Metallocofactors are ubiquitous in nature, serving multiple purposes in proteins. These metallocofactors typically act as the site of catalysis or as an electron relay to move electrons within the protein, or within the cell, and are very energetically costly to manufacture. Yet, in nature it can appear that supernumerary, or ‘auxiliary’ cofactors are apparent, with no clear function. In this thesis, I address the question of what roles additional cofactors play, and why they are retained. The radical S-adenosylmethionine (AdoMet) enzyme superfamily has displayed great diversity in the cofactor requirements for its members. Some members of this family contain only the canonical [4Fe-4S] cluster, which reductively cleaves AdoMet to initiate chemistry, while others have additional [2Fe-2S] or [4Fe-4S] clusters. Even greater cofactor complexity is seen with the B12-dependent subclass, featuring a cobalamin-binding domain in addition to the canonical FeS cluster. The majority of this thesis has focused on using the technique of protein film electrochemistry (PFE) to study members of various subclasses of this superfamily: a dehydrogenase: BtrN, two methylthiotransferases: MiaB and RimO, as well as OxsB and TsrM, two B12-dependent enzymes. By evaluating the redox properties of members of different subclasses, we have been able to shed light on the redox properties of this superfamily, in general, and observed that the redox properties of auxiliary clusters can differ widely between subclasses (e.g. BtrN versus MiaB). PFE has also been used to evaluate five ferredoxins that are possible electron donors for MiaB from Thermotoga maritima. Additionally, bacterial cytochrome c peroxidases (bCCPs) are diheme enzymes catalyzing the detoxification of hydrogen peroxide; however, a novel subclass of bCCPs containing a third heme-binding motif has been identified in enteric pathogens. Protein film electrochemistry has been used to study the redox properties of Escherichia coli YhjA, a member of this subgroup. Further characterization of this novel bCCP was achieved with electron paramagnetic resonance, optical spectroscopy, and steady-state kinetics. Through characterizing YhjA and members of the AdoMet radical enzyme superfamily, we have shed light on the role these additional cofactors play in the mechanism and how these enzymes are tuned for their specific chemistries. / 2018-08-11T00:00:00Z

<p>Three aspects of the electron transport chain have been investigated in the present studies: (<b>i</b>) structure-function studies of the central proton-electron conducting cytochrome <i>b</i>₆<i>f</i>complex, focusing on the effect of lipids in structural stabilization and electron transfer function; (<b>ii</b>) transmembrane electron transfer pathways in the cytochrome <i>b</i>₆<i>f</i>and mitochondrial cytochrome <i>bc</i>₁complexes, determined by heterogeneity in the internal polarity of the membrane protein complexes; and (<b>iii</b>) purification and characterization of a novel ~1 MDa supercomplex, dominated by the presence of photosystem I (PSI), ATP-synthase and ferredoxin-NADP⁺reductase (FNR) from higher plant system <i>Spinacea</i>.<b></b></p>

Neste trabalho foi investigado o potencial de modelos metaloporfirínicos em mimetizar a ação do citocromo P450 na oxidação de um herbicida, a atrazina. Foram utilizadas as metaloporfirinas comerciais de segunda geração solúveis em solvente orgânico, cloreto de 5,10,15,20-tetrakis(2,6-diclorofenil)porfirina metal(III) [M(TDCPP)Cl] e cloreto de 5,10,15,20-tetrakis(pentafluorofenil) porfirina metal(III) [M(TFPP)Cl] (metal = ferro e manganês), tanto em solução homogênea como suportadas em montmorilonita K-10 aminofuncionalizadas; e metaloporfirinas solúveis em água, como a cloreto de 5,10,15,20-tetrakis-(N-metil-4-piridil) porfirina ferro(III), [Fe(TMPy)Cl], e cloreto de [5,10,15,20-tetra(4-carboxifenil)porfirina] ferro(III), [Fe(TCPP)Cl]. Os oxidantes testados foram iodosilbenzeno, ácido metacloroperbenzóico e peróxido de hidrogênio em água, metanol e acetonitrila. Os produtos de oxidação da atrazina foram identificados por cromatografia líquida de alta eficiência (CLAE). Os resultados mostraram que as metaloporfirinas foram capazes de oxidar a atrazina, um herbicida com características de persistência no meio ambiente, e mimetizar a ação da enzima in vivo e in vitro com formação de dois metabólitos: DEA e DIA, resultado da N-desalquilação das cadeias etila e propila do substrato, respectivamente. O DEA correspondeu a um dos principais produtos da reação, e formou-se apenas traços de DIA, mostrando a preferência das metaloporfirinas em oxidar a cadeia etila da atrazina. Verificou-se também a formação de cinco produtos desconhecidos, sendo possível a identificação de apenas um deles por espectrometria de massas, devido à baixa concentração dos demais, o qual corresponde à formação de uma amida na cadeia etila da atrazina (COA). Esse composto correspondeu ao produto de maior rendimento na maioria das reações. O monitoramento das reações em diferentes intervalos de tempo e a variação nas condições reacionais mostraram que os principais produtos de oxidação do herbicida, DEA e COA, são formados por mecanismos independentes e por espécies catalíticas distintas. O DEA é formado via espécie ativa Me(V)OP [Mn(V)OP ou Fe(IV)OP+], enquanto o COA é originado via Me(IV)OP [Mn(IV)OP ou Fe(IV)OP]. Estudos de intermediários por UV-Vis e EPR mostraram que a espécie ferril predomina como intermediário de reação para os sistemas Fe(TFPP)Cl/ACN com os dois oxidantes, iodosilbenzeno e ácido metacloroperbenzóico. Para as metaloporfirinas Fe(TCPP)Cl e Fe(TMPy)Cl o estudo da oxidação do herbicida ficou comprometido devido à baixa solubilidade da atrazina em água, o que provocava sua precipitação e destruição do catalisador. Para as metaloporfirinas suportadas em montmorilonita K-10 aminofuncionalizada também não foi observada formação de produtos, resultado atribuído à dificuldade do substrato, considerado bastante inerte, atingir o sítio catalítico. Todos esses resultados mostraram o potencial de aplicação desses modelos biomiméticos em estudos que buscam elucidar o metabolismo de herbicidas in vivo, tendo em vista a dificuldade de se trabalhar com as enzimas in vitro, e resultaram na proposição de um esquema de reação da oxidação da atrazina catalisada pelas metaloporfirinas nas condições estudadas. / In this work we investigated the ability of metalloporphyrin model systems to mimic the action of cytochrome P450 in the oxidation of a herbicide, atrazine. To this end, we employed the second generation commercially available metalloporphyrins metal (III) 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin chloride [M(TDCPP)Cl] and metal (III) 5,10,15,20- tetrakis(pentafluorophenyl)porphyrin chloride [M(TFPP)Cl] (metal = iron or manganese), all soluble in organic solvents, as well as the water soluble metalloporphyrins iron (III) 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin chloride [Fe(TMPy)Cl] and iron (III) 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin chloride [Fe(TCPP)Cl]. These metalloporphyrins were used both in homogeneous solution and supported on montmorillonite K-10. Iodosylbenzene, metachloroperbenzoic acid, and hydrogen peroxide were tested as oxidants, using one of the following reaction media: water, methanol, and acetonitrile. Products generated during atrazine oxidation were identified by high performance liquid chromatography. Our results show that the metalloporphyrins are able to oxidize atrazine, a highly persistent herbicide in the environment, as well as mimic the action of P450 enzymes both in vivo and in vitro, with formation of two metabolites, namely DEA and DIA, which result from the N-dealkylation of the ethyl and propyl chains of the substrate, respectively. We also detected the formation of five unknown products, and we were able to identify only one of them by means of mass spectrometry, which corresponds to the formation of an amide on the atrazine ethyl chain (COA) and was the compound obtained in highest yields in most of the reactions. The other four unknown products were obtained in very low concentrations, which prevented us from determining their structures. By monitoring the reactions at different time intervals and varying the reactional conditions, we were able to see that the main herbicide oxidation products, DEA and COA, are generated via distinct mechanisms and different active catalytic species. DEA is formed via the species Me(V)OP [Mn(V)OP or Fe(IV)OP.+], while COA results from the action of the species Me(IV)OP [Mn(IV)OP or Fe(IV)OP]. Studies of the reaction intermediates by UV-VIS and EPR showed that the ferryl species is the main reaction intermediate in the case of Fe(TFPP)Cl/ACN systems and the oxidants, iodosylbenzene and metachloroperbenzoic acid. Studies of herbicide oxidation were difficult to carry out in the case of the metalloporphyrins Fe(TCPP)Cl and Fe(TMPy)Cl due to the low solubility of atrazine in water, which led to its precipitation and catalyst destruction. With respect to the metalloporphyrins supported on montmorillonite K-10, no reaction products were obseved because of the difficult diffusion of the inert substrate into the catalytic site. All these results demonstrate the potential application of these biomimetic model systems in studies that pursue the elucidation of herbicide metabolism in vivo, especially when one bears in mind the difficulty in working with enzymes in vitro. Our data enabled the proposition of a scheme for metalloporphyrin-catalyzed atrazine oxidation under the conditions used herein.

A associação lipídio/citocromo c é importante e deve ser estudada, pois repercute na atividade peroxidática da proteína abordada e pode contribuir para o processo apoptótico, ou morte programada da célula, e também desempenha um papel significativo na cadeia respiratória. A natureza e a especificidade da interação do citocromo c com bicamadas lipídicas têm sido bastante investigadas ultimamente, mas informações detalhadas e precisas sobre tais assuntos ainda não existem. É aceito que ocorre primeiramente uma interação eletrostática entre a proteína citocromo c e as membranas fosfolipídicas. Em seguida, há uma interação hidrofóbica. Entretanto, ainda não é bem compreendido o papel da cadeia fosfolipídica. A associação do citocromo c com membranas lipídicas induz mudanças no estado de spin do átomo de ferro. A interação entre as vesículas carregadas e o citocromo c induz mudanças estruturais na proteína, as quais são refletidas no seu centro ativo, ou grupo heme. As mudanças do campo cristalino no sítio do ferro hemínico de forte para fraco são acompanhadas por mudanças do estado de spin de baixo para alto, respectivamente. Neste trabalho, estuda-se sistematicamente a natureza da interação entre o citocromo c e a cadeia fosfolipídica. As mudanças estruturais no grupo heme foram correlacionadas com a natureza do lipídio, ou seja, com a carga da cabeça e com o tamanho e o tipo da cadeia fosfolipídica. Foram utilizados treze lipídios diferentes, naturais e sintetizados, com cabeças polares negativas e neutras e com cadeias carbônicas saturadas e insaturadas de diferentes comprimentos. Para tal investigação, utilizamos as técnicas: Ressonância Paramagnética Eletrônica (RPE) Onda Contínua (CW) e Pulsada (PW) e Dicroísmo Circular Magnético (MCD). As técnicas enunciadas avaliam as mudanças de estado de spin e a simetria do citocromo c nos seus estados férrico e ferroso. A interação lipoprotéica lipídio/citocromo c foi avaliada com lipídios diferentes, inclusive com o lipossomo PCPECL, que mimetiza a membrana interna da mitocôndria nos eucariontes. A partir dos resultados experimentais, sugerimos um modelo para esse tipo de associação. / This association lipid/cytochrome c is interesting to study in order to understand the peroxidase activity of this protein, that plays an important role in the respiratory chain and in the apoptosis process or the programmed cell death. The nature and specificity of the interaction of cytochrome c with lipid bilayers have been major goals in recent studies, but detailed information on that issue is not yet widely available. In this regard, it is generally accepted that the electrostatic interaction is an important factor in the association of cytochrome c with phospholipid membranes, followed by a hydrophobic interaction. However, the role played by the phospholipid chain is not well understood. The association of cytochrome c with negative membranes induces a change in the heme iron spin state. The interaction between the charged vesicles and cytochrome c leads to structural changes in the active central or heme group. The changing of the crystalline field of the heme iron from strong to weak is accompanied by spin states changes from low to high spin, respectively. These facts concerned us to investigate more systematically the nature of the interaction between cytochrome c and the phospholipid chains. The lipid-induced effects in the heme iron crystalline field are correlated to the nature of the charged head group and to the size and type of the phospholipid chain. Thirteen different lipids, nature and synthetic, were used, with negative and neutra1 polar head group and saturated and unsaturated acyl chains with different length. This work investigates the change of heme iron spin state and symmetry of ferric cytochrome c using Continuous Wave (CW) and pulsed (PW) Electron Paramagnetic Resonance (EPR) and Magnetic Circular Dichroism (MCD) techniques. These techniques analyze the spin state change and the symmetry of the iron cytochrome c in its ferric and ferrous states. The effect of the different lipids were analyzed, including PCPECL membrane that mimetics the inner mitocondrial membrane in eukaryotes.

by Wong Che-cheuk, Dobe. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 98-112). / Abstract also in Chinese. / Acknowledgements --- p.i / Abstracts --- p.ii / List of Abbreviation --- p.vi / Table of Contents --- p.vii / Chapter 1. --- Introduction / Chapter 1.1 --- Stages of Cancer Development --- p.1 / Chapter 1.2 --- Comparison of Breast Cancer in Hong Kong & the United States --- p.2 / Chapter 1.2.1 --- Statistics of Breast Cancer in the United States --- p.2 / Chapter 1.2.2 --- Statistics of Breast Cancer in Hong Kong --- p.2 / Chapter 1.3 --- Factors for Breast Cancer --- p.6 / Chapter 1.3.1 --- Genetic Factor --- p.6 / Chapter 1.3.2 --- Hormonal Factor --- p.7 / Chapter 1.3.3 --- Genetic Bias --- p.9 / Chapter 1.3.4 --- Influence of Diet --- p.10 / Chapter 1.3.5 --- Obesity --- p.14 / Chapter 1.3.6 --- Xenoestrogen --- p.14 / Chapter 1.4 --- Hormonal Therapy in Breast Cancer --- p.15 / Chapter 1.4.1 --- Antiestrogen --- p.15 / Chapter 1.4.2 --- Progestin Antagonist --- p.19 / Chapter 1.4.3 --- Aromatase Inhibitor --- p.20 / Chapter 1.4.4 --- Gonadotropin Releasing Hormone (GnRH) Analogue --- p.23 / Chapter 1.5 --- Metabolism of Estrogen --- p.25 / Chapter 1.6 --- Substance with Chemopreventive Properties towards Breast Cancer --- p.29 / Chapter 1.7 --- Aryl Hydrocarbon Receptor --- p.33 / Chapter 1.8 --- Cytochrome P450s --- p.34 / Chapter 1.9 --- Yuehchukene --- p.36 / Chapter 1.10 --- Objectives of the Present Study --- p.38 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Animals --- p.40 / Chapter 2.2 --- Animal Treatment --- p.40 / Chapter 2.3 --- Preparation of Crude Microsomal Fraction --- p.41 / Chapter 2.4 --- Protein Assay --- p.41 / Chapter 2.5 --- Ethoxyresorufm-O-deethylase Assay --- p.41 / Chapter 2.6 --- Methoxyresorufin-O-deethylase Assay --- p.42 / Chapter 2.7 --- Estradiol-2-hydroxylase Assay --- p.42 / Chapter 2.8 --- Progesterone Hydroxylase Assay --- p.43 / Chapter 2.9 --- Hepatic Aromatase Activity Assay --- p.43 / Chapter 2.10 --- Inhibition of Ethoxyresorufm-O-deethylase and Estradiol-2-hydroxylase --- p.44 / Chapter 2.11 --- Free Radicals Scavenging Assay --- p.44 / Chapter 2.12 --- Chemicals --- p.45 / Chapter 3. --- Result / Chapter 3.1 --- Optimization of Condition --- p.47 / Chapter 3.1.1 --- Dosage --- p.47 / Chapter 3.1.2 --- Time for Sacrifice --- p.47 / Chapter 3.2 --- "Effect of Isoprenyl Compounds on the Body Weight, Liver Weight and Hepatic Microsomal Protein Content" --- p.50 / Chapter 3.3 --- Hepatic Enzyme Activities --- p.54 / Chapter 3.3.1 --- Ethoxyresorufm-O-deethylase --- p.54 / Chapter 3.3.2 --- Methoxyresorufm-O-deethylase --- p.57 / Chapter 3.3.3 --- Estradiol-2-hydroxylase --- p.60 / Chapter 3.3.4 --- Progesterone Hydroxylase --- p.62 / Chapter 3.3.5 --- Aromatase --- p.65 / Chapter 3.4 --- Effect of Inhibitors in Ethoxyresorufin-O-deethylase and Estradiol-2-hydroxylase Activity --- p.65 / Chapter 3.5 --- Free Radical Scavenging Activity --- p.72 / Chapter 4. --- Discussion --- p.77 / Chapter 5. --- Conclusion --- p.95 / Chapter 6. --- References --- p.98 / Chapter 7. --- Appendix --- p.113

SHP (Sphaeroides Heme Protein) is a monoheme cytochrome c of unknown function. In general, ligands cannot bind to ferric SHP, but some diatomic molecules, such as O2 or NO, can bind to ferrous SHP. The gene encoding SHP and genes encoding a diheme cytochrome c (DHC) and a b-type cytochrome (Cyt-b) are found in the same chromosome region in different species. In the case of Shewanella oneidensis MR-1, mRNA levels for SHP, DHC, and Cyt-b are up-regulated by nearly 10-fold when grown under anaerobic conditions using nitrate as the electron acceptor. Thus it is possible that the physiological role of SHP may be in nitrate metabolism. However, nitrate is too big to be a candidate substrate for SHP, and some nitrification steps need more than one electron transfer (SHP is a monoheme cytochrome). Therefore, we will focus on the nitrite reductase, nitric oxide reductase and nitric oxide dioxygenase activities of SHP. In this thesis it is shown that SHP can catalyse the reaction between oxygen and nitric oxide to give a nitrate ion as the final product. Thus a possible aerobic function for SHP as a nitric oxide dioxygenase is proposed. Aerobically, SHP is proposed to be a nitric oxide dioxygenase which utilizes the same mechanism as other NO dioxygenases, flavohemoglobin (HMP) and neuroglobin (Ngb). This mechanism is proposed to proceed via an oxy-ferrous complex (SHP2+-O2) which reacts with nitric oxide. A mechanism for the catalytic reaction with ferrous-NO complex is described. SHP2+-NO can be quickly converted back to ferrous SHP by reacting with superoxide liberated by SHP2+-O2 or from another source. In addition it is also found that Shewanella MR-1 wild type reveals a higher NO tolerance than the SHP knockout strain in aerobic conditions. The catalytic mechanism of NO dioxygenase is oxygen-dependent, but the SHP mRNA up-regulation in Shewanella oneidensis MR-1 grown with nitrate under anaerobic conditions indicates that SHP may also perform some anaerobic function and may possibly be involved in nitrate metabolism. This work found that SHP reveals anaerobic nitrite reductase activity. However, the catalytic efficiency of SHP is considerably lower than other nitrite reductases. This infers that although SHP can reduce nitrite in vitro, it is unlikely to function as a nitrite reductase in vivo. Ferrous SHP binds NO with a Kd of less than 1 μM, and does not auto-oxidise. Therefore, under anaerobic conditions SHP2+-NO must be processed by some other mechanism. In addition, biochemical results reveal that the SHP/DHC complex has NO reductase activity under anaerobic conditions. Unfortunately, this function was not proved in vivo. SHP was initially isolated from Rhodobacter sphaeroides and its structure was reported in 2000. Based upon this structure, SHP is clearly a class I cytochrome c with one axial histidine ligand to the heme iron. Unusually, however, it has an asparagine residue as the other axial heme ligand, and as such is unique among cytochromes c. For this reason it may be assumed that the asparagine plays a special role. This study reveals several potential reasons why SHP utilises asparagine as a heme ligand. Firstly, in the ferric form, asparagine 88 binds to the heme iron to prevent small molecules binding. Secondly, in the ferrous form it moves to allow oxygen to bind and form the oxy-ferrous complex, using hydrogen bonding for stability. Thirdly, using asparagine as a heme ligand creates a suitable redox potential for reduction by DHC, thus allowing NO dioxygenation.

Espécies silvestres com grande potencial zootécnico devem ser exploradas de forma racional a fim de se evitar a extinção das mesmas. Assim se dá a importância de pesquisas voltadas à reprodução daquelas criadas em cativeiro, como a cutia (Dasyprocta sp.). Este animal é um mamífero e roedor vivente, em sua maioria, na Caatinga brasileira. A ultraestrutura é a base para determinar os estágios celulares e, assim, facilitar as comparações dos processos entre cutias e roedores silvestres ou outros mamíferos. As enzimas P450 aromatase e P450c17 são responsáveis pela regulagem da produção de estrógenos e andrógenos, respectivamente. Considerando a hipótese de que o comportamento de expressão das enzimas do complexo citocromo P540 permanece o mesmo no testículo e na via espermática de cutias durante as fases de desenvolvimento sexual, objetivou-se observar a atuação das enzimas P450 aromatase e P450c17 (17-&#945;-hidroxilase/17,20-liase) nas diferentes fases do desenvolvimento sexual, detalhar a ultraestrutura dos componentes desta via e constatar o desenvolvimento do processo espermatogênico. Segmentos do ducto deferente, epidídimo e testículo de 28 cutias machos em diferentes idades (um dia, 2-14 meses) foram fixados em paraformoldeído e glutaraldeído. O material foi coletado no Centro de Multiplicação da Universidade Federal Rural do Semiárido, Mossoró, RN (Autorização IBAMA nº 2028236/2008). Foram feitos: histologia, seguindo o protocolo padrão para hematoxilina e eosina; processamento para corte semifino (azul de toluidina); microscopia eletrônica de transmissão e varredura; e imunohistoquímica. Este trabalho foi pioneiro ao observar que o epidídimo de cutias é composto por células basais, células principais, células haloides e, quando impúbere, por células \"limpas\", e por células apicais, quando a partir da puberdade. O ducto deferente de cutias antes da puberdade era caracterizado por duas camadas musculares, possivelmente devido à falta de trânsito espermático. No epitélio germinativo foram encontradas, em sua maioria, células em prófase I, principalmente em paquíteno. A espermiogênese é completa quando na pré-puberdade, entretanto, a espermiação ocorre a partir dos 9 meses de idade. A expressão da enzima P450 aromatase variou ao longo do desenvolvimento sexual, sendo na puberdade seu pico de atividade. A P450c17 não mostrou nenhuma ação em qualquer fase sexual. Pode-se concluir que o epitélio germinativo testicular e intersticial, bem como o epitélio pseudoestratificado estereociliado do epidídimo e do ducto deferente de cutias criadas em cativeiro sofrem mudanças morfológicas e funcionais ao longo do desenvolvimento sexual. As atividades androgênicas preponderantes em cutias criadas em cativeiro ocorrem no período da puberdade. / Wild species with great potential livestock should be explored rationally in order to prevent the extinction of the same. Thus is the importance of research aimed at reproducing those bred in captivity, such as agouti (Dasyprocta sp.). This animal is a mammal and rodent living mostly in the Brazilian Caatinga. The ultrastructure is the basis for determining the stages and thus facilitates comparisons of cases between agouti and wild rodents or other mammals. The enzymes P450 aromatase and P450c17 are responsible for regulating the production of estrogens and androgens, respectively. On the assumption that the behavior of expression of the enzymes of complex cytochrome P540 remains the same in the testis and excurrent canals of the agouti during the stages of sexual development, aimed to observe the activity of the enzymes P450 aromatase and P450c17 (17-&#945;- hidroxilase/17,20-lyase) in different stages of sexual development, detail the ultrastructure of the components of this pathway and observe the development of spermatogenesis. Segments of the vas deferens, epididymis and testis of 28 agouti males at different ages (1 day, 2-14 months) were fixed in glutaraldehyde and paraformoldehyde. The material was collected on Center of Multiplication of Federal Rural University of the Semi-arid, Natal, RN (IBAMA Authorization No. 2028236/2008). Were made: histology following the standard protocol for hematoxylin and eosin; processing to semithin (blue toluidine), electron microscopy of transmission and scanning; and immunohistochemistry. This work was pioneered by observing that the epididymis is composed by basal cells, principal cells, haloids cells; and for clean cells when impubertal and apical cells after puberty in agoutis. The vas deferens before puberty was characterized by two muscle layers, possibly due to the lack of sperm transit. In the germinal epithelium were found mostly cells in prophase I, mainly in pachytene. Spermiogenesis is complete when prepubertal phase; however, spermiation takes place from 9 months of age. The expression of enzymes of the cytochrome complex varied over sexual development and peak activity of P450 aromatase was at puberty. The P450c17 showed no action at any stage of sexual development. It can be concluded that the testicular germinal epithelium and interstitial epithelium as well as the pseudostratified estereociliated epithelium of the epididymis and vas deferens undergo morphological and functional changes during the sexual development. Androgenic activities prevalent in agoutis kept in captivity occur during puberty.

A interação do citocromo c com a cardiolipina ocorre por interações eletrostáticas e hidrofóbicas. A formação do complexo citocromo c/ cardiolipina promove uma pequena mudança estrutural na proteína, que proporciona atividade peroxidásica ao citocromo c e consequentemente capacidade de oxidar substratos orgânicos, incluindo a cardiolipina. A oxidação da cardiolipina acompanhada da inserção de um grupo peróxido vem sendo relacionada à perda da interação hidrofóbica entre o complexo citocromo c/cardiolipina, que resulta no desligamento do citocromo c da membrana e na sua saída do espaço intermembranas para o citosol, onde essa proteína induz a cascata de apoptose. Neste trabalho foi avaliada a ligação do citocromo c a lipossomos contendo cardiolipina oxidada e a reatividade desta proteína com o mono-hidroperóxido da cardiolipina (TLCL(OOH)1) presente na membrana. Nossos dados mostraram que ocorre uma diminuição significativa na ligação do citocromo c a membrana oxidadas apenas quando 100% da cardiolipina presente na membrana está na forma de TLCL(OOH)1, condição que extrapolaria o que seria esperado para o sistema biológico. Análises por SDS-PAGE revelaram que o citocromo c sofre agregação na presença de membranas contendo TLCL(OOH)1, indicando que a proteína reage com este peróxido. De fato, determinamos a velocidade de reação do citocromo c com o TLCL(OOH)1 e com hidroperóxido do ácido linoléico, inseridos em membrana contendo cardiolipina (9,58 ± 0,16 x 102 M-1.s-1 e 6,91 ± 0,30 x 102 M-1.s-1, respectivamente). As velocidades de reação com os peróxidos de lipídio foram pelo menos 10 vezes superiores à velocidade medida com o peróxido de hidrogênio (5,91 ± 0,18 x101 M-1.s-1). Assim, mostramos que o citocromo c liga-se à membrana contendo hidroperóxido de cardiolipina e que reage com o mesmo promovendo a formação de agregado protéico de alto peso molecular / The interaction of cytochrome c with cardiolipin is promoted by electrostatic and hydrophobic interactions. The cytochrome c / cardiolipin complex formation causes structural changes in the protein that activates cytochrome c peroxidase activity, giving it the ability to oxidize organic substrates, including cardiolipin. The oxidation of cardiolipin coupled with a peroxide group insertion has been related to the loss of hydrophobic interactions between the cytochrome c / cardiolipin complex, resulting in cytochrome c release from the membrane and in its translocation from intermembranes space to cytosol, where this protein induces apoptosis cascade. In this work the binding of cytochrome c to liposomes containing oxidized cardiolipin and its reactivity with the membrane mono-hydroperoxides (TLCL(OOH)1) were evaluated. Our data showed a significant decrease in cytochrome c binding to oxidized membranes only when 100% of the membrane cardiolipin is in the TLCL(OOH)1 form, a condition that would extrapolate the expected concentrations that would be found in a biological system. SDS-PAGE analysis revealed that cytochrome c undergoes aggregation in the presence of membranes containing TLCL(OOH)1, indicating that this protein reacts with the peroxide. In fact, we determined the rate of cytochrome c reaction with TLCL(OOH)1 and linoleic acid hydroperoxide inserted into cardiolipin containing membranes (9.58 ± 0.16 x 102 M-1s-1 and 6.91 ± 0.30 x 102 M-1s-1,respectively). The reaction rates obtained with lipid peroxides were at least 10 times higher than that obtained with hydrogen peroxide (5.91 ± 0.18 x 101 M-1s-1).Thus we show that cytochrome c binds to membrane containing cardiolipin hydroperoxides and reacts with it promoting the formation of high molecular weight protein aggregates.

Many droplet microfluidic assays have been described in the literature over the last decade of research, however, there has been little reported industrial use of droplet microfluidics in drug discovery compound screening, and in particular that of P450 enzyme inhibition assays for profiling drug-drug interactions. This is partly for Intellectual Property reasons, since Pharmaceutical companies do not wish to give away trade secrets in a competitive market, but also because the technology is not yet 'proven' and remains in the proof-of-concept stage. In droplet microfluidics, where at least two liquid phases are encountered, it is important that leakage of material between phases is addressed. This effect has been extensively reported in the literature using fluorescent dyes, however there is very little evidence of research using large compound sets of diverse chemistry. This is probably because few researchers have access to the large pharmaceutical libraries necessary for this work. This project assessed the feasibility of translating a widely used microtitre plate-based P450 enzyme inhibition assay to droplet format; determined the extent of partitioning from droplets using a large pharmaceutical library set and attempted to model this behaviour, and thirdly, considered the pharmacological impact the droplet format may have on the assay. The P450 cytochrome 1A2 enzyme type (isoform) was chosen for translation to the micro-droplet format. Assays of this type are often conducted using fluorogenic substrates, making them favourable for relatively easy fluorescent detection in droplet format using simple optical detection assemblies. Oil selection was investigated to determine which oil systems would be better suited in respect of droplet formation. The use of surfactants in the oil phase and its impact on droplet formation was studied and the synthesis, preparation and characterisation of a custom perfluoropolyether (PFPE) surfactant ('AZF') conducted. Droplet chips were designed and fabricated to produce droplets of 200-300 µm diameter using novel channel designs and sealing techniques. The droplets were analysed by fluorescence spectroscopy using bespoke detector apparatus. Partitioning from aqueous to oil phase was studied for a small range of compounds and oils (with and without surfactant for fluorous oils). Partitioning was lowest using fluorous oils alone, and increased substantially when surfactant was included. Results from the large pharmaceutical test set suggested the percentage of compounds that may partition readily to the oil phase is low even when using surfactant. However, attempts to correlate this to known physicochemical properties and to develop a predictive model for fluorous solubility proved largely unsuccessful. Partitioning in the droplet chip using a droplet collection pooling method was difficult to quantify as a consequence of the profound impact turbulence had on partitioning. Miniaturisation of the P450 cytochrome inhibition assay to the droplet format initially gave poorly reproducible low signals. Possible causes included detector insensitivity, partitioning of reagent and/or fluorescent metabolite over longer incubation times, and binding of the 1A2 P450 cytochrome enzyme-protein at the droplet interface. Protein interaction at the droplet-oil boundary was studied by fluorescence labelling a protein contained in 200µm droplets and observing the extent of fluorescence localisation at the interface by epifluorescent and confocal fluorescence microscopy. The data from this work indicates a pronounced localisation of protein at the droplet interface, possibly leading to enzyme deactivation and the loss of signal seen for the assay in the droplet chip. A number of protein titrations were co-added to the droplets as 'blocking proteins' which were found to improve the reaction output, however were also noted to affect the pharmacology of the assay, noted by an order of magnitude shift in the reported IC50 for the test inhibitor used (fluvoxamine). The effects of compound leakage from droplets, and the possible detrimental impact on biological reagents by interaction at the droplet-oil interface, is a challenge that may limit widespread adoption of droplet MF systems in drug screening operations. Appropriate control measures and/or a means to reduce these effects are essential to enable accurate quantification with industrial drug discovery environments. The findings in this work highlight the challenges that have to be addressed for droplet microfluidic technology to be successfully incorporated into key areas of assay screening within drug discovery. In terms of further research, there is a significant requirement for the research community to delve further into these challenges and work closely with the industry sector to understand the beneficial role microfluidics can have and how to develop effective robust strategies the industry can easily adopt to progress this area of science.

Cytochrome P450 enzymes (CYP) are key oxidative enzymes that are crucial in several biological processes, such as metabolism of exogenous and endogenous substances, the biological transformation of drugs and xenobiotics and biosynthesis of steroids and fatty acid. Several CYP have been identified in extra hepatic tissues implying that these enzymes exert other biological functions, which might explain their association with a number of diseases including diabetes, obesity and cancer. Understanding of these functions may provide the platform for the development of new therapeutic approaches and this is the aim of this investigation, namely to delineate the role of CYP in breast carcinogenesis. Cancer cells exhibit high levels of glycolysis even in the presence of high oxygen concentration. Cancer cells have very high proliferating rates so they need more biosynthesis materials like nucleic acids, phospholipids, fatty acids and glycolysis is the main source of biosynthetic precursors. Energy metabolism has recently attracted the interest of several laboratories as targeting the pathways for energy production in cancer cells could be an efficient anticancer treatment. Previous studies have shown that reactive oxygen species (ROS) regulate the energy metabolism in cancer cells. CYP are one of the ROS source. Expression of CYP in extrahepatic implies that these enzymes exert other biological functions which have not yet been elucidated. These findings led us to hypothesise that cytochrome P450 enzymes might be involved in the determination of the pathway of cellular energy metabolism in breast cancer cells and in particular in directing tumour cells to produce energy through glycolysis rather than Oxidative phosphorylation (OXPHOS). To investigate the role of CYP in breast carcinogenesis, we followed the protein levels of CYP1B1, CYP1A1, CYP2E1, CYP2C8, CYP2C9 and CYP3A4 in MCF-7 (Michigan Cancer Foundation-7), T47-D, MDA-MB-231 (MD Anderson series 231 cell line) and MDA-MB-468 (MD Anderson series 468 cell line) breast cancer cells treated with glycolytic inhibitors 3-Bromopyruvate and 2-Deoxyglucose (3BP and 2DG). CYP were differentially expressed in breast cancer cells upon treatment with the glycolytic inhibitors (2DG and 3BP) in breast cancer cell lines bearing different genetic background and migratory capacity. The CYP mediated ROS generation was followed in breast cancer cells overexpressing CYP1B1, CYP2C8, CYP2C9 and CYP2E1 or treated with 3BP, 2DG and CYP1B1 specific inhibitor 2,3',4,5'-Tetramethoxystilbene (TMS) by H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) staining. The functional significance of the CYP1B1, CYP2C8, CYP2C9, CYP2E1 mediated modulation of the cellular redox state was investigated by recording changes of indicators of biological pathways known to be affected by the cellular redox state such as cell cycle, adenosine triphosphate (ATP) level, lactate level, mitochondrial potential, autophagy and endoplasmic reticulum (ER) stress. Furthermore, the effect of CYP1B1 and CYP2E1 induction by their inducers (Benzopyrene and Acetaminophen respectively) and inhibition by their specific inhibitors (TMS and chlormethiazole (CMZ) respectively) on cell survival was investigated. Migratory potential of breast cancer cells was investigated under the treatment of glycolytic inhibitors, CYP1B1 inducer and inhibitors. The results obtained provide evidence that CYP are potentially involved in the regulation of ROS, cell cycle, ATP level, lactate level, mitochondrial potential, autophagy, ER stress and migratory potential in a manner dependent on the genetic background of the cells and the stage of the breast cancer, supporting the notion that CYP are potential breast cancer biomarkers.