Questa tesi descrive lo stato delle conoscenze degli effetti tossici da micotossine su animali d allevamento, ed alcuni esperimenti condotti per valutare gli effetti indotti da esposizione cronica da micotossine sui ruminanti. Uno studio è stato condotto su 15 aziende specializzate in sistemi di produzione intensiva di carni bovine, situate nel Nord Italia (province di Verona e Mantova), con l'obiettivo di individuare i rischi d esposizione a contaminazione da micotossine. Alcuni metodi di laboratorio sono stati sviluppati: un metodo per la determinazione di ocratossina A (OTA) accumulata in tessuti e organi; Un metodo per valutare gli effetti delle fumonisine sulla biosintesi delle basi sfingoidi sfingosina (So) e sfinganina (Sa); Un metodo per rilevare l addotto AFB1-albumina. La razione totale mescolata (TMR) è risultata positiva alla AF e FB. Tra i singoli alimenti, il mais e la semola glutinata di mais sono stati i principali responsabili della contaminazione del TMR. Il livello di contaminazione è positivamente correlato al contenuto di umidità di mais. Il metodo per la determinazione dell OTA nei tessuti ed organi ha mostrato un buon recupero medio. L'analisi del rapporto Sa/So nel sangue non ha mostrato alcun effetto negativo delle fumonisine sulla biosintesi lipidica. L'addotto AFB1-albumina è risultato positivo per il 18% dei campioni totali di sangue. / The thesis describes the state of knowledge about toxic effects of mycotoxins on farm animals, and some experiments conducted to assess effects induced by chronic exposure to mycotoxins on ruminants. A field study for was carried out on 15 farms specialised for intensive beef production system, located in Northern Italy (provinces of Verona and Mantova), with the aim to identify risks of exposure to mycotoxins contamination. Some laboratory methods were performed: a method for the detection of ochratoxin A (OTA) concentration in tissues and organs; a method for evaluating the effects of fumonisin on biosynthesis of the two sphingoid bases sfingosine (So) and sphinganine (Sa); a method to detect the AFB1-albumin adduct. Total mixed rations (TMR) resulted positive for AF and FB contamination. Among single feedstuffs, corn and corn gluten feed were the main responsible for TMR contamination. Level of contamination was positively related to corn moisture content. The method for the determination of OTA in tissue and organ showed a good mean recovery. The analysis of ration Sa/So in blood did not show any negative effect by fumonisin on the lipidic biosynthesis. The AFB1-albumin adduct was positive on 18% of total blood samples.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 96-101).

Thesis (M.S.)--Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.

A novel approach of using near infrared region (NIR) dyes is applied to elucidate the binding interaction between human serum albumin (HSA) and mangiferin (MGF). HSA is a blood carrier protein used for drug delivery, while mangiferin is a natural polyphenol found in mangoes that possesses numerous beneficial health properties. The NIR dyes are used as a probe to investigate MGF binding interaction with HSA via monitoring fluorescence of Trp-214 residue. Molecular modeling is used for docking and semi-empirical analysis. The investigation of the binding interaction between Trp-214 and MGF is significant, for it may offer broader pharmacological insight and applications for the polyphenol. Mangiferin in proposed to bind with a 2:1 stoichiometric ratio with HSA to the Trp-214 residue in subdomain IIA and another possible binding site to be determined in future studies. Spectral changes suggest a stabilized protein conformation upon mangiferin binding with the addition of NIR dye E-06 and MHI-06.

Σκοπός της συγκεκριμένης ερευνητικής εργασίας ήταν η αποσαφήνιση του ρόλου της αλβουμίνης στην πρόκληση νεφρικής ίνωσης, διαμέσου του συστήματος ΕΤ-1/ΝΟ. Για το λόγο αυτό πραγματοποιήθηκαν in vitro πειράματα, στα οποία χρησιμοποιήθηκαν ΗΚ-2 κύτταρα και διερευνήθηκε η επίδραση της αλβουμίνης στην αλλαγή της μορφολογίας, στην προσκόλληση, στη μετανάστευση, στην παραγωγή ΝΟ και cGMP, στην έκφραση των et-1, etr-a, etr-b, enos και inos και τη συσσώρευση των αντίστοιχων πρωτεϊνών, καθώς επίσης και των πρωτεϊνών τουμπουλίνης και ακτίνης και τέλος στην ενζυμική δραστικότητα της μεταλλοπρωτεϊνάσης MMP-2. Προέκυψε ότι η αλβουμίνη τροποποιεί την έκφραση των γονιδίων et-1 και των υποδοχέων της etr-a και etr-b, των συνθετασών του ΝΟ, enos και inos, τη συσσώρευση των αντίστοιχων πρωτεϊνών, του cGMP, καθώς και του παραγόμενου NO. Επίσης, η αλβουμίνη δεν επηρεάζει τη συσσώρευση της ακτίνης, ενώ προκαλεί ελάττωση της συσσώρευσης της τουμπουλίνης και αναδιοργάνωση του κυτταροσκελετού. Τέλος, επάγει την ενζυμική δραστικότητα της MMP-2 και τον πολλαπλασιασμό, ενώ εμποδίζει τη μετανάστευση και την προσκόλληση των HK-2. / The aim of this research was the elucidation of the role of albumin in the induction of renal fibrosis, through the activation of the ET-1/NO system. We performed in vitro experiments using HK-2 cells (human proximal tubular epithelial cells) and we studied the morphological alterations, cell adhesion, migration, production of NO and cGMP, expression of et-1, etr-a, etr-b, enos and inos and the accumulation of the subsequent proteins, as well as tubulin and actin. Finally we studied the enzymic activity of metalloproteinase MMP-2. Albumin was found to alter the expression of et-1, etr-a, etr-b and enos and the accumulation of the subsequent proteins. Moreover, it affects the amount of the synthesized and secreted cGMP and NO, reduces the accumulation of tubulin, while having no effect on the accumulation of actin and participates in the reorganization of the cytoskeleton. Furthermore, albumin alters the enzymic activity of MMP-2, induces cell proliferation and inhibits the adhesion and migration of the HK-2 cells.

With the studies performed in this theses the local drug delivery technique FibMat developed by the biotech company AddBIO, was shown to be applicable to other plasma proteins and drugs than the fibrinogen-bisphosphonate combination that is today being commercialized. Hence the potential for a broader field of application was demonstrated. The application targeted today is as a surface modification giving improved strength to bone around screws used in bone implants. The effect of changing protein and manufacturing conditions was studied with null ellipsometry. It was demonstrated that with changes in incubation temperature, pH and salinity the fibrinogen could be successfully exchanged for the plasma proteins human serum albumin and immunoglobulin G. With liquid scintillation counting it was shown that the developed protein multilayers were able to absorb and release the bone strengthening drug alendronic acid in levels comparable to that of the fibrinogen based ditto. Disk susceptibility tests with the bacteria S. Aureus showed a potential for antibacterial functionalization with gentamicin. The release was, in the case of the fibrinogen multilayer, detectable up to 48 hours. Similar test revealed an inability of silver nanoparticle incorporated protein multilayers to achieve inhibitory levels.

Background and Purpose: Physical activity is significantly lower in the end-stage renal disease (ESRD) population compared with age-matched sedentary controls. Self-efficacy is a significant contributing factor to exercise behaviour in the healthy and various chronic disease populations; however, little information is available on self-efficacy and exercise in the ESRD population. The purpose of this study was to examine the relationship between self-efficacy and physical activity levels in individuals with ESRD and to determine factors which may contribute to this relationship. Participants: Patients (127 hemodialysis and 5 peritoneal dialysis) were recruited from the Kingston General Hospital renal units. Methods: Self-efficacy was measured using the Exercise Self-Efficacy Scale (ESES) and the Chronic Disease Self-Efficacy Scale (CDSES). Physical activity was measured using the Human Activity Profile (HAP). Responses on the HAP resulted in 2 scores: the maximum activity score (MAS) and the adjusted activity score (AAS). Contributing factors, such as age, co-morbidities, length on dialysis (vintage), medications, dialysis efficacy (Kt/V), albumin, prealbumin, total protein, protein catabolic rate (PCR) and hemoglobin (HgB) were collected from patient records. Results: The average MAS and AAS scores were 62.5±15.6 and 49.1± 21.0 (SD), respectively, which are substantively lower than those in the healthy population. Serum albumin was the sole contributing factor to physical activity such that AAS in those with lower levels of albumin (24-30g/L, 31-34 g/L) was less than those with higher levels of albumin (p<0.05). Both MAS and AAS were significantly lower in those with diabetes (n=66) compared to those without (n=66), as was the difference between MAS and AAS. Age and self-efficacy explained 51% of the variance in MAS. Age, self-efficacy and serum albumin level explained 59% of variance in AAS. Discussion and Conclusion: Individuals on dialysis have lower functional capability within their maximal energy ability at any age and this difference is more pronounced among those with diabetes. Physical activity was substantially influenced by an individual’s self-efficacy, age and serum albumin level. These findings indicate that self-efficacy must be taken into account and used to promote greater participation in physical activity in the ESRD population. / Thesis (Master, Rehabilitation Science) -- Queen's University, 2010-04-27 09:47:12.764

Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering. / <p>QC 20140207</p>

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Thesis (M.S.) -- Mississippi State University. Department of Chemistry. / Title from title screen. Includes bibliographical references.