<p>Dissolved organic matter (DOM) in natural waters is a heterogeneous mixture of organic molecules with direct and indirect influences on aquatic organisms. Although the influences are usually attributed to DOM quantity (quantified as Dissolved Organic Carbon, DOC), the role of quality (optical and binding characteristics obtained by absorbance and fluorescence spectroscopy and potentiometric titration, respectively) is not well-understood. Through an initial critical review of the literature, followed by experimental geochemical, toxicological, and physiological investigations, a number of conclusions were reached that improve our knowledge in this area. Freshwater DOM sources exhibit source-dependent protection against metal toxicity, in particular copper (Cu). Generally, for this indirect effect, optically-dark terrestrially-derived or allochthonous DOMs offer better protection than microbially-derived or autochthonous sources. Linear regressions revealed that the better ameliorative effect is principally related to a higher aromatic composition (specific absorption coefficient, SAC₃₄₀) and a greater humic-like fluorescent component as quantified by parallel factor analysis (PARAFAC). In addition, the allochthonous DOMs were shown to have relatively higher magnitudes of titration index (TI), a new summary of chemical reactivity of DOM molecules obtained by titration analysis, and closely related to optical properties. TI was strongly correlated with SAC₃₄₀, suggesting greater binding capacities for DOM molecules with higher SAC₃₄₀. Consequently, a method for incorporation of SAC₃₄₀ as a DOM quality measure into the Biotic Ligand Model (BLM) was developed which improved Cu toxicity predictions in experimental tests with natural DOMs. For direct effects, two basic physiological functions (Na⁺ metabolism and nitrogen excretion) of the adult water flea (<em>Daphnia magna</em>, a cladoceran crustacean) and the zebrafish (<em>Danio </em><em>rerio</em>, a teleost fish) were investigated at circumneutral and acidic pH (≥ 7 and ~ 5, respectively). Three previously characterized, chemically-distinct natural DOM sources as well as a commercial humic acid (AHA) were examined. Regardless of the pH conditions, while Na⁺ regulation of <em>D</em>. <em>magna </em>remained unaffected by the presence of all DOMs, the passive diffusive efflux of Na⁺ in zebrafish was attenuated, indicating ameliorative action against unidirectional Na⁺ loss. In addition, only a distinct allochthonous-autochthonous DOM source stimulated the Na⁺ uptake rate of zebrafish at low pH. Ammonia excretion rates of <em>D</em>. <em>magna </em>were reduced at circumneutral pH by the most highly coloured, allochthonous DOM, and at low pH by all three natural DOMs. Both in <em>D. magna </em>and in <em>D. rerio</em>, urea excretion rates at both pH conditions were not influenced by the presence of the various DOMs, and the same was true for ammonia excretion in the zebrafish. A commercially prepared humic acid (Aldrich humic acid, AHA) exerted anomalous actions relative to those of natural DOMs, and does not appear to be representative of their normal effects. In contrast to the actions of DOM in detoxifying metals, these direct effects of DOMs on freshwater organisms appeared highly unpredictable with variable dependencies on the source, pH and species. This thesis has advanced our understanding of the relationships between DOM quality and its indirect and direct effects on aquatic organisms, and points to new directions for future work.</p> / Doctor of Philosophy (PhD)

[pt] s pontos quânticos de grafeno têm potencial para aplicações biológicas devido às suas propriedades ópticas e de tamanho nanométrico. Este estudo investigou por 28 dias (672 h) as interações de GQDs de diferentes precursores (ácido cítrico + ureia e ácido cítrico + tioacetamida) com biomoléculas modelo (albumina sérica humana - HSA) e DNA do timo de bezerro (ctDNA). Os GQDs-ureia mostraram estabilidade no diâmetro hidrodinâmico (12 nm) e carga superficial (-7 mV). Já os GQDs-tioacetamida apresentaram agregação progressiva de 5,0 nm iniciais para 22,7 nm após 28 dias, sem sedimentação devido à compensação de cargas preservando a dispersão coloidal. Ensaios revelaram supressão da fluorescência da HSA com aumentos na concentração dos GQDs. A constante de interação (Ki) GQDs-ureia oscilou inicialmente, estabilizando após 48 h. Para GQDs-tioacetamida houve menor flutuação de Ki ao longo de 672 h, indicando rearranjos conformacionais das biomoléculas com os GQDs antes do equilíbrio. A interação com o DNA, acompanhada por titulação absorciométrica no UV-Vis mostrou biointeração fraca de natureza hidrofóbica/eletrostática para ambos GQDs, com constantes de ligação aparentes (∼105 L mol−1). Ensaio com brometo de etídio revelou alterações na estrutura do DNA sem intercalação dos GQDs.Testes estatísticos confirmam a reprodutibilidade das interações dos GQDs com proteínas (HSA) e DNA no período de 28 dias (95 por cento de confiança). A estabilidade dos parâmetros de quantificação ao longo do tempo sugere a viabilidade dos GQDs como sondas analíticas após longos períodos de bioconjugação. Assim, o estudo apresenta bases metrologicamente sólidas para aplicação segura de GQDs em tecnologias biomédicas, expandindo o entendimento da relação tempo-estrutura-atividade nesses nanossistemas. / [en] Graphene quantum dots have potential for biological applications due to their optical properties and nanometric size. This study investigated for 28 days (672 h) the interactions of GQDs from different precursors (citric acid + urea and citric acid + thioacetamide) with model biomolecules (human serum albumin - HSA) and Calf thymus DNA (ctDNA). The GQDs-urea showed stability in hydrodynamic diameter (12 nm) and surface charge (- 7 mV). In contrast, GQDs-thioacetamide showed progressive aggregation from 5.0 nm initially to 22.7 nm after 28 days, without sedimentation due to charge compensation preserving colloidal dispersion. Tests revealed quenching of HSA fluorescence with increases in GQD concentration. The GQDs-urea interaction constant (Ki) fluctuated initially, stabilizing after 48 h. For GQDs-thioacetamide there was less fluctuation in Ki over 672 h, indicating conformational rearrangements of the biomolecules with the GQDs before equilibrium. Interaction with DNA monitored by UV-Vis photometric absorption titration showed weak bio-interaction of a hydrophobic/electrostatic nature for both GQDs, with apparent binding constants (∼105 L mol−1). Ethidium bromide assay revealed changes in DNA structure without intercalation of the GQDs. Statistical tests confirm the reproducibility of GQDs interactions with proteins (HSA) and DNA over 28 days (95 percent confidence). The stability of the quantification parameters over time suggests the viability of GQDs as analytical probes after long periods of bioconjugation. Thus, the study presents metrologically sound bases for the safe application of GQDs in biomedical technologies, expanding the understanding of the time-structure-activity relationship in these nanosystems.

<p>Увод: Хронична бубрежна инсуфицијенција (ХБИ) се дефинише као структурно или функционално оштећење бубрега у трајању од најмање три месеца и/или смањење јачине гломеруларне филтрације (ЈГФ) испод 60 мл/мин/1.73м2. У доступној литератури постоје различити подаци о присуству оралних манифестација код пацијената са ХБИ у квантитативном и квалитативном погледу. Стање бубрежне дисфункције праћено је променама у протоку и саставу пљувачке што је у последњој деценији допринело испитивању клиничких и лабораторијских показатеља бубрежне болести. Циљ: Циљ студије је био да се испита објективно стање оралне слузокоже, вредности рН, сијалометрије, концентрације урее, креатинина и секреторног имуноглобулина А пљувачке као и орални микробиолошки статус код пацијената са ХБИ. Материјал и методе: Узорак је био сачињен од 50 предијализних (31 мушкарца и 19 жена просечне старости 59,06&plusmn;14,30) и 25 хемодијализних пацијената (18 мушкараца и 7 жена просечне старости 54,92&plusmn;13,60) са постављеном дијагнозом ХБИ, заједно са 25 системски здравих испитаника компарибилних по полу и старости. Поред клиничког прегледа усне дупље спроведен је тест витроадхезије, одређивање интензитета саливације, рН вредности пљувачке и индекса крварења из интерденталне папиле (PBI). На узорцима сакупљене пљувачке, уз помоћ аутоматизованог система Beckman Coulter АУ480 спроведено је лабораторијско одређивање урее и креатинина методом спектрофотометрије и секреторног имуноглобулина А методом имунотурбидиметрије. За микробилошко испитивање коришћен је брис језика и техника оралног испирка. Резултати: Нису утврђене статистички значајне разлике између група према демографско-социјалним подацима. Предијализни испитаници су имали значајно веће присуство промена оралне слузокоже и оралних симптома. Просечне вредности клиренса креатинина су биле значајно мање код оболелих испитаника са бледилом оралне слузокоже, уремичним задахом, ксеростомијом и измењеним осећајем укуса у поређењу са испитаницима без наведених промена. Код предијализних су утврђене значајно смањене вредности сијалометрије према контролним групама и повећане pH вредности према групи здравих испитаника. Просечне концентрације урее и креатинина су се статистички значајно разликовале између испитиваних група. Умерена позитивна корелација је утврђена између серумских и пљувачних концентрација урее и креатинина код предијализних и креатинина код хемодијализних. Према просечним вредностима секреторног имуноглобулина А није било разлика између група. Код пацијената са ХБИ утврђено је значајно веће присуство гљива из рода Candida са предоминацијом non-albicans Candida врста. Закључак: Резултати истраживања указују на важност утврђивања клиничких карактеристика усне дупље код предијализних пацијената. Интензитет саливације, pH вредност и пљувачне концентрације уремијских токсина могу бити поуздани маркери бубрежног оштећења. Једноставан и неинвазиван приступ приликом узорковања пљувачке и поузданост лабораторијске анализе треба да допринесу широј примени пљувачке као компетитивним дијагностичким флуидом серуму. Техника оралног испирка је прецизна квантитативна метода за одређивање степена гљивичне колонизације.</p> / <p>Uvod: Hronična bubrežna insuficijencija (HBI) se definiše kao strukturno ili funkcionalno oštećenje bubrega u trajanju od najmanje tri meseca i/ili smanjenje jačine glomerularne filtracije (JGF) ispod 60 ml/min/1.73m2. U dostupnoj literaturi postoje različiti podaci o prisustvu oralnih manifestacija kod pacijenata sa HBI u kvantitativnom i kvalitativnom pogledu. Stanje bubrežne disfunkcije praćeno je promenama u protoku i sastavu pljuvačke što je u poslednjoj deceniji doprinelo ispitivanju kliničkih i laboratorijskih pokazatelja bubrežne bolesti. Cilj: Cilj studije je bio da se ispita objektivno stanje oralne sluzokože, vrednosti rN, sijalometrije, koncentracije uree, kreatinina i sekretornog imunoglobulina A pljuvačke kao i oralni mikrobiološki status kod pacijenata sa HBI. Materijal i metode: Uzorak je bio sačinjen od 50 predijaliznih (31 muškarca i 19 žena prosečne starosti 59,06&plusmn;14,30) i 25 hemodijaliznih pacijenata (18 muškaraca i 7 žena prosečne starosti 54,92&plusmn;13,60) sa postavljenom dijagnozom HBI, zajedno sa 25 sistemski zdravih ispitanika komparibilnih po polu i starosti. Pored kliničkog pregleda usne duplje sproveden je test vitroadhezije, određivanje intenziteta salivacije, rN vrednosti pljuvačke i indeksa krvarenja iz interdentalne papile (PBI). Na uzorcima sakupljene pljuvačke, uz pomoć automatizovanog sistema Beckman Coulter AU480 sprovedeno je laboratorijsko određivanje uree i kreatinina metodom spektrofotometrije i sekretornog imunoglobulina A metodom imunoturbidimetrije. Za mikrobiloško ispitivanje korišćen je bris jezika i tehnika oralnog ispirka. Rezultati: Nisu utvrđene statistički značajne razlike između grupa prema demografsko-socijalnim podacima. Predijalizni ispitanici su imali značajno veće prisustvo promena oralne sluzokože i oralnih simptoma. Prosečne vrednosti klirensa kreatinina su bile značajno manje kod obolelih ispitanika sa bledilom oralne sluzokože, uremičnim zadahom, kserostomijom i izmenjenim osećajem ukusa u poređenju sa ispitanicima bez navedenih promena. Kod predijaliznih su utvrđene značajno smanjene vrednosti sijalometrije prema kontrolnim grupama i povećane pH vrednosti prema grupi zdravih ispitanika. Prosečne koncentracije uree i kreatinina su se statistički značajno razlikovale između ispitivanih grupa. Umerena pozitivna korelacija je utvrđena između serumskih i pljuvačnih koncentracija uree i kreatinina kod predijaliznih i kreatinina kod hemodijaliznih. Prema prosečnim vrednostima sekretornog imunoglobulina A nije bilo razlika između grupa. Kod pacijenata sa HBI utvrđeno je značajno veće prisustvo gljiva iz roda Candida sa predominacijom non-albicans Candida vrsta. Zaključak: Rezultati istraživanja ukazuju na važnost utvrđivanja kliničkih karakteristika usne duplje kod predijaliznih pacijenata. Intenzitet salivacije, pH vrednost i pljuvačne koncentracije uremijskih toksina mogu biti pouzdani markeri bubrežnog oštećenja. Jednostavan i neinvazivan pristup prilikom uzorkovanja pljuvačke i pouzdanost laboratorijske analize treba da doprinesu široj primeni pljuvačke kao kompetitivnim dijagnostičkim fluidom serumu. Tehnika oralnog ispirka je precizna kvantitativna metoda za određivanje stepena gljivične kolonizacije.</p> / <p>Introduction: Chronic kidney disease (CKD) is defined as structural and functional kidney damage for a period of at least three months and/or reduction of glomerular filtration rate (GFR) under 60 ml/min/1.73m2. There are different data in the available literature in term of quantitative and qualitative presence of the oral manifestation in patients with CKD. Kidney dysfunction is accompanied by changes in the salivary flow and composition, which is in the last decade contributed by examination of clinical and laboratory markers of renal disease. Aim: The aim of the study was to examine condition of oral mucosa, pH value, salivary flow rate, concentration of salivary urea, creatinine, secretory immunoglobulin A and oral microbiological status in patients with CKD. Materials and Methods: The sample was consisted of 50 predialysis (31 males and 19 females, mean age 59,06&plusmn;14,30) and 25 hemodialysis patients (18 males and 7 females, mean age 54,92&plusmn;13,60) with a diagnosis of CKD, along with 25 age and gender matched healthy controls. In addition of clinical examination, tongue blade adhesion test, sialometry, salivary pH test and determination of papilla bleeding index (PBI) were conducted. Saliva samples were collected for laboratory analysis performed by automated system Beckman Coulter AU480. Levels of uremic toxins (urea and creatinine) and secretory immunoglobulin A were determinated by spectrophotometric and immunoturbidimetric method, respectively. Oral swab and oral rinse method were used for microbiological examination. Results: The sociodemographic characteristics of the patients with CKD and healthy controls showed no significant differences. Predialysis subjects had significantly higher presence of oral mucosa changes and oral symptoms. Mean values of creatinine clearence were significantly lower in patients with oral mucosa pallor, uremic fetor, xerostomia and disguesia, compared to patients without listed symptoms. Predialysis patients showed significantly decreased salivary flow rate compared to both control groups and significantly increased pH values compared to healthy controls. Mean concentrations of salivary urea and creatinine were statistically different between the groups. Moderate positive correlation was determined between serum and salivary levels of urea and creatinine in predialysis patients and creatinine in hemodialysis patients. Statistical analysis showed no differences between groups in mean concentration of secretory immunoglobulin A. The rate of oral fungal colonisation was significantly higher in CKD patients with predominance of non-albicans Candida species. Conclusion: The results of the present study indicate the importance of determining the clinical characteristics of oral cavity in predialysis patients. Saliva flow rate, pH value and salivary concentration of uremic toxins could be reliable markers of kidney disease. Simple and non-invasive approach due to saliva sampling and reliability of laboratory test should contribute to a wider application of saliva as a competitive diagnostic fluid. Oral rinse technique is an accurate quantitative method for determining the rate of fungal colonization.</p>

Diesel engines produce large amounts of nitrogen oxides (NOX) while running. Nitrogen oxides are highly toxic and also contribute towards the formation of tropospheric ozone. Increasingly stringent legislation regarding the amount of nitrogen oxides that are allowed to be emitted from diesel-powered vehicles has forced manufacturers of diesel-engines to develop after-treatment systems that reduce the amount of nitrogen oxides in the exhaust. One of the main components in such a system is selective catalytic reduction (SCR), where nitrogen oxides are reduced to diatomic nitrogen and water with the help of ammonia. A vital part of this process is the spraying of a urea-water-solution (UWS), which is needed in order to produce the reducing agent ammonia. UWS spraying introduces the risk of solid deposits (such as biuret, ammelide and ammeline) forming in the after-treatment system, should the flow conditions be unfavourable. Risk factors include high temperatures, but also low dynamics and high thickness of the resulting liquid film that forms as the UWS spray hits the surfaces of the after-treatment system. It is thus essential that manufacturers of SCR after-treatment systems have correct data on how much UWS that should be sprayed into the exhaust for any given flow condition. Experimental tests are thoroughly used to assess this but are very expensive and are thus limited to prototype testing during product development. When assessing a wider range of concepts and geometries early on in the product development stage, simulation tools such as computational fluid dynamics (CFD) are used instead. One of the most computationally heavy processes to simulate within a SCR after-treatment system is the UWS spray and its interaction with surfaces inside the after-treatment system, where correct prediction of the formation of solid deposits are of great importance. Most CFD models used for this purpose hold a relatively good level of accuracy and are utilized throughout the whole industry where SCR aftertreatment is applied. Despite this, these models are limited in the fact that they are only able to cover timescales in the scope of seconds to minutes while using a tolerable amount of computational power. However, the time spectrum for solid deposit formation is minutes to hours. Scania is one of Sweden’s biggest developers of SCR after-treatment, with the technology being incorporated directly into its silencers. AVL Fire is the main UWS spray simulation tool for engineers at Scania at the moment. One major drawback of using AVL Fire for UWS spray simulations is that it is deemed too time-consuming to set up new cases and too unstable during simulation, which makes it too costly in terms of expensive engineering hours. This project has investigated the potential of using STAR-CCM+ for UWS spray simulations at Scania instead. A standard method has been evaluated, as well as parameters that will prove useful in further investigations of a potential speedup method. The studied method in STAR-CCM+ is easy to setup and the simulation process is robust and stable. Various other perks come from using STAR-CCM+ as well, such as: a user-friendly interface, easy and powerful mesh-generation and great post-process capabilities. Several different parameters have been investigated for their impact on the studied method, such as mesh refinement of the spray injector area and the number of parcels injected every time-step through the spray injector (simply put the resolution of the spray). A possible speedup by freezing the momentum equations when allowed and lowering the amount of inner iterations has also been investigated. A handful of operating conditions have been studied for two different geometries. The attained simulation results display correlations with physical measurements, but further assessment for identifying the risk of solid deposit needs to be performed on the studied cases to assess the full accuracy of solid deposit prediction of the studied method. Recommendations for future work includes fully implementing and evaluating the speedup method available for spray simulations in STAR-CCM+ as well as directly comparing how the accuracy and performance of the method relates to that of the method used in AVL Fire for spray simulations. / Dieselmotorer producerar under körning stora mängder kväveoxider (NOx). Kväve-oxider är starkt giftiga föreningar som även bidrar till att öka mängden marknära ozon. Allt strängare lagstiftning gällande mängden kväveoxider som får släppas ut från fordon med dieselmotorer har lett till att tillverkare av dieselmotorer blivit tvingade att utveckla efterbehandlingssystem som renar avgasen från motorn. En av huvudkomponenterna i ett sådant system idag är selective catalytic reduction (SCR; på svenska selektiv katalytisk reduktion), där kväveoxider omvandlas till kvävgas och vatten med hjälp av ammoniak. För att producera ammoniak används en lösning av urea och vatten (t.ex. AdBlue®), som introduceras till efterbehandlingssystemet via spray. Denna process har dock en stor nackdel, då det under omvandlingsprocessen kan finnas risk för klumpbildning av ämnen som biuret, ammelid och ammelin ifall flödesförhållandena är ogynnsamma. Riskfaktorer för klumpbildning inkluderar höga temperaturer samt låg dynamik och hög tjocklek för den vätskefilm som bildas när sprayen med urea-lösning kommer i kontakt med ytor i efterbehandlingssystemet. Det är därför av stor vikt för tillverkare av efterbehandlingssystem som använder SCR att känna till hur mycket urealösning som kan sprayas in för varje givet flöde. Experimentella tester används till stor del för att utvärdera detta, men är väldigt dyra och kan endast göras för ett fåtal prototyper under en produkts utveckling. För att kunna utvärdera ett större antal koncept och geometrier tidigare i utvecklingsstadiet av en ny produkt används därför ofta datorkraft med simuleringsverktyg som CFD (Computational Fluid Dynamics). En av de mest beräkningstunga processerna att simulera i ett efterbehandlingssystem med SCR är sprayandet av urea-lösning och dess interaktion med ytor, där korrekta förutbestämmelser av huruvida det finns risk för klumpbildning eller inte är av stor betydelse. De flesta CFD modeller som används i detta syfte har förhållandevis god noggrannhet och används i stor utsträckning i den bransch där efterbehandling med SCR tillämpas. Däremot är dessa modeller begränsade i att de endast kan åstadkomma simuleringar (med en acceptabel mängd datorkraft) som sträcker sig i tidsintervallet sekunder till minuter. Bildningen av klump är dock en process som kan ta upp till flera timmar. Scania är en av Sveriges största tillämpare av SCR, då tekniken används i de efterbehandlingssystem som finns inbyggda i tillverkarens ljuddämpare. Scania använder främst AVL Fire för simulering av spray med urea. AVL Fire anses dock vara för tidskrävande vid skapelsen av nya simuleringsfall och för instabilt under simulering. Detta projekt har därför undersökt möjligheten att använda STAR-CCM+ för simulering av spray med urea hos Scania. Den metod i STAR-CCM+ som utvärderats är enkel att använda då nya simuleringsfall ska skapas, samtidigt som den är robust och stabil under simulering. Relevanta parametrar för en potentiell uppsnabbningsmetod har också undersökts. STAR-CCM+ i sin helhet är användarvänligt, där verktyget för att skapa och generera mesh är enkelt att använda såväl som kraftfullt när mer avancerade operationer krävs. Möjligheterna för postprocessing är väldigt smidiga för transienta förlopp, vilket är ett stort plus för simuleringar med urea-spray, vars injektion och resulterande processer är väldigt transienta skeenden i sig. Flera olika parametrar har undersökts, för att granska hur stor påverkan de har på prestandan och noggrannheten hos den studerade metoden. Två av dessa är tätheten av beräkningsnoder i den region där spray-munstycket är placerat samt antalet paket med urea-vatten lösning som injiceras varje tidssteg via spray-munstycket. En möjlig uppsnabbning av metoden, som går ut på att frysa ekvationerna för bevarelse av rörelsemängd (eng - momentum equations) när det är tillåtet och samtidigt minska antalet inre iterationer för varje tidssteg, har också undersökts. Ett flertal olika flödesförhållanden har också undersökts för två olika geometrier. De erhållna resultaten tyder på korrelation med data från fysiska experiment. Dock bör ytterligare hydrodynamiska utvärderingar tillämpas för att ordentligt kunna redogöra för hur väl STAR-CCM+ kan användas för att förutse risken för klump- bildning i en spray-process med urea-vatten lösning. Framtida arbete borde fokusera på att utvärdera den uppsnabbningsmetod som finns för spray-simuleringar i STAR-CCM+, samt direkt jämföra hur väl metodens noggrannhet och prestanda står sig gentemot den metod som används i AVL Fire för spray-simuleringar.

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Tesis por compendio / [EN] Carbamoyl phosphate synthetase 1 (CPS1), a 1462-residue mitochondrial enzyme, catalyzes the entry of ammonia into the urea cycle, which converts ammonia, the neurotoxic waste product of protein catabolism, into barely toxic urea. The urea cycle inborn error and rare disease CPS1 deficiency (CPS1D) is inherited with mendelian autosomal recessive inheritance, being due to CPS1 gene mutations (>200 mutations reported), and causing life-threatening hyperammonemia. We have produced recombinantly human CPS1 (hCPS1) in a baculovirus/insect cell expression system, isolating the enzyme in active and highly purified form, in massive amounts. This has allowed enzyme crystallization for structural studies by X-ray diffraction (an off-shoot of the present studies). This hCPS1 production system allows site-directed mutagenesis and enzyme characterization as catalyst (activity, kinetics) and as protein (stability, aggregation state, domain composition). We have revealed previously unexplored traits of hCPS1 such as its domain composition, the ability of glycerol to replace the natural and essential CPS1 activator N-acetyl-L-glutamate (NAG), and the hCPS1 protection (chemical chaperoning) by NAG and by its pharmacological analog N-carbamyl-L-glutamate (NCG). We have exploited this system to explore the effects on the activity, kinetic parameters and stability/folding of the enzyme, and to test the disease-causing nature, of mutations identified in patients with CPS1 deficiency (CPS1D). These results, supplemented with those obtained with other non-clinical mutations, have provided novel information on the functions of three non-catalytic domains of CPS1. We have introduced three CPS1D-associated mutations and one trivial polymorphism in the glutaminase-like domain of CPS1, supporting a stabilizing and an activity-enhancing function of this non-catalytic domain. Two mutations introduced into the bicarbonate phosphorylation domain have shed light on bicarbonate binding and have directly confirmed the importance of this domain for NAG binding to the distant (in the sequence) C-terminal CPS1 domain. The introduction of 18 CPS1D-associated missense mutations mapping in a clinically highly eloquent central non-catalytic domain have proven the disease-causing nature of most of these mutations while showing that in most of the cases they trigger enzyme misfolding and/or destabilization. These results, by proving an important role of this domain in the structural integration of the multidomain CPS1 protein, have led us to call this domain the Integrating Domain. Finally, we have examined the effects of eight CPS1D-associated mutations, of one trivial polymorphism and of five non-clinical mutations, all of them mapping in the C-terminal domain of the enzyme where NAG binds, whereas we have re-analyzed prior results with another four clinical and five non-clinical mutations affecting this domain. We have largely confirmed the pathogenic nature of the clinical mutations, predominantly because of decreased activity, in many cases due to hampered NAG binding. A few mutations had substantial negative effects on CPS1 stability/folding. Our analysis reveals that NAG activation begins with a movement of the final part of the ß4-¿4 loop of the NAG site. Transmission of the activating signal to the phosphorylation domains involves helix ¿4 from this domain and is possibly transmitted by the mutually homologous loops 1313-1332 and 778-787 (figures are residue numbers) belonging, respectively, to the carbamate and bicarbonate phosphorylation domains. These two homologous loops are called from here on Signal Transmission Loops. / [ES] La carbamil fosfato sintetasa 1 (CPS1), una enzima mitocondrial, cataliza la entrada del amonio en el ciclo de la urea, que convierte esta neurotoxina derivada del catabolismo de las proteínas en urea, mucho menos tóxica. El déficit de CPS1 (CPS1D) es un error innato del ciclo de la urea, una enfermedad rara autosómica recesiva, que se debe a mutaciones en el gen CPS1 (>200 mutaciones descritas) y que cursa con hiperamonemia. Hemos producido CPS1 humana recombinante (hCPS1) en un sistema de expresión de células de insecto y baculovirus, y la hemos aislado en forma activa, muy pura y en cantidad elevada. Este sistema de producción de hCPS1 permite la realización de mutagénesis dirigida y la caracterización de la enzima como catalizador (actividad, cinética) y como proteína (estabilidad, estado de agregación y composición de dominios). Hemos revelado características de la hCPS1 antes no exploradas como es la composición de dominios, la capacidad que tiene el glicerol para reemplazar al activador natural y esencial de la CPS1, N-acetil-L-glutamato (NAG), y la protección de la hCPS1 por NAG y por su análogo farmacológico N-carbamil-L-glutamato (NCG) (chaperonas químicas). Hemos utilizado este sistema para explorar los efectos en actividad, parámetros cinéticos y estabilidad/plegamiento de la enzima, y para comprobar la naturaleza patogénica de mutaciones identificadas en pacientes con CPS1D. Estos resultados, junto con los obtenidos con otras mutaciones no clínicas, han aportado información novedosa sobre tres de los dominios no catalíticos de CPS1. Las observaciones realizadas tras introducir en el dominio de tipo glutaminasa de la enzima tres mutaciones asociadas a CPS1D y un polimorfismo trivial, apoyan la contribución de este dominio no catalítico a la estabilidad y a aumentar la actividad de la enzima. Dos mutaciones introducidas en el dominio de fosforilación de bicarbonato han arrojado luz sobre el modo de unión del bicarbonato (un sustrato). Los resultados de estas mutaciones también han confirmado la contribución de este dominio para la unión de NAG, cuyo sitio de unión se encuentra en el dominio C-terminal de CPS1, bastante alejado (en la secuencia) del dominio de fosforilación de bicarbonato. Además, hemos introducido 18 mutaciones de cambio de sentido asociadas a CPS1D, las cuales están localizadas en un dominio no catalítico, central y de elevada elocuencia clínica. Estos resultados han demostrado la naturaleza patogénica de estas mutaciones, ya que en la mayoría de los casos estas mutaciones producen un mal plegamiento o/y desestabilización de la enzima. Debido a que estos resultados han puesto de manifiesto el importante papel de este dominio en la integración estructural de la proteína multidominio CPS1, lo hemos llamado Dominio Integrador. Finalmente, hemos examinado los efectos de 8 mutaciones asociadas a CPS1D, de un polimorfismo trivial y de 5 mutaciones no clínicas, todas localizadas en el dominio C-terminal de la enzima, donde se une NAG. Además, hemos reanalizado resultados anteriores con otras 4 mutaciones clínicas y 5 no clínicas afectando a este dominio. Hemos confirmado el carácter patogénico de las mutaciones clínicas, las cuales predominantemente causan una disminución en la actividad enzimática, en muchos casos debida a que la unión de NAG se encuentra obstaculizada. Unas pocas mutaciones mostraron efectos negativos en la estabilidad/plegamiento de CPS1. Nuestros análisis revelan que la activación por el NAG empieza con un movimiento de la parte final del bucle ß4-¿4 del sitio de NAG. La transmisión de la señal activadora a los dominios de fosforilación implica a la hélice ¿4 de este dominio y posiblemente se transmite a través de los bucles homólogos 1313-1332 y 778-787 (numeración de residuos) pertenecientes, respectivamente, a los dominios de fosforilación de carbamato y bicarbonato. Por ello, hemos llamado a ambos bucles Bucles de / [CA] La carbamil fosfat sintetasa 1 (CPS1), un enzim mitocondrial, catalitza l'entrada d'amoni en el cicle de la urea, que convertix l'amoni, producte neurotòxic del catabolisme de les proteïnes, en urea, una molècula molt poc tòxica. El dèficit de CPS1 (CPS1D) és un error innat del cicle de la urea, una malaltia rara autosòmica recessiva, que es deu a mutacions en el gen CPS1 (>200 mutacions descrites) i que cursa amb hiperamonièmia. Hem produït CPS1 humana recombinant (hCPS1) en un sistema d'expressió de cèl·lules d'insecte i baculovirus, i l'hem aïllada en forma activa, molt pura i en gran quantitat. Això ha permés la cristal·lització de l'enzim per a estudis estructurals amb difracció de raios-X (treball no inclòs en esta tesi Aquest sistema de producció de hCPS1 permet la realització de mutagènesi dirigida i la caracterització de l'enzim com a catalitzador (activitat, cinètica) i com a proteïna (estabilitat, estat d'agregació i composició de dominis). Hem revelat característiques de la hCPS1 no explorades abans com és la composició de dominis, la capacitat que té el glicerol per a reemplaçar l'activador natural i essencial de CPS1, N-acetil-L-glutamat (NAG), i la protecció de la hCPS1 per NAG i pel seu anàleg farmacològic N-carbamil-L-glutamat (NCG) (xaperones químiques) . Hem utilitzat aquest sistema per a explorar els efectes en l'activitat, els paràmetres cinètics i l'estabilitat/plegament de l'enzim, i per a comprovar la naturalesa patogènica de mutacions identificades en pacients amb CPS1D. Aquestos resultats, junt amb els obtinguts amb altres mutacions no clíniques, han aportat informació nova sobre tres dels dominis no catalítics de la CPS1. Les observacions, després d'introduir tres mutacions associades a CPS1D i un polimorfisme trivial en el domini tipus glutaminasa de CPS1, recolzen la contribució d'aquest domini no catalític a l'estabilitat i a l'optimització de l'activitat enzimàtica. Dues mutacions introduïdes en el domini de fosforilació de bicarbonat han esclarit el mode d'unió de bicarbonat. Els resultats d'aquestes mutacions també han confirmat la contribució d'aquest domini per a la unió de NAG, el lloc d'unió de la qual es troba en el domini C-terminal de CPS1, prou allunyat (en la seqüència) del domini de fosforilació de bicarbonat. A més, hem introduït 18 mutacions de canvi de sentit associades a CPS1D, les quals estan localitzades en un domini no catalític, central i d'elevada eloqüència clínica. Aquestos resultats han demostrat la naturalesa patogènica d'aquestes mutacions, ja que, en la majoria dels casos produïxen un mal plegament o/i desestabilització de l'enzim. Pel fet que aquestos resultats han posat de manifest l'important paper d'aquest domini en la integració estructural de la proteïna multidomini CPS1, l'hem anomenat Domini Integrador. Finalment, hem examinat els efectes de huit mutacions associades a CPS1D, un polimorfisme trivial i cinc mutacions no clíniques, totes elles localitzades en el domini C-terminal de l'enzim, on s'unix NAG. A més, hem reanalitzat resultats anteriors amb altres quatre mutacions clíniques i cinc no clíniques que afecten aquest domini. Hem confirmat el caràcter patogènic de les mutacions clíniques, les quals predominantment causen una disminució en l'activitat enzimàtica, en molts casos pel fet que la unió de NAG es troba obstaculitzada. Unes poques mutacions van mostrar efectes negatius substancials en l'estabilitat/plegament de CPS1. Les nostres anàlisis revelen que l'activació de NAG comença amb un moviment de la part final del bucle ß4-¿4 del lloc de NAG. La transmissió del senyal activadora als dominis de fosforilació involucra l'hèlix ¿4 d'aquest domini i es transmet, possiblement, a través dels bucles homòlegs 1313-1332 i 778-787 (numeració dels residus), pertanyents, respectivament, als dominis de fosforilació de carbamato i bicarbonat. Per això, hem anomenat a ambd / Díez Fernández, C. (2015). USING RECOMBINANT HUMAN CARBAMOYL PHOSPHATE SYNTHETASE 1 (CPS1) FOR STUDYING THIS ENZYME'S FUNCTION, REGULATION, PATHOLOGY AND STRUCTURE [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/52855 / Compendio

For the first time, successful synthesis and characterisation of the missing (Z)-isomer of thallium(I) methanediazotate has been accomplished, utilising low-temperature NMR monitoring analysis. The title compound was synthesised from N-methyl-N-nitrosourea and thallium(I) propoxide, under sub-ambient temperature conditions, as a highly moisture sensitive entity. Quantum chemical calculations, performed at the CCSD(T) level, depict excellent conformity to experimental results. Indeed, compared to its (E) counterpart, the formation of the title compound is thermodynamically less favoured, but preferred by means of kinetic control owing to a hindered isomerisation.

Le squelette 1,3,5-triazépane-2,6-dione est un hétérocycle à sept chainons dérivé de dipeptides et accessible en quatre étapes en solution. Une voie de synthèse en parallèle sur support solide de cet hétérocycle a été élaborée. Cette synthèse, qui repose sur les principes de "catch and release" et de cyclo-clivage, a permis la création d’une chimiothèque de plus d’une centaine de composés. Pour augmenter la diversité du squelette 1,3,5-triazépane-2,6-dione, des modifications post-cyclisation peuvent avoir lieu telles que des réactions de N-mono-alkylation ou de N,N-di-alkylation de l’urée, des réactions d’acylation ou bien des réactions de thionation des fonctions carbonyles. De même, la synthèse des cycles analogues aux 1,3,5-triazépane-2,6-diones des tailles plus importantes a été examinée conduisant à l’obtention de plusieurs macrocycles.Les 1,3,5-triazépane-2,6-diones présentent un fort potentiel pour la recherche de molécules d’intérêt thérapeutique puisque le cycle est rigide, non-planaire et possède une bonne capacité de distribution des pharmacophores dans l’espace. Des molécules de cette famille présentent une activité inhibitrice modérée mais spécifique sur les phospholipases A2 secrétées humaines de type V et X. La recherche de nouveaux inhibiteurs de sPLA2 par une étude de relation structure/activité, par création d’une pince à calcium ou par simulation moléculaire a conduit à l’identification de nouveaux composés actifs. / The 1,3,5-triazepane-2,6-dione scaffold is a seven membered heterocycle derived from dipeptides and accessible in a four steps synthesis in solution. A parallel solid phase synthesis of this heterocycle was developed. This strategy, based on "catch and release" and cyclo-cleavage processes, had created a library containing more than one hundred compounds. To increase the diversity of 1,3,5-triazepane-2,6-dione moieties, some post-cyclisation modifications were performed, e.g. urea N-mono-alkylation or N,N-di-alkylation, acylation, and carbonyl thionation. Synthesis of larger cycles was also investigated and several macrocycles were obtained.The 1,3,5-triazepane-2,6-diones have a strong pharmacological interest, because their cycle is rigid, non-planar and can allow multiple presentation of pharmacophores in space. Some 1,3,5-triazepane-2,6-diones have shown a small but specific activity on the groups V and X of the human secreted phospholipases A2. Structure/activity relationships, clamp synthesis to bind calcium or virtual screening were the strategies used to identify new active compounds.

Espécies nitrogenadas vem sendo adicionadas ao meio ambiente de forma intensa desde o desenvolvimento do processo Haber-Bosch para transformação de N2 em NH3, alterando significativamente o ciclo biogeoquímico do nitrogênio no ambiente. Uma vez na atmosfera, o nitrogênio reativo é depositado de volta à superfície da Terra por processos de deposição úmida e seca. A importância de levar em conta as formas orgânicas de nitrogênio para estimar a deposição de nitrogênio atmosférico pela chuva (úmida) já é conhecida, no entanto, ainda há poucos trabalhos que avaliam essa fração devido às dificuldades e incertezas impostas pelos métodos analíticos disponíveis. Este trabalho traz o desenvolvimento de um novo método, simples e de baixo custo, para a determinação de nitrogênio orgânico (N-org) em água de chuva utilizando o processo foto-Fenton e um foto-reator construído de forma artesanal. Por meio da adição de solução de Fenton (50 µmol L-1 Fe2+ e 2 mmol L-1 H2O2) e 90 min de radiação UV (85 °C) foi possível obter em média 106 ± 8% de recuperação de nitrogênio para soluções contendo 50 µmol N L-1 das moléculas modelos: ureia, serina, glicina e histidina. No caso da arginina, 90 min de radiação foi suficiente para degradar soluções contendo 10 µmol N L-1. O reator comercial se mostrou mais eficiente na degradação dos compostos testados (30 min), no entanto, com um tempo de 75 min, o reator artesanal atingiu os mesmos resultados. Com o uso de 0,2 g L-1 TiO2 e 120 min de radiação UV, também foi possível obter resultados satisfatórios. Porém, esse método possuiu valores de branco elevados, havendo necessidade de filtrar as amostras irradiadas antes da análise, adicionando tempo e custo ao procedimento analítico. O método desenvolvido utilizando foto-Fenton foi aplicado para a determinação de N-org em amostras de água de chuva coletadas na cidade de Ribeirão Preto (SP) de 2013 a 2017. A concentração de N-org variou de 3,5 a 195 µmol N L-1 com concentração média ponderada pelo volume (MPV) de 17,7 ± 1,0 µmol N L-1 (n=236). Essa média foi maior que aquelas obtidas em água de chuva de várias partes do mundo, podendo ser atribuída a elevada queima de biomassa na região de estudo. As concentrações de aminoácidos livres dissolvidos (AA) representaram em média 15 ± 12% (n=144) em relação à fração orgânica de nitrogênio na chuva, enquanto as concentrações de ureia foram próximas ou inferiores ao limite de quantificação do método (0,5 µmol N L-1). Considerando toda série temporal iniciada no mesmo sítio amostral desde 2005, as concentrações MPV calculadas para os íons NH4+ foi de 22,2 ± 1,1 µmol L-1 (n=460), NO3- de 13,3 ± 0,6 µmol L-1 (n=466), sendo que a concentração de NO2- foi irrelevante. Foram obtidas concentrações significativamente mais elevadas (teste-t, P=0,05) de NH4+, NO3-, N-org e AA no período de safra da cana (seco) com relação à entressafra (chuvoso), para todos os anos avaliados. Apesar da colheita manual da cana ter sido drasticamente reduzida, o fato de manter a mesma tendência sazonal desde 2005, demonstra que a prática da queima de biomassa ainda é intensa na região. O uso de fogo para manejo na área rural ainda é comum, além de haver grandes áreas queimadas de forma acidental. A deposição úmida de nitrogênio (N-org + NH4+ + NO3-) para Ribeirão Preto foi de 10,5 kg (N) ha-1 ano-1, sendo que a fração orgânica representou 33% dessa deposição, demonstrando a importância de se determinar N-org para melhor estimar os fluxos atmosféricos de deposição. A massa estimada de nitrogênio depositada pela chuva é cerca de 16% do nitrogênio aplicado por meio de fertilizantes em culturas de cana. Somando as deposições de nitrogênio pelo material particulado, pela fase gasosa, e úmida, esta última representa 71% do fluxo atmosférico de nitrogênio. Nesse contexto, a fração orgânica corresponde a 24% da deposição total, sendo que este valor ainda pode estar sendo subestimado, pois as concentrações de N-org no material particulado não foram determinadas. A simplicidade e exatidão do método aqui proposto pode facilitar a aquisição de dados de N-org na chuva de outras partes do mundo, melhorando assim o conhecimento sobre o ciclo biogeoquímico global do nitrogênio. / Nitrogen species have been intensively added to the environment since the development of Haber-Bosch process for N2 transformation to NH3, significantly altering the biogeochemical cycle of nitrogen. Once in the atmosphere, reactive nitrogen is deposited back to the Earth\'s surface by wet and dry deposition processes. The importance of taking into account the organic forms of nitrogen to estimate atmospheric nitrogen deposition by rain is already known. However, few studies evaluated this fraction due to the difficulties and uncertainties imposed by the available analytical methods. This work presents the development of a new, simple and low-cost method for the determination of organic nitrogen (N-org) in rainwater using photo-Fenton process and a homemade photo-reactor. By adding Fenton solution (50 mol L-1 Fe2+ and 2 mmol L-1 H2O2) and keeping the reaction under UV radiation for 90 min (85 °C), it was possible to obtain an average of 106 ± 8% nitrogen recovery for solutions containing 50 mol N L-1 of the model molecules: urea, serine, glycine and histidine. In the case of arginine, 90 min of radiation was sufficient to degrade solutions containing 10 mol N L-1. A commercial reactor showed to be more efficient in degradation of the tested compounds (30 min). However, with a time of 75 min, the homemade reactor achieved the same results. With 0.2 g L-1 TiO2 and 120 min of UV radiation, it was also possible to obtain satisfactory results. Nevertheless, this method had high blank values, and filtration of irradiated samples before the analysis was necessary, which increased time and cost to the analytical procedure. The developed method using photo-Fenton was applied to determine N-org in rainwater samples collected in Ribeirão Preto city (SP) from 2013 to 2017. N-org concentrations ranged from 3.5 to 195 mol N L-1 with a volume-weighted mean concentration (VWM) of 17.7 ± 1.0 mol N L-1 (n = 236). This value was higher than those reported for rainwater from different parts of the world, and this fact can be attributed to the high biomass burning in the study region. Dissolved free amino acids (AA) mean concentrations represented 15 ± 12% (n = 144) of the organic nitrogen fraction in rain, while urea concentrations were close to or below the limit of quantification of the method (0.5 mol N L-1). Considering all temporal series, initiated in 2005 at the same sampling site, the VWM concentration calculated for NH4+ ions was 22.2 ± 1.1 mol L-1 (n = 460) and for NO3- was 13.3 ± 0.6 mol L-1 (n = 466), while NO2- mean concentration was irrelevant. Significantly higher concentrations (t-test, P = 0.05) of NH4+, NO3-, N-org and AA were obtained during the harvest period (dry season) in relation to the non-harvest one (rainy season), for all evaluated years. Although manual harvesting was drastically reduced, the fact that the same seasonal trend has been maintained since 2005 demonstrates that the practice of biomass burning is still intense in the region. In rural area, using fire for land management is still common, in addition to large areas burned by unintentional fires. Nitrogen deposition (N-org + NH4+ + NO3-) in Ribeirão Preto was 10.5 kg (N) ha-1 year-1, and the organic fraction represented 33% of this deposition, demonstrating the importance of determining N-org to better estimate the atmospheric deposition fluxes. The estimated mass of nitrogen deposited by rain represents approximately 16% of the nitrogen introduced by fertilizers in sugarcane crops. Summing up the nitrogen deposition by particulate matter, by gas phase, and by rain, the latter represents 71% of the atmospheric nitrogen flux. In this context, the organic fraction corresponds to 24% of the total deposition, and this value may still be underestimated, since N-org concentrations in particulate matter were not determined. The simplicity and accuracy of the method proposed here may facilitate acquisition of N-org data in rainwater from other parts of the world, thus improving the knowledge on the global biogeochemical cycle of nitrogen.

Lisozima mostrou-se capaz, quando dispersa em certos meios orgânico-aquosos, de gerar sistemas pseudoplásticos que evoluem espontaneamente para redes de caráter viscoelástico. Esse fenômeno foi investigado neste estudo, para lisozima dispersa em uma série de misturas binárias contendo derivados de uréia como componente orgânico. Devido aos notáveis efeitos observados para um de tais derivados, a saber, tetrametiluréia (TMU), especial atenção foi dedicada aos sistemas contendo esse composto. O enfoque experimental incluiu espectroscopia Raman, microcalorimetria, reologia e espalhamento de raios X a baixo ângulo (SAXS). O trabalho envolveu o estudo dos sistemas orgânico-aquosos isolados e na presença de proteína. No primeiro caso, foi feita uma investigação espectroscópica e microcalorimétrica dos sistemas quanto a aspectos relativos à segregação de microdomínios na fase liquida e sua conseqüente relação com a deflagração da transição viscoelástica na proteína. Foram observadas descontinuidades nos valores de entalpias de excesso de mistura para o sistema TMU/água em torno de wTMU = 0,6, assim como padrões peculiares de comportamento espectral em torno dessa concentração da mistura binária. No segundo caso, os sistemas complexos de proteínas gerados foram investigados sob o ponto de vista morfológico e dinâmico, através das técnicas reológicas e de SAXS. Energias de ativação de fluxo determinadas na região sub-crítica, de comportamento Newtoniano, mostraram uma dependência exponencial direta com wTMU, indicando que mudanças estruturais nos fluidos complexos já ocorrem em regiões de composição do solvente bem abaixo da região de transição em wTMU = 0,6. Na região viscoelástica, 0,6<wTMU<0,9, ensaios de relaxação indicaram a presença de duas populações distintas. A tangente de perda (tg &#948; = G\"/G\') apresentou valores menores que a unidade para todos os casos, indicando o caráter \"solid-like\" das redes nas condições de ensaio. Apesar de seu caráter sólido, as redes mostram-se bastante flexíveis, suportando grandes deformações antes da ruptura, como inferido a partir da larga região viscoelástica linear. A variação nos módulos elástico (G\') e de perda (G\") com a composição do solvente indica a dependência do caráter viscoelástico com a fração de massa de TMU na mistura binária. Na região de baixo conteúdo de água (wTMU = 0,9), um aumento em G\" após a região viscoelástica linear é observado, indicando aumento da estruturação antes da ruptura da rede. As curvas de SAXS foram modeladas em seus fatores de forma e de interferência com uma equação unificada para objetos aleatoriamente distribuídos em um continuum. Os resultados permitiram a construção de um modelo, compatível com as demais evidências experimentais, segundo o qual as moléculas de lisozima encontram-se em duas conformações distintas nas matrizes: uma de conformação estendida e caráter fractal, predominante em wTMU > 0,6, com dimensão máxima de ca. de 160 &#197;, responsável pela interdigitação com espécies fractais vizinhas e uma espécie compacta, minoritária em wTMU > 0,6 (porém predominante em wTMU <0,6), com raio de giro de ca. 48 &#197;, presente nos microdomínios intersticiais aquosos da matriz. Verificou-se ainda a viabilidade de incorporação homogênea de uma metaloproteína, o citocromo-c, às matrizes de lisozima, sem perturbação significativa de sua morfologia, o que constitue um evento de potencial interesse biotecnológico. Os resultados deste trabalho trazem novos suportes experimentais à hipótese de correlação entre inversão na microconfiguração do meio solvente binário e deflagração do processo de transição sol-gel da proteína. / Lysozyme was found to be able, when dispersed in certain organic/aqueous media, to generate pseudoplastic systems that spontaneously evolve to three-dimensional networks with viscoelastic character. This phenomenon was investigated in this study, for lysozyme dispersed in a series of binary mixtures containing urea derivatives as the organic component. Due to the remarkable effects obtained in one of such derivatives, namely, tetramethylurea (TMU), special attention was given to systems containing that compound. The experimental approach included Raman spectroscopy, microcalorimetry, rheology and small angle X ray scattering (SAXS). The work involved the study of the organic/aqueous systems on their own and in the presence of protein. The former consisted of binary liquid mixtures that were spectroscopically and microcalorimetrically investigated as to aspects concerning microdomain segregation in the liquid phase and its consequent relationship with the threshold of the protein viscoelastic transition. Discontinuities in the excess enthalpy of mixture were observed for TMU/water system around wTMU=0.6, as well as peculiar spectral patterns around that same binary mixture concentration. The latter comprised complex protein systems that were investigated both under a morphological and dynamical point of view. Flow activation energies determined in the sub-critical (Newtonian) region showed an exponential increase with wTMU, indicating that structural changes in the comp]ex fluids are under way at solvent concentration regions well below that of the transition, at wTMU = 0.6. In the viscoelastic region, 0.6<wTMU<0.9, relaxation studies indicated the presence of two distinct populations. The loss tangent (tan &#948; = G\"/G\') presented values lower than the unity for all cases, indicating the solid-like character of the matrices, in the assay conditions. Despite their solid character the networks are quite flexible, standing large strains before rupture, as inferred from the large linear viscoelastic region. The variation in elastic (G\') and loss (G\") moduli with solvent composition indicates a dependence of the viscoelastic character on TMU mass fraction in the binary mixture. In the region of low water content (wTMU = 0.9), an increase in G\" after the LVR is observed, indicating increase in network structuring before rupture. SAXS curves were modeled with a unified equation for randomly distributed objects in a continuum. Results allowed the proposal of a model, which is compatible with the experimental evidence obtained through the other techniques in this work, according to which lysozyme molecules occur in two distinct conformations: one expanded and with fractal character, prevailing at wTMU > 0.6, with maximum dimension ca. 160 &#197; and interdigitated with neighbouring fractal species; and a compact conformation, of minor prevalence at wTMU>0.6 (but prevailing at wTMU < 0.6), with radius of gyration ca. 48 &#197;, present in the matrix microdomain interstices. It has also been verified the feasibility of homogeneous incorporation of cytochrome-c into the lysozyme matrices, an event of potential biotechnological interest. Results from this work bring further experimental support to our hypothesis on the correlation between microconfigurational inversion in the binary solvent medium and the sol-gel transition in the protein.