<p>Bilirubin is a degradation product of haem, which is constantly formed in all</p><p>mammals. Increased levels of bilirubin in humans lead to jaundice, a condition</p><p>that is very common during the first days after birth. This neonatal</p><p>jaundice can routinely be treated by phototherapy without any serious side</p><p>effects. During this treatment, bilirubin undergoes a photoreaction to isomers</p><p>that can be excreted. The most efficient photoreaction is the isomerisation</p><p>around a double bond (Z-E-isomerisation), which results in more soluble</p><p>photoproducts.</p><p>The work presented in this thesis shows results of a femtosecond optical</p><p>spectroscopy study, combined with quantum-mechanical investigations, of</p><p>the mechanism of isomerisation of bilirubin. The spectroscopic research was</p><p>conducted with bilirubin in organic solvents, and in buffer complexed by</p><p>human serum albumin. This albumin complex is present in the blood, and</p><p>has thus medical importance. Quantum-chemical calculations (CASSCF) on</p><p>a bilirubin model were used to explain experimental results.</p><p>The fluorescence decay observed with femtosecond spectroscopy shows an</p><p>ultrafast component (~120 fs), which is explained by exciton localisation,</p><p>followed by processes with a lifetime of about 1-3 ps. These are interpreted</p><p>as the formation of a twisted intermediate, which decays with a lifetime of</p><p>10-15 ps back to the ground state, as observed by absorption spectroscopy.</p><p>CASSCF calculations, in combination with the experimental results, suggest</p><p>the ca. 1-3 ps components to be relaxation to the twisted S1 minimum, followed</p><p>by the crossing of a barrier, from where further relaxation takes place</p><p>through a conical intersection back to the ground state.</p><p>Time-dependent DFT calculations were utilised to analyse the absorption</p><p>spectrum of bilirubin. Good agreement with the measured spectrum was</p><p>achieved, and low-lying states were observed, that need further investigation.</p><p>The theoretically obtained CD spectrum provides direct evidence that</p><p>bilirubin preferentially binds to human serum albumin in the enantiomeric</p><p>P-form at neutral pH.</p> / <p>Bilirubin är en nedbrytningsprodukt av hem som ständigt bildas hos alla</p><p>däggdjur. En förhöjd bilirubinkoncentration i den mänskliga kroppen kan</p><p>leda till gulsot, något som är mycket vanligt under de första dagarna efter</p><p>födelsen (neonatal gulsot). Fototerapi används rutinmässigt som säker behandlingsmetod,</p><p>under vilken bilirubin genomgår en fotoreaktion till en</p><p>isomer som kan utsöndras. Den mest effektiva fotoreaktionen är en Z-Eisomerisation,</p><p>vilken leder till lösligare fotoprodukter.</p><p>Arbetet som presenteras i denna avhandling visar resultaten av en kombinerad</p><p>femtosekund optisk-spektroskopisk och kvantmekanisk undersökning</p><p>av mekanismen bakom bilirubins isomerisation. Den spektroskopiska</p><p>studien genomfördes med bilirubin, löst i organiska lösningsmedel och i</p><p>buffert i komplex med humant serumalbumin. Detta albuminkomplex finns i</p><p>blodet, och är därför av medicinskt intresse. Kvantmekanistiska CASSCFberäkningar</p><p>på en bilirubinmodell användes för att förklara de experimentella</p><p>resultaten.</p><p>Det uppmätta fluorescence sönderfallet visar ultrasnabba komponenter</p><p>(~120 fs). Dessa tolkas som excitonlokalisering, som följs av bildandet av</p><p>ett vridet intermediat med en hastighetskonstant på ca. 1 ps-1(beroende på</p><p>lösningsmedlet). Absorptionsmätningar visar att detta intermediat sönderfaller</p><p>tillbaka till grundtillståndet med en livstid på 10-15 ps.</p><p>CASSCF beräkningar, i kombination med de experimentella resultaten, tyder</p><p>på att sönderfallet med livslängden på ca. 1 ps är en relaxation till det</p><p>vridna S1-tillståndet. Reaktionsvägen därifrån antas passera en barriär till en</p><p>konisk genomskärning, som möjliggör snabb relaxation till grundtillståndet.</p><p>Tidsberoende DFT-beräkningar användes för att analysera bilirubins absorptionsspektrum,</p><p>vilket gav bra överensstämmelse med uppmätta data. Dessutom</p><p>hittades ett tillstånd med låg excitationsenergi, som kräver ytterligare</p><p>studier. Med hjälp av det beräknade CD-spectret kunde det visas att bilirubin</p><p>binder till albumin i P-formen vid neutralt pH.</p>

Electron transfer phenomena in proteins represent one of the most common types of biochemical reactions. They play a central role in energy conversion pathways in living cells, and are crucial components in respiration and photosynthesis. These complex biochemical reaction cascades consist of a series of proteins and protein complexes that couple a charge transfer to different forms of chemical energy. The efficiency and sophisticated optimisation of signal transfer in these natural redox chains has inspired engineering of artificial architectures mimicking essential properties of their natural analogues. Implementation of direct electron transfer (DET) in protein assemblies was a breakthrough in bioelectronics, providing a simple and efficient way for coupling biological recognition events to a signal transducer. DET avoids the use of redox mediators, reducing potential interferences and side reactions, as well as being more compatible with in vivo conditions. However, only a few haem proteins, including the redox protein cytochrome c (cyt.c), and blue copper enzymes show efficient DET on different kinds of electrodes. Previous investigations with cyt.c have mainly focused on heterogeneous electron transfer of monolayers of this protein on gold. An important advance was the fabrication of cyt.c multilayers by electrostatic layer-by-layer self-assembly. The ease of fabrication, the stability, and the controllable permeability of polyelectrolyte multilayers have made them particularly attractive for electroanalytical applications. With cyt.c and sulfonated polyaniline it was for the first time possible that fully electro-active multilayers of the redox protein could be prepared. This approach was extended to design an analytical signal chain based on multilayers of cyt.c and xanthine oxidase (XOD). The system does not need an external mediator but relies on an in situ generation of a mediating radical and thus allows a signal transfer from hypoxanthine via the substrate converting enzyme and cyt.c to the electrode. Another kind of a signal chain is based on assembling proteins in complexes on electrodes in such a way that a direct protein-protein electron transfer becomes feasible. This design does not need a redox mediator in analogy to natural protein communication. For this purpose, cyt.c and the enzyme bilirubin oxidase (BOD, EC 1.3.3.5) are co-immobilized in a self-assembled polyelectrolyte multilayer on gold electrodes. Although these two proteins are not natural reaction partners, the protein architecture facilitates an electron transfer from the electrode via multiple protein layers to molecular oxygen resulting in a significant catalytic reduction current. Finally, we describe a novel strategy for multi-protein layer-by-layer self-assembly combining cyt.c with an enzyme sulfite oxidase (SOx) without use of any additional polymer. Electrostatic interactions between these two proteins with rather separated pI values during the assembly process from a low ionic strength buffer were found sufficient for the layer-by-layer deposition of the both biomolecules. It is anticipated that the concepts described in this work will stimulate further progress in multilayer design of even more complex biomimetic signal cascades taking advantage of direct communication between proteins. / Elektronentransferphänomene in Proteinen stellen den häufigsten Typ biochemischer Reaktionen dar. Sie spielen eine zentrale Rolle bei der Energieumwandlung in der Zelle und sind entscheidende Komponenten in der Atmung und Photosynthese. Diese komplexen Kaskaden biochemischer Reaktionen setzen sich aus einer Reihe von Proteinen und Proteinkomplexen zusammen, die den Energietransfer an verschiedene Formen chemischer Energie koppeln. Die große Effektivität und Selektivität des Signaltransfers in diesen natürlichen Redoxketten war Vorbild für die Entwicklung künstlicher Architekturen, die die wesentlichen Eigenschaften ihrer natürlichen Analoga nachahmen. Die Implementierung des direkten Elektronentransfers (DET) von Proteinen mit Elektroden war ein Durchbruch im Bereich der Bioelektronik. Sie lieferte einen einfachen und effizienten Weg für das Koppeln biologischer Erkennungsereignisse an einen Signalumwandler. Durch den DET können Redoxmediatoren vermieden und damit potentielle Grenzflächen und Nebenreaktionen reduziert werden. Ebenso wird damit die Kompatibilität für in vivo Bedingungen erhöht. Jedoch zeigen nur einige Hämproteine wie das Redoxprotein Cytochrom c (Cyt c) und blaue Kupferproteine einen effizienten DET auf verschiedenen Elektrodentypen. Bisherige Untersuchungen mit Cyt c konzentrierten sich hauptsächlich auf den heterogenen Elektronentransfer von Monoschichten dieses Proteins auf Gold. Ein wichtiger Fortschritt war die Herstellung von Cyt c Multischichten durch die elektrostatische Layer-by-Layer-Technik. Die einfache Herstellung, die Stabilität sowie die kontrollierbaren Permeationseigenschaften von Polyelektrolyt-Multischichten machte sie besonders attraktiv für elektroanalytische Anwendungen. So gelang es auch zum ersten Mal vollständig elektroaktive Multischichten aus Cyt c und Polyanilinsulfonsäure zu präparieren. Dieser Ansatz wurde hier erweitert, um eine analytische Signalkette auf der Basis von Multischichten aus Cyt c und Xanthinoxidase zu entwerfen. Das System bedarf keinen externen Mediator, es hängt jedoch von der in situ Generierung eines vermittelnden Radikals ab und erlaubt daher einen Signaltransfer von Hypoxanthin über ein substratumwandelndes Enzym und Cyt c zur Elektrode. Eine andere Art von Signalketten basiert auf der Assemblierung von Proteinen in Komplexen auf Elektroden in solcher Art und Weise, daß ein direkter Protein-Protein-Elektronentransfer möglich wird. Dieser Ansatz benötigt keinen Redoxmediator in Analogie zu Beispielen aus dem biologischen Signaltransfer. Zu diesem Zweck werden Cyt c und das Enzym Bilirubinoxidase mit einem selbst-assemblierenden Polyelektrolyten auf einer Goldelektrode koimmobilisiert. Obwohl diese zwei Proteine keine natürlichen Reaktionspartner sind, unterstützt die Protein-Architektur einen Elektronentransfer von der Elektrode über mehrere Proteinschichten zu molekularem Sauerstoff und ergibt einen signifikanten katalytischen Reduktionsstrom. Schließlich wird eine neue Strategie beschrieben für eine Selbstassemblierung von Proteinen ohne zusätzlichen Polyelektrolyten - am Beispiel der Kombination von Cyt c mit Sulfitoxidase. Es stellte sich heraus, daß die elektrostatische Wechselwirkung zwischen diesen zwei Proteinen mit ziemlich weit voneinander entfernt liegenden pI-Werten während des Assemblierungsprozesses durch einen Puffer mit geringer Ionenstärke ausreicht um die beiden Biomoleküle nach dem Layer-by-Layer-Prinzip auf einer Elektrode abzuscheiden. Es wird erwartet, daß das entwickelte Konzept von Multiprotein-Assemblaten auf Elektroden weitere Fortschritte bei dem Entwurf von Multischichten und sogar noch komplexeren biomimetischen Signalkaskaden anregen wird und dabei der Vorteil der direkten Kommunikation zwischen Proteinen genutzt wird.

Bilirubin is a degradation product of haem, which is constantly formed in all mammals. Increased levels of bilirubin in humans lead to jaundice, a condition that is very common during the first days after birth. This neonatal jaundice can routinely be treated by phototherapy without any serious side effects. During this treatment, bilirubin undergoes a photoreaction to isomers that can be excreted. The most efficient photoreaction is the isomerisation around a double bond (Z-E-isomerisation), which results in more soluble photoproducts. The work presented in this thesis shows results of a femtosecond optical spectroscopy study, combined with quantum-mechanical investigations, of the mechanism of isomerisation of bilirubin. The spectroscopic research was conducted with bilirubin in organic solvents, and in buffer complexed by human serum albumin. This albumin complex is present in the blood, and has thus medical importance. Quantum-chemical calculations (CASSCF) on a bilirubin model were used to explain experimental results. The fluorescence decay observed with femtosecond spectroscopy shows an ultrafast component (~120 fs), which is explained by exciton localisation, followed by processes with a lifetime of about 1-3 ps. These are interpreted as the formation of a twisted intermediate, which decays with a lifetime of 10-15 ps back to the ground state, as observed by absorption spectroscopy. CASSCF calculations, in combination with the experimental results, suggest the ca. 1-3 ps components to be relaxation to the twisted S1 minimum, followed by the crossing of a barrier, from where further relaxation takes place through a conical intersection back to the ground state. Time-dependent DFT calculations were utilised to analyse the absorption spectrum of bilirubin. Good agreement with the measured spectrum was achieved, and low-lying states were observed, that need further investigation. The theoretically obtained CD spectrum provides direct evidence that bilirubin preferentially binds to human serum albumin in the enantiomeric P-form at neutral pH. / Bilirubin är en nedbrytningsprodukt av hem som ständigt bildas hos alla däggdjur. En förhöjd bilirubinkoncentration i den mänskliga kroppen kan leda till gulsot, något som är mycket vanligt under de första dagarna efter födelsen (neonatal gulsot). Fototerapi används rutinmässigt som säker behandlingsmetod, under vilken bilirubin genomgår en fotoreaktion till en isomer som kan utsöndras. Den mest effektiva fotoreaktionen är en Z-Eisomerisation, vilken leder till lösligare fotoprodukter. Arbetet som presenteras i denna avhandling visar resultaten av en kombinerad femtosekund optisk-spektroskopisk och kvantmekanisk undersökning av mekanismen bakom bilirubins isomerisation. Den spektroskopiska studien genomfördes med bilirubin, löst i organiska lösningsmedel och i buffert i komplex med humant serumalbumin. Detta albuminkomplex finns i blodet, och är därför av medicinskt intresse. Kvantmekanistiska CASSCFberäkningar på en bilirubinmodell användes för att förklara de experimentella resultaten. Det uppmätta fluorescence sönderfallet visar ultrasnabba komponenter (~120 fs). Dessa tolkas som excitonlokalisering, som följs av bildandet av ett vridet intermediat med en hastighetskonstant på ca. 1 ps-1(beroende på lösningsmedlet). Absorptionsmätningar visar att detta intermediat sönderfaller tillbaka till grundtillståndet med en livstid på 10-15 ps. CASSCF beräkningar, i kombination med de experimentella resultaten, tyder på att sönderfallet med livslängden på ca. 1 ps är en relaxation till det vridna S1-tillståndet. Reaktionsvägen därifrån antas passera en barriär till en konisk genomskärning, som möjliggör snabb relaxation till grundtillståndet. Tidsberoende DFT-beräkningar användes för att analysera bilirubins absorptionsspektrum, vilket gav bra överensstämmelse med uppmätta data. Dessutom hittades ett tillstånd med låg excitationsenergi, som kräver ytterligare studier. Med hjälp av det beräknade CD-spectret kunde det visas att bilirubin binder till albumin i P-formen vid neutralt pH.

Eine Erhöhung des Bilirubins über 2 mg/dl betrifft 90 % aller Neugeborenen. Sie ist meist physiologisch und tritt optisch sichtbar bei 60-70 % dieses Kollektivs auf. In der pathologischen, exzessiv erhöhten Form ist sie der häufigste Grund für eine stationäre Wiederaufnahme während der ersten sieben Lebenstage. Ihre schwerste Komplikation, der Kernikterus, scheint - trotz allgemein verfügbarer, preiswerter und sicherer Therapiemöglichkeiten - wieder vermehrt aufzutreten. Die Gründe liegen im Überwachungsdefizit bei früher Entlassung von schlecht aufgeklärten Eltern, Nichtbeachtung der Besonderheiten der Neugeborenen ≤ 38 Schwangerschaftswochen und der zunehmenden Tendenz zum Stillen bei häufig unzureichender Anleitung. Ferner werden ikterische Kinder nur zu oft lediglich visuell bezüglich des Grades der Bilirubinämie eingeschätzt und die Therapie somit erheblich verzögert. Gegenstand dieser Arbeit ist die Frage, ob aus der Dynamik des Serumbilirubinspiegels von der Geburt bis zum 3. Lebenstag die Wahrscheinlichkeit des Auftretens einer phototherapiepflichtigen Hyperbilirubinämie abgeschätzt werden kann. Dazu wurde der Serumbilirubinspiegel direkt postnatal aus dem Nabelschnurblut, bzw. am 3. Lebenstag gleichzeitig mit dem Stoffwechselscreening ermittelt und der Phototherapiebedarf im Verlauf festgehalten. Um die Aussage zu präzisieren, wurde die Studienpopulation aus 2573 Kindern weiter unterteilt in 2180 reife tAGA- (hier Eu- und Hypertrophe), 267 reife tSGA-Kinder (Hypotrophe) und 126 FG (Frühgeborene). In allen 3 Gruppen korrelierten das Nabelschnurbilirubin und der Serumbilirubinwert vom 3. Lebenstag positiv mit der Entwicklung einer Hyperbilirubinämie. Anhand dieser Ausgangswerte konnten Grenzen für Hoch-, Mittelhoch-, Mittelniedrig- und Niedrigrisikogruppen definiert werden, welche die Entwicklung einer Hyperbilirubinämie mit einer Wahrscheinlichkeit von ≥ 20 %, 5-20 %, 0 < x <5 % und 0 % voraussagen. Damit kann man bereits früh eine Vorabselektion entsprechend dem Gefährdungspotential treffen und die Verlaufskontrollen entsprechend terminieren. Als Risikofaktoren einer therapiepflichtigen Hyperbilirubinämie wurden außerdem Frühgeburtlichkeit, seltener tSGA, geringes Geburtsgewicht und niedriges Gestationsalter (in der vorliegenden FG-Gruppe nicht signifikant) gefunden. Im Falle einer Sectiogeburt und bei Zuhilfenahme von Hilfsmitteln im Rahmen einer vaginalen Entbindung nahm der Bedarf an Phototherapie in der tAGA- und tSGA-Gruppe zu.

Stroke is the third largest cause of death, and the leading cause of disability worldwide. Treatments are sought to reduce mortality, and increase survival time following an ischaemic stroke. Hypoxia-ischaemia (HI) is the combination of cerebral ischaemia and global hypoxia that can lead to neuronal damage, particularly perinatally. The complex neurodegenerative cascade following ischaemic stroke and HI activates many stress pathways, including heme oxygenase (HO). HO metabolises free heme to release iron, carbon monoxide, and biliverdin, which is subsequently metabolised to bilirubin. This thesis aims to elucidate the role HO plays following HI, and assess any neuroprotective mechanisms using HO modulators. The 26 day old rat model of HI was used to induce the neurodegenerative cascade. All animals were sacrificed 3 days post-insult. Immunohistochemistry and Western blotting demonstrated that HO-1 was increased in the ipsilateral hemisphere of both HI (by 1.7 � 0.1 fold: p = 0.016, n = 4) and middle cerebral artery occlusion (MCAO) brains (by 1.6 � 0.1 fold: p = 0.037, n = 4), compared to controls. HO-2 was constitutively expressed throughout the control brain, but HI upregulated HO-2 expression (by 1.7 � 0.2 fold: p = 0.027, n = 4) ipsilaterally, whereas MCAO did not alter HO-2 expression. Administration of the HO inhibitor tin protoporphyrin (SnPP; 30[mu]mol/kg intraperitoneally) daily, beginning 1 day prior to HI until sacrifice, reduced infarct volume to 50% � 10 of saline-treated animals (p = 0.039, n = 6-8). The HO inducer ferriprotoporphyrin (FePP; 30[mu]mol/kg) had no effect on infarct volume. HO activity and protein expression were not significantly altered following treatment with SnPP. Therefore, the neuroprotective actions of SnPP may be through alternative mechanisms. SnPP treatment increased HI + saline-induced total nitric oxide synthase (NOS) activity by 1.5 � 0.06 fold (p < 0.001, n = 6-8). Conversely, SnPP inhibited both inducible NOS (50% � 7 of HI + saline; p = 0.045, n = 7-8) and cyclooxygenase (COX) activity (32% � 6 of HI + saline; p = 0.049, n = 4-8). SnPP treatment also increased mitochondrial complex I activity by 1.6 � 0.25 fold (p = 0.04, n = 4-8) and complex V activity by 1.7 � 0.26 fold (p = 0.046, n = 4-8) in the ipsilateral hemisphere. It appears that SnPP is acting on inflammatory and mitochondrial enzymes to produce neuroprotection. In vitro analysis of cultured RAW264.7 macrophages exposed to lipopolysaccharide (LPS; 10[mu]g/mL) treated with SnPP (dose range: 10⁻�⁰M - 10⁻⁵M) did not alter nitrite levels or cell viability. However, high dose SnPP (10⁻⁵M) in the absence of LPS increased nitrite levels from control cells by 2.7 � 0.7 fold (p = 0.043, n = 6), complementing the in vivo total NOS data. Other mechanisms such as NMDA receptor activation were not affected by 100[mu]M SnPP or 100[mu]M SnCl₂ in patch clamped cortical pyramidal neurons. Overall, the role that HO plays following HI remains unclear, but this thesis provides definitive evidence that SnPP (an established HO inhibitor) provides neuroprotection. This neuroprotection may be due to its effects on inducible pathways such as NOS and COX. Therefore, further experimentation is required to fully elucidate the role that HO plays following cerebral ischaemia, and additional in vivo evidence will be necessary to establish HO inhibitors as a putative candidate for cerebral ischaemia neuroprotection.

Stroke is the third largest cause of death, and the leading cause of disability worldwide. Treatments are sought to reduce mortality, and increase survival time following an ischaemic stroke. Hypoxia-ischaemia (HI) is the combination of cerebral ischaemia and global hypoxia that can lead to neuronal damage, particularly perinatally. The complex neurodegenerative cascade following ischaemic stroke and HI activates many stress pathways, including heme oxygenase (HO). HO metabolises free heme to release iron, carbon monoxide, and biliverdin, which is subsequently metabolised to bilirubin. This thesis aims to elucidate the role HO plays following HI, and assess any neuroprotective mechanisms using HO modulators. The 26 day old rat model of HI was used to induce the neurodegenerative cascade. All animals were sacrificed 3 days post-insult. Immunohistochemistry and Western blotting demonstrated that HO-1 was increased in the ipsilateral hemisphere of both HI (by 1.7 � 0.1 fold: p = 0.016, n = 4) and middle cerebral artery occlusion (MCAO) brains (by 1.6 � 0.1 fold: p = 0.037, n = 4), compared to controls. HO-2 was constitutively expressed throughout the control brain, but HI upregulated HO-2 expression (by 1.7 � 0.2 fold: p = 0.027, n = 4) ipsilaterally, whereas MCAO did not alter HO-2 expression. Administration of the HO inhibitor tin protoporphyrin (SnPP; 30[mu]mol/kg intraperitoneally) daily, beginning 1 day prior to HI until sacrifice, reduced infarct volume to 50% � 10 of saline-treated animals (p = 0.039, n = 6-8). The HO inducer ferriprotoporphyrin (FePP; 30[mu]mol/kg) had no effect on infarct volume. HO activity and protein expression were not significantly altered following treatment with SnPP. Therefore, the neuroprotective actions of SnPP may be through alternative mechanisms. SnPP treatment increased HI + saline-induced total nitric oxide synthase (NOS) activity by 1.5 � 0.06 fold (p < 0.001, n = 6-8). Conversely, SnPP inhibited both inducible NOS (50% � 7 of HI + saline; p = 0.045, n = 7-8) and cyclooxygenase (COX) activity (32% � 6 of HI + saline; p = 0.049, n = 4-8). SnPP treatment also increased mitochondrial complex I activity by 1.6 � 0.25 fold (p = 0.04, n = 4-8) and complex V activity by 1.7 � 0.26 fold (p = 0.046, n = 4-8) in the ipsilateral hemisphere. It appears that SnPP is acting on inflammatory and mitochondrial enzymes to produce neuroprotection. In vitro analysis of cultured RAW264.7 macrophages exposed to lipopolysaccharide (LPS; 10[mu]g/mL) treated with SnPP (dose range: 10⁻�⁰M - 10⁻⁵M) did not alter nitrite levels or cell viability. However, high dose SnPP (10⁻⁵M) in the absence of LPS increased nitrite levels from control cells by 2.7 � 0.7 fold (p = 0.043, n = 6), complementing the in vivo total NOS data. Other mechanisms such as NMDA receptor activation were not affected by 100[mu]M SnPP or 100[mu]M SnCl₂ in patch clamped cortical pyramidal neurons. Overall, the role that HO plays following HI remains unclear, but this thesis provides definitive evidence that SnPP (an established HO inhibitor) provides neuroprotection. This neuroprotection may be due to its effects on inducible pathways such as NOS and COX. Therefore, further experimentation is required to fully elucidate the role that HO plays following cerebral ischaemia, and additional in vivo evidence will be necessary to establish HO inhibitors as a putative candidate for cerebral ischaemia neuroprotection.

As biocélulas a combustível enzimáticas (BFCs) são dispositivos eletroquímicos que convertem energia química em energia elétrica, utilizando enzimas como biocatalisadores. Quando miniaturizada, uma BFC pode ser implantada em animais vertebrados e invertebrados, vislumbrando-se sua utilização na produção de energia elétrica para alimentar microdispositivos biomédicos e microssensores em pequenos insetos. No entanto, ainda é um desafio obter BFCs implantáveis e miniaturizadas, com uma potência suficiente (dezenas de microwatts) para alimentar microcircuitos eletrônicos de maneira estável e em longo prazo. Diante do exposto, esta tese de doutorado apresenta um estudo das propriedades eletroquímicas de eletrodos enzimáticos, visando a aplicação em BFCs de glicose/O2 miniaturizadas e implantáveis. Para isso, utilizaram-se fibras flexíveis de carbono (FCFs) modificadas com as enzimas bilirrubina oxidase (BOx) no cátodo e glicose desidrogenase (GDh) NAD-dependente no ânodo, a fim de se obter a redução de O2 e a oxidação de glicose, respectivamente. Os resultados obtidos mostram que FCFs previamente submetidas a um tratamento químico de oxidação com permanganato de potássio e com posterior eletrodepolimerização do mediador vermelho neutro produzem bioânodos estáveis e robustos. Estes eletrodos, combinados com biocátodos compostos por FCFs na ausência de mediadores redox, foram utilizados em BFCs miniaturizadas, que foram implantadas em formigas da espécie Atta sexdens rubrupilosa. A potência máxima da BFC operando in vivo foi 13,5 &plusmn; 3,8 &micro;W cm-2 em 190 &plusmn; 58,9 mV, com corrente máxima de 143 &plusmn; 40,2 &micro;A cm-2 e a voltagem de circuito aberto de 260 &plusmn; 99,6 mV. Acredita-se que estes valores ainda possam ser otimizados e este trabalho contribui para mostrar que a flexibilidade das FFC, a presença de um mediador de elétrons polimérico no ânodo, o uso do tratamento químico de oxidação com permanganato de potássio das fibras e a miniaturização dos eletrodos são elementos importantes, e que podem ser considerados no desenvolvimento de biocélulas a combustível implantáveis. / Enzymatic biofuel cells (BFCs) are electrochemical devices that convert chemical energy into electrical energy using enzymes as biocatalysts. When miniaturized, BFCs can be implanted in vertebrate and invertebrate animals and, their use to produce electrical energy to feed biomedical microdevices and micro-sensors in small insects can be observed. However, it is still challenging to obtain implantable and miniaturized BFCs, with sufficient power (tens of microwatts) to power electronic microcircuits in a stable and long-term manner. In view of the above, this PhD thesis presents a study of the electrochemical properties of enzymatic electrodes, aiming to use them in miniaturized and implantable glucose/O2 BFCs. In order to obtain a reduction in O2 and oxidation of glucose, flexible carbon fibers (FCFs) modified with bilirubin oxidase (BOx) enzymes in the cathode and glucose dehydrogenase (GDh) at the anode, respectively, were used. The results show that FCFs previously submitted to a chemical treatment of oxidation with potassium permanganate and, subsequently, electropolymerization of the neutral red mediator produce stable and robust bioanodes. These electrodes, combined with biocathodes consisting of FCFs in the absence of redox mediators, were used in miniaturized BFCs, which were implanted in Atta sexdens rubrupilosa ant species. The BFC maximum power source, operating in vivo, was 13.5 &plusmn; 3.8 &mu;W cm-2 at 190 ± 58.9 mV, with a maximum current of 143 &plusmn; 40.2 &mu;A cm-2 and the open circuit voltage was 260 &plusmn; 99.6 mV. Although these values can be optimized, this research shows that the flexibility of the FCF, the presence of a polymer electron mediator on the anode, using the chemical treatment of oxidation with potassium permanganate of the fibers and electrode miniaturization are important elements, which can be considered in the development of implantable biofuels.

Bilirrubina oxidase de Myrothecium verrucaria é uma multicobre oxidase capaz de reduzir O2 pela oxidação de fenóis, aminas aromáticas e polipirróis. Eletroquimicamente, essa reação de redução ocorre pela transferência de elétrons entre a enzima e um eletrodo. Nesta tese, foi investigada a eficiência da enzima como agente redutor de O2 na superfície de eletrodos modificados pela função orgânica naftil-2-carboxilato por acoplamento de diazônio. Essa modificação na superfície do eletrodo aumenta em até quatro vezes a atividade do filme catalítico em relação à obtida por eletrodos em que a adsorção da enzima foi feita de forma convencional, sem a modificação. Foram estudados os efeitos da temperatura sobre a atividade da enzima para a redução de O2, sendo observado um aumento linear da atividade do eletrodo com o aumento da temperatura até 30 &deg;C, de tal forma que temperaturas mais altas proporcionaram o aumento da inativação natural das moléculas de enzima. Esse efeito de inativação foi confirmado pela diminuição da atividade com o tempo na presença de O2, por cronoamperometria, sendo a atividade interrompida pela inserção de argônio e retomada do mesmo ponto pela reinserção de O2, descartando a idéia da queda de corrente proveniente da dessorção de enzima. Foi estudado também o efeito do pH na máxima atividade da bilirrubina oxidase, conduzidos entre pH 5,0 e 8,0, e verificando-se que a máxima atividade da enzima foi obtida entre pH 5,5 e 6,0 e, além disso, verificou-se que a corrente catalítica em baixos valores de pH aumenta diretamente com o aumento do sobrepotencial aplicado. Porém, em altos valores de pH, a curva de redução toma a forma sigmoidal e passa a ser independente do sobrepotencial aplicado, sendo a reação governada por etapas químicas de transferência de prótons. O uso de eletrodos de disco rotatório possibilitou resolver parâmetros de Michaelis-Menten para a cinética do filme catalítico de forma mais precisa (a resposta de corrente é menos dependente do transporte de massa de reagentes) e esses dados foram obtidos dentro de um intervalo de pH importante para aplicações práticas. O sobrepotencial da reação de redução de O2 catalisada por bilirrubina oxidase foi comparado com o sobrepotencial obtido para a mesma reação catalisada por Platina eletrodepositada sobre a superfície de grafite pirolítico, onde foi observado um sobrepotencial de 140 mV para a catálise enzimática, demasiado menor que o valor de 415 mV obtidos para a Platina, sob as mesmas condições experimentais, em pH neutro. A metodologia proposta para a construção de um cátodo para aplicação em células a combustível enzimáticas e os subsequentes estudos possibilitaram uma investigação minuciosa para caracterizar a enzima bilirrubina oxidase como talvez o catalisador mais eficiente na redução eletroquímica de oxigênio molecular em células a combustível até o momento. / Bilirubin oxidase from Myrothecium verrucaria is a multicopper oxidase reducing O2 at the expenses of phenols, aromatic amines and polypyrrols oxidation. Electrochemically, this reduction reaction undergoes through the electron transfer between enzyme and electrode. In this thesis, the enzyme was investigated as an efficient O2 reducing agent on electrode surfaces modified by naphthil-2-carboxylate functionalities through diazonium coupling. This modification of the electrode surface increases the activity of the catalytic film up to four times comparing to that obtained by electrodes in which the enzyme molecules were adsorbed conventionally, without modification. It was studied the effect of temperature on O2 reduction, in which catalysis increased linearly with temperature up to 30 &deg;C, and higher temperatures increased the natural enzyme inactivation. This inactivation was confirmed by the activity drop off with time in the presence of O2, by chronoamperometry, ceased out when argon was inserted into the cell and re-established from the same point when argon was purged out by insertion of O2. These results cast aside the idea of activity drop off caused by enzyme desorption. It was also investigated the pH effect on the maximum activity of bilirubin oxidase, carried out between pH 5.0 and 8.0, being the highest activity obtained at pH 5.5-6.0. Furthermore, it was observed that the catalytic current directly increases with applied overpotential, at low pH values, and the reduction wave shape becomes sigmoidal and independent on applied overpotential at high pH values. The reaction is then governed by chemical steps, as the proton transfer. The use of rotating-disc electrodes favored solving the Michaelis-Menten kinetics for the catalytic film in a much greater accuracy (the current response is much less dependent on reagent mass transport) and these data were obtained for pH interval important for practical applications. The overpotential for the O2 reduction reaction catalyzed by bilirubin oxidase was compared to the overpotential obtained by the same reaction catalyzed by Platinum electrodeposited onto a pyrolytic graphite electrode. An overpotential of only 140 mV was observed for the enzymatic catalysis, much lower compared to the 415 mV obtained for the Platinum electrode, under the same experimental conditions, at neutral pH. The proposed method for constructing a cathode for enzymatic fuel cell application and subsequent investigation described allowed an in-depth study of bilirubin oxidase characterization as perhaps the most efficient catalysts for the electrochemical reduction of molecular oxygen in fuel cells to date.

in English The concise summary of literary information about bilirubin and its structure derivative ranarubin is the topic of this thesis. The experimental part is dedicated to investigation of some properties of this substances and their comparison. The rate of degradation and acid- base behaviour was monitored under the laboratory conditions using capillary zone electrophoresis. The results point to differences in their behaviour.

Surface-enhanced Raman scattering (SERS)-active systems based on macroscopic Ag nanosponge aggregates as well as the conditions of SERS spectral measurements were optimized for selective and sensitive detection of a biomedically important, amphiphilic bile pigment bilirubin (BR) in alkaline aqueous solutions and in its solutions in a selected water miscible solvent, namely dimethylsulfoxide (DMSO) and/or water-immiscible solvent, namely CH2Cl2. Ag nanosponges assembled by using HCl as a pre-aggregation agent were found to be the optimal SERS-active systems for a reliable detection of BR in all the above-mentioned solvents. In all cases, the protonated form of adsorbed BR has been detected upon BR incorporation into Ag nanosponges, and its marker bands have been established by SERS spectral probing at excitation wavelengths in the 445-780 nm range. The sensitivity of SERS spectral detection was evaluated in terms of the concentration values of SERS spectral detection limits (SERS LODs) of BR incorporated into Ag nanosponges. In particular, the SERS LOD for BR incorporated from its alkaline aqueous solution is 1 x 10-8 M (at 532 nm excitation), for BR incorporated from its solution in DMSO, its value is also 1 x 10-8 M (at 532 and/or 633 nm excitations), and for incorporation from the solution of BR...