Protein kinases are major drug targets for many diseases. Among these are the STE20/SPS1-related proline/alanine-rich kinase (SPAK) and the oxidative- stress-responsive kinase 1 (OSR1), which are two related serine/threonine protein kinases. Both kinases are key components of the WNK-SPAK/OSR1 signalling pathway that has emerged as a key regulator of electrolyte homeostasis, body fluid and blood pressure. Various knock-in and knock-out SPAK and OSR1 mouse models exhibited reduced blood pressure. This highlighted SPAK and OSR1 kinases as promising targets in the treatment of hypertension. Encouraged by this, this project was initiated to discover specific WNK-signalling inhibitors by targeting SPAK and OSR1 kinases as potential novel antihypertensive agents. My work led to the identification of an allosteric pocket located in the highly conserved C-terminal domains of SPAK and OSR1, which influences their kinase activities. Using in silico screening, Rafoxanide, an anti-parasitic agent, was identified as a novel allosteric inhibitor of SPAK and OSR1. Additionally, high throughput screening led to the discovery of the clinically used agent, Verteporfin, as a novel and potent WNK-signalling inhibitor. Moreover, several fragment-binders to the C-terminal domain of OSR1 kinase were identified using NMRfragment based screening. In addition, the NMR backbone assignments of the C-terminal domain of OSR1 kinase were determined and used to map the previously unknown binding site of different OSR1 and SPAK inhibitors. Collectively, these findings have significantly advanced the field of SPAK and OSR1 kinase inhibition and provided key tools that will facilitate the future discovery of other SPAK and OSR1 kinase inhibitors.

Solos com horizontes húmicos e hísticos de ecossistemas tropicais e subtropicais de altitude desempenham importante função no estoque do C atmosférico, na regulação climática e dos fluxos hidrológicos, na retenção de água no solo e na atuação enquanto filtro ambiental. Além disso, esses solos que acumulam naturalmente C funcionam como arquivos da história natural, pois sua gênese está diretamente ligada às condições ambientais vigentes durante o processo de sua formação. O presente estudo teve como objetivo investigar a origem, composição e estabilidade da MOS e o efeito da matéria orgânica (MOS) sobre a estabilidade de agregados em perfis de solo com horizontes húmicos e hísticos sob floresta de Araucária. Amostras indeformadas foram coletadas em diferentes profundidades em uma topossequência sob Floresta Ombrófila mista na bacia sedimentar de Curitiba / PR - área de proteção ambiental do Iraí. Os solos foram classificados como Latossolo Bruno Alumínico rúbrico, Gleissolo Melânico Ta Distrófico organossólico e Organossolo Háplico Sáprico típico. Em cada perfil também foram coletadas amostras de serapilheira. No Estudo 1 nós determinamos os estoques de C e N, a estabilidade desses estoques por ensaios de mineralização de C e monitoramos a alteração da composição química da MOS antes e depois da incubação. Os resultados mostraram que o enriquecimento de C na topossequência cresceu do topo da vertente para a planície aluvial e que as diferenças observadas no teor e na composição química da MOS foram definidas pelo regime hídrico do solo. Aproximadamente 70% do estoque de C encontra-se armazenado em horizontes sub-superficiais, sendo a estabilidade desses estoques maior quando comparada à superfície. Em condições anóxicas os açúcares são relativamente mais degradados, havendo enriquecimento relativo de compostos aromáticos e alifáticos. No Estudo 2 nós investigamos a influência da composição química da MOS na estabilidade de agregados dos perfis, avaliada pela dispersão por ultrassom. Os resultados mostraram que os compostos alifáticos e estruturas do tipo carboidrato foram relevantes para a estabilidade dos agregados. No solo mais intemperizado, as interações organo-minerais entre os grupos C-O-alquil e a superfície dos argilas foi o mecanismo mais importante na agregação, ao passo que no gleissolo e no organossolo o mecanismo predominante foi a proteção hidrofóbica conferida pela autoassociação das zonas hidrofóbicas da MOS, envolvendo principalmente os grupos alifáticos da estrutura. No Estudo 3 nós utilizamos a MOS como um traçador de paleo-mudanças ambientais, com objetivo de identificar alterações vegetacionais em função de mudanças climáticas. Os resultados mostraram que no topo da vertente a ciclagem da MOS foi mais intensa, havendo maior contribuição da floresta (vegetação C3) para a composição da MOS. Entretanto, nas posições mais baixas da paisagem, a composição da MOS é derivada principalmente de vegetação C4 (campo), que teria predominado no passado. Esses resultados corroboram a mudança de um clima seco para um clima mais úmido na região, que teria ocorrido durante o Holoceno. / Soils with humic and histic horizons of tropical and subtropical ecosystems of altitude play an important role in stock of atmospheric C, climate regulation, water retention and environmental filter. Furthermore, these C-rich soils are archives of natural history, because their genesis is directly linked to environmental conditions during the process of their formation. This study aimed to investigate the origin, composition and stability of organic matter (SOM) and the effect of SOM composition on aggregate stability in soil profiles with humic and histic horizons under Araucaria forest. Undisturbed samples were collected from different depths in a toposequence under rain forest in the sedimentary basin of Curitiba, Brazil, located in the environmental protected area of Iraí. The soils were classified as Oxisol, Gleysol and Histosol. In each profile we collected different litter fractions. In the first study we determined the C and N stocks, the stability of these stocks by C mineralization experiment and we monitored the changes in the SOM chemical composition before and after incubation. The results showed that the C content increased from the top to the flood plain and that the differences in content and SOM chemical composition were defined by soil water regime. Approximately 70% of C stock is stored in subsurface horizons, with greater stability of these stocks when it is compared to the surface horizon. In anoxic conditions sugars are relatively more degraded, with greater relative enrichment of aromatic and aliphatic compounds. In the second study we investigated the influence of the SOM chemical composition on aggregate stability measured by ultrasound energy application. The results showed that aliphatic compounds and the carbohydrate structures are important for the structural stability. In the oxisol the organo-mineral interactions between the CO-alkyl groups and the clays surface was the most important mechanism of aggregation, while in the gleysol and in the histosol the predominant mechanism iwas the hydrophobic protection by the self-association of the hydrophobic zones of SOM, involving the aliphatic structures. In the third study we use the SOM as a paleoenvironmental tracer to try to identify the vegetation changes due to climate change in the past. The results showed thatin the top the SOM turnover was more effective, eith higher contribution of the forest (C3 vegetation) to the SOM composition. However, at the lower landscape positions, the SOM composition is mainly derived from the C4 vegetation (grassland) ) that would have prevailed in the past. Our results support the change of a dry to a more humid climate in that region, occurred during the Holocene.

Um modelo para redes de neurônios é estudado onde a função energia E é dada pelo produto do quadrado das distâncias no espaço de fase entre o estado S da rede e os P padrões armazenados.Os tempos de convergência e as correspondentes dispersões também foram estudados numericamente e estão relacionados ao número de estados espúrios ao redor de cada configuração armazenada. Ambas quantidades sâo pequenas na fase de recuperaçâo , indicando a homogeneidade e suavidade do espaço de fase. / A model for neural networks is studied through a multineuron interaction energy function E given by the product of the squared distances in phase space between the state S of the net and the P stored patterns. Convergence times and the corresponding dispersions have also been studied numerically a.s para.meters to measure the efficiency of this model. These quantities are related to the number of spurious sLates for ea.ch configuration of stored pa.tterns. Both the average convergence time and the dispersions are small in the retrieval phase, indicating the homogeneity and smoothness of the phase space.

No Brasil, o acúmulo de carbono orgânico no solo (COS) tem sido obtido em plantio direto (NT), que faz parte do plano de agricultura de baixo carbono para a mitigação da emissão de gases do efeito estufa. Quando combinado com leguminosas de cobertura, o NT pode resultar ainda em maiores taxas de acúmulo de COS. Baseado num experimento de 30 anos, seis estudos foram realizados com o objetivo geral de avaliar o efeito de dois métodos de preparo (preparo convencional –CT e NT) com três sistemas de culturas [aveia/milho (O/M), ervilhaca/milho (V/M) e o consórcio aveia+ervilhaca/milho+caupi (OV/MC)] sobre os mecanismos de estabilização do C e consequente potencial de sequestro de COS em camadas superficias e subsuperficias de um Argissolo subtropical do Sul do Brasil. O primeiro estudo engloba uma metaanálise global avaliando o potencial do NT em acumular COS. Os resultados deste estudo mostraram um grande potencial de acumular COS em regiões tropicais e subtropicais, o que foi explicado pela alta precipitação média anual nessas regiões. O segundo e terceiro estudos mostraram um importante mas diferenciado papel do plantio direto e das leguminosas de coberturas sobre a estabilização do C no solo, sendo que o não revolvimento favoreceu a oclusão da matéria orgânica em macroagregados enquanto que as leguminosas favoreceram a associação organomineral em microagregados Neste estudo, a importância dos macroagregados sobre a associação organomineral foi enaltecida. Além disso, o quarto estudo revelou forte contribuição das leguminosas de cobertura sobre o potencial de acumular COS em NT, sendo que altas taxas de acúmulo de COS ocorreram durante um período mais longo em camadas subsuperficias do solo. No quinto estudo, uma combinação de fracionamento densimétrico e granulométrico com análises de carboidratos e n-alcanos foi realizada objetivando maior entendimento do papel das leguminosas de cobertura sobre o acúmulo de C em associações organomineral. Este estudo deu suporte à hipótese que o acúmulo de C adicional na fração argila em camadas superficiais e subsuperficias do NT com leguminosas de cobertura é microbiologicamente processado. No sexto estudo, encontramos que o maior teor de C na fração leve do solo sob plantio direto e leguminosas de cobertura favoreceu a comunidade de fungos que, por sua vez, mediou a melhora na agregação de solo. O acúmulo de constituintes da parede celular de fungos contribuiu, portanto, ao acúmulo de COS e agregação de solo sob plantio direto e leguminosas de cobertura. / In Brazil, soil organic carbon (SOC) accumulation have been observed under notillage (NT) which is part of a low carbon agriculture plan for mitigation of greenhouse gases emissions. When combined with legume cover crops, NT could result in even greater soil organic carbon (SOC) sequestration rate than NT alone. Using a 30-year experiment, we performed six studies for which the general objective was to evaluate the effect of two tillage (conventional system – CT and NT) with three cropping systems [oat/maize (O/M), vetch/maize (V/M) and the consortium oat+vetch/maize+cowpea (OV/MC)] on stabilization mechanisms of C and consequent SOC sequestration potential in both the superficial and sub-superficial soil layers of a subtropical Acrisol of Southern Brazil. Encompassing a meta-analysis evaluating the potential of NT in accumulate SOC around the world, the first study showed a great potential in tropical and subtropical regions to accumulate SOC that was explained by the high mean annual precipitation. In addition, the second and third studies showed the meaningful but differential roles of no-tillage and legume cover crops on C stabilization with the former favoring occlusion in soil macroaggregates and the latter, mineral-organic association in soil microaggregates. In these studies, we supported the importance of macroaggregates to the mineral-organic association The fourth study revealed the strong contribution of legume on the potential of SOC sequestration in NT, and high rates of C accumulation occurred over a longer period in sub-superficial soil layers. In the fifth study, we used density and particle size fractionation in combination with carbohydrate and nalkane analyses to provide better understanding of the effect of legume cover crop on C accumulation in mineral-organic association. This study gives support to the hypothesis that the additional clay-bound SOC accumulation at depth under NT with legume cover crops is microbially processed. In the sixth study, we found that the greater C content in light fraction under NT and legume cover crops favoured the fungal community which, in turn, mediated the improvement in soil aggregation. The accrual of fungal cell-wall constituents contributed therefore to SOC accumulation and soil aggregation under NT and legume cover crops.

The objective of this work was to develop suitable delivery systems for biological agents that have antimicrobial activities using biocompatible polymers, aiming to reduce their toxicity when administered. Two biological agents, colistin as an antibacterial agent and nystatin (Nys) as an antifungal agent, are the focus of this thesis as they are potent treatments for current pathogen infections, especially to the multidrug-resistant (MDR) bacteria/fungi, but have potential toxicity to human. Polymeric drug delivery systems, including prodrug, hydrogel and micelle formulations, have been developed and discussed for their potential as topical and systemic regimes. The majority of the work was focused on the effect of the covalently attachment of synthetic polymers onto the biological agents upon their antimicrobial activities and the toxicity. The conjugation between colistin and polymers was achieved successfully through either irreversible or releasable linkages. Although irreversible polymer modifications on colistin showed no antimicrobial activity (chapter 2), an acceptable antibacterial activity was observed from the polymer-colistin conjugates with a releasable linkage through either ‘grafting-to’ (chapter 3) or ‘grafting-from’ (chapter 4) approaches. On the other hand, even though the pure polymer-Nys conjugate with a releasable imine linkage cannot be obtained due to the nature of the labile imine bond, the crude conjugate showed an excellent antifungal activity and a reduced toxicity compared to the native Nys (chapter 6). Other polymeric delivery systems were also discussed in this thesis. The incorporation of colistin within a developed hydrogel delivery system as an antibacterial patch for burn infections was investigated through in vitro and in vivo studies, showing a similar antibacterial activity as the native colistin solution against MDR Gram-negative bacteria with no systemic toxicity (chapter 5). Finally, an amphiphilic polymer containing boronic acid groups on the side chains was synthesised and used to target the hydroxyl groups on Nys, expecting to build up an environmental responsive micelle through dynamic boronate ester bond (chapter 7). Although more work is still needed, this system showed a potential to improve Nys solubility.

Natural α,β-epoxyketone proteasome inhibitors produced primarily by Streptomyces species, such as TMC-86A, are potent anticancer compounds. The enzymes responsible for biosynthesis of their key α,β-epoxyketone pharmacophore had yet to be determined at the onset of this research. Elucidation of the responsible enzymes and their putative substrate would allow for stereoselective completion of a synthetically challenging epoxidation reaction, responsible for the high cost of epoxyketone pharmaceuticals produced by industry. The cytochrome P450 TmcI and the flavin-dependent TmcF enzymes from the TMC- 86A biosynthetic gene cluster in Streptomyces chromofuscus, believed to have a role in epoxyketone biosynthesis, were cloned, expressed and overproduced. Potential substrates were produced through a peptide coupling synthetic route. In vitro assays demonstrated that EpnF, a TmcF homologue, could produce α,β-epoxyketone compounds with an α-dimethyl-β-keto carboxylic acid substrate. These substrates were prone to decarboxylative degradation, therefore stable methyl ester precursors were synthesised as an alternative. Using an esterase, methyl ester precursors were hydrolysed to reproduce the previously observed epoxyketone compound in a one pot reaction with EpnF. Substrate specificity of EpnF was probed by in vitro assay using an alanine derived analogue, which was found to be a viable substrate for the enzyme. Lastly, the EpnF catalytic cycle was interrogated with a synthesised authentic standard of a proposed intermediate, which was observed to co-elute with the desired intermediate from a quenched EpnF assay using UHPLC-MS chromatography.

Chapter 1 begins with an introduction to azetidines, including a discussion of the methodologies for their synthesis, their applications, relevance in natural products and as building blocks in medicinal chemistry. It then describes the development of a new asymmetric route to 2-substituted azetidin-3-ones using Enders’ SAMP/RAMP auxiliary. A one-pot process was developed involving the metalation of SAMP hydrazones of N-Boc-azetidin-3-one, alkylation and subsequent in situ hydrolysis to give the substituted products. Various bases and reaction conditions were explored to find optimal conditions for maximal yield and enantioselectivity. A representative range of electrophiles were screened including alkyl, allyl and benzyl halides and carbonyl compounds, producing enantioselectivities of up to 85% ee. Multiple substitution on the azetidin-3-one ring was briefly explored by repetition of the alkylation/hydrolysis sequence. Derivitisation by way of Pictet-Spengler reactions was used to confirm the absolute configuration at the newly created stereocentre. Chapter 2 begins with an introduction to 1,2-diazetidines outlining methods for their synthesis, before introducing the relevance of these nitrogen spirocycles. This chapter then describes two routes for the synthesis of these novel spirocyclic 1,2- diazetidines by (i) formation of the diazetidine ring and (ii) functionalisation of a range of 3-methylene-1,2-diazetidines including differentially protected variants. The diazetidines were subjected to dichloro- and difluorocyclopropanation with the latter achieved in high yields. Additionally, reactions with tetracyanoethylene by way of highly asynchronous [2π+2π] cycloadditions proceeded in near quantitative yield. In this way, a range of novel 4,5-diazaspiro[2.3]hexane and 1,2- diazaspiro[3.3]heptane spirocycles were produced. Chapter 3 details the experimental procedure and characterisation for all the novel compounds synthesised.

The natural compound cordycepin (3’-deoxyadenosine) causes a reduction in breast cancer cell viability. Microarray analysis showed that growth related genes are down-regulated by cordycepin. Indeed, mTOR, ERK and AMPK signalling was shown to be altered by cordycepin, but the effect was too fast to be mediated by transcriptional changes. It was hypothesised that cordycepin affected signal transduction through translation. However, polysome profiling did not identify clear candidates for the effects of cordycepin on signal transduction but unveiled that cordycepin leads to translation repression on 5’ terminal oligopyrimidine (TOP) mRNAs. As TOP mRNAs are known to be regulated by mTOR signaling, this result consistently suggests mTOR signaling is inhibited by cordycepin treatment. To test if it is possible that cordycepin affects gene expression via signal transduction, we compared its effects to various signal transduction inhibitors and an activator. So far, Pictilisib, a pan-PI3K inhibitor, is the only inhibitor that mimics both the gene expression and signal transduction effects of cordycepin, indicating the PI3K-PDK1-AKT axis is affected by cordycepin. The RNAs upregulated by cordycepin were highly enriched in a group of non-coding RNAs, which are also appeared to induce during serum withdrawal. Knockdown of poly(A) polymerases induced these RNAs, indicating that they probably are degraded by the PABPN1 and poly(A) polymerase dependent nuclear RNA decay pathway. Thus the data suggest that cordycepin affects gene regulation by two distinct pathways, one affecting signal transduction and growth related mRNA expression and another affecting polyadenylation mediated decay of non-coding mRNAs.

Neste trabalho, é feito um estudo de sistemas de valência intermediária e sistemas Kondo, utilizando um método diagramático para calcular as funções de Green, necessárias ao cálculo das densidades de estado, para estes sistemas. / We studied intermediate valence and Kondo systems using a diagrammatic technique to calculate the Green functions.

Cordycepin (3′-deoxyadenosine) is a medicinal bioactive component of the caterpillar fungi (Cordyceps and Ophicordyceps). It is reported to have nephroprotective, antiapoptotic, anti-metastatic, hepatoprotective (Yue et al. 2013), inflammatory effects, antioxidant, anti-tumor, immunomodulatory and vasorelaxation activities. Cordycepin is well known to terminate and inhibit polyadenylation, both in vitro and in vivo. Other proposed mechanisms of action of cordycepin include activation of adenosine receptors, activation of AMP dependent kinase (AMPK) and inhibition of PARP1. The purpose of this study is to elucidate the biological and pharmacological effects of cordycepin on cancer cell lines such as MCF-7 cells. In this study I found that cordycepin reduces the cell proliferation in all examined cell lines without always exerting an effect on 4EBP phosphorylation and protein synthesis rates. Therefore, the effects on protein synthesis via inhibition of mTOR, which were previously reported, are not only the sole reason for the effect of cordycepin on cell proliferation. Knockdown of poly (A) polymerases reduces cell proliferation and survival, indicating that poly (A) polymerases are potential targets of cordycepin. I studied different adenosine analogues and found that 8 aminoadenosine, the only one that also consistently inhibits polyadenylation, also reduces levels of P-4EBP. It also inhibits the expression of specific genes indicating that the effects on polyadenylation, mTOR signalling and gene expression are linked. Also consistent with polyadenylation inhibition as the major mode of action is the fact that the effects of cordycepin on gene expression are predominantly post-transcriptional. However, knockdown of poly (A) polymerases did not have the same effects on gene expression or on polyadenylation, indicating that cordycepin may act as a dominant negative rather than as a null mutant. This is consistent with the fact that cordycepin is known to arrest a normally transient polyadenylation complex. We performed microarray analysis of cordycepin treated MCF-7 cells and found that the downregulated mRNAs were predominantly involved in transcriptional regulation, cell proliferation, cell cycle and cell migration. These data show that cordycepin is a promising new drug for cancer and indicates that the mode of action it is likely to be through the inhibition of polyadenylation.