Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.

The thesis deals with the problem of friction during rapid transient 1-D flows in a pipe caused by water hammers. The evolution of the wall shear stress is interpreted in terms of two steps. The first step is the dramatic change of the wall shear stress during the passage of the pressure wave; the corresponding new value of the shear stress is much greater than the value predicted in steady-state. The second step, which begins after the passage of the pressure wave, is a relaxation process; here the shear stress decreases, tending to the new steady-state value corresponding to the new average velocity. The Extended Irreversible Thermodynamics theory is proposed as a tool to model the wall shear stress during the relaxation process. The Friction Relaxation Model presented in this thesis describes both steps of the evolution of the wall shear stress during water hammers, and therefore it enables to take into account the information about the velocity gradient at the wall, which is otherwise not available in 1D modelling.

Irreversible thermodynamics is a widely-applicable toolset that extends thermodynamics to describe systems undergoing irreversible processes. It is particularly useful for describing macroscopic flow of system components, whether conserved (e.g., particle number) or non-conserved (e.g., spin). We give a general introduction to this toolset and calculate the entropy production due to bulk and interfacial flow. We compare the entropy production and heating rate of bulk and interfacial transport, as well as interfacial charge and spin transport. We then demonstrate the power and applicability of this toolset by applying it to three systems. We first consider metal oxide growth, and discuss inconsistency in previous theory by Mott. We show, however, that Mott's solution is the lowest order of a consistent asymptotic solution, with the ion and electron concentrations and fluxes going as power series in t^-k/2, where k = 1, 2, .... We find that this gives corrections to the "parabolic growth law" that has oxide thickness going as t^1/2; the lowest order correction is logarithmic in t. We then consider the effect on spin of electric currents crossing an interface between a ferromagnet (FM) and non-magnetic material (NM). Previous theories for electrical potential and spin accumulation neglect chemical or magnetic contributions to the energy. We apply irreversible thermodynamics to show that both contributions are pivotal in predicting the spin accumulation, particularly in the NM. We also show that charge screening, not considered in previous theories, causes spin accumulation in the FM, which may be important in ferromagnetic semiconductors. Finally, we apply irreversible thermodynamics to thermal equilibration in a thin-film FM on a substrate. Recent experiments suggest that applying a thermal gradient across the length of the system causes a spin current along the thickness; this spin current is present much farther from the heat sources than expected. We find that, although the interaction between the separate thermal equilibration processes increases the largest equilibration length, thermal equilibration does not predict a length as large as the experimentally measured length; it does predict, however, a thermal gradient along the thickness that has the shape of the measured spin current.

Very little work has been done to study and understand the internal mechanisms that provide goose down with its resiliency under repeated compression. We have employed low magnification optical microscopy to identify some of these important mechanisms. Microscopy showed that a small tertiary structure exists on most goose down fibers and creates an important point of contact when two fibers interact. This tertiary contact mechanism has been coupled with fiber orientation and incorporated into a unique strain-energy function. The principal stresses for an initial compression cycle can be determined from this strain-energy function according to the hyperelastic constitutive theory. Irreversible deformation and hysteresis necessitate another means to determine the stresses during unloading and reloading. For these stages, the framework used by Beatty et al. (2002) for an ideal Mullins material will be utilized in conjunction with a shift in the stress-free state to determine the principal stresses. The proposed model is then evaluated for uniaxial compression and shown to capture the general behavior of goose down in compression including the irreversible deformation and hysteresis.

Cysteine proteases are a class of proteolytic enzymes, which are involved in a series of metabolic and catabolic processes, such as protein turnover, digestion, blood coagulation, apoptosis, fertilization and cell differentiation, and the immune response system. The development of novel potent and selective inhibitors for cysteine proteases has therefore gained increasing attention among medicinal chemists. In this thesis we have reported the design, synthesis, and evaluation of several peptidyl inhibitors for clan CA and clan CD cysteine proteases. We have continued the investigation of dipeptidyl vinyl sulfones as potent and selective inhibitors for dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease, which is involved in the processing of intracellular proteases, such as granzymes. We have found that DPPI tolerates negatively charged amino acid residues in the P2 position with inhibition rates of 7,600 M-1s-1. Dipeptidyl vinyl sulfones with positively charged amino acid residues at the P1 position, however, do not inhibit DPPI at all. A second project focused on the epoxidation of the double bond of the vinyl sulfone moiety of the dipeptidyl vinyl sulfones. Instead of epoxidizing the double bond, we found that an isomerization had occurred. The newly formed compounds were determined to be allyl sulfones. We tested this new class of inhibitors with clan CA proteases and obtained inhibition rates of 560 M-1s-1 for Cbz-Leu-Phe-AS-Ph with calpain I. Two new classes of compounds for the clan CD protease S. mansoni legumain were designed, synthesized, and evaluated. Aza-peptidyl epoxides were found to be potent and selective inhibitors of S. mansoni legumain with IC50’s as low as 45 nM. Aza-peptide Michael acceptors were derived from the aza-peptide epoxide design and synthesized in an analogous fashion. The aza-peptide Michael acceptors inhibited S. mansoni legumain with even lower IC50’s, as low as 10 nM. However, the aza-peptide Michael acceptors react with thioalkylating agents contained in the buffer, such as DTT. The rates of degradation were determined spectroscopically, and half-lives of 3 to 20 minutes were measured. This observation gave us insights into the enzymatic mechanism and allowed us to determine the point of attack for the legumain active site cysteine thiol.

Both clan CA and clan CD proteases have a variety of physiological and pathological roles. In particular, both clans have members who have been implicated in cell death pathways, including apoptosis. Caspases are members of clan CD. Many of the caspase inhibitors used in apoptotic studies have shown cross reactivity with clan CA proteases. Thus, the anti-apoptotic effect of these inhibitors could be due to the broad-spectrum inhibition of a variety of cysteine proteases. Recently, the Powers laboratory designed a new class of inhibitors highly specific for clan CD proteases, aza-peptide epoxides. Initial data showed that this high selectivity could be due to the presence of the aza-residue, and not simply an artifact of substrate specificities. E-64c, an epoxysuccinyl inhibitor, is known to be a highly potent inhibitor of cathepsin B and calpain I. Thus, to determine if these clan CA proteases could tolerate an aza-residue, aza-E-64c and its analogues were synthesized. These inhibitors, termed epoxysuccinyl aza-peptides, were found to be significantly less potent for cathepsin B, calpain I, and papain than their non-aza counterparts, including E-64c. Previous findings have shown that the reactivity and selectivity of aza-peptide epoxides with caspases were significantly influenced by epoxide stereochemistry and the prime side substituent. Thus, this second project involved the systematic study of epoxide stereochemistry effects, prime side substituent effects, and the combined effect of these two variables. All inhibitors were tested with the seven apoptotic caspases: caspases-2, -3, -6, -7, -8, -9, and -10. We found that epoxide stereochemistry, prime side substituent, and also the peptidyl sequence have combined effects on potency and selectivity. In general, the (S,S) stereoisomer is the most potent relative to the (R,R) and (cis) stereochemistries. Modeling studies were done to determine why this is true. Aza-peptide epoxides were also briefly compared to aza-peptide Michael acceptors, another class of inhibitors highly specific for clan CD proteases

The possible existence of extraterrestrial life forms has been of interest to humans for many millennia. In the past few decades space travel has provided an opportunity to search life outside of Earth. Chiral molecules are critical molecules in Earth-based life and are among the first chemical molecules sought after as proof of potential extraterrestrial life; however, identification of these chiral molecules is difficult due the lack of sensitive instruments. The objective of this work is to develop a benchmark reaction to be used as a guide in the development of instrumentation, such as a polarimeter, to be used in the search for extraterrestrial life. To achieve this objective, to investigate the enzyme mandelate racemase (MR), which catalyzes the racemization between the enantiomers of mandelate. MR is a member of the enolase superfamily, which contains a (alpha/beta)7-b barrel domain, the fold most frequently found among all known protein structures. Activity of the enzyme was measured at low temperatures and in non-aqueous media, as these are the conditions that represent extraterrestrial terrain. We find that mandelate racemase (MR) is active in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between 30C to 70C; however, the enzyme is not active in several organic cryosolvents. The stability of the structure of MR was also explored. Using differential scanning calorimetry (DSC) we observe the unfolding of the enzyme was irreversible and therefore kinetically controlled. We also found proof for divergent evolution of the enolase superfamily, providing evidence for divergent evolution across the MR and muconate lactonizing enzyme (MLE) subfamilies has been demonstrated. However, we also conclude that reactions yielding a polarimetric signal, such as racemizations employed in this work, are suitable as a tool to find signs of life.

This dissertation research was aimed at investigating an interesting class of 1,2-dialkynylimidazoles as: 1. irreversible p38 MAP kinase α-isoform (p38α) inhibitors; 2. p38α docking site probes; 3. anti-cancer agents. Based on the mild, thermal rearrangement of 1,2-dialkynylimidazoles to reactive carbene or diradical intermediates, a series of 1,2-dialkynylimidazoles was designed as potential irreversible p38α inhibitors. The synthesis of these dialkynylimidazoles and their kinase inhibition activity were reported. Interestingly, one of the 1-ethynyl-substituted dialkynylimidazoles is a potent (IC50 = 200 nM) and selective inhibitor of p38α. Additionally, this compound covalently modifies p38α as determined by ESI-MS after 12 h incubation at 37 °C. The unique kinase inhibition, covalent kinase adduct formation, and minimal CYP450 2D6 inhibition by this compound demonstrate that dialkynylimidazoles are a new, promising class of p38α inhibitors. Blocking docking interactions between kinase network partners is a promising alternative approach for selectively inhibiting kinases. The second project involves the identification of a new class of small molecules, covalent p38α MAP kinase docking site probes. We proposed that the mechanism may involve the addition of a thiol to the N-ethynyl group. Moreover, we demonstrated that such probes can be used fluorescently to label p38α both in vitro and in cells via azide-alkyne “Click” chemistry. This serves as the basis of an assay that can be used to identify inhibitors that specifically target the substrate docking site of p38α. The last project was focused on evaluating a new class of 1,2-dialkynylimidazoles as anti-cancer agents. One 1,2-dialkynylimidazole analog was found to be cytotoxic against a range of human cancer lines and to induce apoptosis in the human non-small cell lung cancer cell line A549. In order to elucidate the relationship between the structural basis and role of the thermal generation of diradical or carbene intermediates, a series of dialkynylimidazoles and related N-alkynylimidazoles was prepared and their cytotoxicity was determined against A549 cell line. Although the experimentally determined activation energy is in excellent agreement with that predicated from the DFT calculation, there is no correlation between the rate of Bergman cyclization and cytotoxicity to A549 cells. An alternative mechanism was proposed involving the unexpected selective thiol addition to the N-ethynyl group of certain 1,2-dialkynylimidazoles. / text

No trabalho estuda-se o crescimento de trincas em carga monotônica e cíclica nos casos de materiais quase-frágeis, introduzindo uma lei de dano cíclico. Revisam-se conceitos sobre modelos coesivos, leis de carga-descarga, leis de evolução de dano e efeito de escala. É seguido o modelo coesivo irreversível proposto por Wang e Siegmund (2006). Em particular se dá ênfase aos efeitos de escala não estatísticos. O modelo de zona coesiva irreversível apresenta uma formulação de dano e considera carregamento em fadiga. Quando o tamanho estrutural é reduzido (ou as trinca se extendem), a fratura por fadiga não mais ocorre por propagação de trinca, mas sim por uma decoesão uniforme. O objetivo desde trabalho é implementar este modelo e verificar sua potencialidade na captura de efeitos de escala, comparando com experimentos e dados disponíveis na literatura. / At present work is intended to study crack growth in cyclic and monotonic loading in the case of quasi-brittle materials, introducing a damage mechanism, is reviewed concepts of cohesive models, loading-unloading laws, damage evolution laws and effect of scale. The irreversible cohesive zone model proposed by Wang e Siegmund (2006) is followed. In particular emphasizes in the not statistical size effects. The irreversible cohesive zone model, presents a damage formulation and considers fatigue loading. It is demonstrated in this study that, when the structure size is reduced (or extend cracks), the fatigue fracture no longer occurs by crack propagation, then occurs by uniform decohesion . The objetive of this work is implementing this model and verify its capability to capture the scale effect compared with experiments and data available in literature.

Neste estudo, randomizado, duplo cego, avaliamos a eficácia anestésica em quarenta e dois pacientes, do Setor de Urgência da Faculdade de Odontologia da Universidade de São Paulo, com pulpite irreversível, que receberam mepivacaína 2% (n=21) ou lidocaína 2% (n=21) associadas à epinefrina 1:100.000 para bloqueio do nervo alveolar inferior (BNAI). O sinal subjetivo de anestesia do lábio e língua, a presença de anestesia pulpar e ausência de dor durante o procedimento de pulpectomia foram avaliados, respectivamente, por indagação ao paciente, pelo testador elétrico pulpar (TEP) e pela escala de dor verbal (VAS). Pela técnica pterigomandibular indireta das três posições, foi injetado primeiramente um tubete (1,8mL), e depois de 10 minutos, foi testada a anestesia pulpar (AP), pelo TEP, por duas leituras negativas ao estímulo máximo (80A) do aparelho. Quando não instalada a AP, um segundo tubete (mais 1,8mL) era reinjetado. Confirmada a anestesia pulpar, iniciava-se a pulpectomia. O sucesso do BNAI foi definido como a capacidade de acessar a câmara pulpar e a realização da pulpectomia sem relato de dor (VAS) pelo paciente (escore 0 ou 1), enquanto o insucesso foi caracterizado pelo incomodo/dor (escore 2 ou 3), que impedisse a continuação. Nesse caso, um terceiro e último tubete foi dado por técnicas complementares (intraligamentar ou intrapulpar) para finalizar o procedimento. Na análise estatística utilizou-se o teste Exato de Fisher e ANOVA com nível de significância fixado em 5%. Obtivemos que no grupo Mepivacaína com 1,8mL, a taxa de anestesia pulpar (AP) foi de 52% (11/21), e sucesso no BNAI de 55% (6/11); a injeção de mais 1,8mL (2º tubete) aumentou a AP para 86% (18/21) e o sucesso no BNAI para 55% (10*/18). No Grupo Lidocaína, com 1.8mL, a taxa da AP foi de 33% (7/21), o BNAI foi de 0%; com mais 1,8mL (2º tubete), a AP aumentou para 67% (14/21) e sucesso no BNAI para 14% (2*/14) (*com diferença estatística onde p0,05). A mepivacaína com volume menor proporcionou, clinicamente, maior índice de anestesia pulpar e sucesso do BNAI (pulpectomia total), e permitiu chegar mais próximo da polpa quando comparada a lidocaína. Concluímos que a mepivacaína obteve melhores resultados no sucesso do BNAI para a realização da pulpectomia em molares inferiores com pulpite irreversível. / The aim of this, double blind randomized study, was to evaluate the anesthetic efficacy of inferior alveolar nerve block (IANB) using 2% mepivacaine and 2% lidocaine both associated with adrenaline 1:100,000 in molars with irreversible pulpitis. The sample of these study consisted of forty-two healthy patients diagnosed with irreversible pulpitis actively experiencing pain. For blocking the IAN was established the following protocol: injection of one cartridge (1.8 mL) by the technique pterigomandibular of three indirect positions, expected 10 minutes and electric pulp test (EPT) Vitality Scanner®-SybronEndo, USA was accomplishment. If tooth sensitivity pain persisted another cartridge (plus 1.8 mL) was given by the same technique and the same methodology was performed. The pulpectomy was continued after the confirmation of pulpal anesthesia, which was established as the lack of response at maximum stimulation (80A) of EPT. In cases where the patient reported pain during pulpectomy even confirmed the blockage, a third cartridge was given by complementary techniques (intraligamentary or intrapulpal) to complete the endodontic procedure. The anesthetic efficacy in IANB was established when the pulpectomy was performed without report of pain and without complementation. For analysis and comparison of results we used the Fisher exact statistical test and ANOVA with significance level set at 5%. Regarding the effectiveness of the anesthetic with 1,8 mL mepivacaine, determined pulpar anesthesia (PA) was 52% (11/21), and success in IANB (pulpectomy) 55% (6/11), the most injection of 1,8 mL, increased 86% (18/21) for AP and success in IANB to 55% (10*/18). In the lidocaine group, with 1.8mL, AP rate was 33% (7/21), the IANB was 0%, with a further 1.8 mL (cartridge 2) the AP increased to 67% (14/21) and success was to IANB 14 % (2*/14), (*statistically difference p 0.05). Mepivacaine with smaller volume clinically provided a higher rate of pulpal anesthesia and most successful of IANB (pulpectomy total), allowing to reach nearest dental pulp than lidocaine. Concluded that mepivacaine performed better in success of IANB (pulpectomy) that lidocaine in molars with irreversible pulpitis