MALDI Mass Spectrometry Imaging for the Discovery of Prostate Carcinoma Biomarkers

The elucidation of new biological markers of prostate cancer (PCa) should aid in the detection, and prognosis of this disease. Diagnostic decision making by pathologists in prostate cancer is highly dependent on tissue morphology. The ability to localize disease-specific molecular changes in tissue would help improve this critical pathology decision making process. Direct profiling of proteins in tissue sections using MALDI imaging mass spectrometry (MALDI-IMS) has the power to link molecular detail to morphological and pathological changes, enhancing the ability to identify candidates for new specific biomarkers. However, critical questions remain regarding the integration of this technique with clinical decision making. To address these questions, and to investigate the potential of MALDI-IMS for the diagnosis of prostate cancer, we have used this approach to analyze prostate tissue for the determination of the cellular origins of different protein signals to improve cancer detection and to identify specific protein markers of PCa. We found that specific protein/peptide expression changes correlated with the presence or absence of prostate cancer as well as the presence of micro-metastatic disease. Additionally, the over-expression of a single peptide (m/z = 4355) was able to accurately define primary cancer tissue from adjacent normal tissue. Tandem mass spectrometry analysis identified this peptide as a fragment of MEKK2, a member of the MAP kinase signaling pathway. Validation of MEKK2 overexpression in moderately differentiated PCa and prostate cancer cell lines was performed using immunohistochemistry and Western Blot analysis. Classification algorithms using specific ions differentially expressed in PCa tissue and a ROC cut-off value for the normalized intensity of the MEKK2 fragment at m/z 4355 were used to classify a blinded validation set. Finally, the optimization of sample processing in a new fixative which preserves macromolecules has led to improved through-put of samples making MALDI-IMS more compatible with current histological applications, facilitating its implementation in a clinical setting. This study highlights the potential of MALDI-IMS to define the molecular events involved in prostate tumorigenesis and demonstrates the applicability of this approach to clinical diagnostics as an aid to pathological decision making in prostate cancer

The metabolomic response to severe thermal injury and the impact of age

Severe thermal injury results in a profound hypermetabolic response and is associated with increased morbidity, mortality and delayed rehabilitation of burn survivors. Serum 1H-NMR metabolomics was used to examine early global metabolic changes in the response to severe thermal injury (>15% TBSA) in young adults (16-64 years) and older adults (<15% TBSA). Early changes in the metabolome reflected hypoxic metabolism, hyperglycaemia, increased ketogenesis, peripheral lipolysis and increased energy production in both cohorts. Early metabolic profiles from the young adult group were used to construct OPLSDA models that could discriminate with high accuracy between outcome groups. Models from 0-24hrs serum samples predicted survival (AUC 0.92), whilst models from 24-96hrs samples predicted Multiple organ failure (MOF) (AUC 0.92) and sepsis (AUC 0.89). Untargeted LC-MS metabolomics was applied to study the longitudinal changes in the serum metabolome after severe thermal injury in 13 young adults, from admission until 6-months post-injury. Univariate ANOVA analysis revealed significant changes in 432 metabolite features, affecting 35 distinct classes, representing global metabolic disturbance. Changes in 300 lipid metabolite features may represent a ‘lipid storm’ in serum after severe thermal injury. Novel areas of metabolism and metabolites were identified as putative biomarkers warranting further targeted study

Alterations in Myocardial Function and Electrocardiology in Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiomyopathy with a highly variable phenotype. The assessment of arrhythmogenic potential in HCM patients and identification of early signs of the disease in relatives of HCM patients is challenging. The aim of this thesis was to characterize the mechanical and electrical changes in the left ventricle of carriers of either the MYBPC3-Q1061X or TPM1-D175N mutation for hypertrophic cardiomyopathy and to identify novel imaging and electrocardiographic parameters with the potential to enhance sudden cardiac death risk stratification and follow-up. A total of 140 subjects carrying a pathogenic variant for HCM were recruited for these studies from three centers in Finland, divided into two groups: those with left ventricular hypertrophy (G+/LVH+ n = 98) and those without hypertrophy (G+/LVH- n = 42). We studied the association of ventricular arrhythmias on 24h ambulatory electrocardiograms to 2D strain echocardiographic findings and cardiac magnetic resonance imaging variables in 31 G+/LVH+ HCM patients. Mechanical dispersion was significantly increased in HCM patients with episodes of ventricular arrhythmia on ambulatory ECGs and was a better predictor of these episodes than global longitudinal strain or late gadolinium enhancement. Mechanical dispersion may be a useful marker of arrhythmogenic potential in HCM patients. We evaluated conventional and novel ECG parameters in the whole cohort of mutation carriers. An abnormal ECG was present in 97% of G+/LVH+ and 86% of G+/LVH- subjects. The combination criteria of RV1RV3 + Q waves and septal remodeling identified G+/LVH- subjects with a 64% sensitivity and 97% specificity. The proposed novel ECG criteria may increase the efficacy of using electrocardiography in identification of G+/LVH- subjects. A group of 46 HCM patients was assessed with a 24h ambulatory ECG with comprehensive repolarization analysis and these findings were compared with imaging findings. Rate adapted QTe interval was prolonged in HCM patients. Maximal wall thickness was associated with longer maximal QTe and median T wave peak to T wave end interval. HCM patients with late gadolinium enhancement had a steeper QTe/RR slope compared to HCM patients without LGE and control subjects. The presence of LGE may independently affect the repolarization dynamics in HCM. The metabolome of carriers of the MYBPC3-Q1061X mutation was investigated with comprehensive laboratory assays. Concentrations of branched chain amino acids, triglycerides and ether phospholipids were increased in mutation carriers with hypertrophy as compared to controls and non-hypertrophic mutation carriers, and correlated with echocardiographic LVH and signs of diastolic and systolic dysfunction.Hypertrofinen kardiomyopatia (HCM) on yleisin perinnöllinen sydänlihassairaus. Sairauteen liittyy äkkikuoleman riski osalla potilaista. Tämän riskin arvioiminen on tunnetusti vaikeaa myös moderneilla riskinarviomenetelmillä ja tarve diagnostisille työkaluille, jotka tätä parantaisivat, on ilmeinen. Taudin periytyvyyden arvioimiseksi tarvitaan geenidiagnostiikan lisäksi hyviä menetelmiä varhaisten tautimuotojen havaitsemiseen seurannan kohdentamiseksi. Tämän väitöskirjan tavoite on ollut sydänlihaksen mekaanisten ja sähköisten muutosten arvioiminen ja parempien diagnostisen työkalujen löytäminen rytmihäiriöriskin ja sairauden esimuutosten todentamiseen. Potilasmateriaali koostui 140 suomalaisesta, jotka kantavat joko MYBPC3-Q1061X tai TPM1-D175N geenimutaatiota, jotka aiheuttavat hypertrofista kardiomyopatiaa. Potilaat jaettiin kahteen ryhmään: Ensimmäisessä ryhmässä olivat mutaatiokantajat, joilla todettiin vasemman kammion seinämähypertrofia eli hypertrofinen kardiomyopatia (n = 98) ja toisessa ryhmässä mutaatiokantajat, joilla ei ole hypertrofiaa (n = 42). Ensimmäisessä osatyössä tutkittiin lyhyiden kammioarytmioiden esiintymisen korrelaatiota kuvantamislöydöksiin 31:llä HCM-potilaalla. Totesimme ultraäänitutkimuksella mitatun mekaanisen dispersion olevan selvästi suurentunut HCM-potilailla, joilla esiintyi kammioarytmioita holter-seurannassa. Mekaaninen dispersio ennusti rytmihäiriöitä paremmin kuin pitkittäinen strain ultraäänessä tai jälkitehostuma MRI:ssä. Toisessa osatyössä kartoitimme koko mutaatiokantajakohortin EKG-löydöksiä ja niiden yhteyttä kuvantamislöydöksiin. Ehdottamamme uudet EKG-parametrit RV1RV3 ja väliseinän uudelleenmuovautuminen olivat hyvin spesifejä mutaatiokantajille ja erottelivat hyvin ei-hypertrofiset mutaatiokantajat kontrolliryhmästä. Yhdistelemällä näitä uusia EKG-löydöksiä vanhojen hyväksi havaittujen mittausten kanssa päästiin aikaisempaa parempaan erottelukykyyn ei-hypertrofisten mutaatiokantajien ja kontrolliryhmän välillä. Käyttämällä uusia EKG-löydöksiä voidaan yksilöiden seurantaa mahdollisesti kohdentaa paremmin. Kolmannessa osatyössä tutkimme holter-rytminseurannan repolarisaatiomuutosten yhteyttä kuvantamislöydöksiin 46:lla HCM-potilaalla. Totesimme QT-ajan olevan pidentynyt HCM-potilailla ja se piteni progressiivisesti matalammilla syketaajuuksilla suhteessa terveisiin verrokkeihin. QT-ajan pitenemä oli yhteydessä vasemman kammion seinämäpaksuuteen. Potilailla, joilla oli myöhäistehostumaa vasemmassa kammiossa, oli jyrkempi QT/RR kulmakerroin. Myöhäistehostuma voi olla itsenäinen tekijä repolarisaatiomuutoksissa ja vaikuttaa tätä kautta myös rytmihäiriöherkkyyteen. HCM-potilaiden metabolomia ei ole juurikaan aikaisemmin tutkittu. Selvitimme laajalla analyysillä HCM-potilaiden metabolomista profiilia. Löydöksenä oli haaraketjuisten aminohappojen, triglyseridien ja fosfolipidien konsentraatiomuutoksia verrattuna terveisiin kontrolleihin. Nämä muutokset assosioituivat kuvantamislöydöksiin eli taudin vaikeusasteeseen

Proteomic, circulating and functional biomarkers of cardiovascular disease

Cardiovascular disease is the leading cause of morbidity and mortality in the Western world, mainly through cerebrovascular and coronary artery related events. Cardiovascular disease is a chronic progressive disease with different stages. These stages can be assessed by a variety of biomarkers. Biomarker quantification can be used for different purposes: screening, prediction of disease recurrence, therapeutic monitoring, diagnosis and prognostication. Noninvasive, inexpensive diagnostic tests currently applied in clinical practice have a relative high rate of false positive and false negative results. Therefore further refinement of the diagnostic process could improve clinical care. Regarding prognostication the need for improvement also remains as current risk models only predict a small quantity of occurring cardiovascular events. The concept of the cardiovascular continuum postulates that cardiovascular disease consists of a chain of events, is initiated by numerous cardiovascular risk factors and subsequently progresses through pathophysiological processes, ultimately leading to end-stage heart failure. For that reason cardiovascular diseases are chronic progressive conditions and can be divided into different stages, such as early tissue dysfunction or subclinical atherosclerosis prior to development of clinically overt disease. Biomarkers suitable for prognostication and diagnosis can differ at each stage. The general aim of this thesis was therefore the investigation of a variety of biomarkers in diagnosis and prediction of cardiovascular disease at different stages of the cardiovascular continuum, as covered by three different study cohorts contributing to this thesis. This included several approaches: the comparison of central and peripheral pulse pressure in middle aged hypertensive patients in regards of their prognostic potential; the application of established circulating, functional and structural biomarkers to the diagnostic process of coronary artery disease in stable angina patients; the development/refinement of a urinary proteomic biomarker for coronary artery disease and the examination of its diagnostic potential in stable angina patients. Biomarkers successful in the diagnosis of coronary artery disease were included in multiple biomarker models. Aside from biomarker development for the general population, investigations of specific cohorts, such as patients with certain diseases and belonging to certain age groups or sharing specific biochemical features provided advances in the past. To estimate the potential of a biomarker in risk prediction association studies with surrogate biomarkers are applicable. We collected a cohort of middle-aged hypertensive patients to assess if central pulse pressure, derived from non-invasive assessment of arterial stiffness, could improve risk prediction. Central pulse pressure has been previously shown to have prognostic value in populations with end-stage renal failure, coronary artery disease and high prevalence of diabetes mellitus. Considering the prognostic information of peripheral pulse pressure in the elderly, the hypothesis that central pulse pressure could improve risk prediction is comprehensive and was investigated as part of this thesis. This was accomplished by comparing the strength of correlation between central or peripheral pulse pressure and these surrogate biomarkers. When compared to peripheral pulse pressure, central pulse pressure had stronger associations with aortic pulse wave velocity, carotid intima-media thickness, and left ventricular mass index, but equal association with the albumin:creatinine ratio. In contrast, after adjustment for age, mean arterial pressure, heart rate and hypertension status there was no significant difference between central and peripheral pulse pressure for prediction of listed surrogate biomarkers in multivariate analysis. These results suggested that central pulse pressure is unlikely to provide more prognostic information than peripheral pulse pressure in middle-aged hypertensive patients. The diagnosis of coronary artery disease is clinically relevant in symptomatic patients, either acute or stable. The diagnosis of stable flow limiting coronary artery disease is especially challenging as non-cardiac as well as other cardiac conditions can mimic symptoms. Non-invasive diagnostic tools have either moderate sensitivities or specificities, or are not widely available. Therefore new biomarkers for the diagnosis of flow limiting coronary artery disease have the potential to improve current diagnostic strategies. This could be accomplished adjacent to existing biomarkers or by replacement of such, due to cost effectiveness, better discriminatory etc. As part of this thesis, a biomarker identification and validation study was conducted into urinary proteomics of coronary artery disease. First we tried to replicate a study conducted by our research group in the past. Therein, an established coronary artery disease specific polypeptide pattern was unable to differentiate between patients with severe coronary artery disease and healthy controls despite strong cohort similarities to the original study. We therefore recalibrated the urinary polypeptide pattern using an enlarged biomarker discovery cohort and adjusted the pattern for lipid lowering and angiotensin converting enzyme inhibitor treatment effects. We calculated a score from the resulting polypeptide pattern, which identified coronary artery disease patients with a sensitivity of 79% and a specificity of 88% in a biomarker validation cohort. As the next step of biomarker development we performed a diagnostic validation study. The investigated clinical cohort consisted of stable angina patients with or without coronary artery disease. The new polypeptide pattern score was unable to differentiate between these two groups. The score however correlated strongly with coronary artery disease extent as measured by the Gensini score, implying that urinary proteomics in the diagnosis of coronary artery disease is promising, yet requires further effort before clinical employment. In addition to the urinary proteomic biomarker development second diagnostic approach was selected. As coronary artery disease is a complex chronic disease, the combination of different biomarkers should result in a better discrimination between stable angina patients with or without coronary artery disease. This approach attempts to position the individual as precisely as possible on the cardiovascular continuum including serologic, functional vascular and imaging biomarkers of subclinical atherosclerosis. Serologic markers thereby present a plasma proteomic approach covering pathophysiological processes with known correlation or causative for coronary artery disease. Functional and structural changes of the peripheral vasculature resemble the coronary artery system. We investigated circulating biomarkers and vascular biomarkers separately. A variety of circulating biomarkers differentiated patients with severe coronary artery disease from healthy control subjects. When patients with stable angina and with or without coronary artery disease as diagnosed by coronary angiography were investigated no statistically significant differences could be detected for circulating biomarkers. In the same study a microvascular biomarker, the reactive hyperaemia index, and a macrovascular biomarker, the carotide plaque score, were able to differentiated between cases and controls. Both markers either added separately or together improved the risk classification of exercise treadmill test results. This suggests that a multiple biomarker approach in the diagnosis of coronary artery disease in stable angina patients could be successful. Different aspects of the cardiovascular continuum can be applied to diagnosis and prognostication of cardiovascular disease. In this regard we were able to show, that early processes such as endothelial dysfunction or later processes such as plaque formation can support the diagnostic process. However, randomly collected circulating biomarkers might be unable to do this. Our finding that central pulse pressure is unlikely to have more prognostic value in middle aged hypertensive patients underlines that biomarkers can be useful in specific patient collectives but not necessarily in all cohorts. Instead of applying established biomarkers, also new biomarkers can be developed. Urine proteomics showed great promise in this regard, as specific polypeptide patterns reflect coronary artery disease and are strongly correlated to its extent

Applications of MATLAB in Science and Engineering

The book consists of 24 chapters illustrating a wide range of areas where MATLAB tools are applied. These areas include mathematics, physics, chemistry and chemical engineering, mechanical engineering, biological (molecular biology) and medical sciences, communication and control systems, digital signal, image and video processing, system modeling and simulation. Many interesting problems have been included throughout the book, and its contents will be beneficial for students and professionals in wide areas of interest

ACARORUM CATALOGUS IX. Acariformes, Acaridida, Schizoglyphoidea (Schizoglyphidae), Histiostomatoidea (Histiostomatidae, Guanolichidae), Canestrinioidea (Canestriniidae, Chetochelacaridae, Lophonotacaridae, Heterocoptidae), Hemisarcoptoidea (Chaetodactylidae, Hyadesiidae, Algophagidae, Hemisarcoptidae, Carpoglyphidae, Winterschmidtiidae)

The 9th volume of the series Acarorum Catalogus contains lists of mites of 13 families, 225 genera and 1268 species of the superfamilies Schizoglyphoidea, Histiostomatoidea, Canestrinioidea and Hemisarcoptoidea. Most of these mites live on insects or other animals (as parasites, phoretic or commensals), some inhabit rotten plant material, dung or fungi. Mites of the families Chetochelacaridae and Lophonotacaridae are specialised to live with Myriapods (Diplopoda). The peculiar aquatic or intertidal mites of the families Hyadesidae and Algophagidae are also included.Publishe