Human disease processes are often characterized by a deviation from the normal physiological concentration of critical biomarkers. The detection of disease biomarkers requires the development of novel sensing methods which are sensitive, specific, efficient and low-cost. To address this need, a novel conductive and recognitive hydrogel composite material has been developed. This work investigated the fabrication methods, the chemical and physical composition, the sensing capabilities, and the biocompatibility of the proposed conductive and recognitive hydrogel composite materials. The conductive polymer was found to respond by changing conductivity in the presence of biomolecules. Specificity can then be incorporated into the system by integrating the conductive polymer with a molecularly imprinted hydrogel. The demonstration of a conductive and recognitive hydrogel composite is a step towards the integration of these materials into close-loop sensing and drug delivery systems. / text

There is an unmet need for novel biomarker discovery in Inflammatory Bowel Diseases (IBD) to aid clinical management in several clinical settings including diagnosis and prognosis. With an ever-advancing repertoire of biological therapies on the horizon, it is important to personalise treatments at an early stage. The aim of this thesis is to explore the clinical utility of novel blood-based biomarkers in diagnosis, disease classification and prognosis in 2 cohorts: newly diagnosed IBD and acute severe colitis. Investigating the circulating methylome, 290 probes exhibited Holm significant IBD-associated methylation differences, including VMP1/MIR21 (p=7.5×10-14) and RPS6KA2 (1.1×10-19) and were consistent within the European cohort. 11 Differentially methylated positions (DMPs) predicted treatment escalation after Holm adjustment (top probe p=0.003). A panel of 6 probes identified 2 patient subgroups that have significantly different disease courses (Hazard Ratio (HR) 10.5, 95%CI: 4.3-25.6; logrank p=1.5×10-24). The 6 probe marker outperformed conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7-5.8), logrank p=0.0004 and Alb < 36g/L, HR 2.9(1.5-5.6), p=0.0001). Within the same cohort, a novel proximity extension assay (PEA) was then utilised to identify novel diagnostic and prognostic protein markers. 61 proteins were significantly associated with IBD including MMP12 (Holm-adjusted p=4.1×10-26). A total of 9 proteins predicted disease course in this cohort. Using a panel of 7 randomly selected top prognostic probes, 2 patient groups were identified that had significantly different disease courses: logrank p=2.2×10-10, HR 5.6(2.0-15.6), outperforming conventional biomarkers in predicting treatment escalation (hsCRP > 4mg/L, HR 3.2(1.7- 5.8), logrank p=0.0003 and Alb < 36g/L, HR 2.7(1.4-5.2), p=0.0004). In a subcohort, serum calprotectin (SC) and conventional blood markers were investigated for their utility in diagnosis and prognosis in IBD. SC performed at par with CRP at differentiating IBD from controls with an area under the curve (AUC) of 0.87 (CI 0.81-0.92). For prognostication, both albumin and SC remained significant predictors of treatment escalation in IBD (logrank test p=5.1×10-5). MicroRNAs (miRNA) are small non-coding nucleic acids that have the capacity to modulate gene expression. Using small RNA sequencing in acute severe colitis (ASUC) and healthy controls (HC), 10 serum-based miRNA markers were significantly associated with acute severe colitis, including miR-30a-5p. Validating the findings using qPCR, miR-30a-5p was downregulated in ASUC (p=0.003). Furthermore, miR30a-5p remained a significant predictor of eventual colectomy in acute colitis (logrank test p=0.0014). These data highlight the translational potential for methylation, miRNA and proteomic biomarkers in diagnosing and prognosticating in IBD.

Metabolomics is a powerful science that can be applied for the discovery of disease biomarkers, and investigation of altered metabolomes due to abiotic and biotic perturbations. This dissertation is focused on untargeted metabolomic applications to investigate fungal metabolite alterations associated with pathogenicity, fungal disease propagations, and symbiosis. This dissertation employs qualitative analysis of metabolite mixtures using HS-SPME coupled GC-MS and TMS derivatization followed by GC-MS analytical platforms. In the first study, we discovered a biomarker combination to diagnose fungal soft tissue disease in sweet potato at an early stage of disease propagation. We used an HS-SPME GC-MS untargeted metabolomics workflow to analyze the VOC associated with Rhizopus stolonifer infected and healthy sweet potatoes in situ and simulated warehouse environments. A single combination of 4 biomarkers was able to diagnose R. stolonifer fungal soft tissue disease (AUC = 0.980, 95% C.I. 0.937-1) and the early stage of the fungal soft rot disease (AUC = 0.999, 95% C.I. 0.978-1). We were able to detect the biomarkers: 1- propanol, ethyl alcohol, ethyl propionate and 3-methyl-3- buten-1-ol during disease progression in a simulated warehouse environment. Therefore, this study shows the feasibility of early diagnosis of fungal soft tissue disease by a real-time screening of volatile profiles of sweet potato in post-harvest storage. When considering the study of a particular species metabolome, it is crucial to develop a metabolite extraction protocol. In the second study, the performance of the six different metabolite extraction solvents mixtures was tested with the preferred mix being: butanol:methanol:water (2:1:1, v/v at -20 °C) which was used as a single solvent mix to extract both polar and relatively non-polar metabolites simultaneously in a single extraction step. The Macrophomina phaseolina fungal metabolome was investigated using the solvent mix. Finally, fungal mutualism was studied using untargeted metabolomics. Most often mycorrhizal metabolomics workflows are based on analyzing the Arbuscular Mycorrhizae colonized root metabolome. But here, we used hyphal materials to examine the mutualistic symbiotic association of the AM fungi. All untargeted metabolomic studies included chemometric data analysis and specific biomarkers and or metabolites were determined using multivariate statistics or prediction model building and validating.

Xanthene dyes are common fluorophores which have been widely used as molecular probes. The xanthene fluorophores can be used as highly selective optical sensors to detect disease biomarkers. A new fluorogenic dye containing an alpha, beta-unsaturated aldehyde moiety exhibits selective fluorescent signal enhancement in the presence of cysteine or peptides containing N-terminal cysteine residues. The mechanism is based on synergistic covalent and supramolecular interactions. A unique rhodamine boronic acid indicator is used in an optimized data collection protocol for wavelength- and time-dependent selectivity of various saccharides and nucleosides. One indicator is thereby capable of selectively distinguishing structurally related analytes in mixtures. Moreover, the rhodamine-based boronic acid responds linearly to increasing riboside concentrations in urine samples, potentially enabling the screening for inborn purine metabolism disorders.

Purpose of Review: The incidence of diabetes is increasing worldwide. Diabetes mellitus is characterized by hyperglycemia, which in the long-term damages the function of many organs including the eyes, the vasculature, the nervous system, and the kidneys, thereby imposing an important cause of morbidity for affected individuals. More recently, increased bone fragility was also noted in patients with diabetes. While patients with type 1 diabetes mellitus (T1DM) have low bone mass and a 6-fold risk for hip fractures, patients with type 2 diabetes mellitus (T2DM) have an increased bone mass, yet still display a 2-fold elevated risk for hip fractures. Although the underlying mechanisms are just beginning to be unraveled, it is clear that diagnostic tools are lacking to identify patients at risk for fracture, especially in the case of T2DM, in which classical tools to diagnose osteoporosis such as dual X-ray absorptiometry have limitations. Thus, new biomarkers are urgently needed to help identify patients with diabetes who are at risk to fracture. - Recent Findings: Previously, microRNAs have received great attention not only for being involved in the pathogenesis of various chronic diseases, including osteoporosis, but also for their value as biomarkers. - Summary: Here, we summarize the current knowledge on microRNAs and their role in diabetic bone disease and highlight recent studies on miRNAs as biomarkers to predict bone fragility in T1DM and T2DM. Finally, we discuss future directions and challenges for their use as prognostic markers.

No description available.

Inflammatory bowel disease (IBD) represents a group of multifactorial illnesses with increasing incidence worldwide. Crohn's disease (CD) and ulcerative colitis (UC) are the two most thoroughly defined phenotypes of IBD. IBD associated with primary sclerosing cholangitis (PSC) - a progressive biliary disease leading to cirrhosis and liver failure - is considered as specific IBD phenotype (also referred to as 'PSC - IBD') due to its clinical and pathophysiological characteristics. The aim of the experimental part of this thesis was to define specific features of PSC - IBD in the key areas of IBD pathogenesis. These are: microbiota composition, gut - barrier failure, genetic predisposition and aberrant cellular and antibody immune response. Furthermore, the other goals were to describe relation of IBD status and activity to liver transplantation (LTx) and carcinogenesis based on thorough analysis of clinical data in patients under surveillance at the liver transplantation unit. Using the next-generation parallel sequencing technology, we discovered specific bacterial and mycobial features of gut microbiota composition in PSC - IBD which significantly differed from UC and healthy controls recruited from Czech general population. Moreover, we identified numerous seral biomarkers distinguishing CD, UC...

<p>Uvod: Pojam &quot;sekretor&rdquo; ili &bdquo;nesekretor&quot; se odnosi na sposobnost pojedinca da luči antigene krvnih grupa ABO sistema u telesnim tečnostima. Određivanje ABO krvne grupe i sekretornog statusa, testom inhibicije hemaglutinacije pljuvačke i Lewis fenotipa na eritrocitima su važni u kliničkoj i forenzičkoj medicini, u odnosu na etiopatogenezu mnogih bolesti. Nesekretorstvo ABO krvnogrupne supstance je udruženo sa če&scaron;ćom pojavom autoimunih inflamatornih oboljenja među kojima su i seronegativne spondiloartropatije. Veća učestalost seronegativnih spondiloartropatija među osobama koje imaju HLA-B27 antigen predstavlja polaznu osnovu stanovi&scaron;ta da genetski faktori u kombinaciji sa faktorima sredine utiču na pojavu seronegativnih spondiloartropatija u genetski predisponiranih individua. Teza istražuje značaj testa inhibicije hemaglutinacije plijuvačke i određivanja ABO i Lewis fenotipa na eritrocitim u dijagnostici seronegativnih spondiloartropatija. Ciljevi i hipoteze: Cilj istraživanja je bio da se utvrdi učestalost nesekretora i sekretora ABO krvnogrupne supstance i Lewis fenotipa u grupi obolelih od seronegativnih spondiloartropatija i izvr&scaron;iti poređenje rezultata u odnosu na grupu zdravih ispitanka. Pretpostavljeno je da postoji značajno veći broj nesekretora ABO krvnogrupne supstance u obolelih od seronegativnih spondiloartropatija u odnosu na zdrave ispitanike. Pretpostavljeno je i da postoji značajno veća učestalost nesekretora ABO krvnogrupne supstance u obolelih od seronegativnih spondiloartropatija sa negativnim HLA-B27 antigenom. Pretpostavljeno je da kod osoba obolelih od seronegativnih spondiloartropatija dolazi do promene Lewis fenotipa na eritrocitima u odnosu na sekretorni status u pljuvačci. Metode: Sprovedena je longitudinalna prospektibvna studija. Ispitanici stariji od &scaron;est godina oba pola podeljeni su u dve randomizovane grupe. Eksperimentalnu grupu sačinjavalo je 110 ispitanika sa dijagnozom oboljenja iz grupe seronegativnih spondiloartropatija. Kontrolnu grupu sačinjavalo je 103 dobrovoljna davaoca krvi, bez dijaghnoze oboljenja iz grupe seronegativnih spondiloartropatija. Ispitanicima kontrolne i eksperimentalne grupe određena je pripadnosti ABO krvnogrupnom sistemu, sekretorni status i Lewis fenotip, dok je osobama eksperimentalne grupe određen i fenotip HLA-B27. Uključujući kriterijumi osim navedenog bili su da ispitivane osobe ženskog pola nisu trudinice i da ispitanici obe grupe nisu primali transfuziju krvi tri meseca pre uključivanja u istraživanje. Rezultati: Rezultati istraživanja ukazuju da nesekretori ABO krvnogrupne supstance imaju 1,63 puta veći rizik (veću učestalost), oboljevanja od seronegativnih spondiloartropatija u odnosu na zdravu populaciju ispitanika, kao i da smanjena ekspresija Lewis (b) antigena predstavlja doprinoseći faktor razvoja seronegativnih spondiloartropatija. Ustanovljeno je da pod uticajem seronegativnih spondiloartropatija dolazi do izmene Lewis fenotipa na eritrocitima obolelih. Verovatnoća dokazivanja oboljenja iz grupe seronegativnih spondilartropatija među obolelima veća je za 11% kod nesekretora fenotipa HLA-B27- u odnosu na obolele nesekretore fenotipa HLA-B27+. Zaključak: Sekretorni status i Lewis fenotip predstavljaju zasebne dijagnostičke biohemijske markere nezavisne od HLA-B27 antigena koji doprinose ranijem otkrivanju osoba koje imaju predispoziciju razvoja oboljenja iz grupe seronegativnih spondiloartropatija.</p> / <p>Introduction:The term secretory state referes to ability of individual to secrete ABO blood group antigens in body fluids. Determination of the ABO blood group antigens and secretory status by hemagglutionation inhibition test using saliva as well as Lewis phenotype on erythrocytes are important in clinical and forensic medicine, in relation to the etiopathogenesis of many diseases. Non secretory status of ABO blood groupantegens is related with higher incidence of autoimmune inflammatory disease which include seronegative spondyloarthropathyes. Increased frequency of seronegative spondyloarthropathies among people who have the HLA-B27 antigen is starting point of the view that genetic factors in combination with environmental factors influence the occurence of seronegative spondyloarthropathies in genetically predisposed individuals. The tesis explores the significance of the hemagglutionation inhibition test of saliva and determination of ABO and Lewis antigens in diagnostics of seronegativespondyloarthropathyes. Goals and hypothesis: The aim of this study was to determine the frequency of non secretors and secretors of ABO blood group antigens and Lewis phenotype in a group of patient with seronegative spondyloarthropathies and make comparsion to the healthy examined group. It was assumed that there is a significantly higher number of non secretors of ABO blood group antigens among the patients with seronegative spondyloarthropathies compared to healthy examined persons. It was assumed that there is a significantly higher number of non secretors of ABO blood group antigens among the patients with seronegative spondyloarthropathies who do not have HLA-B27 antigen. It was assumed that in patients with seronegative spondyloarthropathies Lewis phenotype on erythrocits can be changed in relation to the secretory status in the saliva. Methods: We performed a prospective longitudinal study. Respondants older than six years of both gender were divided into two randomized groups. Experimental group is consisted of 110 patients diagnosed with seronegative spondyloarthropathy. The control group consisted of 103 blood donors who did not have diagnosed disease from the group of seronegative spondyloarthropathies. To the subjects of the control and experimental groups was determined ABO blood group antigens, secretory status and Lewis phenotype, while to the subjects of experimental group also was designated HLA-B27 phenotype. Including criteria other than the above were that among female respondants were not pregnant, and that both groups of respondants did not receive a blood transfusion three months before joining the study. Results: The resuts of the study showed that non secretors of ABO blood group antigens have a 1,63 times higher rise (higher incidence) of developing disease from the group of seronegative spondyloarthropathies compared to a healthy population of subjects, as well as that decreased expression of Lewis antigens (b) represents a contributing factor for development of seronegativespondyloarthropathies. It was found that under the influence of seronegative spondyloarthropathies there are changes in Lewis phenotype on erythrocytes of patients. It was found that under the influence of seronegative spondyloarthropathies there are changes in Lewis phenotype on erythrocytes of patients. Probability for confirmation seronegative spondyloarthropathies is higher for 11% among non secretors who have HLA-B27 negative phenotype in comparison to non secretors who have HLA-B27 positive phenotype. Conclusion: Secretory status and Lewis phenotype are separate diagnostic biochemical markers independent of HLA-B27 antigen that contribute to the early detection of people who have a predisposition of the disease from the group of seronegative spondyloarthropathies.</p>

<p>Napredak na polju molekularne biologije omogućio je identifikaciju molekularnih markera za karcinom bronha sa vrednim prognostičkim i prediktivnim značajem i njihova uloga kod uznapredovalog, metastatskog oblika bolesti je u velikoj meri istražena, dok kod ranih stadijuma bolesti jo&scaron; uvek nije sasvim jasna. Cilj ovog istraživnja bio je da se utvrdi učestalost najče&scaron;ćih genskih alteracija u tumorskim ćelijama bolesnika sa ranim stadijumom adenokarcinoma bronha, da se utvrdi pojedinačna zavisnost ispitivanih genskih alteracija u tumorskim ćelijama sa određenim kliničko-patolo&scaron;kim karakteristikama i da se utvrdi potencijalni prognostički značaj pojedinačne genske alteracije u tumorskim ćelijama na vreme preživljavanja bez povratka bolesti i ukupno vreme preživljavanja. Istraživanje je obuhvatilo 161 bolesnika sa adenokarcinomom bronha, stadijuma bolesti od I do IIIA, kod kojih je sprovedena radikalna hirur&scaron;ka resekcija u Institutu za plućne bolesti Vojvodine u periodu izmedju 2007 i 2014 godine. U tumorskim uzorcima fiksiranim u parafinu odredjivane su mutacije EGFR, KRAS i PIK3CA gena, ALK i ROS1 rearanžman i PD1 i PD-L1 ekspresija. Kliničkopatolo&scaron;ke karakteristike su preuzete iz registra za karcinom bronha Instituta za plućne bolesti Vojvodine. Ukupno preživljavanje je računato od dana operacije do dana smrti, a preživljavanje bez povratka bolesti je računato od dana operacije do momenta ponovne pojave bolesti. Od 161 testiranog tumorskog uzorka, prisustvo mutacija detektovano je kod 96 uzoraka (59.6%). Prisustvo mutacije KRAS gena detektovano je kod 69 (42.9%), mutacije EGFR gena kod 10 (6.2%), a mutacije PIK3CA gena kod 7 (4.3%) tumorskih uzoraka. ALK rearanžman je detektovan kod 3 (1.9%), a ROS1 rearanžman kod 7 (4.3%) tumorskih uzoraka. PD-1 ekspresija detektovana je u 71 tumorskom uzorku (45%), dok je PD-L1 ekspresija detektovana u 59 tumorskih uzoraka (36.6%). PD-1 ekspresija nije bila značajno povezana ni sa jednim od klinčko-patolo&scaron;kih karakteristika (uključujući KRAS, EGFR, ALK, ROS1 i PI3KCA status). PD-L1 ekspresija je bila značajno povezana sa tipom hirurgije (P = 0.01) i sa prisustvom KRAS mutacije (P = 0.02). Mutacioni status u domenu KRAS gena je bio značajno povezan sa godinama starosti (P = 0.004), polom (P = 0.006) i pu&scaron;ačkim statusom (P = 0.004). Mutacioni status u domenu EGFR gena je bio značajno povezan sa pu&scaron;enjem (P &lt; 0.001) i sa godinama starosti (P = 0.013). Mutacioni statusi u domenu gena za ALK, ROS1 i PI3KCA nisu bili značajno povezani ni sa jednom od ispitivanih kliničko-patolo&scaron;kih karakteristika. Prisustvo PD-1 ekspresije je bilo značajno povezano sa preživljavanjem bez povratka bolesti (P = 0.03) i ukupnim preživljavanjem (P = 0.01). PD-L1 ekspresija, KRAS, EGFR, ALK, ROS1 i PIK3CA mutacioni status nisu bili značajno opvezani sa preživljavanjem bez povratka bolesti i ukupnim preživljavanjem. Najče&scaron;će detektovane genske alteracije su mutacije u domenu KRAS i EGFR gena. Prisustvo KRAS mutacije je značajno povezano sa godinama starosti ispitanika, polom i pu&scaron;ačkim statusom dok je prisustvo EGFR mutacije značajno povezano sa godinama starosti ispitanika i pu&scaron;ačkim statusom. Prisustvo PD-L1 ekspresije je značajno povezano sa vrstom hirur&scaron;kog lečenja i sa prisustvom KRAS mutacija. Jedino prisustvo PD-1 ekspresije u tumorskim ćelijama predstavlja nezavistan prognostički faktor za preživljavanje bez povratka bolesti i ukupno preživljavanje bolesnika sa ranim stadijumom adenokarcinoma bronha.</p> / <p>Advances in the field of molecular biology gave us insight into biomarkers for lung cancer with great prognostic and predictive value and their role in advanced stage disease is well known while in early stage disease is yet to be proven. The aim of this study was to determine the frequencies of the most common gene alterations in patients with early stage lung adenocarcinoma, to determine the relationship between gene alterations in tumor cells and clinicopathologial characteristics and to determine prognostic value of each gene alteration regarding overall survival and disease free survival. One hundred sixty-one patients diagnosed with lung adenocarcinoma clinical stage I-IIIA who underwent radical surgical resection at the Institute for Pulmonary Diseases of Vojvodina between 2007 and 2014 were included in this study. Mutations in EGFR, KRAS and PIK3CA gene, ALK and ROS1 rearrangement and PD-1 and PD-L1 expression were determined in representative formalin-fixed, paraffin-embedded (FFPE) tumor block from each patient. Clinical data were extracted from the institutional lung cancer registry of the Institute for Pulmonary Diseases. Overall survival was calculated as time from the day of surgery to the day of death. Disease free survival was calculated as time from the day of surgery to the day of disease relapse. Among 161 tested tumor tissue, presence of mutation was found in 96 (59.6%) of them. There were 69 (42.9%) mutations in KRAS gene, 10 (6.2%) in EGFR gene and 7 (4.3%) in PIK3CA gene. ALK and ROS1 rearrangement were present in 3 (1.9%) and 7 (4.3%), respectively. PD-1 expression was determined in 71 (45.0%) tumor sample while PD-L1 expression was determined in 59 (36.6%). PD-1 expression was not correlated with any of the clinicopathologial characteristics (including KRAS, EGFR, ALK, ROS1 and PIK3CA mutational status). PD-L1 expression correlated with type of surgery (P = 0.01) and KRAS positivity (P = 0.02). KRAS mutation status correlated with age (P = 0.004), sex (P = 0.006) and smoking status (P = 0.004). EGFR status correlated with smoking status (P &lt; 0.001) and age (P = 0.013). ALK, ROS1 and PIK3CA status were not correlated with any of the clinicopathologial characteristics. PD-1expression was significantly associated with disease free survival (P = 0.03) and overall survival (P = 0.01). PD-L1 expression, KRAS, EGFR, ALK, ROS1 and PIK3CA status were not associated with disease free survival and overall survival. The most frequent gene alteration are mutations in KRAS and EGFR gene. Presence of KRAS mutation is in correlation with patients age, sex and smoking status while presence of EGFR mutation is in correlation with patients age and smoking status. PD-L1 expression is in correlation with type of surgery and KRAS mutational status. Only presence of PD-1 expression represent an independent prognostic factor for disease free survival and overall survival.</p>