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Problematyka artykuły dotyczy doświadczeń biograficznych początkujących nauczycieli związanych z ich startem zawodowym. W tekście zostanie przybliżony procesualny charakter rozwoju zawodowego nauczyciela, scharakteryzowany fenomen startu zawodowego, a na zakończenie w odwołaniu do empirycznych ilustracji będzie zarysowana mapa wsparcia, z jakiego korzystają nowicjusze, aby lepiej radzić sobie z wymaganiami szkoły jako miejsca pracy. Analizie poddane będą narracje nauczycieli uzyskane w swobodnych wywiadach ukierunkowanych.

Background Pulmonary hypertension constitutes a rare disease characterized by a severe development and a high risk of premature death. One of its main clinical types is pulmonary arterial hypertension (PAH), in which the highest percentage of patients are affected by idiopathic PAH. The pathogenesis of this disease has not completely been discovered and elucidated so far, and non-specific clinical symptoms make the diagnosis of PAH a serious problem. Currently, PAH is confirmed with invasive examination based on right heart catheterization. Purpose The main aim of the study was to evaluate and compare both plasma and urine fingerprints of PAH patients and control group with the use of an untargeted metabolomics approach. The study also focused on correlation analysis between the observed metabolic changes and the clinical parameters to select specific indicators of PAH disease. Methods An untargeted metabolomics approach was applied to the plasma and urine samples with the use of gas chromatography coupled to triple quadrupole mass spectrometry (GC-QqQ/MS) and advanced statistical tests were applied to evaluate the potential metabolic indicators of PAH. The PAH patients (n=40) and healthy controls (n=39) were matched for age, sex, BMI and included in the study. The obtained raw datasets were properly processed (data deconvolution, signal correction using QCSVR method and PQN normalization) and subsequently subjected to uni- and multivariate statistical tests (Student's t-test, Welch's test, U Mann-Whitney test, PCA and OPLS-DA). The identification of the statistically significant metabolites was performed using universal libraries, such as: Fiehn's and NIST11. Results The statistically significant metabolites (n=10 and 11 for urine and plasma samples, respectively)originate from various biochemical pathways associated with the carbohydrate, amino acid, lipid, fatty acid and pyrimidine metabolism. The metabolic changes observed in the urine samples of PAH patients (compared to the control group) included different concentrations in: threonic acid, hippuric acid, acetic acid, sorbitol, butanoic acid and propionic acid. The metabolic alterations in the plasma samples covered changes in the levels of valine, leucine, lactic acid, hydroxybutanoic acid, nonanoic acid, cholesterol and octadecanoic acid. Metabolites representing the highest correlation with mean pulmonary arterial pressure include, for instance: propionic acid (r=0.85), valine (r=0.75) and lactic acid (r=0.63) Conclusions The observed metabolic changes are related to various biological processes that are disturbed in the course of PAH, namely: proliferation of pulmonary vascular cells or the functions of cardiomyocytes and the right ventricle of the heart. The obtained results confirmed the potential of a metabolomics approach to uncover and explain the underlying molecular mechanisms of PAH. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministry of Science and High Education of Poland

Bladder cancer (BCa) is ninth amongst the most common types of cancer in the human population worldwide. The statistics of incidence and mortality of BCa are alarming and the currently applied diagnostic methods are still not sensitive enough. This leads to a large number of undiagnosed BCa cases, usually among patients in the early stages of the disease. Despite the fact that many risk factors of BCa have been recognized, the pathomechanism of development of bladder cancer has not been fully explained yet. Therefore, in the present study, multiplatform urinary metabolomics has been implemented in order to scrutinize potential diagnostic indicators of BCa that might help to explain its pathomechanism and be potentially useful in diagnosis and determination of stage of the disease. Urine samples collected from muscle-invasive high grade BCa patients (n = 24) and healthy volunteers (n = 24) were matched in terms of most common BCa risk factors i.e. gender, age, BMI and smoking status. They were analyzed by high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) using RP and HILIC chromatography, gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in scan mode, and proton nuclear magnetic resonance (1H NMR). The six datasets obtained were submitted to univariate and multivariate statistical analyses. 17 metabolites significantly discriminated urinary profiles of BCa patients from urinary profiles of healthy volunteers. These metabolites are mainly involved in amino acid metabolism, pyrimidine and purine metabolism, as well as energy metabolism and might play a crucial role in the pathogenesis of BCa.

In addition to chronic anovulation and clinical signs of hyperandrogenism women with polycystic ovary syndrome (PCOS) are insulin resistant and therefore, develop central obesity with its long term consequences such as dyslipidaemia, hypertension, atherosclerosis and type 2 diabetes mellitus (T2DM), which all lead to the development of cardiovascular disease (CVD). Due to the polysymptomatic nature of this syndrome and lack of consensus on its diagnostic criteria there is a strong need of finding a reliable biochemical or molecular marker, which would facilitate making the accurate diagnosis of PCOS. Therefore, the aim of our study was to perform a metabolomics analysis with the use of two complementary techniques: gas chromatography and liquid chromatography coupled with mass spectrometry, of the serum samples from women with PCOS (n = 30) and to compare them with healthy age and BMI matched controls (n = 30). Obtained results were subjected to one-dimensional statistical analysis (student’s t-test or its non-parametric equivalent U Mann-Whitney test) and multivariate statistical analysis (the principal component analysis [PCA], variable importance into projection [VIP] and selectivity ratio [SR]). The results of our study showed that women with PCOS are characterised by metabolic disorders of the amino acids, carbohydrates, steroid hormones, lipids and purines. Compared to control subjects, women with PCOS had increased serum levels of phospholipids, aromatic amino acids, organic acids, hormones and sphinganine and decreased total cholesterol. Among the identified compounds, total cholesterol, phenylalanine and dehydroepiandrosterone sulfate, uric and lactic acid were the compounds with the strongest discriminating power.

Bladder cancer (BC) is a common malignancy of the urinary system and a leading cause of death worldwide. In this work, untargeted metabolomic profiling of biological fluids is presented as a non-invasive tool for bladder cancer biomarker discovery as a first step towards developing superior methods for detection, treatment, and prevention well as to further our current understanding of this disease. In this study, urine samples from 24 healthy volunteers and 24 BC patients were subjected to metabolomic profiling using high throughput solid-phase microextraction (SPME) in thin-film format and reversed-phase high-performance liquid chromatography coupled with a Q Exactive Focus Orbitrap mass spectrometer. The chemometric analysis enabled the selection of metabolites contributing to the observed separation of BC patients from the control group. Relevant differences were demonstrated for phenylalanine metabolism compounds, i.e., benzoic acid, hippuric acid, and 4-hydroxycinnamic acid. Furthermore, compounds involved in the metabolism of histidine, beta-alanine, and glycerophospholipids were also identified. Thin-film SPME can be efficiently used as an alternative approach to other traditional urine sample preparation methods, demonstrating the SPME technique as a simple and efficient tool for urinary metabolomics research. Moreover, this study’s results may support a better understanding of bladder cancer development and progression mechanisms.

Prostate cancer (CaP) is a common cancer in men. Its late detection and inefficient diagnosis are a challenge for researchers who are currently searching for new cancer-related indicators that would facilitate better detectability of CaP and explain its pathogenesis. In the present preliminary study, endogenous volatile metabolites were detected in plasma and urine samples by using the metabolic fingerprinting approach. The analyses were performed using the GC-QqQ/MS technique in the scan mode. The detected and putatively identified metabolites were statistically analyzed using advanced univariate and multivariate statistical methods. Eleven urinary and three plasma metabolites were selected as statistically significant in patients with CaP as compared to those in healthy controls. Supervised methods such as logistic regression and quadratic support vector machine were applied to obtain the classification models. The accuracy, sensitivity, and specificity of the models were above 83%, 85%, and 81%, respectively. The putatively identified metabolites were associated with biochemical pathways such as tricarboxylic acid cycle, glycolysis, carbohydrate conversion, and steroidal lipid metabolism that are mainly involved in energy production for cell growth and proliferation.

The investigation of innovative label-free &alpha, amino acids detection methods represents a crucial step for the early diagnosis of several diseases. While 1,8-diazafluoren-9-one (DFO) is known in forensic application because of the fluorescent products by reacting with the amino acids present in the papillary exudate, its application for diagnostic purposes has not been fully investigated. The stabilization of DFO over a transparent substrate allows its complexation with biomolecules for the detection of &alpha, amino acids. In this study, DFO was immobilized into a titanium dioxide (TiO2) matrix for the fluorescence detection of glycine, as a target &alpha, amino acid (a potential marker of the urogenital tract cancers). The DFO/TiO2 composite was characterized by atomic force microscopy, spectroscopic ellipsometry, fluorescence spectroscopy and fluorescence microscopy. The performed fluorescent studies indicate spectacular formation of aggregates at higher concentration. The measurements performed using various fluorescence and microscopic techniques together with the suitable analysis show that the aggregates are able to emit short-lived fluorescence.

The discovery of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, has opened a new frontier in the study of signal transduction and understanding cell-to-cell communication. EVs play a key role regulating various biological processes such as tissue regeneration, blood coagulation, immunomodulation or carcinogenesis. Therefore, they gain much attention as potentially therapeutic and non-invasive diagnostic targets. In this aspect, a comprehensive characterization of the EVs molecular content and understanding EVs functions are especially relevant. However, routine isolation of a usually small amount of high purity EVs is challenging, and analysis requires highly sensitive advanced analytical solutions. The utility of EVs in clinical settings is still limited due to the lack of standardization in existing protocols. This review focuses on the implication of the state-of-the-art mass-spectrometry based untargeted metabolomics approach for the molecular profiling of EVs, with the emphasis on the analytical methodologies, highlighting potential challenges and limitations.

Imidazol(in)e derivatives, having the chemical structure similar to clonidine, exert diverse pharmacological activities connected with their interactions with alpha2-adrenergic receptors, e.g. hypotension, bradycardia, sedation as well as antinociceptive, anxiolytic, antiarrhythmic, muscle relaxant and mydriatic effects. The mechanism of pupillary dilation observed after systemic administration of imidazol(in)es to rats, mice and cats depends on the stimulation of postsynaptic alpha2-adrenoceptors within the brain. It was proved that the central nervous system (CNS)-localized I1-imidazoline receptors are not engaged in those effects. It appeared interesting to analyze the CNS-mediated pharmacodynamics of imidazole(in)e agents in terms of their chromatographic and calculation chemistry-derived parameters. In the present study a systematic determination and comparative pharmacometric analysis of mydriatic effects in rats were performed on a series of 20 imidazol(in)e agents, composed of the well-known drugs and of the substances used in experimental pharmacology. The eye pupil dilatory activities of the compounds were assessed in anesthetized Wistar rats according to the established Koss method. Among twenty imidazol(in)e derivatives studied, 18 produced diverse dose-dependent mydriatic effects. In the quantitative structure-activity relationships (QSAR) analysis, the pharmacological data (half maximum mydriatic effect - ED50 in μmol/kg) were considered along with the structural parameters of the agents from molecular modeling. The theoretically calculated lipophilicity parameters, CLOGP, of imidazol(in)es, as well as their lipophilicity parameters from HPLC, logkw, were also considered. The attempts to derive statistically significant QSAR equations for a full series of the agents under study were unsuccessful. However, for a subgroup of eight apparently structurally related imidazol(in)es a significant relationship between log(1/ED50) and logkw values was obtained. The lack of "predictive" QSAR for the whole series of the structurally diverse agents is probably due to a complex mechanism of the ligand-alpha2-adrenergic receptor interactions, which are predominantly of a highly structurally specific polar nature. Such interactions are difficult to quantify with the established chemical structural descriptors, contrary to the less specific, molecular bulkiness-related interactions.

Arterial stiffening is a hallmark of early vascular aging (EVA) syndrome and an independent predictor of cardiovascular morbidity and mortality. In this case-control study we sought to identify plasma metabolites associated with EVA syndrome in the setting of hypertension. An untargeted metabolomic approach was used to identify plasma metabolites in an age-, BMI-, and sex-matched groups of EVA (n = 79) and non-EVA (n = 73) individuals with hypertension. After raw data processing and filtration, 497 putative compounds were characterized, out of which 4 were identified as lysophosphaditylcholines (LPCs) [LPC (18:2), LPC (16:0), LPC (18:0), and LPC (18:1)]. A main finding of this study shows that identified LPCs were independently associated with EVA status. Although LPCs have been shown previously to be positively associated with inflammation and atherosclerosis, we observed that hypertensive individuals characterized by 4 down-regulated LPCs had 3.8 times higher risk of EVA compared to those with higher LPC levels (OR = 3.8, 95% CI 1.7–8.5, P < 0.001). Our results provide new insights into a metabolomic phenotype of vascular aging and warrants further investigation of negative association of LPCs with EVA status. This study suggests that LPCs are potential candidates to be considered for further evaluation and validation as predictors of EVA in patients with hypertension.

Changes in metabolites composition can reflect currently present pathological processes in a living organism and constitute a basis for diagnosis and treatment improvements. Thus, the multiplatform metabolomics approach was applied for the investigation of molecular mechanisms of chronic kidney disease (CKD) progression. The high-performance liquid chromatography coupled with time-of-flight mass spectrometry (HPLC-TOF-MS) and gas chromatography coupled with triple quadrupole mass spectrometry (GC-QqQ/MS) serum metabolic fingerprinting followed by uni- and multivariate statistical analysis was carried out to determine metabolic pattern differentiating CKD patients and healthy controls. Furthermore, metabolites changes between stage 3 and 4 of the disease, as well as health status were investigated. The progression of the disease was found to be related to alterations in acylcarnitine, amino acid, lysophospholipid and carbohydrate metabolism. Elevated levels of serum acylcarnitines, sugar alcohols, and organic acids, as well as decreased levels of lysophospholipids, and amino acids, were found to be statistically significant for CKD progression. The obtained results confirm the utility of metabolomics approach as a tool for an explanation of molecular processes underlying CKD development.

Plasma untargeted metabolomics is a common method for evaluation of the mechanisms underlying human pathologies and identification of novel biomarkers. The plasma proteins provide the environment for transport of hydrophobic metabolites. The current sample preparation protocol relies on the immediate precipitation of proteins and thus leads to co-precipitation of a significant fraction of hydrophobic metabolites. Here we present a new simple procedure that overcomes the co-precipitation problem and improves metabolome coverage. Introducing an additional step preceding the protein precipitation, namely limited digestion with proteinase K, allows release of associated metabolites through the relaxation of the native proteins tertiary structure. The modified protocol allows clear detection of hydrophobic metabolites including fatty acids and phospholipids. Considering the potential involvement of the hydrophobic metabolites in human cardiovascular and cancer diseases, the method may constitute a novel approach in plasma untargeted metabolomics.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and localize mainly in the stomach or small intestine. The metabolomic signatures of GISTs driven by different KIT gene mutations remain undiscovered and unexplored. The main aim of this pilot study was to determine and compare metabolomic profiles in GIST xenograft models with different genetic backgrounds. Metabolomic profiling using gas chromatography coupled with mass spectrometry followed by univariate and multivariate statistical analyses was applied to select metabolites that differentiated the GIST models studied. The significant differences observed in the metabolites were mainly derived from glycolysis, the citric acid cycle and glutamine and lipid metabolism. The obtained results may suggest variable metabolomic signatures of tumours, possibly related to the different underlying, specific KIT gene mutations and with potential implications for the biological behaviour and natural course of this rare disease. This study constitutes a proof of concept in GISTs and reveals the potential of the metabolomic approach in orphan malignancies.