines, mainly, but also by amino acid metabolism in cells in general. Understanding to importance of single amino acids in malignant and non-malignant cell pathways can significantly contribute to effective targeted therapy without side effect on healthy tissue. Prostate cancer cells in presence of sarcosine increase their migration potential, malignancy by increased doubling time, and concentration of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and sarcosine oxidase. Through this biochemical pathway is increased synthesis of main methylation donor S-adenosilmethyonine (SAM) ad cellular methylation potential.

Patients on haemodialysis are highly susceptible to different forms of heart failure. To date, the benefit of Mineralocorticoid-receptor antagonist (MRA) administration in haemodialysis patients remains subject to discussion. Biomarkers play an important role in therapy guidance and pose a promising tool to detect pathological processes of heart failure in an earlier stage. The randomised-controlled Mineralocorticoid-Receptor Antagonists in End-Stage Renal Disease (MiREnDa) trial was set up to investigate the effect of 50 mg of spironolactone once daily on left ventricular mass index in haemodialysis patients and several secondary endpoints. This dissertation reports findings from the MiREnDa trial on (a) the efficacy of spironolactone to influence serum levels of biomarkers of heart failure, fibrosis and inflammation and electrolytes and (b) the ability of N-terminal pro-B-type natriuretic peptide (NT-proBNP), Galectin-3 and soluble source of tumorigenicity 2 (sST2) to reflect left ventricular hypertrophy and diastolic dysfunction assessed by imaging characteristics. Treatment of spironolactone over a 40-week period did not alter serum levels of biomarkers of heart failure, fibrosis and inflammation including NT-proBNP, Galectin-3 and sST2. A small but significant effect on serum sodium but not potassium was observed. NT-proBNP was significantly different in the presence or absence of left ventricular hypertrophy (LVH) (normal vs. LVH (median [IQR]): 2,120 [810; 5,040] vs. 6,340 [2,410; 15,360] pg/ml, p<0.01) or moderate and severe diastolic dysfunction (DD) (normal diastolic function and DD grade I vs. DD grade II and DD grade III: 2,300 [850; 6,050] vs. 12,260 [3,340; 34,830] pg/ml, p=0.02). NT-proBNP further showed a significant correlation at baseline with LVMi (Spearman’s rho=0.41, p<0.001), LAVi (Spearman’s rho=0.55, p<0.001) and septal E/e’ (Spearman’s rho=0.45, p<0.001). No correlation was observed between Galectin-3 and the investigated functional and morphological parameters. sST2 was mildly correlated to LVMi at baseline (Spearman’s rho=0.21, p=0.05) and NT-proBNP at baseline (Spearman’s rho=0.37, p<0.001). In conclusion, spironolactone did not affect the investigated parameters but NT-proBNP proved to be significantly correlated to cardiac imaging measurements. / Dialysepatienten erkranken häufig an Formen der Herzinsuffizienz. Zugleich ist der Nutzen von Mineralkortikoidrezeptorantagonisten bei Dialysepatienten bis heute umstritten. Biomarkermessungen ermöglichen es, pathologische Prozesse am Herzen in einem möglichst frühen Stadium zu erkennen. In der randomisiert-kontrollierten "Mineralocorticoid-Receptor Antagonists in End-Stage Renal Disease" (MiREnDa) Studie wurde der Effekt der täglichen Einnahme von 50 mg Spironolacton auf den linksventrikulären Massenindex bei Dialysepatienten zusammen mit verschiedenen sekundären Endpunkten untersucht. Diese Arbeit beleuchtet Ergebnisse der MiREnDa-Studie zur Wirkung von Spironolacton auf Serumspiegel von Biomarkern für Herzinsuffizienz, Fibrose und Entzündung sowie von Elektrolyten. Darüber hinaus wird der Zusammenhang zwischen N-terminalen natriuretischen Peptid Typ B (NT-proBNP), Galectin-3 und Soluble source of tumorigenicity 2 (sST2) und Veränderungen in den wichtigsten bildgebenden Merkmalen linksventrikulärer Hypertrophie und diastolischer Dysfunktion beschrieben. Die Einnahme von Spironolacton über 40 Wochen hatte keinen Effekt auf Biomarker für Herzinsuffizienz, Fibrose und Entzündung wie NT-proBNP, Galectin-3 und sST2. Lediglich die Natriumspiegel, nicht aber die Kaliumspiegel, wurden signifikant beeinflusst. NT-proBNP unterschied sich signifikant zwischen Patient*innen mit und ohne links-ventrikulärer Hypertrophie (LVH) (normal vs. LVH (Median [IQR]): 2.120 [810; 5.040] vs. 6.340 [2.410; 15.360] pg/ml, p<0,01) beziehungsweise mit und ohne relevanter diastolischer Dysfunktion (DD) (normale diastolische Funktion und DD Grad I vs. DD Grad II und DD Grad III: 2.300 [850; 6.050] vs. 12.260 [3.340; 34.830] pg/ml, p=0,02). NT-proBNP korrelierte außerdem signifikant mit LVMi (Spearman's rho=0,41, p<0,001), LAVi (Spearman's rho=0,55, p<0,001) und E/e' (Spearman's rho=0,45, p<0,001). Galectin-3 war unabhängig von allen untersuchten Parametern. sST2 korrelierte mäßig mit LVMi (Spearman's rho=0,21, p=0,05) und deutlich mit NT-proBNP (Spearman's rho=0,37, p<0,001). Zusammenfassend beeinflusste Spironolacton keinen der untersuchten Parameter relevant und lediglich NT-proBNP wies eine signifikante Korrelation zu kardialen Bildgebungsparameters auf.

Background: Respiratory tract infections are responsible for millions of deaths annually. Interferon-stimulated genes (ISGs) play a significant role in fighting off viral respiratory tract infections in the antiviral defence system. Measuring extracellular protein products of ISGs could be potential biomarkers of viral infection. Although, the feasibility and performance of ISGs as functional and robust clinical biomarkers from a non-invasive sample format remains unknown. Methods: Three ISGs, CXCL10, CXCL11, and TNFSF10, were examined in in-vivo and in-vitro gene expression datasets (RNA-sequencing and microarray) infected with common respiratory tract infections (Rhinovirus, Respiratory syncytial virus, influenza A and SARS-CoV-2) samples and compared to negative controls. Using qualitative selection criteria of 1) elevated presence in at least one dataset with viral infection, 2) secreted protein product, and 3) commercially available antibodies for detection, CXCL10, CXCL11 and TNFSF10 gene expression levels were assessed. A correlation analysis was performed with SARS-CoV-2 infection severity and gene expression kinetics. CXCL10 was subsequently validated at the protein level in saliva as a prerequisite for developing a host-response LFA. Results: CXCL10 and CXCL11 upregulation were positively correlated with RSV compared to control (p < 0.05). CXCL10/CXCL11/TNFSF10 were not different between samples collected from RV infected subjects relative to controls (p > 0.05). No significant association was found with influenza A for all three genes. CXCL10/CXCL11/TNFSF10 upregulation was positively correlated with SARS-CoV-2 infection compared to control (p < 0.001). CXCL10 expression correlated with COVID-19 viral load. CXCL10 was chosen as a lead biomarker candidate based on these analyses that included different virus infections, time-courses, and measures of severity. CXCL10 was not detected at the protein level in healthy saliva but was elevated in saliva from COVID-19 patients. A CXCL10 LFA was developed with a sensitivity of 2 ng/ml in a buffer and artificial saliva. Conclusion: We establish and validate the potential of developing rapid test techniques to examine host immune response from a bioinformatic approach to developing a prototype rapid test with capabilities to be used in point-of-care settings. / Thesis / Master of Science (MSc) / Respiratory tract infections are a leading cause of death and one of the main reasons to seek primary care. Both historically and in the present day, respiratory tract infections remain a massive socioeconomic burden. Current diagnostics fail to quickly identify a respiratory tract infection's etiology, and prognosis, leading to suboptimal patient care and the over prescription of antibiotics. Advanced tools used in academia and research, including next-generation -omics sequencing and metagenomics, have capabilities to identify all nucleic acid material in a sample - including host RNA- which offers potential to improve the diagnosing of respiratory tract infections. However, these technologies have not been integrated into routine care due to economic, technical, and logistical barriers. We explored host RNA (transcriptomics), looking at antiviral interferon-stimulated genes for their potential as a biomarker of viral infection amenable to point-of-care testing platforms from non-invasive sample types.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder associated with aging. AD is by far the best understood and most studied neurodegenerative disease. Substantial advances have been made over the last decade, however it is debatable how much closer we are to a clinically useful therapy. A long standing goal in the AD field has been to improve the accuracy of early detection, with the assumption that the ability to intervene earlier in the disease process will lead to a better clinical outcome. Major facets of this effort have been the continued development and improvement of AD biomarkers, with a strong focus on developing imaging modalities. AD is accompanied by two pathological hallmarks in the brain: extracellular neuritic plaques composed of the beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein. Evidence of Aβ as the driving force behind the progression of AD (the amyloid cascade hypothesis) was first published by Hardy & Higgins in 1992, and this peptide has been the focus of therapeutic and diagnostic testing for decades. Significant technological advances in recent years now allow imaging of amyloid pathology in vivo. These methods evaluate Aβ burden in a living person, and could potentially serve as both a biomarker, and as a diagnostic tool to detect disease. Pittsburgh Compound B (PiB) is currently the best studied of these imaging agents, however, our current knowledge of the quantitative relationship between PiB binding and amyloid pathology in the brain is limited. A better understanding of how these variables relate to one another is essential for the continued development of reliable diagnostic biomarkers for AD. We analyzed increasingly insoluble pools of Aβ to quantify their relative contributions to the overall Aβ burden, and to determine if any of these measures could be used to predict disease status. We found that the amount of PiB binding in a cortical region of the brain could distinguish cases of mild cognitive impairment (MCI) when corrected to the amount of PiB binding in the cerebellum. As the Aβ peptide ages, the amino acid aspartate may spontaneously convert to an isoaspartate residue through a succinimide intermediary. The presence of iso-Asp Aβ has been used to indicate the presence of aged plaques in AD and Down syndrome cases. We sought to investigate the potential relationship between levels of ‘aged’ Aβ in the plasma as indicated by iso-Asp Aβ and disease state, as a potential biomarker for the presence of AD pathology. We found that AD cases had lower levels of all forms of Aβ in plasma when standardized to the group average, and that plasma levels of Aβ and iso-Asp Aβ were reversed between disease groups. A follow up study is required, however, these initial data are a promising step towards utilizing aged iso-Asp Aβ plasma levels as a potential biomarker to indicate disease state.

The Proximity Ligation Assay (PLA) is a relatively new method which utilizes the strength of both immunoassays and DNA detection. PLA has the capacity of high multiplexing due to the high specificity achieved with both dual protein-binding and dual primer binding during detection with Real-Time PCR. We developed a multiplexed PLA protocol that can measure 28 biomarkers in human EDTA plasma. The method was tested on 46 individuals diagnosed with colorectal cancer and 48 age matched healthy controls. The results are very promising as we re-discover the most well-known biomarkers for colorectal cancer and also find some potential new markers (significance tested with students T-test with p&lt;0.05). Further improvements of the protocol are needed to decrease the variation.

The primary aim of the work described in this thesis is to establish a foundation for the applicability of a biochemical biomarker, cholinesterase (ChE) activity in food animal species, as an instrument for evaluating exposure to pollutants as well as predicting high-level effects on public health. Secondary aims are to increase the awareness of pesticide users of anti-ChE exposure, to decide whether poisoning episodes involve anti-ChE by measuring residual effects in tissues, and to identify sources of contamination in food animal tissues. The ChE are specialized carboxylic ester hydrolases that break down esters of choline. They are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE). Both AChE and BChE activities were found to be higher in cattle than in sheep and higher in erythrocytes than in plasma and serum. The anticoagulant heparin significantly affects AChE activity in plasma compared with EDTA. Of the different tissue tested, the mean of ChE activities was found to be highest in tissue from the liver, followed by lung, muscle, kidney and heart for sheep and cattle. In pigs, the ChE activities tested higher in kidney, liver, lung, muscle and heart. The effect of freezing on ChE activities in liver and muscle tissues was significant inhibition after 6 months at -80 °C, whereas decreased after 3 months at -20 °C. A technique to improve the purification of AChE in sheep tissue was developed. BW284c51 strongly reduced acetylthiocholine iodide (AcTChI) and propionylthiocholine iodide (PrTChI) hydrolysis and slightly affected that of butyrylthiocholine iodide (BuTChI) in the liver, while iso-OMPA had no significant effect for muscle BuTChI of sheep and pigs. Histochemical study of liver tissue found AChE localised mainly in the cytoplasm of the cell lining in the sinusoids. The optimal pH values of AChE and BChE in liver and muscle ranged between 7.8 and 8.5. Both AChE and BChE activities increased when increase the time course and temperature. The half maximal inhibitory concentration (IC50) was found to be higher for carbaryl than dichlorvos (DDVP) and diazinon (DZN). Very little residual AChE activity was seen in the liver, but more was found in muscles. In general, the rate constants of inhibition (ki) values for liver and muscles were increased in different pHs according to the rank order of 8.5 > 7.5 > 6.5, while in plasma it was decreased in different temperatures as follows: 20 °C > 30 °C > 40 °C. The final experiments were carried out at the rate of spontaneous reactivation (ks) of inhibited AChE by DDVP and DZN from liver and muscle was found to be higher in sheep compared to cattle and pig, while the aging of phosphorylated AChE (ka) was found to be higher in cattle compared to sheep and pig. In addition, this study indicated that the developed bispyridinium symmetric (K048) oxime seems to be promising reactivated to DDVP-inhibited AChE for sheep and pigs while HI-6 was effective in cattle.

Background: Proteomic approaches identifying biomarkers have been applied to asthma to only a very limited extent. Methods: With an antibody array (RayBiotech, Norcross, GA, USA), the relative intensity and rank differences of 444 proteins were compared in 24 plasma samples obtained at age 3, 11 from children with and 12 without asthma diagnoses at ages 5 and 9. Protein candidates identified by antibody array were quantitated by ELISA in an enlarged sample. Proteins found to differentiate children with and without asthma were also examined for association with known Year 1 asthma risk factors, eczema, and wheeze. Results: In the antibody array, four proteins had rank differences between asthma and non-asthma groups (FDR < 0.1). By ELISA, mean log (+/- s.e.m.) erythropoietin (EPO) level (IU/l) was lower (0.750 +/- 0.048 vs. 0.898 +/- 0.035; p = 0.006) and mean (+/- s.e.m.) soluble GP130 (sGP130) level (ng/ml) was higher in the asthma vs. the non-asthma group (302 +/- 13 vs. 270 +/- 8; p = 0.041). The other 2 array proteins (galactin-3 and eotaxin-3) did not differ by ELISA by asthma. EPO related to the asthma risk factor, first year eczema, whereas sGP130 related to first year wheeze. Conclusions: Through two independent assessments, age 3 plasma levels of EPO and sGP130 were found related to childhood asthma.

The purposes of this dissertation were to investigate the response of salivary alpha-amylase to a single resistance training session and to a week-long resistance training over-reaching protocol. The major findings of this dissertation are as follows: Study 1 – A single resistance training session consisting of 5 sets of 10 repetitions of squat and bench press at 95 percent of repetition maximum creates a statistically significant increase in salivary alpha-amylase concentrations. Study 2 – Two resistance training sessions consisting of 5 sets of 10 repetitions of squat and bench press at 95 percent of repetition maximum within 5 days does not create a statistically significant change in resting baseline salivary alpha-amylase concentrations. These results are corroborated by not causing statistically significant change in perceived stress, as measured by Total Mood Disturbance, calculated from the Profile Of Mood States questionnaire, nor causing a change in perceived stress calculated from the Daily Analysis of Life Demands for Athletes survey.