Chemistry and Biochemistry

Proteins play essential roles in every cellular process. Biomarkers have the potential to identify key disease indicators at the individualised level, as well as to detect a disease at the early-stages before the development/advance of a serious illness. The emergence of innovative, post-genomic technology, like proteomics, led to the development of strategies aimed at identifying altered, sensitive biomarkers specific to the disease among thousands of molecules present in biological samples. Blood and urine contains multitude, unstudied, and unknown biomarkers, particularly in low-molecular region of blood and urine proteome. Babesiosis, idiopathic dilated cardiomyopathy (iDCM), and chronic valvular disease (CVD-MV) in dogs are common and serious disease. The aims of the project is: to discover novel, early-stage biomarkers of kidney and cardiac disease in dogs which could lead toward more precise and adequate diagnose, prognosis, and monitoring of these diseases; to perform a proteomic analysis on the sera and urine of dogs with uncomplicated and complicated form of babesiosis, as well as dogs with diagnosed iDCM and CVD-MV in order to identify novel, early-stage diagnostic and prognostic biomarkers; to expand the pathway signalling research in order to improve the understanding of pathogenesis for studied disease; to validate new biomarkers for studied diseases. Tthese expected scientific outcomes will contribute in practice: (a) in a short term view, to detect individualised molecular fingerprints; to improve the diagnosis and prognosis of iDCM, CVD-MV, and babesiosis, as well as potential targets for novel approaches to monitor the therapies of these diseases; to enable the ability to personalise the treatment of individual patients resulting in enhanced patient care; and (b) in a long-term view, to allow entry of proteomics in clinical veterinary medicine in Croatia and transfer of knowledge in –omics- technologies in other parts of veterinary medicine.

Faculty of Veterinary Medicine University of Zagreb (FVM) is well established research institution in Croatia with more than 178 staff members who have excellent education and scientific experience. In order to conduct structural changes in veterinary medicine, scientists at FVM have accepted ERA Chair initiative, aiming at fostering structural changes, synergies between cohesion and research funding, and to raise research quality in the application of molecular technologies to international levels of excellence.

The project proposal aims to reinforce research performance of FVM through enhancing its capacities in molecular veterinary medicine, by attracting outstanding experienced researchers, transfer of knowledge and research potential, especially in the fields of proteomics and metabolomics.

The exchange of know-how and experience with esteemed outstanding experienced researchers will be accomplished through coherent set of measures: training, workshops and trans-national two-way secondments of research staff. The workshops on different aspects in molecular medicine organised in Zagreb and will mostly cover three chosen fields: comparative tumour analysis, post-genomic technology and wildlife research at the FVM departments. By the upgrading of its research performance and excellence FVM will improve performance in competitive research funding, prevent brain drain, enhance its competiveness and visibility in broader region and become internationally recognized partner.

The proposed activities should increase research performance in specific field of molecular veterinary medicine and FVM will be able to increase its involvement in international research projects with EU partners and to be better integrated into the ERA. The ERA Chair Initiative will be unique in croatian veterinary science with potential benefits for the broader research community and neighbouring countries.

Fascioloidosis is an invasive disease caused by the non-native European species of bisexual fluke Fascioloides magna. For now, it is possible to distinguish three types of final wild carriers of this fluke in Europe: typical (red deer and fallow deer), “dead end” (wild boar) and atypical (roe deer, mouflon and chamois). Although the appearance of this parasite has stimulated a relatively large interest of the scientific public, resulting in the publication of numerous scientific articles, host-parasite interactions are still not clarified. At the same time, fascioloidosis of atypical carriers is often a fatal disease, and a decline in the number of roe deer was recorded in the invaded areas. The goal is to understand the host-parasite interaction in different types of hosts. The assumptions of the project are: i) permanent migration of flukes through the carrier’s liver is a consequence of the carrier’s inadequate immune response, and ii) permanent migration of flukes through the carrier’s liver is a consequence of the impossibility of sexual maturation of the fluke. The goals of the project are: a) to study the macroscopic and microscopic changes on and in the livers of different types of carriers, b) to understand the immune response to the fluke F. magna in different carriers, c) to compare the diversity/existence of specific MHC gene alleles in different types of carriers, d) to analyze different fluke stages in different carriers. Blood samples, whole livers and flukes will be collected during regular hunting operations. We will classify individuals into negative, invaded, re-invaded and cured. Animals from areas where fascioloidosis has not been established will serve as completely negative controls. We will analyze the collected samples by parasitological, omics methods, NGS and classic MHC gene analysis, by dissection of flukes. The analyses will be carried out by a team formed during the implementation of the establishment research project with the help of members of the ERA Chairs project for the proteomics part. We expect that the results of the project will provide answers about the significant interactions between the carrier and the parasite on the example of fascioloidosis.

Mastitis (inflammation of the mammary gland) is recognized as the biggest threat to the health and productivity of dairy cows, and causes great economic damage in the production and processing of milk and dairy products. In most cases, only clinical mastitis is treated, while subclinical mastitis goes unnoticed causing great losses. While clinical mastitis is easy to detect in most cases, the subclinical form is a big problem on farms, so that the incidence in a herd can be from 2 to 20 times higher than the clinical form. According to some studies, subclinical mastitis represents approximately 90-95% of total mastitis cases, with a prevalence in herds of 15-75%. Detecting subclinical mastitis is difficult because clinical symptoms are not visible, udder and milk look normal, and the disease very often turns into a clinical form. A large number of somatic cells (BSS) results in a reduced amount of milk produced per cow (10-25%) and a reduction in milk fat by 5-12%. The project is focused on the detection, characterization and quantification of the proteomic profile of the milk of cows with subclinical mastitis, with the aim of applying advanced, innovative and state-of-the-art technologies in the development and creation of a new diagnostic test for subclinical mastitis. For this, it will be necessary to perform profiling of biomarkers for the differential diagnosis of mastitis using a quantitative “bottom-up” proteomic approach, to perform bioinformatic analysis of the collected data, biological validation of potential biomarkers, to produce appropriate antibodies, to produce multiple tests, and finally to adapt the validated biomarkers to the format of immunochromatographic tests.

Docosahexaenoic acid (DHA) is a fatty acid that is highly concentrated in brain, cerebral cortex, skin, sperm, testicles and retina. The biochemical pathway for DHA synthesis was enigmatic for a long time and current model suggests that after the series of desaturation and elongation processes in endoplasmic reticulum C24:6n3 is synthesized, transported to the peroxisomal matrix and then shortened to 22:6n-3 via one cycle of β-oxidation followed by its transport back to the endoplasmic reticulum for specific esterification into the appropriate phospholipids.

Deficiency in DHA metabolism could lead to severe diseases in humans and animals, from lethal autosomal-recessive to behavioral and learning deficits. Moreover, DHA may play important role in a lot of diseases that are currently under investigation: neurodegenerative, diabetes and diabetic retinopathy, metabolic syndrome and tumor cells apoptosis. In this project, we will investigate the influence of dietary n-3/n-6 ratio and direct DHA supplementation in insulin dependent and non-insulin dependent diabetes mellitus on DHA metabolism, mitochondrial and peroxisomal oxidation and DHA metabolome. Our assumption is to prove that nutritional modulation can override insulin influence on diabetic dyslipidemia which could have profound influence on the treatment of diabetes. Additionally, we will try to find key enzymes of mitochondrial and peroxisomal β-oxidation which could influence DHA metabolism by nutritional modulation.                Investigations will be performed on Sprague–Dawley rats which will be randomly divided into groups with different dietary treatments which will consist of different n3/n6 ratios and increased DHA content. Induction of diabetes will be performed by streptozotocin (IDDM) or by dietary treatment with high fructose/high fat diet (NIDDM). Integration of results from experiment 1 and 2 will define the behaviour of DHA in different types of diabetes. Investigations on subcellular level will allow us to determine the influence of diabetes on peroxisomal and mitochondrial beta oxidation and DHA metabolism. Investigation of DHA metabolome will link nutrition, diabetes and bioactive DHA products. Finally, data from the whole project will result in integration of gained knowledge and better understanding of DHA biosynthesis.

Metabolic syndrome refers to a combination of several metabolic risk factors including insulin resistance, central obesity, dyslipidaemia and hypertension. Dysfunction of lipid metabolism is a key component in the development of metabolic syndrome and the prevalence of metabolic syndrome is strongly associated with the severity of obesity. Therefore, dietary intervention could be important strategy for the prevention of metabolic syndrome and associated diseases. Obesity arises from a sustained positive energy balance which triggers a pro-inflammatory response. The enhanced secretion of pro-inflammatory peptides promotes penetration of inflammatory cells such as macrophages and lymphocytes resulting in alteration of microenvironment within adipose tissue. This causes law-grade chronic inflammation and lipid spill-over from the adipose to the liver, muscle and pancreas resulting in glucotoxicity and lipotoxicity. These disturbances culminate in impaired insulin signaling, dysregulated glucose homeostasis and consequent development of insulin resistance. Chronic inflammation and insulin resistance are now recognized as contributing factors to many modern diseases such as insulin non dependent diabetes mellitus, cardiovascular diseases, Alzheimer disease and cancer. To achieve project objectives we will investigate several interactions that take place in metabolic syndrome pathogenesis: A) Influence of dietary n3/n6 ratio and DHA as a preventive mechanism on the severity of metabolic syndrome with sex as additional variable, B) Influence of dietary n3/n6 ratio and DHA on therapy of induced metabolic syndrome with sex as additional variable, C) Parental transmission of predisposition to obesity-related disorders to their offspring and influence of offspring diet on severity of metabolic disorders before and after sexual maturity, D) Interaction between age and metabolic syndrome with sex and dietary energy (HFD vs. low calorie) as an additional variables and E) Cancer lipid metabolism and cancer markers of lipid origin as well as possible link between obesity, metabolic syndrome, low grade chronic inflammation and cancer. Finally, the integration of gained knowledge from all experiments will allow us to suggest new approaches to dietary prevention and therapy of metabolic syndrome and to better understand underlying link between obesity related chronic inflammation and cancer.