Isolation, characterisation and the role of complexes formed between transferrin and insulin-like growth factor-binding protein 3

Sistem faktora rasta, po strukturi i funkciji, sličnih insulinu („insulin-like growth factor“- IGF) obuhvata elemente sa primarnom ulogom u regulaciji rasta, razvoja, diferencijacije ćelija kako u fiziološkim, tako i u patofiziološkim uslovima. Pored dva funkcionalna peptidna hormona, IGF-I i IGF-II, ovaj sistem sadrži molekule koji regulišu ispoljavanje njihove fiziološke funkcije. Regulatorni elementi IGF sistema su: vezujući proteini (IGFBP-1 do -6), receptori za IGF molekule: IGF-1R, IGF-2R, insulinski receptor (IR), hibridni receptor IGF-R/IR, kao i specifične proteaze. Nakon proteolize IGFBP, funkcija IGF-I/II se ispoljava interakcijom sa IGF specifičnim receptorima na površini ćelija.U kompleksu sa IGFBP-3 se nalazi 70-75% IGF peptida. Pored primarne uloge nosača IGF molekula, IGFBP-3 učestvuje i u brojnim interakcijama nezavisno od IGF. IGFBP-3 poseduje više strukturnih domena što ga čini sposobnim za vezivanje za druge molekule i pokretanje signalnih puteva i mehanizama nezavisnih, a nekad i suprotnih, dejstvu IGF peptida poput stimulacije rasta ćelija ili pokretanja apoptotskih procesa. Jedan od partnera koji interaguje sa IGFBP-3 je transferin (Tf), glavni transporter jona gvožđa u krvi.Iako se za postojanje kompleksa IGFBP-3/Tf znalo i ranije, rezultati ove disertacije predstavljaju prvi prikaz strukturnih i funkcionalnih osobina ovih kompleksa, kao i sagledavanje njihove moguće uloge. U ovoj disertaciji je opisan optimizovan postupak za izolovanje kompleksa iz fizioloških uzoraka (seruma i tkiva), navedene su strukturne karakteristike kompleksa IGFBP-3/Tf, faktori koji utiču na njihovo formiranje, određena je koncentracija kompleksa u serumu zdravih odraslih osoba i osoba sa poremećajem u metabolizmu gvožđa, dokazana je neophodnost jona gvožđa za formiranje kompleksa i analizirana je subćelijska raspodela kompleksa u tkivu debelog creva (zdravog i tumorskog). U radu je ispitan uticaj: anemije, visoke koncentracije gvožđa i tumora debelog creva, na koncentraciju i strukturu kompleksa. Za tumor debelog creva je karakteristična sistemska anemija, praćena akumulacijom gvožđa u samom tumorskom tkivu. Na model sistemu tumora debelog creva ispitana je važnost puta internalizacije IGFBP-3 preko kompleksa sa Tf, odnosno posredstvom transferinskog receptora (TfR).Nađeno je da je koncentracija kompleksa IGFBP-3/Tf kod zdravih ljudi 241 ± 62 μg/L i da se u ovoj formi nalazi 5-7% ukupnog IGFBP-3. Kod različitih patofizioloških stanja sa poremećajem u metabolizmu gvožđa utvrđeno je da formiranje kompleksa primarno zavisi od koncentracije gvožđa, potom koncetracije IGFBP-3, ali i proteina uključenih u metabolizam gvožđa, poput feritina. Kod IGFBP-3/Tf kompleksa analiziranih u uzorcima seruma dobijenih od pacijenata sa tumorom debelog creva primećen je izmenjen način...The insulin-like growth factor (IGF) system plays an important role in the regulation of cell growth, development and differentiation, in both physiological and pathophysiological condition. Beside two peptide hormones IGF-I and IGF-II, this system includes the following regulatory elements: six IGF-binding proteins (IGFBP-1 to -6), specific receptors for IGF (IGF-1R and IGF-2R), insulin receptor (IR), hybrid receptor (IGF-R/IR) as well as IGFBP specific proteases. When IGFBPs are proteolytically cleaved, IGF-I/II are able to exert their physiological function by interacting with specific receptors on the cell surface.Approximately 70-75% of the total IGFs, are transported in circulation in the form of ternary complexes with IGFBP-3. Besides being the principal carrier of IGF molecules, IGFBP-3 was reported to exert a number of activities which are IGF-independent. Due to its structural sub-domain organisation, this molecule is able to interact with binding partners other than IGFs, and consequently activate mechanisms and signaling cascades with independent or even opposite effects to those of IGF-I/II, such as apoptosis. One of these binding partners is transferrin (Tf), the principal iron transporter in the blood.Although the existence of IGFBP-3/Tf complexes has already been reported, the results presented in this thesis offer the first complete structural and functional characterisation of the complexes, together with the analysis of their potential role. The fully optimised method for the isolation of intact IGFBP-3/Tf complexes from serum and tissue samples has been described, some structural characteristics have been defined, the effect of iron and other factors on the formation of complexes was studied, their concentration in sera from healthy persons and patients with impaired iron metabolism disorders was measured, as well as their subcellular distribution in colon tissue (healthy and tumor tissue). The formation and concentration of IGFBP-3/Tf complexes in persons with anemia, persons with very high iron concentration or patients with colorectal carcinoma (CRC) have also been investigated. The emphasis was made on samples from patients with CRC, a disease accompanied by systemic anemia and increased iron accumulation in cancer cells. CRC was a model system for the analysis of IGFBP-3 internalisation via Tf-TfR pathway.The concentration of IGFBP-3/Tf complexes in healthy adults was measured to be 241 ± 62 μg/L, which makes up to 5-7% of the total IGFBP-3. The results have shown that in impaired iron metabolism, the formation of complexes is highly dependent on the iron concentration, then IGFBP-3 concentration, as well as the concentration of other proteins involved in iron metabolism, such as ferritin. IGFBP-3/Tf complexes isolated form serum..

Modelne karakteristike telesne strukture studentkinja Fakulteta za specijalnu edukaciju i rehabilitaciju (FASPER) izmerene metodom multikanalne bioelektrične impedance

The goal of this research was to define the initial body structure model of female students of Faculty of Special Education and Rehabilitation (FASPER), University of Belgrade, as a person whose professional and work commitment is working with disabled people and it's perceived as a very responsible, stressful and atypical work profile. The sample was made of 125 participants (female students) of III and IV year of Bachelor studies of FASPER. Research was conducted in a Methodical-research laboratory (MRL) in Faculty of sport and physical education, University of Belgrade in a time period 2012-2017, in accordance with standardized procedure of applying electrical multichannel bioimpedance method with body structure analyzer - InBody 720. It contained twenty (20) variables, eight (8) were basic and twelve (12) were derived. Based on BMI it's claimed that 77.60% participants has a normal body status, but the prevalence of overweight students (BMI lt 25.00) were 7.20%, apropos the prevalence of obesity (BMI lt 30.00) was 4.00%. It's also established that the prevalence of underweight students (BMI>18.50) is 11.20%, with 4.00% participants who were significantly thinness underweight i.e. poorly nourished (BMI>17.50). According to the results it was concluded that even 96.8% of sample was not in line with ideal or optimal body structure profile. For example, 63.2% participants has some form of body mass deficits (average of 5.25 kg) with mostly muscle component deficit 78.4% (average muscle mass deficit is 4.28 kg) and with body fat component deficit 43.2% (average deficit of body fat is 2.01 kg). On the other hand, 33.6% participants has a form of surplus (average surplus in body structure is 9.44 kg) where even 54.4% has a surplus of body fat component (average level of surplus of body fat 7.48 kg). The results of 4D body structure model of female students from FASPER can be defined like this: 31.77 L of Total Body Water (53.37%), 8.50 kg of proteins (14.26%), 16.98 kg of body fat (27.10%) and 3.10 kg of minerals (5.18%). Beside the probability that the reason of bad nutrition behaviour of these students is the cause of their increasing fat depots it's also a fact that 69.6% of these students are physically inactive and 21.6% just occasionally active, which mean that 91.2% of students was physically non active person, could indicate a most possible reason of body fat component suficit and body muscle component deficit as a main body structure characteristics.El objetivo de la investigación es definir las características de modelo de la estructura corporal de las estudiantes de la FASPER de la Universidad en Belgrado, como personas cuya orientación laboral futura es el trabajo con las personas inválidas, lo que representa UN perfil laboral profesional muy responsable, estresado y atípico. La muestra ha sido compuesta de las 125 examinadas. Las mediciones se han hecho en el Laboratorio metódico-investigativo de la Facultad de Deporte y Educación Física de la Universidad en Belgrado, utilizando el analizador de la estructura corporal - InBody 720. La investigación ha abarcado veinte (20) variables. Los resultados han mostrado que los 77,60 % de las examinadas están en el estatus de nutrición normal, que la prevalencia de las examinadas con sobrepeso (BMI lt 25.00) es el 7,20 %, es decir, con obesidad (BMI lt 30.00) el 4.00 %. También se ha establecido que la prevalencia de desnutrición (BMI lt 18.50%) es el 11,20%, donde incluso el 4,00% de las examinadas es considerablemente desnutrido (BMI lt 17.50%). Los resultados demostraron que incluso los 96.8% de las examinadas se desvían del perfil de la nutrición ideal. En relación con la desviación hacia el déficit, los 63% de las examinadas tiene alguna forma de déficit de la masa corporal (el promedio del déficit de la masa corporal es de 5.20 kg) y la mayoría de ellas tiene el déficit del componente de músculos los 78,4% (el déficit de 4.28 kg), así como también en relación con el déficit de componente graso los 43,2% (déficit de 2.01 kg). Con el perfil de alguno de los excedentes están los 33,6% de las examinadas (excedente de la masa corporal de 9.44 kg), donde incluso los 54,4% tiene el excedente del componente graso (excedente de 7.48 kg). Además de la probabilidad que la forma de nutrición es la razón para el aumento del componente graso y el hecho que incluso los 69,6% de las examinadas no están físicamente activas de manera adecuada, los 21.6 % solo de vez en cuando, es decir los 91.2% en total, indica la posible razón porque en ellas se ha encontrado el excedente del componente graso y el déficit del componente muscular.Cilj istraživanja je definisanje modelskih karakteristika telesne strukture studentkinja FASPERA Univerziteta u Beogradu, kao osoba čije je buduće radno opredeljenje rad sa invalidnim licima, što predstavlja veoma odgovoran, stresan i atipičan profesionalni radni profil. Uzorak je bio sastavljen od 125 ispitanica. Merenja su izvršena u Metodičko-istraživačkoj laboratoriji Fakulteta sporta i fizičkog vaspitanja Univerziteta u Beogradu, pomoću analizatora telesne strukture - InBody 720. Istraživanjem je obuhvaćeno dvadeset (20) varijabli. Rezultati su pokazali da je 77.60 % ispitanica u statusu normalne uhranjenosti, da je prevalencija ispitanica sa prekomernom težinom (BMI lt 25.00) 7.20%, odnosno gojaznosti (BMI lt 30.00) 4.00%. Takođe, utvrđeno je i da je prevalencija nedovoljne uhranjenosti (BMI > 18.50) 11.20%, gde je čak 4.00% ispitanica i značajno neuhranjeno (BMI >17.50). Rezultati su pokazali da čak 96.8 % ispitanica odstupa od profila idealne uhranjenosti. U odnosu na odstupanje ka deficitu, 63.2% ispitanica ima neki oblik deficita telesne mase (prosek deficita telesne mase od 5.20 kg) i to ih ima najviše sa deficitom mišićne komponente 78.4% (deficit od 4.28 kg), kao i u odnosu na deficit masne komponente 43.2% (deficit od 2.01 kg). Sa profilom nekog od suficita ima 33.6% ispitanica (suficit telesne mase od 9.44 kg), gde čak 54.4% ima suficit masne komponente (suficit od 7.48 kg). Pored verovatnoće da je način ishrane razlog za povećane masne komponente, i činjenica da čak 69.6% nije, a 21.6% samo povremeno, odnosno ukupno 91.2% ispitanica nije adekvatno fizički aktivno, ukazuje na mogući razlog zašto je kod njih utvrđen suficit masne, odnosno deficit mišićne komponente

Physicochemical characterisation of dihydro-alpha-lipoic acid interaction with human serum albumin by multi-spectroscopic and molecular modelling approaches

The binding of a popular food supplement and well-known antioxidant, dihydro-alpha-lipoic acid (DHLA) to human serum albumin (HSA) was characterised. The binding was monitored by several spectroscopic methods together with the molecular docking approach. HSA was able to bind DHLA with moderate affinity, 1.00±0.05×104 M-1. Spectroscopic data demonstrated that the preferential binding site for DHLA on HSA is IIA (Sudlow I). Both experimental and molecular docking analysis identified electrostatic (salt bridges) and hydrogen bonds as the key interactions involved in DHLA binding to HSA. Molecular docking confirmed that the Sudlow I site could accommodate DHLA and that the ligand is bound to the protein in a specific conformation. The molecular dynamic simulation showed that the formed complex is stable. Binding of DHLA does not affect the structure of the protein, but it thermally stabilises HSA. Bound DHLA had no effect on the susceptibility of HSA to trypsin digestion. Since DHLA is a commonly used food supplement, knowledge of its pharmacokinetics and pharmacodynamic properties in an organism is very important. This study further expands it by providing a detailed analysis of its interaction with HSA, the primary drug transporter in the circulation

Simple two-step semi-preparative isolation and purification of transferrin from human serum

Human transferrin (Tf) is a bilobal 76 kDa iron-binding glycoprotein present in human serum. Each lobe has the ability to bind one ferric ion (Fe3+) and a single synergistic bicarbonate anion. The main role of Tf is to transport Fe3+ ions through the circulation to cells, via interaction with transferrin receptor (TFR) on the cell surface. Previously described methods for Tf isolation and purification are either very time-consuming or provide Tf of lower final purity. Here we describe a fast and simple FPLC method for the isolation and purification of Tf from human serum. Serum samples were prepared by precipitation, while protein purification was performed on FPLC system, using an anionexchange column. Several different buffers at the same pH were tested. Tf purified by this method was analyzed by Western blot, followed by immunodetection, as well as with silver staining after SDS PAGE. Its functionality was tested with respect to iron-binding capacity (ferozzine method) and its ability to interact with TFR by immunofluorescent staining. The conformation of purified Tf was analyzed by recording intrinsic fluorescent emmision spectra originating from Trp residues. The method itself is highly reproducible (intra- and interday), easy to perform (only two steps) and fast (under an hour), yielding 98% to 99% pure Tf with all buffers. Purified Tf was shown to have retained its ironbinding capacity, as well as the ability to interact with TFR. Purified Tf also retained its native three-dimensional structure. Described method for the isolation and purification of Tf is fast, simple and highly reproducible, yielding a functional Tf of high purity in its native state while offering the flexibility of using different buffer systems. All of these features make this protocol a method of choice for the isolation and purification of Tf on a semi-preparative scale

Proteomic profiling of anti-transferrin pull-down in patients with underlying oxidative stress

Human serum tansferrin (hsTf) is a major circulatory protein crucial for the transport/metabolism of Fe3+ ions. By sequestering and delivering ferric ions to target tissues/cells hsTf maintains redox homeostasis. Oxidative stress (OS), one of the hallmarks of (patho)physiological conditions, alters protein structure and function. The main role of hsTf hinges on specific interaction with cellular Tf receptor (TfR) while other interactions contribute to diverse functions. The aim of this study was to profile interacting partners of hsTf in the samples of serum coming from patients diagnosed with a wide range of pathological conditions with underlying OS status. Anti-hsTf pull-down samples were analysed using mass spectrometry. Data went through analysis by appropriate bioinformatic tools. Results reveal differences in expression of hsTf interacting proteins in sample groups of patients suffering from kidney insufficiency subjected to dialysis treatment (peritoneal-PD or hemo-HD) also with patients with gestational diabetes compared to respective healthy sample groups. Colorectal cancer stage T3 versus T2 stage shows an inverse distribution of expression profiles in comparison to healthy samples. Most prominent differences are seen in hsTf interacting partners involved in the complement and coagulation cascades and cholesterol metabolic pathways, suggesting a multifaceted role of hsTf in these processes throughout the course of the disease

Application of different Lactobacillus strains in production pea seed protein hydrolysates with antioxidative activity

In recent years antioxidant properties of plant derived protein hydrolysates were established. Antioxidative activity of these hydrolysates largely depends on specificity of the proteinase used for hydrolysis, duration of proteinase treatment, as well as composition and molecular weight of hydrolysis derived peptides. In order to explore potential use of nine proteinase containing Lactobacillus strains in production of antioxidative peptides we tested their ability to hydrolyze lyophilized hydrosoluble protein fraction from dehulled and defatted dry pea seeds (Pisum sativum), cultivar NS Mraz. We also developed plant protein based liquid medium for cultivation of lactobacilli which was later used as starter for hydrolysis reaction in larger volume. We found that all nine strains could hydrolyze plant hydrosoluble fraction of pea seed proteins but with different efficiency. We also examined the effect of duration of hydrolysis on antioxidative activity in <10kDa fraction of obtained peptide extracts, using most proteolyticaly active strain, human vaginal isolate Lactobacillus rhamnosus T10. General conclusion was that antioxidative activity was positively correlated with increase of hydrolysis duration and amount of small peptides, which reached up to 34% of total proteins after 24h of hydrolysis treatment

Trend promene osnovnih antropometrijskih karakteristika studenata Kriminalističko-policijske akademije u toku studija

By applying transversal method of research on a sample of 267 male and 88 female students of the Academy of Criminalistic and Police Studies, Belgrade determination of basic anthropometrical characteristics in the function of the year of study was conducted. Basic anthropometrical characteristics were expressed in following variables: body weight - BW, expressed in kg; body height - BH, expressed in cm; body mass index - BMI, expressed in kg/m2. The obtained results showed statistically significant tendency of change in students of the ACPS during the course of studies, namely in increase of BM as basic measure for estimation of body voluminosity (1.2 kg per a study year, F ratio 4287, p = 0.039), as well as in BMI, as a measure for the assessment of the body status, that is, the state of nourishment (0.42 kg/m2 per a study year, F ratio 7.191, p = 0.008). As for female students, the obtained results showed statistically significant tendency of change, namely in decrease of BMI, as a measure for the assessment of the body status, that is, the state of nourishment (- 0.56 kg/m2 per a study year, F ratio 8.290, p = 0.005). At this point, based on the research results, we can not say for sure what could be the cause of the determined trend of increase of voluminosity and body nourishment in male students, and decrease of body nourishment in female students, which should be determined in future researches.Na uzorku od 267 ispitanika muškog pola i 88 ispitanika ženskog pola studenata Kriminalističko-policijske akademije (KPA) iz Beograda primenom transverzalne metode istraživanja izvršeno je dijagnostikovanje osnovnih antropometrijskih karakteristika u funkciji godine studija. Osnovne antropometrijske karakteristike ispitanika su bile predstavljene sledećim varijablama: telesna masa - TM, izražena u kg; telesna visina - TV, izražena u cm; i telesno-maseni indeks (BMI), izražen u kg/m2. Dobijeni rezultati su pokazali da je kod studenata KPA u toku studija došlo do statistički značajnog trenda promene, i to povećanja kod TM, kao osnovne mere za procenu voluminoznosti tela (1.20 kg po godini studija, F odnos 4.287, p = 0.039) kao i kod BMI, kao mere za procenu telesnog statusa, tj. stanja uhranjenosti (0.42 kg/m2 po godini studija, F odnos 7.191, p = 0.008). U odnosu nastudentkinje, dobijeni rezultati su pokazali da je u toku studija KPA došlo do statistički značajnog trenda promene, i to smanjenja kod BMI, kao mere za procenu telesnog statusa, tj. stanja uhranjenosti (- 0.56 kg/m2 po godini studija, F odnos 8.290, p = 0.005). U ovom trenutku se na osnovu rezultata istraživanja ne može sa sigurnošću tvrditi šta je posledica utvrđenog trenda povećanja voluminoznosti i telesne uhranjenosti kod studenata odnosno smanjenja telesne uhranjenosti kod studentkinja, što treba utvrditi u narednim istraživanjima

Glycosylation and Characterization of Human Transferrin in an End-Stage Kidney Disease

Chronic kidney disease (CKD) is a global health concern affecting approximately one billion individuals worldwide. End-stage kidney disease (ESKD), the most severe form of CKD, is often accompanied by anemia. Peritoneal dialysis (PD), a common treatment for ESKD, utilizes the peritoneum for solute transfer but is associated with complications including protein loss, including transferrin (Tf) a key protein involved in iron transport. This study investigated Tf characteristics in ESKD patients compared to healthy individuals using lectin microarray, spectroscopic techniques and immunocytochemical analysis to assess Tf interaction with transferrin receptors (TfRs). ESKD patients exhibited altered Tf glycosylation patterns, evidenced by significant changes in lectin reactivity compared to healthy controls. However, structural analyses revealed no significant differences in the Tf secondary or tertiary structures between the two groups. A functional analysis demonstrated comparable Tf-TfR interaction in both PD and healthy samples. Despite significant alterations in Tf glycosylation, structural integrity and Tf-TfR interaction remained preserved in PD patients. These findings suggest that while glycosylation changes may influence iron metabolism, they do not impair Tf function. The study highlights the importance of a glucose-free dialysis solutions in managing anemia exacerbation in PD patients with poorly controlled anemia, potentially offering a targeted therapeutic approach to improve patient outcomes

Isolation, characterisation and the role of complexes formed between transferrin and insulin-like growth factor-binding protein 3

Sistem faktora rasta, po strukturi i funkciji, sličnih insulinu („insulin-like growth factor“- IGF) obuhvata elemente sa primarnom ulogom u regulaciji rasta, razvoja, diferencijacije ćelija kako u fiziološkim, tako i u patofiziološkim uslovima. Pored dva funkcionalna peptidna hormona, IGF-I i IGF-II, ovaj sistem sadrži molekule koji regulišu ispoljavanje njihove fiziološke funkcije. Regulatorni elementi IGF sistema su: vezujući proteini (IGFBP-1 do -6), receptori za IGF molekule: IGF-1R, IGF-2R, insulinski receptor (IR), hibridni receptor IGF-R/IR, kao i specifične proteaze. Nakon proteolize IGFBP, funkcija IGF-I/II se ispoljava interakcijom sa IGF specifičnim receptorima na površini ćelija. U kompleksu sa IGFBP-3 se nalazi 70-75% IGF peptida. Pored primarne uloge nosača IGF molekula, IGFBP-3 učestvuje i u brojnim interakcijama nezavisno od IGF. IGFBP-3 poseduje više strukturnih domena što ga čini sposobnim za vezivanje za druge molekule i pokretanje signalnih puteva i mehanizama nezavisnih, a nekad i suprotnih, dejstvu IGF peptida poput stimulacije rasta ćelija ili pokretanja apoptotskih procesa. Jedan od partnera koji interaguje sa IGFBP-3 je transferin (Tf), glavni transporter jona gvožđa u krvi. Iako se za postojanje kompleksa IGFBP-3/Tf znalo i ranije, rezultati ove disertacije predstavljaju prvi prikaz strukturnih i funkcionalnih osobina ovih kompleksa, kao i sagledavanje njihove moguće uloge. U ovoj disertaciji je opisan optimizovan postupak za izolovanje kompleksa iz fizioloških uzoraka (seruma i tkiva), navedene su strukturne karakteristike kompleksa IGFBP-3/Tf, faktori koji utiču na njihovo formiranje, određena je koncentracija kompleksa u serumu zdravih odraslih osoba i osoba sa poremećajem u metabolizmu gvožđa, dokazana je neophodnost jona gvožđa za formiranje kompleksa i analizirana je subćelijska raspodela kompleksa u tkivu debelog creva (zdravog i tumorskog). U radu je ispitan uticaj: anemije, visoke koncentracije gvožđa i tumora debelog creva, na koncentraciju i strukturu kompleksa. Za tumor debelog creva je karakteristična sistemska anemija, praćena akumulacijom gvožđa u samom tumorskom tkivu. Na model sistemu tumora debelog creva ispitana je važnost puta internalizacije IGFBP-3 preko kompleksa sa Tf, odnosno posredstvom transferinskog receptora (TfR). Nađeno je da je koncentracija kompleksa IGFBP-3/Tf kod zdravih ljudi 241 ± 62 μg/L i da se u ovoj formi nalazi 5-7% ukupnog IGFBP-3. Kod različitih patofizioloških stanja sa poremećajem u metabolizmu gvožđa utvrđeno je da formiranje kompleksa primarno zavisi od koncentracije gvožđa, potom koncetracije IGFBP-3, ali i proteina uključenih u metabolizam gvožđa, poput feritina. Kod IGFBP-3/Tf kompleksa analiziranih u uzorcima seruma dobijenih od pacijenata sa tumorom debelog creva primećen je izmenjen način...The insulin-like growth factor (IGF) system plays an important role in the regulation of cell growth, development and differentiation, in both physiological and pathophysiological condition. Beside two peptide hormones IGF-I and IGF-II, this system includes the following regulatory elements: six IGF-binding proteins (IGFBP-1 to -6), specific receptors for IGF (IGF-1R and IGF-2R), insulin receptor (IR), hybrid receptor (IGF-R/IR) as well as IGFBP specific proteases. When IGFBPs are proteolytically cleaved, IGF-I/II are able to exert their physiological function by interacting with specific receptors on the cell surface. Approximately 70-75% of the total IGFs, are transported in circulation in the form of ternary complexes with IGFBP-3. Besides being the principal carrier of IGF molecules, IGFBP-3 was reported to exert a number of activities which are IGF-independent. Due to its structural sub-domain organisation, this molecule is able to interact with binding partners other than IGFs, and consequently activate mechanisms and signaling cascades with independent or even opposite effects to those of IGF-I/II, such as apoptosis. One of these binding partners is transferrin (Tf), the principal iron transporter in the blood. Although the existence of IGFBP-3/Tf complexes has already been reported, the results presented in this thesis offer the first complete structural and functional characterisation of the complexes, together with the analysis of their potential role. The fully optimised method for the isolation of intact IGFBP-3/Tf complexes from serum and tissue samples has been described, some structural characteristics have been defined, the effect of iron and other factors on the formation of complexes was studied, their concentration in sera from healthy persons and patients with impaired iron metabolism disorders was measured, as well as their subcellular distribution in colon tissue (healthy and tumor tissue). The formation and concentration of IGFBP-3/Tf complexes in persons with anemia, persons with very high iron concentration or patients with colorectal carcinoma (CRC) have also been investigated. The emphasis was made on samples from patients with CRC, a disease accompanied by systemic anemia and increased iron accumulation in cancer cells. CRC was a model system for the analysis of IGFBP-3 internalisation via Tf-TfR pathway. The concentration of IGFBP-3/Tf complexes in healthy adults was measured to be 241 ± 62 μg/L, which makes up to 5-7% of the total IGFBP-3. The results have shown that in impaired iron metabolism, the formation of complexes is highly dependent on the iron concentration, then IGFBP-3 concentration, as well as the concentration of other proteins involved in iron metabolism, such as ferritin. IGFBP-3/Tf complexes isolated form serum..