Hydrogen sulfide (H2S) is an endogenous gasotransmitter that regulates vascular function and blood pressure, and also protects the heart from injury associated with myocardial infarction (MI). The mitochondrial enzyme thiosulfate sulfurtransferase (TST) has a putative role in the breakdown of H2S but its role in the cardiovascular system is unknown. I hypothesised that TST reduces cardiovascular H2S availability and that inhibiting TST activity may therefore ameliorate cardiovascular pathology. In the heart, TST was expressed by cardiomyocytes and vascular smooth muscle cells. Tst-/- mice all survived to adulthood and had normal cardiac structure and function. Cardiac and hepatic H2S breakdown rates were reduced and H2S levels were higher in the blood of Tst-/- mice. However, in heart tissue, protein levels for the H2S-activated Nrf2 downstream targets, thioredoxin (Trx1) and heme oxygenase-1 (HO-1) were comparable. In contrast, protein levels for the cardiac specific H2S-synthetic enzyme, cystathionine gamma lyase (CSE) was reduced, suggesting a homeostatic negative feedback mechanism to maintain H2S at non-toxic levels. Respiration, measured using an oxygen-sensing electrode was normal in isolated mitochondria from whole Tst-/- compared to control C57BL6 hearts. Endothelial nitric oxide synthase (eNOS) protein expression was lower in Tst-/- hearts, highlighting potential cross talk between H2S and nitric oxide (NO) signalling. TST was expressed in whole aorta homogenates and in isolated endothelial cells from aorta and small intramuscular vessels of the hindlimb from C57BL/6N control mice. Myography and western blotting revealed a greater influence of NO in aorta from Tst-/- mice that was associated with increased phosphorylation of the activating serine1177 residue of eNOS (PeNOSSer1177). NO plays a lesser role in resistance arteries, but in comparison to control vessels, small mesenteric vessels from Tst-/- mice was more reliant on small and intermediate calcium activated potassium channels for relaxation. Tst-/- mice were normotensive, despite this alteration in the regulation of vascular tone. However, metabolic cage experiments identified that Tst-/- mice presented with diuresis, polydipsia, and increased urinary electrolyte excretion of sodium, potassium and chloride, possibly to compensate for increased vascular tone in order to maintain stable blood pressure. To investigate the role of TST in regulating the response to pathological challenge, MI was induced by coronary artery ligation (CAL). In control mice, gene expression of CSE was downregulated by 2 days after CAL, but TST expression was 12-fold increased, suggesting regulation of H2S bioavailability during the acute MI-healing phase. Tst-/- male mice had a 40% greater incidence of cardiac rupture during infarct healing and surviving Tst-/- mice had greater left ventricular dilatation and impaired function compared to controls. Ex vivo, isolated perfused hearts from Tst-/- mice were more susceptible to ischaemia/ reperfusion injury, suggesting an additional role of TST in determining cardiomyocyte susceptibility to injury. In conclusion, these data indicate that cardiovascular H2S bioavailability is regulated through degradation by TST. The data presented here provide evidence for significant tissue specific crosstalk between H2S synthetic and degradative mechanisms and between H2S and other local regulatory mechanisms, including ion channels and NOS. We infer TST has a physiological role in the kidney where its loss leads to changes in renal electrolyte and water handling, although other compensatory mechanisms prevent a change in blood pressure. Under conditions of pathological challenge following MI, loss of TST is detrimental, illustrating its key role in removal of H2S. The data refute the original hypothesis that TST inhibition would be protective against cardiovascular pathology. Further studies in mice with tissue specific deletion of TST are now required to more fully reveal the cardiovascular role of TST.

Hydrogen sulfide (H2S) is an endogenously produced signaling gas involved in numerous cellular functions. At the appropriate concentration, exogenous administration of this gasotransmitter regulates vasodilation, promotes angiogenesis of endothelial cells, and generally exhibits beneficial effects as an anti-inflammatory and antioperoxidative agent. H2S is also capable of acting as a gaseous chemotherapeutic agent. Therefore, the therapeutic potential of exogenous delivery of H2S is vast. The delivery of H2S is complicated by its gaseous nature. Under physiologically relevant conditions, H2S is rapidly depleted from solution by oxidation and/or degassing. Therefore, direct exogenous delivery is difficult. To date, most studies have employed Na2S as a convenient H2S source. However, the rapid surge in H2S concentration upon Na2S dissolution followed by its rapid decline poorly mimics the sustained production of low concentrations of H2S that occurs in biological systems. We synthesized a library of S-aroylthiooximes (SATOs)—H2S-releasing compounds that more aptly mimic in vivo H2S concentrations. SATOs are synthesized via reaction of a S-aroylthiohydroxylamine and an aldehyde or ketone. SATOs release H2S in response to a thiol functionality. H2S release from SATOs could be controlled, with H2S release half-lives on the order of minutes to hours. SATO chemistry was utilized to prepare H2S-releasing polymers. Copolymers prepared using RAFT polymerization could be functionalized with SATOs with conversions > 99%, and these polymers released H2S on a similar timescale to our small molecule donors, confirming the viability of SATO formation as a post-polymerization modification strategy. SATO-functionalized polymer amphiphiles were prepared that self-assembled into micelles or vesicles based on their composition. H2S was released from these polymer assemblies more slowly than from the small molecules and statistical polymers. These H2S-releasing micelles were employed in in vitro cytotoxicity studies. H2S released from the micelles was found to be selectively toxic to human colon cancer cells compared with healthy fibroblasts. These polymeric micelle donors outperformed existing H2S donors in terms of their toxicity towards cancer cells. The observed enhanced toxicity was suspected to arise from the slow and sustained release of H2S from the micelles. / Ph. D.

Self-assembly is a fundamental process in the field of nanotechnology, where molecules organize into complex structures spontaneously or induced by environmental factors. Peptides, short chains of amino acids, can self-assemble into many types of nanostructures. The self-assembly of peptides is governed by noncovalent interactions, including electrostatic interactions, hydrogen bonding, hydrophobic interactions, aromatic-aromatic interactions, and van der Waals forces. By varying the amino acid sequences and manipulating environmental parameters, these interactions can be modulated to obtain diverse supramolecular nanostructures, exhibiting a wide range of physical, chemical, and biological properties. Furthermore, the ability to control these properties opens up a world of possibilities in biomedical and biocatalytic applications. From drug delivery systems to enzyme mimics, as well as cancer treatments, the potential of these self-assembling peptides is vast and continues to be a vibrant area of research. Exploiting this potential, this dissertation delves into the design, synthesis, and investigation of self-assembling peptides for a range of applications. The introductory chapters of this document lay the groundwork, providing a comprehensive overview of self-assembly and its potential in biocatalytic and biomedical domains. The focus shifts in the later chapters to drug delivery applications, particularly in the delivery of hydrogen sulfide (H2S), and its implications in cardioprotection and cancer treatment. Finally, this document details an evaluation of self-assembled peptides in the context of biocatalysis using a combined experimental and computational approach. Chapter 3 discusses the design and synthesis of peptide-H2S donor conjugates (PHDCs) with an unusual adamantyl group. Several of PHDCs studied in this chapter self-assembled into novel nanocrescent structures observed under both conventional transmission microscopy (TEM) and cryogenic TEM (cryo-TEM). By varying the C-terminal amino acid with cationic, nonionic, or anionic amino acids, the PHDC morphologies remained unaffected, offering a robust peptide design for crescent-shaped supramolecular nanostructures. Chapter 4 discusses an extension of this project, introducing a cyclohexane in PHDCs instead of an adamantyl group. In this work, we designed and fabricated four constitutional isomeric PHDCs, which self-assembled into nanoribbons with different dimensions and large nanobelts. These morphologies exhibited varying cellular uptake and in vitro H2S release amounts, influencing their protective effects against oxidative stress induced by H2O2. With the knowledge of the impact of subtle changes in PHDC structures, Chapter 5 discusses our further design of three more PHDCs with the variation of side chain capping group, from an aromatic phenyl ring to a cyclohexane unit, to an aliphatic n-hexyl chain. In this chapter, we studied how changes in the hydrocarbon tail can influence the supramolecular nanostructures and their potential ability for colon cancer treatment. A final aspect of H2S delivery in Chapter 6 involves the creation of a stable PHDC with an extended H2S release profile. By integrating the H2S donor into a β-sheet forming peptide sequence with a Newkome-like poly(ethylene glycol) dendron, this PHDC self-assembles into spherical or fibril nanostructures with or without stirring. The H2S release was further studied by triggering release with various charged thiol molecules. Finally, another facet of this document focuses on three constitutional isomeric tetrapeptides containing a catalytic functional amino acid, His. Chapter 7 discusses these tetrapeptides, which self-assembled into nanocoils, nanotoroids, and nanoribbons based on the position of the His residue in the peptide sequence. Computational studies simulating the self-assembling process revealed the distribution of His residues and hydrophobic pockets, reminiscent of natural enzyme binding sites. A tight spatial distribution of His residues and hydrophobic pocket in nanocoils provided a picture for why this morphology exhibited the highest rate enhancement in catalyzing a model ester hydrolysis reaction. This study demonstrated how subtle molecular-level changes impact supramolecular nanostructures and catalytic efficiency. The final chapter details conclusions on all the research in this dissertation and discusses further directions of self-assembling peptides in the application of drug delivery and design of catalyst mimics. / Doctor of Philosophy / Self-assembly is a fascinating process in nanotechnology, where molecular building blocks come together to form complex structures. Peptides, which are short chains made up of amino acids, can play a crucial role in this process. They can organize themselves into various shapes due to different forces acting between their amino acid building blocks. By changing the arrangement of amino acids and adjusting the environment, scientists can create a wide range of nanoscale structures with unique properties from peptides. These self-assembling peptides have enormous potential in fields like medicine and catalysis. This dissertation describes how to design and make self-assembling peptides for various uses. Chapter 1 describes the general structure of the document, and Chapter 2 discusses the basics of self-assembly and how it can be applied in medicine and other areas. Chapters 3-6 focus on using self-assembling peptides to deliver hydrogen sulfide (H2S), a noxious gaseous molecule that is now recognized as a vital signaling molecule involved in various physiological processes. Several classes of peptide-H2S donor conjugates (PHDCs) are discussed in these chapters, including PHDCs that form nanoscale crescents, twisted ribbons, fibers, and other structures. These nanostructures show promise in protecting cells from harmful substances or can act as drugs in cancer treatment. We also investigate how different modifications affect their performance in biomedical applications. The final research chapter, Chapter 7, involves using self-assembling peptides as catalysts, molecules that speed up chemical reactions. By arranging the amino acids in different ways, peptides that form nanoscale coils, toroids, or ribbons-like structures were created. These different shapes influenced how well they catalyzed reactions. Computational modeling studies helped explain how small differences in molecular design led to big impacts on their catalytic abilities. The final chapter discloses conclusions on all the research in this dissertation and discusses the further directions of self-assembling peptides as medicines and catalysts.

Peptide-based biomaterials have gained much interest in various applications in drug delivery and tissue engineering in recent years, in large part due to their typically excellent biocompatibility and biodegradability. Composed of different amino acids, peptides can be designed with numerous sequences, providing flexibility and tunability in biomaterials. Peptides are easy to modify with small molecule drugs, inorganic components, and polymer chains to access multiple functions and tune properties relevant to biology and medicine. Stimuli-responsive peptide-based biomaterials can respond to environmental stimuli, such as light and ultrasound, in addition to local environmental factors, such as temperature, enzyme activity, and pH. Under environmental changes, these materials can be triggered to release therapeutic payloads, change conformations, or induce self-assembly in the target sites. In this work, I introduce the design, synthesis, and potential applications of several stimuli-responsive peptide-based biomaterials. The first half of this dissertation is based on enzyme-responsive, peptide-based biomaterials as extracellular matrix (ECM) mimics in tissue engineering. We synthesized linear and dendritic elastin-like peptides (ELPs) as crosslinkers and conjugated them with hyaluronic acid (HA) to form hydrogels. Trypsin was used as the enzyme trigger for cleaving the C-terminal lysine and to study how crosslinker topology affects enzymatic degradation. Hydrogels with dendritic ELPs degraded more slowly than linear ELPs, providing a novel strategy to tune the degradation rate of hydrogels as ECM mimics by the molecular design of crosslinker topology. Building on this peptide-polysaccharide platform for synthetic ECM design, we subsequently prepared hydrogels embedded with bioactive cryptic sites. These novel polymeric hydrogels mimicked native ECM cryptic sites by using depsipeptides that undergo an enzyme-triggered molecular rearrangement, "switching" from a non-functional epitope to a bioactive sequence. Mass spectrometry, 1H and 13C NMR spectroscopy, and fluorescence studies were applied to track structural changes in the peptide. SEM was used to image these polymer-peptide hybrid hydrogels. Finally, in vitro studies were conducted to evaluate cell interactions with the hydrogels. Switch peptide-modified alginate hydrogels showed increased cell adhesion upon induction of enzymatic activity, which provided a "gain of function" of the synthetic ECM. Critically, enzymes associated with the cells themselves could trigger the peptide switch and change in synthetic ECM behavior. With knowledge of stimuli-responsive peptide-based biomaterials applied in tissue engineering, I then studied how this system could be used in drug delivery by designing peptide-hydrogen sulfide (H2S) donor conjugates (PHDCs). H2S is a gasotransmitter that is produced endogenously, which has been explored in recent years with many potential therapeutical applications. We studied H2S release profiles in dual-enzyme-responsive PHDCs, with a further investigation into PHDC–Fe2+ complexes for potential tumor treatments via chemodynamic therapy. The PHDC–Fe2+ complexes were examined in a C6 glioma cell line, exhibiting an improved cell-killing effect compared with controls, by inducing toxic hydroxyl radical generation (•OH) via a Fenton reaction. To this end, we further discovered how side chains influence self-assembling nanostructures, H2S release profiles, and biological activities via three constitutionally isomeric PHDCs. Different morphologies and varied H2S release rates were observed, paving the way for tuning the properties of PHDCs by simple changes in molecular design. Finally, this dissertation discloses conclusions and future directions on stimuli-responsive peptide-based biomaterials using similar platforms with different designs in the drug delivery and tissue engineering fields. / Doctor of Philosophy / Peptides, short sequences of two or more amino acids linked by chemical bonds, are smaller versions of proteins. Forming naturally in nature, peptides are promising candidates in the design of biocompatible and biodegradable materials. To make these peptide-based materials "smart", certain sequences or functional groups are installed in the peptides, making them responsive to environmental changes, or stimuli. These external stimuli include light, ultrasound, temperature, enzyme activity, and pH changes. In this work, we have explored the design and synthesis of stimuli-responsive peptide-based biomaterials and their potential applications in tissue engineering and drug delivery. The first half of this dissertation focuses on the design and synthesis of two enzyme-responsive, peptide-based materials that function as extracellular matrix (ECM) mimics. The ECM is a three-dimensional microenvironment where cells reside, providing structural support and adhesive anchor points for cells. In the first system, we synthesized peptide-polysaccharide hydrogels with different peptide crosslinkers, comparing their enzymatic degradation performance to evaluate how peptide topology (architecture) influences degradation. A more branched topology led to a slower hydrogel degradation rate. To introduce biofunctionality into the ECM mimics, we embedded the second system with a "switchable" peptide sequence, which transformed from a non-functional peptide into a functional, bioactive epitope after being triggered by an enzyme. The functional peptide after the switch provided cell adhesion and increased cell spreading. The latter half of this dissertation explores the possibility of stimuli-responsive peptide-based biomaterials in drug delivery. We designed peptides that release hydrogen sulfide (H2S), a signaling gas is commonly known for its foul smell and toxicity, and studied the biological behaviors in cells. The peptide-H2S donor conjugates (PHDCs) were activated by the enzyme legumain, which cancer cells overproduce, leading to H2S release. With the combined treatment with Fe2+, the PHDC-Fe2+ system reduced cancer cell viability due to the high amount of hydroxyl radicals (•OH) generated by the Fenton reaction. This system may be a potential design platform for precise tumor treatments.

Hydrogen sulfide (H2S) is a gasotransmitter that is produced endogenously and freely permeates cell membranes. It plays important roles in many physiological pathways, and by regulating these pathways, it provides many therapeutic effects. For example, H2S dilates vascular vessels, promotes angiogenesis, and protects cells from oxidative stress. Due to its therapeutic effects, H2S has been used as a potential treatment for diseases like diabetes, ischemia-reperfusion injuries, lung diseases, ulcers and edemas, among others. To apply H2S for therapeutic applications, two challenges need to be addressed. The first challenge is the H2S donor, which not only provides H2S but must be stable enough to avoid side effects caused by overdose; and the second challenge is the delivery strategies, which transport the H2S to the target sites. A series of S-aroylthiooximes (SATOs), an H2S releasing compound, were synthesized and conjugated to peptide sequences to form H2S-releasing aromatic peptide amphiphile (APA) hydrogels. APAs formed nanofibers, which were stabilized by beta-sheets and aromatic stacking. The self-assembled structures were affected by the substituents on the aromatic rings of SATOs, leading to the formation of twisted nanofibers. After the addition of cysteine, H2S was released from the APAs with half-lives ranging from 13 min to 31 min. The electron-donating groups slowed down the H2S release rate, while the electron-withdrawing groups accelerated the release rate. Therefore, the release rates of H2S were controlled by electronic effects. When self-assembled structures were formed, the H2S release rate was slowed down even more, due to the difficulties in cysteine diffusion into the core of the structures. Antimicrobial effects were also discovered using the H2S releasing APA hydrogels. The H2S-releasing dipeptides S-FE and S-YE formed self-assembled twisted nanoribbons and nanotubes, respectively. The non H2S-releasing control oxime dipeptides C-FE and C-YE were also synthesized. The C-FE formed nanoribbons while the C-YE only showed non-specific aggregates. S-FE and S-YE released H2S with peaking times of about 41 and 39 min. Both the self-assembled structures and the release rates were affected by their packing differences. In vitro and ex vivo experiments with Staphylococcus aureus (Xen29), a commonly found bacterium on burn wounds, showed significant antimicrobial effects. APAs S-FE and C-FE eliminated Xen29 and inhibited the biofilm formation, while S-FE always showed better effects than C-FE. These antimicrobial H2S-releasing APA hydrogels provide a new approach to treat burn wound infections, and provide healing benefits due to the therapeutic effects of H2S. / Doctor of Philosophy / Hydrogen sulfide (H₂S) is a signaling gas that produced in our body. It regulates physiological pathways, and can be a potential treatment for diseases like diabetes, ischemia-reperfusion injuries, lung diseases, ulcers and edemas, among others. However, two issues need to be addressed before applying H₂S for disease treatments. The first issue is to choose an H₂S donor, which is stable enough to avoid side effects caused by overdose. The second issue is the delivery methods, which transport the H₂S to target sites. A series of S-aroylthiooximes (SATOs), an H₂S releasing compound, were synthesized and attached to peptide sequences to form H₂S-releasing self-assembled aromatic peptide amphiphile (APA) hydrogels. The APA hydrogels were found to be affected by the substituents on the SATO structures. For example, the H₂S released from APAs had halflives ranged from 13 min to 31 min, which were controlled by the substituents. When hydrogels were formed, the H₂S release was slowed down even more, due to the difficulties in cysteine diffusion into the SATO structures. The antimicrobial effects were also discovered using the H₂S releasing APA hydrogels. Two H₂S-releasing APA hydrogels, S-FE and S-YE, were formed. At the same time, two non H₂S-releasing oxime dipeptides, C-FE and C-YE, were also synthesized as controls. The H₂S-releasing peptides, S-FE and S-YE, released H₂S with peaking times of about 41 and 39 min, while no H₂S was released from C-FE and C-YE. The self-assembled structures and the release rates were affected by their structural differences. In vitro and ex vivo experiments with Staphylococcus aureus (Xen29), a commonly found bacterium on burn wound, showed significant antimicrobial effects. Both H₂S-releasing S-FE and non H₂S-releasing C-FE eliminated Xen29 and inhibited the biofilm formation, indicating the potential use of the designed peptides as antimicrobial treatment for wounds. The S-FE always showed better effects than C-FE, suggesting the benefit of H₂S during the elimination of bacteria. These antimicrobial H₂S-releasing APA hydrogels provide a new approach to treat burn wound infection and provide healing benefits due to the therapeutic effects of H₂S.

H2S is a biologically relevant signaling gas that is endogenously produced throughout the body. The (patho)physiological roles of H2S have led researchers to develop various compounds that decompose to release H2S (H2S donors) for exogenous H2S administration. However, many small molecule H2S donors suffer from poor solubility, low stability, and lack of control over H2S release rates. As a result, there has been an increasing interest in utilizing supramolecular materials for exogenous H2S delivery. With growing potential applications of supramolecular H2S-releasing materials, it is important to explore their properties, e.g., solubility and stability under physiological conditions. We investigated the hydrolytic stability over a range of pH conditions of a series of peptides containing H2S-releasing S-aroylthiooximes (SATOs). The SATO-peptides showed structure–reactivity relationships with SATO ring substituents playing a crucial role in hydrolysis rates. Electron-donating substituents accelerate the rate of hydrolysis while electron-withdrawing substituents slows it down. We also explored their hydrolysis mechanisms at different pH values. SATO-peptides were then used to form hydrogels at 1 wt.% triggered by Ca2+. Hydrogels can be applied directly at a site of interest, potentially improving the efficacy of H2S compared with small molecule donors that diffuse away. We developed a H2S-releasing hydrogel capable of slowly releasing H2S locally to test its efficacy on intimal hyperplasia. The hydrogel delivered H2S over the period of several hours and inhibited the proliferation of human vascular smooth muscle cells (VSMCs) significantly better than fast-releasing NaSH salts. This study shows a promising application of supramolecular H2S-releasing materials over widely used sulfide salts. The macroscopic properties of peptide hydrogels could be further modulated to achieve additional control over the H2S release properties. We synthesized a series of peptide hydrogels incorporating different linker segments to study their effects on hydrogelation properties. Most peptides formed hydrogels but with significantly different rheological behavior. We found that peptides with flexible linkers such as ethyl, substituted O-methylene, and others, formed stronger hydrogels compared to those with more rigid linkers. Interestingly, we found that stiffer hydrogels released H2S over longer periods than softer ones by retarding the diffusion of a thiol trigger, likely due to bulk degradation of the soft gels but surface erosion of the stiff gels as they release H2S. / Doctor of Philosophy / H2S has long been known as a foul smelling gas until it was discovered that it is endogenously produced throughout the body and plays many (patho)physiological roles. Therapeutic benefits of H2S have led researchers to develop various compounds that release H2S (H2S donors) for exogenous H2S administration. However, many small molecule H2S donors suffer from poor solubility, low stability, and unregulated H2S release. As a result, there has been an increasing interest in utilizing materials for exogenous H2S delivery. With growing potential applications of H2S-releasing materials, it is important to explore their properties, e.g., solubility and stability under physiological conditions. We investigated the stability of a series of peptides containing H2S-releasing S-aroylthiooximes (SATOs) over a range of pH conditions. The stability of SATO-peptides was dependent on chemical makeup of the SATO part of the peptides. We also explored their hydrolysis mechanisms at different pH values. SATO-peptides were then used to form hydrogels triggered by Ca2+. Hydrogels can be applied directly at a site of interest, potentially improving the efficacy of H2S compared with small molecule donors that diffuse away. We developed a H2S-releasing hydrogel capable of slowly releasing H2S locally to test its efficacy on intimal hyperplasia. The hydrogel delivered H2S over the period of several hours and inhibited the proliferation of human vascular smooth muscle cells (VSMCs) significantly better than fast-releasing NaSH salts. This study shows a promising application of supramolecular H2S-releasing materials over widely used sulfide salts. The macroscopic properties of peptide hydrogels could be further modulated to achieve additional control over the H2S release properties. We synthesized a series of peptide hydrogels incorporating different linker segments to study their effects on hydrogelation properties. Most peptides formed weak to strong hydrogels with calcium chloride.We found that peptides with flexible linkers formed stronger hydrogels compared to those with more rigid linkers. Interestingly, we found that stiffer hydrogels released H2S over longer periods than softer ones.

As respostas termorregulatórias ao lipopolissacarídeo (LPS) são influenciadas por moduladores que aumentam (febrigênicos) ou diminuem (criogênicos) a temperatura corporal (Tb). Entre eles, o neurotransmissor gasoso sulfeto de hidrogênio (H2S) modula a inflamação sistêmica induzida por endotoxina em ratos, agindo como uma molécula anti-inflamatória e criogênica, embora os mecanismos subjacentes ainda sejam pouco compreendidos. Considerando que a endotoxina é um ligando para o Toll-like receptor 4 (TLR4) e que evidências recentes revelam um cross-talk entre a via de sinalização TLR e fosfo-Akt (p-Akt), o objetivo do presente estudo foi investigar se o H2S atua como um mediador antiinflamatório e antipirético durante as fases termorregulatórias que ocorrem no choque endotoxêmico (hipotermia e febre) induzido por lipopolissacarídeo bacteriano (LPS, 2,5 mg / kg intraperitoneal (ip)) através da modulação sobre a produção de prostaglandina D2 (PGD2) e a ativação de Akt na área pré-óptica ântero-ventral do hipotálamo (AVPO). A Tb profunda de ratos mantidos a uma temperatura ambiente de 25 °C foi registrada antes e depois da inibição farmacológica da enzima cistationina ?-sintase (CBS - responsável pela produção endógena de H2S no cérebro) usando aminooxiacetato (AOA, 100 pmol, intracerebroventricular (icv)), combinado ou não com administração de LPS. Para esclarecer os mecanismos responsáveis por esses ajustes da resposta imune, foram determinados na AVPO os níveis de H2S, a produção de PGD2 e o perfil de expressão das proteínas CBS, p-Akt e p-CREB. Além disso, foi analisada a concentração de citocinas plasmáticas (IL-1?, IL-6, IL-10, TNF?, IFN-? , E IL-4). A injeção ip de LPS causou hipotermia típica seguida de febre. Os níveis de AVPO H2S aumentaram significativamente durante a hipotermia quando comparado com ratos eutérmicos e febris. A microinjeção icv de AOA não causou nenhuma alteração na Tb nem na produção basal de PGD2 durante a eutermia. Em ratos tratados com LPS, o AOA causou uma atenuação na queda da Tb durante a fase de hipotermia e uma febre exacerbada, simultaneamente com o aumento na produção de PGD2 e abolição do aumento induzido pela endotoxina na atividade de Akt. Durante a fase de febre, a expressão relativa de CBS esteve significativamente diminuída enquanto a expressão relativa de p-Akt esteve aumentada, quando comparado com ratos eutérmicos e hipotérmicos. As citocinas plasmáticas aumentaram durante a inflamação sistêmica, mas apenas a IL-4 mostrou um padrão semelhante em relação à Akt. Estes dados são consistentes com a noção de que o neurotransmissor gasoso H2S modula as fases de hipotermia e febre durante o choque endotoxêmico, atuando como uma molécula criogênica. Este papel anti-inflamatório durante a inflamação sistémica envolve uma regulação positiva da PGD2, de Akt e da IL-4 plasmática. / Thermoregulatory responses to lipopolysaccharide (LPS) are affected by modulators that increase (pro-pyretic) or decrease (cryogenic) body temperature (Tb). Among them, the gaseous messenger hydrogen sulfide (H2S) modulates endotoxin-induced systemic inflammation being an anti-inflammatory and cryogenic molecule, although the underlying mechanisms are still poorly understood. Since endotoxin is a Toll-like receptor 4 (TLR4) ligand and recent evidence indicates that there is a possible a cross-talk between the TLR and phospho-Akt (p-Akt) signaling pathway, the current study aimed to investigate whether H2S acts as an anti-inflammatory and anti-pyretic mediator during thermoregulatory phases of endotoxic shock (hypothermia and fever) induced by bacterial lipopolysaccharide (LPS, 2.5 mg/kg intraperitoneal (ip)) through the modulation of prostaglandin D2 (PGD2) production and activation of Akt in the anteroventral preoptic region of the hypothalamus (AVPO). Deep Tb in rats kept at an ambient temperature of 25 °C, was recorded before and after pharmacological inhibition of the enzyme cystathionine ?-synthase (CBS - responsible for H 2S endogenous production in the brain) using aminooxyacetate (AOA; 100 pmol/1 ?l intracerebroventricular (icv)) combined or not with endotoxin administration. To clarify the mechanisms responsible for these adjustments on immune response were verified in the AVPO H 2S levels, PGD2 production and expression profiles of CBS, p-Akt and p-CREB. In addition, plasma cytokines concentration (IL-1?, IL-6, IL-10, TNF?, IFN-?, and IL-4) was analyzed. Intraperitoneal injection of LPS caused typical hypothermia followed by fever. Intracerebroventricular microinjection of AOA neither affected Tb nor basal PGD2 production during euthermia. Levels of AVPO H2S were significantly increased during hypothermia when compared to both euthermic and febrile rats. In LPS-treated rats, AOA increased Tb values during hypothermia and fever, along with enhanced PGD2 production and abolition of endotoxin-induced increase in Akt activity. During fever, CBS relative expression was significantly decreased whereas p-Akt was significantly increased when compared to both euthermic and hypothermic rats. Plasma cytokines were increased during systemic inflammation, but only IL-4 showed a similar pattern in relation to Akt. These data are consistent with the notion that the gaseous messenger H2S modulates hypothermia and fever during endotoxic shock, acting as a cryogenic molecule. This anti-inflammatory role during systemic inflammation involves a H2S-induced up-modulation of PGD2, Akt and plasma IL-4.

Com a descoberta de novas fontes de petróleo e gás, em regiões remotas e de difícil acesso, tem-se a necessidade do desenvolvimento de novas tecnologias para garantir a eficácia da exploração destes recursos. Essa exploração e extração muitas vezes se dão em ambientes altamente corrosivos e os equipamentos devem apresentar propriedades que garantam um fator de segurança em serviço. Os aços de alta resistência e baixa liga (ARBL) são utilizados em tubulações para o transporte de gás natural e petróleo. Estes estão constantemente expostos a ambientes ácidos os quais são compostos de umidade e sulfeto de hidrogênio (H2S), podendo causar falha induzida pela presença de hidrogênio (Hydrogen Induced Cracking HIC). Este tipo de falha é normalmente abordado na literatura através de ensaios em solução contendo ácido acético e/ou sais (cloreto de sódio, entre outros), sempre com a injeção de H2S. Há vários mecanismos propostos, no entanto, o assunto não está totalmente resolvido. As alterações de composição química dos aços, processos de refino do aço e processos de conformação mecânica são responsáveis pela microestrutura final e determinantes da resistência à fragilização por hidrogênio. O objetivo deste trabalho é analisar e comparar o comportamento quanto à resistência à corrosão e resistência à HIC de quatro materiais: tubo X65 sour, sua região de solda, tubo X65 não-sour e uma chapa destinada a confecção de tubo X65. Os eletrólitos empregados foram: solução A (ácido acético contendo cloreto de sódio) e a solução B (água do mar sintética), os quais correspondem às soluções recomendadas pela norma NACE TM0284-2003. Os materiais foram submetidos a: ensaios de polarização (Polarização Linear para determinação da Resistência de Polarização - Rp) e ensaios de resistência a HIC segundo a norma NACE TM0284-2003; exames em microscópio óptico e eletrônico de varredura para caracterização da morfologia da corrosão e do trincamento. Os ensaios de Rp revelaram que a solução A é mais agressiva do que a solução B, sendo isso explicado pela diferença de pH entre estas duas soluções. Os resultados mostraram ainda que a máxima resistência à corrosão sempre é obtida para o tubo sour, enquanto a mínima ocorreu para o tubo não-sour. Após o ensaio de resistência a HIC os exames em microscópio óptico revelaram que, em ambas as soluções, o tubo de X65 sour, e a sua solda não apresentaram trincas, bem como a chapa destinada a tubo X65; já o tubo de X65 não-sour apresentou trincamento principalmente na região central. Os exames das trincas revelaram que a presença de cementita intergranular e a estrutura bandeada foram as causas do trincamento. No caso do tubo sour, o bom desempenho foi discutido em termos da microestrutura de ferrita poligonal, acicular e microconstituinte M/A. Já o comportamento distinto encontrado para a chapa (para tubo X65), foi discutido levando-se em conta que esta chapa apresentou menor quantidade de cementita intergranular, uma vez que, sua microestrutura é bandeada e não foi encontrado trincamento. Os resultados também revelaram que a solução B, como no caso da resistência à corrosão, é uma solução menos agressiva, pois o trincamento obtido foi muito menor. / The discovery of new oil and gas reserves, at remote and hard to reach locations, makes imperative the development of new technologies to ensure effective exploitation of these resources. This exploitation is often performed at highly corrosive environments and equipment such as pipelines should have special mechanical and corrosion properties to guarantee safety levels in service. High-Strength Low Alloy (HSLA) steels are used in pipelines for transporting gas and oil. These steels are in constant exposure to acid environments containing hydrogen sulfide (H2S) and water, that can cause pipeline failures due to Hydrogen-Induced Cracking - HIC. The literature reports that Hydrogen-Induced Cracking in steels is normally tested in solutions containing acetic acid and/or, salts (sodium chloride and others) with addition of H2S. Chemical composition, steel refining processes and metal forming processes are responsible for the final microstructure of the steel and have effect on the hydrogen embrittlement resistance. The purpose of this work is to analyze and compare the corrosion resistance and HIC resistance, and compare of four materials: pipeline steel API 5L X65 for sour service, its welded junctions, pipeline steel API 5L X65 for non-sour service and pipeline steel plate API 5L X65. The materials were submitted to linear polarization test (Rp) and HIC resistance test according to NACE TM0284-2003 standard. Both tests were carried out with two different electrolytes: the solution A (acetic acid and sodium chloride) and solution B (synthetic seawater). Subsequently; the surface of the steels were evaluated by optical microscope and scanning electron microscopy in order to characterize the cracking modes and corrosion morphology. The Rp tests showed that the solution A is more aggressive than solution B, behavior attributed to the pH difference between solutions. Steel API 5L X65 for sour service had the highest corrosion resistance and pipeline steel API 5L X65 for non-sour service had the lowest. The HIC test and the surface examination revealed that in both solutions, pipeline steel API 5L X65 for sour service, the welded junctions and the pipeline steel plate API 5L X65 showed no cracks. On the other hand, pipeline steel API 5L X65 for non-sour service presented cracking mainly in the central region. The tests revealed that the cracks nucleated at the intergranular cementite in the banded structure. The good performance of the pipeline steel API 5L X65 for sour service was discussed in terms of the microstructure, formed by polygonal ferrite, acicular ferrite and M/A microconstituent. The performance of steel plate (for pipeline API 5L X65) was different. This material did not exhibit cracks in the matrix in spite of its banded microstructure. This result was discussed taking into account that the plate studied had a small amount of intergranular cementite. The results also showed that the solution B, as in the case of corrosion resistance tests, was less aggressive than solution A, because the cracks produced were smaller.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.

Neste trabalho investigamos o efeito de doadores de H2S no prurido agudo mediado por PAR-2 em camundongos. A injeção i.d. do agonista PAR-2 SLIGRL-NH2, induziu prurido que não foi afetado pelo pré-tratamento com o antagonista H1 pirilamina. A coinjeção dos doadores de H2S GYY4137 (lento) ou NaHS (espontâneo) com SLIGRL-NH2 reduziu significativamente o prurido (P<0,05). A glibenclamida (bloqueador de canais KATP) e o SNP (doador de NO), mas não o ODQ (inibidor da sGC), evitaram estes efeitos. O antagonista TRPA1 HC-030031 reduziu significativamente o prurido induzido pelo SLIGRL-NH2 (P<0,05), mas o prurido induzido pelo agonista TPRA1 AITC não foi afetado por NaHS. Ensaios de Western blot mostraram que ambos PAR-2 e TRPA1 são expressos constitutivamente na pele de camundongos. Nossos dados mostram que o prurido secundário à ativação do PAR-2 pode ser reduzido por H2S, atuando via a abertura dos canais KATP e ativação da via NO-GMPc. Ademais, o receptor TRPA1 pode mediar o prurido induzido por SLIGRL-NH2, mas o H2S não interfere nesta via. / In this study we investigated the effect of H2S donors in PAR-2-mediated acute pruritus in mice. The i.d. injection of the PAR-2 agonist SLIGRL-NH2 induced itching that was unaffected by pre-treatment with the H1 antagonist pyrilamine. Co-injection of the H2S donors GYY4137 (slow) or NaHS (spontaneous) with SLIGRL-NH2 significantly reduced pruritis (P <0.05). Glibenclamide (a KATP channel blocker) and SNP (a NO donor), but not ODQ (a sGC inhibitor) prevented these effects. The TRPA1 antagonist HC-030031 significantly reduced SLIGRL-NH2-induced pruritus (P<0.05), but the pruritus induced by the TPRA1 agonist AITC was unaffected by NaHS. Western blot assays showed that both TRPA1 and PAR-2 are constitutively expressed in the mouse skin. Our data show that itching secondary to PAR-2 activation can be reduced by H2S which acts via the opening of KATP channels and activation of the NO-cGMP pathway. Furthermore, TRPA1 receptors may mediate SLIGRL-NH2-induced pruritus, however, H2S does not interfere with this pathway.