This thesis investigates the structure and thermal behaviour of LiBH4, NaBH4, KBH4, LiK(BH4)2, Ca(BH4)2 and Sr(BH4)2. LiK(BH4)2 is the first mixed alkali metal borohydride and was synthesised and characterised during this work. The crystal structures of these borohydrides were studied using variable temperature neutron and synchrotron X-ray diffraction. The synthesis of isotopically enriched samples of 7Li11BD4, Li11BD4, Na11BD4 and K11BD4 allowed high quality neutron diffraction data to be collected. Particular attention was paid to the exact geometry of the borohydride ions which were generally found to be perfect tetrahedra but with orientational disorder. New structures of Ca(BH4)2 were identified and the first crystal structure of Sr(BH4)2 was determined from synchrotron X-ray diffraction data. Solid state 11B NMR and Raman spectroscopy provided further information about the structure of these borohydrides. The thermal behaviour of the borohydrides was investigated using thermogravimetric analysis with mass spectrometry of the decomposition gas products. Hydrogen is the main decomposition gas product from all of these compounds but small amounts of B2H6 and BH3 were also detected during decomposition. Thermogravimetic analyses of Na11BD4 and K11BD4 were completed whilst collecting in-situ neutron diffraction data allowing information about structural changes and mass losses to be combined in order to better understand the decomposition process.

The crystal structure of a new bismuth-based light-absorbing material for the application in solar cells was determined by single crystal X-ray diffraction for the first time. (CH3NH3)3(Bi2I9) (MBI) is a promising alternative to recently rapidly progressing hybrid organic–inorganic perovskites due to the higher tolerance against water and low toxicity. Single crystal X-ray diffraction provides detailed structural information as an essential prerequisite to gain a fundamental understanding of structure property relationships, while powder diffraction studies demonstrate a high degree of crystallinity in thin films.

Candida parapsilosis is an opportunistic fungal pathogen of humans causing a variety of infections. Immunocompromised individuals represent the most threatened group of patients. The increasing frequency of infections and occurrence of drug resistant strains are the main reasons for research focused on novel antimycotic compounds. Inhibition of secreted aspartic proteases (Sap) of pathogenic Candida spp. appears to be a potential target of therapeutic intervention. The genome of C. parapsilosis contains at least three genes coding for secreted aspartic proteases, denominated SAPP1-3. Protease Sapp1p has been well biochemically and structurally characterized, whereas Sapp2p and Sapp3p have been given less attention. The first part of the thesis is focused on structural analysis of Sapp1p complexes with selected peptidomimetic inhibitors binding to the active site of the enzyme. In addition, complex of the isoenzyme Sapp2p with the well-known secreted aspartate inhibitor Pepstatin A has been analyzed. The second part is related to the fact that C. parapsilosis belongs to the Candida spp. with the unique ability to translate standard leucine CUG codon mostly as serine. Even though it is a non-conservative substitution of hydrophobic amino acids for a hydrophilic one, this unique ability is maintained for more...

Sem resumo / Sem abstract

Este trabalho enquadra-se dentro dos objetivos da engenharia de cristais moleculares para a obtenção de novas formas sólidas que possam apresentar propriedades farmacêuticas aprimoradas, especificamente de um dos fármacos mais utilizados e comercializados na terapia antirretroviral, contra o HIV: lamivudina, β-L-2\',3\'-didesoxi-3\'-tiocitidina (3TC). As formas cristalinas apresentadas correspondem aos sais dos ácidos inorgânicos: bromidrato (3TCH+-Br-), difluoridrato de hidrogênio (3TCH+-F-HF) e nitrato de lamivudina (3TCH+-NO3-). Estes novos sais cristalizaram no grupo espacial não-centrossimétrico P21, com um par iônico por unidade assimétrica. Os sais halogenados (3TCH+-Br- e 3TCH+-F-HF) apresentaram arranjos supramoleculares isoestruturais inclusive com o sal anidro do cloridrato de lamivudina (3TCH+-Cl-), reportado em trabalhos anteriores no nosso grupo de pesquisa, e cuja solubilidade no equilíbrio apresentou um aumento em relação à forma farmacêutica da 3TC. A característica principal dos arranjos cristalinos destes sais está relacionada com o ordenamento supramolecular das unidades catiônicas 3TCH+, a qual é constante, observando-se a formação de vacâncias entre elas ao longo do eixo cristalino a, decorrente da simetria helicoidal característica do grupo espacial. Desta forma, os ânions se acomodam nos interstícios destas vacâncias estabilizando o arranjo cristalino. Entretanto, o sal 3TCH+NO3- apresentou um comportamento conformacional e supramolecular diferente do observado nos sais halogenados. Neste caso observaram-se a formação de fitas helicoidais ao longo do eixo b, as quais vão se acoplando por simetria translacional na direção horizontal no plano [10-1] por meio de ligações de hidrogênio clássicas do tipo N–H•••O entre os fragmentos citosinicos e O–H•••O dos grupos hidroxilas e os ânions nitrato correspondentemente. Portanto, há a formação de planos moleculares em ziguezague, que posteriormente vão se arquitetando paralelamente na direção [1 0 -1] através de interações de curto alcance. Tanto as características conformacionais e supramoleculares, quanto a pureza exibida pelos sais foram também corroboradas com a ajuda de outras técnicas de análise no estado sólido, como a difração de raios X por pó (DRXP), a análise vibracional no infravermelho (IV) e Raman, e a análise térmica: calorimetria exploratória diferencial (DSC), termogravimetria (TG) e microscopia termo-óptica (Hot-stage). Cálculos de single-point em nível da teoria do funcional da densidade (DFT) foram realizados com o intuito de auxiliar na compreensão de algumas interações intermoleculares. Comparações das propriedades estruturais dos sais sintetizados com algumas formas já reportadas da 3TC (por exemplo, a 3TCH-Cl) permitiram inferir possíveis propriedades farmacêuticas. / This work falls within the main goals of crystal engineering, the improvement of pharmaceutical properties, through the design of new solid forms of the lamivudine, β-L-2 ´, 3´-dideoxy-3´-tiocytidine (3TC), one of the most used and marketed drug in the antiretroviral therapy against HIV. The crystalline forms herein presented correspond to inorganic acid salts: Lamivudine hydrobromide (3TCH+-Br-), hydrogen difluoride (3TCH+-F-HF) and nitrate (3TCH+-NO3-). These new salts crystallized in non-centrossymetric space group P21, with an ionic pair per asymmetric unit. The halogenated salts (3TCH+-Br- and 3TCH+-F-HF) exhibited isostructural supramolecular assemblies, similar to the anhydrous salt of lamivudine hydrochloride (3TCH+-Cl-) reported in a previous studies performed in our research group, and whose equilibrium solubility showed an increase when compared with 3TC pharmaceutical form. The main feature of the salt crystalline assemblies is related to the supramolecular ordering of the 3TCH+ cationic units, which is constant, by observing the formation of vacancies between them along the a crystalline axis due to the helical symmetry, characteristic of their space group. In this way, the anions accommodate themselves into the interstices of these vacancies, stabilizing the crystalline assemblies. Meanwhile, the 3TCH+NO3- salt showed a conformational and supramolecular behavior different from that observed in the halogenated salts. In this case it was observed the formation of helical strands along the b axis, which will be engaging by translational symmetry in the horizontal direction in the [10-1] plane through N–H•••O e O–H•••O classical hydrogen bonds, between the cytosine and hydroxyl fragments and the nitrate anions. Therefore, they form molecular zigzag plans which will subsequently architect parallel with the [10-1] direction by short-contact interactions. Both conformational and supramolecular characteristics as well as the purity exhibited by these salts were also supported with the help of other solid state techniques such as X-ray powder diffraction (XRDP), vibrational analysis as Infrared (IR) and Raman spectroscopy and thermal analysis as differential scanning calorimetry (DSC), thermogravimetry (TG) and hot-stage microscopy. Single point theoretical calculations at the level of density functional theory (DFT) were performed in order to assist in the understanding of some intermolecular interactions. Comparison of the structural properties of the synthesized salts with some forms already reported (e.g. 3TCH+-Cl-) allowed to infer some possible pharmaceutical properties.

DNA-Restriktions und -Modifikationssysteme sind in Prokaryoten weit verbreitet und stellen einen wirksamen Schutz gegen das Eindringen mobiler genetischer Elemente dar. Sie kodieren für eine Restriktionsendonuklease (REase) und eine DNA-Methyltransferase (MTase) gleicher Nukleotidsequenz Spezifität. Die MTase methyliert die zelluläre DNA und schützt sie durch diesen epigenetischen Marker vor der Wirkung der REase. Die REase verhindert die Aufnahme fremder, unmethylierter DNA durch sequenzspezifische Spaltung. EcoRII ist eine REase, die für die effiziente DNA-Spaltung mindestens zwei Kopien ihrer Erkennungssequenz benötigt. Untersuchungen der EcoRII-Struktur und -Funktion offenbarten, dass das Protein aus zwei stabilen Domänen aufgebaut ist, wobei die N-terminale Domäne wie ein Repressor die C-terminale Domäne sterisch blockiert und deren katalytische Aktivität verhindert. Dieser als Autoinhibition bezeichnete und von eukaryotischen Proteinen gut bekannter Regulationsmechanismus wurde erstmals für eine REase vorgeschlagen. In dieser Arbeit konnten wir die Regulation der EcoRII-Enzymaktivität durch Autoinhibition auf molekularer Ebene beweisen. Wir identifizierten ß-Strang 1 (B1: 18YFVYIKR24) und a-Helix 2 (H2: 26SANDT30) als essenzielle inhibitorische Elemente der N-terminalen Domäne des EcoRII-Moleküls. Die Deletion von B1 oder H2 führte zu einer vollständigen Aufhebung der Autoinhibition. Darüber hinaus ist es uns gelungen, die 3D-Röntgenkristallstruktur von EcoRII mit 1,9 Å zu lösen und mit Hilfe von Computermodellen neue Interaktionen des Enzyms mit der DNA „minor groove“ zu beschreiben sowie eine Mg2+-Bindungstasche zu charakterisieren. Die Untersuchung der EcoRII-MTase durch limitierte Proteolyse zeigte, dass das Enzym in Abhängigkeit von der DNA-Sequenz und von seinen Kofaktoren, DNA auf unterschiedliche Weise binden kann. Kristallisierungsversuche der EcoRII-MTase in Anwesenheit der hemi-methylierten DNA-Erkennungssequenz ergaben erste diffraktierende Kristalle, deren Qualität optimiert werden muss und zur Strukturlösung führen soll. / Restriction and modification systems are wide spread among prokaryotes and pre-sent an efficient protection against invasion of mobile genetic elements. In general, they code for a restriction endonuclease (REase) and a DNA-methyltransferase (MTase) of the same DNA specificity. The MTase methylates the cellular DNA and by this epigenetic marker protects it against the action of the REase. The REase pre-vents the entry of foreign unmethylated DNA by site-specific cleavage. EcoRII is an REase which needs at least two copies of the recognition sequence for efficient cleavage. Investigations of the EcoRII structure and function revealed that the pro-tein is composed of two stable domains: the N-terminal domain acts as a repressor by sterically blocking the C-terminal domain and thereby inhibiting its catalytic activity. This regulatory mechanism is known as autoinhibition and has been often described for eukaryotic proteins, but for the first time was proposed for a REase. In this work, we verified the regulation of the EcoRII enzyme activity by autoinhibition at the molecular level. We identified ß-strand 1 (B1: 18YFVYIKR24) and a-helix 2 (H2: 26SANDT30) as essential inhibitory elements of the N-terminal domain. Deletion of B1 or H2 caused a complete abolishment of the autoinhibition. Fur-thermore, we were able to solve the 3D-X-ray crystal structure of EcoRII at 1.9 Å. Based on computer modelling we discovered new interactions between EcoRII and the DNA minor groove and defined the position of the Mg2+ binding pocket. Investigations of the EcoRII MTase by limited proteolysis showed that the enzyme binds DNA depending on DNA sequence and cofactors in different manners. Crystallography experiments with EcoRII MTase in the presence of hemimethylated recognition site DNA showed for the first time diffracting crystals which need further optimisation to create high quality crystals which allow structure solution.

<div>The papain-like protease (PLP) domain of nonstructural protein 3 (nsp3) of the coronavirus (CoV) genome promotes viral replication by processing the CoV polyprotein (protease) and also antagonize innate immune responses by deubiquitinating (DUB) and deISGylating (deISG) host substrates. Selectively removing the DUB/deISG activities of PLP while keeping the protease activity intact is a potential strategy for designing a live attenuated virus. However, it is unclear in the literature the precise mechanism by which PLPs support CoV evasion of the innate immune system. Deciphering the substrate specificity of PLPs for host ubiquitin (Ub) and interferon stimulated gene 15 (ISG15) can therefore help in the design of PLP mutants that selectively lack one activity for evaluating the DUB and deISG mechanism in CoV pathogenesis and replication. </div><div> In this dissertation, we investigate the structure and function of the single PLP (PLpro) from beta-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which are dangerous viral pathogens that emerged from a zoonotic source to cause infectious disease in the human population. Additionally, we translate the knowledge gained to the equivalent PLP2 from alpha-CoV porcine epidemic diarrhea virus (PEDV) and feline infectious peritonitis virus (FIPV), which cause fatal disease in suckling piglets on industrial pork farms and household cats, respectively. The primary objective of this work is to rationally design PLP mutants across beta- and alpha-CoVs to help attenuate CoV infection, as no antiviral or vaccine exist for human CoVs and the efficacy of PEDV vaccines are an ongoing research topic. </div><div><br></div><div>In Chapter 1, different human, animal, and the bat origin CoV strains are introduced. The CoV life-cycle and virion structure are outlined, along with the replicase complex for viral replication. The multidomain nsp3 from alpha- and beta-CoV genomes are also described with a focus on the PLP domain and its proposed cleavage sites of the viral polyprotein. The discovery of the first viral protease DUB and the multiple activities of PLPs are defined, which includes a proposed model of how DUB versus deISG activities may act in the innate immune response. This leads into the therapeutic potential of PLP for an antiviral or live attenuated vaccine, which is followed by the introduction of live attenuated vaccines and the reverse genetics system. Next, proof of concept studies on PLP2 mutants are described and the introduction is concluded by stating the ultimate goal for the design of PLP mutants.</div><div><br></div><div>In Chapter 2, we hypothesize that the flanking ubiquitin-like (Ubl2) domain of MERS-CoV PLpro is not required for its enzymatic function. We characterize the specific activity, kinetics, substrate specificity, and inhibition of the PLpro enzyme with and without the Ubl2 domain and reveal that the Ubl2 domain does not significantly alter PLpro function. We determine the structure of the core PLpro, smallest catalytic unit to 1.9 Å resolution and observed no structural changes compared to the wild-type. Additionally, we demonstrate that a purported MERS-CoV PLpro inhibitor is nonselective in non-reducing conditions and should not be pursed for therapeutic use. We show that the core PLpro enzyme i.e. without the Ubl2 domain is a stable and robust construct for crystallization and is also thermally stable based on thermal melting studies with utility for structure-based drug design. </div><div><br></div><div>In Chapter 3, we shed light on the specificity of SARS-CoV PLpro towards Ub versus ISG15 by characterizing the specific activity and kinetic parameters of SARS-CoV PLpro mutants. In addition, the structure of SARS-CoV PLpro in complex with the C-terminal domain of ISG15 is determined and compared with the Ub-bound structure. Based on the structure and kinetic results, the altered specificities of SARS-CoV PLpro mutants Arg167Glu, Met209Ala, and Gln233Glu are compared with the wild-type. Arg167Glu mutant exhibits DUB hyperactivity and is expected to adopt a more favorable interaction with the Arg42 of Ub. At the same time, ARG167GLU contains a shorter side-chain that hinders interaction with the unique Trp123 of ISG15 for deISG activity compared to the wild-type. These results aid in the development of SARS-CoV PLpro mutants that have directed shifts in substrate specificity for Ub versus ISG15. </div><div><br></div><div>In Chapter 4, the process and antiviral activity of ISGylation is reviewed and how viruses can modulate host-derived versus virus-derived machineries to counteract ISGylation for viral infection. MERS-CoV PLpro is cross-reactive for Ub, but less is known about its specificity towards ISG15. In this study, we determine the structure of MERS-CoV PLpro bound with ISG15 to 2.3 Å resolution and reveal a small hydrophobic pocket of ISG15 that consists of P130 and W123, which differs from Ub hydrophobic patch. We design and determine the kinetic parameters for 13 PLpro mutants and reveal that MERS-CoV PLpro only has a single ubiquitin recognition (SUb1) site. Kinetic studies show that removing the charge of the R1649 greatly enhances DUB/protease activity while mutating in an Arg near R42 of Ub or ISG15 hydrophobic region is detrimental to both DUB/deISG activities. Kinetic experiments and probe-reactivity assays showed that Val1691Arg, Val1691Lys, and His1652Arg mutants are drastically reduced DUB/deISG activities compared to the wild-type. Overall, MERS-CoV PLpro mutants with alter kinetic profiles will be useful for discovery tools and DUB/deISG deficient mutants are great candidates for removing host cell antagonism activity by PLpro for live attenuated vaccines.</div><div><br></div><div>In Chapter 5, the goal is to translate the knowledge gained in Chapters 2-4 on beta-CoVs PLpro and evaluate the substrate specificity of alpha-CoVs FIPV and PEDV PLP2 for mutagenesis experiments. First, we design and purify the core PLP2 enzymes for kinetics. PLP2s are efficient DUBs that prefer Ub to ISG15 in vitro, and this preference is conserved in beta-CoV MHV PLP2 as well as alpha-CoV NL63 PLP2. We determine the structure of alpha-CoV PEDV PLP2 to 1.95 Å resolution and reveal the unique Zn-finger coordinating Cys3-His arrangement of the alpha-CoV genus that differs from past beta-CoV PLP crystal structures. To determine residues of the SUb1 site, we generate a homology model of FIPV PLP2 and overlay our PLP2 structures with MERS-CoV PLpro bound with Ub. In addition, we create electrostatic surface maps across coronaviral PLP subfamilies to evaluate the charge distribution of the SUb1 for the rational design of several FIPV and PEDV PLP2 mutants. We evaluate the turnover of PLP mutants for FRET-based substrates and reveal that His101ArgFIPV and Asn101ArgPEDV are drastically reduced in Ub-AMC activity while their peptide activities are within 2-fold of the wild-type. These mutants show delayed reactivity for Ub probes and no longer cleave Ub-chains displaying isopeptide bonds compared to the wild-type. Results from this study reveal a hot spot in both alpha- and beta-CoVs that can be used to selectively remove DUB activity of PLPs for generating a DUB deficient PLP enzyme. </div><div><br></div><div>In this dissertation, we investigate the substrate specificity of PLPs across alpha- and beta-CoVs and develop a fingerprint for Ub and also shed light on ISG15 recognition. Specifically, hot spots were identified in the SUb1 site of different PLPs, which recognize R42 and hydrophobic Ile44 of Ub. Position 97-98 of PLPs can be used to remove DUB activity by substituting an Arg, but usually effect protease function. Substituting an Arg at position 101 and 136 of coronaviral PLPs serve as the best strategy to remove DUB function while not hindering active site functionality. The DUB/deISG deficient mutants described will be useful for inhibiting the ability of PLPs to function in the innate immune response. Ultimately, this work provides a guide for identifying attenuating mutants in existing CoVs for live attenuated vaccines and also a blueprint for engineering PLPs from new emerging CoVs. </div>

As aminoacil tRNA sintetases (AaRSs) são enzimas essenciais na síntese de proteínas assegurando a correta relação entre os aminoácidos e seus tRNA cognatos. O genoma mitocondrial dos tripanossomatídeos perdeu os genes codificantes dos tRNAs, assim os tRNA mitocondriais são codificados no núcleo e importados do citoplasma. O código genético do kinetoplasto desvia do código genético pela utilização do códon de terminação UGA para a decodificação do códon do triptofano. Um único gene codificando o tRNATrp(CCA) observado no genoma de Leismania é responsável pela incorporação do aminoácido triptofano durante a síntese proteíca na mitocôndria. Para decodificar os dois códons do Trp (UGA e UGG) a base na posição 34 do tRNATrp(CCA) passa por um evento de editoração, convertendo o ribunuclotídeo C34 em U34, produzindo o tRNATrp(UCA) capaz de decodificar o códon UGA. Nesse trabalho foram caracterizadas duas triptofanil tRNA sintetases de Leishmania major. De acordo com experimentos de ?western blotting? e análises ?in silico? das seqüências de aminoácidos, uma enzima tem localização citoplasmática (LmTrpRS1) enquanto a outra mitocondrial (LmTrpRS2). Os mRNAs dos dois genes foram definidos por experimentos de 5? e 3? RT-PCR. As duas enzimas foram clonadas em diversos vetores de expressão procariotos e eucariotos. A LmTrpRS1 foi obtida somente na fração insolúvel, já a LmTrpRS2 foi obtida na fração solúvel quando clonada no vetor de expressão pET28a. Esta porém mostrou-se instável precipitando rapidamente após sua purificação. Os ensaios enzimáticos realizados com a mesma mostraram que ela é capaz de reconhecer os tRNAsTrp editado e não editado. Modelagem molecular por homologia com as duas proteínas foi realizada usando a proteína citoplasmática humana como molde, para estudar a interação entre a proteína e o tRNATrp. Xylella fastidiosa é um bactéria gram negativa limitada ao xilema, responsável por um grande número de doenças economicamente importantes, como a doença de Pierces em videiras, Clorose variegata do Citrus (CVC) e a doença da requeima das folhas em outras plantas incluindo, amendoeira, ameixeira, louro, amoreira e café. Em todos os casos a X. fastidiosa afeta o xylema da planta causando redução na produção de frutos. Nesse trabalho nós mostramos a estrutura da Xylellaína, uma cisteíno protease desse patógeno. A estrutura foi resolvida por dispersão anômala a um único comprimento de onda, utilizando cristais de xylellaína selenometionina substituídos. A estrutura da Xylellaína foi refinada até 1,65 Å de resolução, mostrando enovelamento similar às proteínas da família da papaína, porém algumas características interessantes como uma região N-terminal composta por 38 aminoácidos cobrindo o sulco ativo da enzima, um intrigante ribonucleotídeo encontrado fora do sítio ativo da enzima e um ?loop? semelhante ao ?loop? de oclusão presente na catepsina B. / The aminoacyl tRNA synthetases (aaRSs) are essential enzymes in protein synthesis that ensure the correct match between amino acids and their cognate tRNAs. The mitochondrial (kinetoplast) genome of trypanossomatids lacks tRNA genes, and therefore nucleus-encoded tRNAs are imported from the cytoplasm, the kinetoplast genetic code deviates from the universal code in that UGA instead of UGG encodes for tryptophan. A single nucleus-encoded tRNATrp(CCA) is responsible for Trp insertion during organellar protein synthesis. To decode both Trp codons (UGA and UGG), tRNATrp(CCA) undergoes a single C to U editing event at position 34 of the anticodon yielding to versions of the tRNA in the mitochondria with anticodon CCA and UCA, permitting UGA decoding. This work have characterized two Leishmania major tryptophanyl-tRNA synthetase, acording western blotting experiments and ?in silico? sequence analisis one of cytoplasmatic localization (LmTrpRS1) and another from mitochondria localization (LmTrpRS2). The mature mRNA transcripts for both genes were defined by 5? and 3? RT-PCR. Both enzymes were cloned into several expressions vectors. LmTrpRs1 was obtained as an insoluble protein and LmTrpRs2 expressed into the soluble fraction in pET28a expression system. LmTrpRS2 protein, however, is unstable precipitating shortly after purification. The enzymatic assay showed that this enzyme is able to recognize both tRNATrp. Molecular modeling for LmTrpRS1 and LmTrpRS2 were constructed using the cytoplasmatic human tryptophanyl tRNA synthetase as a model, to study the interaction between proteins and tRNATrp. Xylella fastidiosa is a xylem-limited, gram-negative bacteria responsible for a large number of economically important plant diseases, such as Pierces disease in grapevines, citrus variegated chlorosis (CVC) in sweet oranges and leaf scorch diseases in other plants, including almond, plum, oleander, mulberry and coffee. In all cases, X. fastidiosa infects the plant xylem and impairs fruit production. Here, we report the crystal structure of xylellain, a cystein protease from X. fastidiosa. The structure was solved by single-wavelength anomalous dispersion (SAD) using seleno-methionine containing xylellain crystals. The final structure of Xylellaína was refined against the best native data set (1.65 Å) showing R/Rfree= 17/21. Xylellain shares fold similar to Papain like Family, but contains some interesting features, like a 38 N-terminal tail covering the active site cleft; one intriguing ribonucleotide found outside the active site and one loop that resemble the ocluding loop from cathepsin B.

Polymer-based piezoelectric and triboelectric generators form the basis of well-known energy harvesting methods that are capable of transforming ambient vibrational energy into electrical energy via electrical polarization changes in a material and contact electrification, respectively. However, the low energy conversion efficiency and limited thermal stability of polymeric materials hinder practical application. While nanostructured polymers and polymer-based nanocomposites have been widely studied to overcome these limitations, the performance improvement has not been satisfactory due to limitations pertaining to long-standing problems associated with polymeric materials; such as low crystallinity of nanostructured polymers, and in the case of nanocomposites, poor dispersion and distribution of nanoparticles in the polymer matrix. In this thesis, novel functional polymeric nanomaterials, for stable and physically robust energy harvesting applications, are proposed by developing advanced nanofabrication methods. The focus is on ferroelectric polymeric nanomaterials, as this class of materials is particularly well-suited for both piezoelectric and triboelectric energy harvesting. The thesis is broadly divided into two parts. The first part focuses on Nylon-11 nanowires grown by a template-wetting method. Nylon-11 was chosen due to its reasonably good ferroelectric properties and high thermal stability, relative to more commonly studied ferroelectric polymers such as polyvinylidene fluoride (PVDF) and polyvinylidene fluoride-trifluoroethylene (P(VDF-TrFE)). However, limitations in thin-film fabrication of Nylon-11 have led to poor control over crystallinity, and thus investigation of this material for practical applications had been mostly discontinued, and its energy harvesting potential never fully realised. The work in this thesis shows that these problems can be overcome by adopting nanoporous template-wetting as a versatile tool to grow Nylon-11 nanowires with controlled crystallinity. Since the template-grown Nylon-11 nanowires exhibit a polarisation without any additional electrical poling process by exploiting the nanoconfinement effect, they have been directly incorporated into nano-piezoelectric generators, exhibiting high temperature stability and excellent fatigue performance. To further enhance the energy harvesting capability of Nylon-11 nanowires, a gas -flow assisted nano-template (GANT) infiltration method has been developed, whereby rapid crystallisation induced by gas-flow leads to the formation of the ferroelectric δʹ-phase. The well-defined crystallisation conditions resulting from the GANT method not only lead to self-polarization but also increases average crystallinity from 29 % to 38 %. δʹ-phase Nylon-11 nanowires introduced into a prototype triboelectric generator are shown to give rise to a six-fold increase in output power density as observed relative to the δʹ-phase film-based device. Interestingly, based on the accumulated understanding of the template-wetting method, Nylon-11, and energy harvesting devices, it was found that thermodynamically stable α-phase Nylon-11 nanowires are most suitable for triboelectric energy generators, but not piezoelectric generators. Notably, definitive dipole alignment of α-phase nanowires is shown to have been achieved for the first time via a novel thermally assisted nano-template infiltration (TANI) method, resulting in exceptionally strong and thermally stable spontaneous polarization, as confirmed by molecular structure simulations. The output power density of a triboelectric generator based on α-phase nanowires is shown to be enhanced by 328 % compared to a δʹ-phase nanowire-based device under the same mechanical excitation. The second part of the thesis presents recent progress on polymer-based multi-layered nanocomposites for energy harvesting applications. To solve the existing issues related to poor dispersion and distribution of nanoparticles in the polymer matrix, a dual aerosol-jet printing method has been developed and applied. As a result, outstanding dispersion and distribution. Furthermore, this method allows precise control of the various physical properties of interest, including the dielectric permittivity. The resulting nanocomposite contributes to an overall enhancement of the device capacitance, which also leads to high-performance triboelectric generators. This thesis therefore presents advances in novel functional polymeric nanomaterials for energy harvesting applications, with improved performance and thermal stability. It further offers insight regarding the long-standing issues in the field of Nylon-11, template-wetting, and polymer-based nanocomposites.

Propriedades de estado sólido estão diretamente relacionadas ao desempenho de um fármaco. Entre todas as propriedades físicas e químicas dependentes da fase cristalina de um fármaco, estabilidade e solubilidade são as que mais alteram sua biodisponibilidade. Neste sentido, a engenharia de cristais moleculares é uma estratégia para aperfeiçoar as propriedades de estado sólido relacionadas às eficácias dos fármacos. Neste trabalho, nove novas formas cristalinas de fármacos antirretrovirais inibidores nucleosídeos de transcriptase reversa, a saber, lamivudina, zalcitabina e didanosina, foram preparadas e suas estruturas cristalinas foram elucidadas por difração de raios X por monocristal. Parte das modificações cristalinas preparadas foi também caracterizada por microscopia eletrônica de varredura, difração de raios X por pó, espectroscopia vibracional no infravermelho e Raman, calorimetria exploratória diferencial e termogravimetria. As solubilidades aquosas e purezas das modificações cristalinas de lamivudina preparadas como amostras monofásicas foram determinadas por espectrofotometria de absorvância no ultravioleta e cromatografia líquida de alta eficiência, respectivamente. A solubilidade de lamivudina nas modificações cristalinas preparadas pode ser tanto aumentada quanto reduzida quando comparada com a solubilidade da fase cristalina do fármaco incorporada em formulações farmacêuticas. As solubilidades foram também correlacionadas às características estruturais e calorimétricas, o que permitiu o estabelecimento de relações entre estrutura/energia de rede cristalina e propriedade de estado sólido. Ainda, duas modificações cristalinas de lamivudina, em que moléculas do fármaco estão pareadas através de seus fragmentos de citosina, sendo estes pares helicoidalmente sobrepostos, mimetizando uma estrutura polimérica de ácido desoxirribonucléico, revelaram que nucleosídeos têm a informação estrutural necessária para arquitetar duplas hélices de ácidos nucléicos. / Solid state properties are directly related to drug performance. Among all physical and chemical properties dependent on the crystal phase of a drug, stability and solubility are the main ones that alter its bioavailability. In this way, molecular crystal engineering is a strategy to improve solid state properties of drugs related to their efficacies. In this work, nine new crystal forms of antiretroviral nucleoside reverse transcriptase inhibitor drugs, namely, lamivudine, zalcitabina and didanosine, were prepared and their crystal structures were elucidated by single crystal X-ray diffraction. Some of the prepared crystal modifications were also characterized by scanning electron microscopy, powder X-ray diffraction, infrared and Raman vibrational spectroscopy, differential scanning calorimetry and thermogravimetry. The water solubilities and purities of the lamivudine crystal modifications prepared as monophasic samples were determined by ultraviolet absorbance spectrophotometry and high performance liquid chromatography, respectively. The solubility of lamivudine in the prepared crystal modifications can be either increased or decreased when compared to the solubility of the drug crystal phase incorporated into pharmaceutical formulations. The solubilities were also correlated to calorimetric and structural features, which allowed the establishment of relationships between crystal lattice energy/structure and solid state property. In addition, two crystal modifications of lamivudine, in which drug molecules are paired through their cytosine fragments, being these pairs helically stacked, mimicking a polymeric structure of deoxyribonucleic acid, have revealed that nucleosides possess the structural information necessary to assemble double stranded helices of nucleic acids.