Glyoxalase II is one of two metalloenzymes found in the glyoxalase pathway and is responsible for catalyzing the hydrolysis of a thioester substrate. Its bimetallic active site is found to contain a variety of metal combinations, including Fe(III)Zn(II). A recent report indicates that human glyoxalase II, while containing a Fe(II)Zn(II) center, is catalytically active as a mononuclear Zn(II) enzyme. Detailed mechanistic studies of glyoxalase II enzymes are limited due to uncertainty in the metal ion content of recombinantly prepared samples. The research presented in this thesis is focused on gaining mechanistic insight into thioester hydrolysis promoted by well-characterized metal complexes The initial research is focused on studies involving a Fe(III)Zn(II) complex supported by the 2-{[bis(2-pyridylmethyl)amino]methyl}-6-[{[2-hydroxyphenyl)methyl]-(2- pyridylmethyl)amino}methyl]-4-methylphenol) ligand. Thioester hydrolysis reactions were examined by following the loss of a deuterium-labeled thioester (hydroxyphenyl thioacetic acid S-methyl(d3) ester) over time using 2H NMR as the monitoring method. Based on kinetic data and spectroscopic investigations (UV-vis and EPR), a reaction pathway for thioester hydrolysis promoted by the aforementioned Fe(III)Zn(II) complex has been proposed. An important feature of this pathway is the formation of a precursor complex wherein the deprotonated α-hydroxy group of the thioester coordinates to the Zn(II) center prior to nucleophilic attack by an Fe(III)-OH moiety. Of relevance to human glyoxalase II, the thioester hydrolysis reactivity of a mononuclear zinc complex containing the N,N-bis(2-pyridylmethyl)-tert-butylamine ligand, (bpta)Zn](ClO4)2⋅0.5H2O, has been examined. Based on kinetic data, it is proposed that thioester hydrolysis promoted by this complex proceeds via a bimolecular pathway, with a Zn-OH moiety being the nucleophile for attack on the thioester carbonyl. Activation parameters are reported for the zinc complex-promoted thioester hydrolysis reaction and are compared to those of OH- promoted thioester hydrolysis reactions. In a separate area of investigation, the chromium chloride complex {(C6H11N2)[CrCl3]}n has been isolated and characterized by elemental analysis and X-ray crystallography. This complex has been proposed as the catalyst responsible for high yield conversion of glucose to 5- hydroxymethylfurfural (HMF), which is an important reaction toward using renewable resources as feedstock chemicals.

Abstract Heparan Sulfate, A New Target for Platinum in Metastatic TNBC Author: Samantha J. Katner, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Virginia Commonwealth University, 2018 Advisor: Dr. Nicholas Farrell, Professor, Chemistry Department Heparan sulfate proteoglycans (HSPGs), composed of the linear polysaccharide heparan sulfate (HS) conjugated to a protein core, are located on the cell surface and extracellular matrix. The HS chains display varying degrees of sulfation, which constitutes the molecular recognition motif for many HS-protein interactions. HSPGs, associated growth factors, and heparanase promote tumor progression by facilitating invasion, angiogenesis, and metastasis.1 Sulfate clusters on the glycan backbone also mediate the interaction of polynuclear platinum complexes (PPCs) with HSPG through a “sulfate clamp.” Such PPC-HS interactions can be conceptualized as “polyarginine” mimics. Strong HS-PPC binding protects the oligosaccharide against sulfate loss through metalloshielding.2 The biological consequences of metalloshielding will in principle affect HS interactions with relevant enzymes and proteins such as heparanase and growth factors, similar in concept to the inhibition of DNA-protein binding through modification of DNA structure and conformation. The end-point of functional modulation of HS interactions is inhibition of angiogenesis and metastasis. PPCs are dual-function agents through their interactions with both nucleic acids and HS. The novel Pt-HS interactions open up new areas of metalloglycomics and potential anti-angiogenic activity. Here, we report PPC interactions with HS-like models: Fondaparinux (FPX)3 and heparin4. We demonstrate TriplatinNC high affinity to heparin in biophysical studies and compare HS interactions with DNA and HS using competition assays.3,4 these approaches may be extended to a range of metal-ammine compounds.4 The biological consequences of PPC-HS interactions include modulation of heparanase cleavage of FPX,3 growth factor binding to HS, and growth factor-induced migration and signaling in breast cancer and endothelial cells, as potential anti-metastatic and anti-angiogenic effects in vivo. We report proof-of-principle of strong in vivo anti-metastatic activity of PPCs in triple negative breast cancer (TNBC) models.5–7 Already, PPC-HS interactions have major biological consequences in the aggressive metastatic TNBC mouse models. Impressively, PPCs reduce overall tumor metastases with emphasis in lung, bone, and liver locations in both immunocompetent and immunosuppressive mouse models. PPCs demonstrated permeability through the blood brain barrier (BBB) implying further applications for PPCs. PPCs represent a novel class of intrinsically dual-function agents combining platinum cytotoxicity through DNA targeting with anti-angiogenic effects through glycan targeting. Together, these results suggest that strong PPC-HS interactions have a significant role in the inhibition of breast cancer metastases, particularly in metastatic TNBC patients. 1. Peterson, E. J. et al. Antiangiogenic platinum through glycan targeting. Chem. Sci. 8, 241–252 (2017). 2. Mangrum, J. B. et al. A new approach to glycan targeting: enzyme inhibition by oligosaccharide metalloshielding. Chem. Commun. (Camb). 50, 4056–8 (2014). 3. Gorle, A. K. et al. Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate. Chem. Eur. J (2018). doi:10.1002/chem.201706030 4. Katner, S. J., Johnson, W. E., Peterson, E. J., Page, P. & Farrell, N. P. Comparison of Metal–Ammine Compounds Binding to DNA and Heparin. Glycans as Ligands in Bioinorganic Chemistry. Inorg. Chem. acs.inorgchem.7b03043 (2018). doi:10.1021/acs.inorgchem.7b03043 5. Katsuta, E., Peterson, E. J., Katner, S. J., Farrell, N. P. & Takabe, K. Triplatin preferably suppress lung metastasis of breast cancer, and peritoneal carcinomatosis of colon and pancreatic cancer. Proc. AACR Washingt. D.C. Abstract #5117 (2017). 6. Katner, S. J. et al. Heparan sulfate , a new target for platinum in metastatic TNBC. Proc. AACR Chicago, Abstract #3941 (2018). 7. Katner, S. J. et al. Anti-metastatic platinum through glycan targeting in breast cancer. Proc. AACR Washingt. D.C. Abstract #17 (2017). 8. Silva, H. et al. Heparan sulfate proteoglycan-mediated entry pathway for charged tri-platinum compounds. Differential cellular accumulation mechanisms for platinum. Mol. Pharmacol. 9, 1795–1802 (2012). 9. Peterson, E. J. et al. Nucleolar targeting by platinum: P53-independent apoptosis follows rRNA inhibition, cell-cycle arrest, and DNA compaction. Mol. Pharm. 12, 287–297 (2015).

Made available in DSpace on 2014-12-17T14:53:16Z (GMT). No. of bitstreams: 1 WellingtonA_DISSERT.pdf: 14102064 bytes, checksum: 78702039b725e34f918e73accb37c546 (MD5) Previous issue date: 2013-05-10 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The general aim of this research was to analyze the factors that determine the environmental management strategies in the industry Millennium Inorganic Chemicals: Crystal Global, but also analyze which environmental management strategies are used by industry. For the achievement of the proposed objective, we conducted a case study organization based in twenty-eight environmental management strategies raised in the literature. Following this survey, it was possible to ascertain which of these strategies are used in the industry studied, as well as which factors are decisive for the implementation of these. The research is characterized as qualitative and applied, as the goals is exploratory and descriptive, with regard to the data, these were obtained through interviews with the directors of the organization and also documents made available by the industry. As a main result, it was realized that the industry practices twenty-eight twenty of environmental management strategies proposed in this study, the scale Waste Management that encompasses more strategies. Regarding the determinants realized that the endogenous factors that are most pressing for the organization creating environmental strategies, and these determinants, since the exogenous, with the exception of environmental legislation, not put pressure on the organization / O objetivo geral dessa pesquisa foi analisar os fatores que determinam as estrat?gias de gest?o ambiental na ind?stria Millennium Inorganic Chemicals: Cristal Global, como tamb?m analisar quais estrat?gias de gest?o ambiental s?o usadas pela ind?stria. Para o atingimento do objetivo proposto, foi realizado um estudo de caso na organiza??o baseado em vinte e oito estrat?gias de gest?o ambiental levantadas na literatura. Ap?s esse levantamento, p?de-se verificar quais dessas estrat?gias s?o usadas na ind?stria estudada, como tamb?m quais fatores s?o determinantes para implanta??o destas. A pesquisa caracteriza-se como qualitativa e de natureza aplicada, quanto aos objetivos ? explorat?ria e descritiva, no que se refere aos dados, estes foram obtidos por meio de entrevistas junto aos diretores da organiza??o e tamb?m documentos disponibilizados pela ind?stria. Como principal resultado, percebeu-se que a ind?stria pratica vinte das vinte e oito estrat?gias de gest?o ambiental propostas nesse estudo, sendo a dimens?o Gest?o de Res?duos que mais engloba estrat?gias. Quanto aos fatores determinantes percebeu-se que os fatores end?genos s?o os que mais pressionam a organiza??o para cria??o de estrat?gias ambientais, sendo estes determinantes, j? os ex?genos, com exce??o da legisla??o ambiental, n?o exercem press?o sobre a organiza??o

Glia are non-excitable cells found in nervous tissue, and have an important role in synaptic plasticity and the maintenance of neuronal environment, as well as the activity, development, degeneration, and repair of neurons. Glial cells are interconnected via gap junctions to form a multicellular syncytium and utilize intercellular and intracellular Ca2+signals to regulate their functions. Glial Ca2+ signals regulate important cell functions that include gene expression, cell proliferation, metabolism, ion transport systems, release of cell products, and cell death. Consequently, significant alterations of glial Ca2+ signals are associated with pathological processes such as epilepsy, Alzheimer's disease and stroke. Two major forms of Ca2+ signals, intercellular Ca2+ waves and intracellular Ca2+ oscillations occur within glia. Intercellular Ca2+ waves consist of the propagation of elevations in intracellular calcium concentration ([Ca2+]i) between neighboring cells, while intracellular Ca2+ oscillations consist of repetitive elevations in [Ca2+]i that remain confined to single cells. Ca2+ signals are initiated by either a localized chemical, mechanical and electrical stimuli. However, the exact mechanism of their initiation, propagation and modulation is not fully understood. Previous studies have led to the hypothesis that mechanically-induced intercellular Ca2+ waves in glia are mediated by the diffusion of second messenger inositol (1,4,5)-trisphosphate (IP3) through the gap junctions (GJ). However, intracellular Ca2+ may also diffuse between cells during the spread of intercellular Ca2+ wave. Alternatively, Ca2+ waves may be mediated by the release of extracellular messengers, e.g. ATP, that act via phospholipase C (PLC) -linked receptors, e.g. P2y receptors. It is also unknown if the propagation of Ca2+waves requires the regeneration of the signaling message by each cell. An interesting consequence of the propagation of an intercellular Ca2+ wave in glia is that they induce intracellular Ca2+ oscillations in cells that participate in its propagation. These intracellular Ca2+ oscillations may serve to resolve information contained in the position and strength of a local stimulus that induces intercellular Ca2+ wave propagation. Although the mechanism by which Ca2+ waves initiate Ca2+ oscillations is unknown it would seem likely that the mechanism of wave propagation is linked to the mechanism of initiation of Ca2+ oscillations. Guided by previous findings, I hypothesized that intercellular Ca2+ waves propagate by the diffusion of IP3 via gap junctions between neighboring cells to establish an intercellular gradient of IP3 concentration ([IP3]i that within individual cells initiates distinct intracellular Ca2+ oscillations. Two specific aims were investigated to test this hypothesis. The First Specific Aim was to determine if intercellular Ca2+ waves in glia are initiated by the generation of IP3 within a stimulated cell, and propagated by diffusion of IP3 molecules between neighboring cells via gap junctions. The Second Specific Aim, was to determine if intercellular Ca2+ waves induce distinct intracellular Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The initiation and propagation of intercellular Ca2+ waves and intracellular Ca2+ oscillations were examined in primary cultures of rat neonatal cortical glia, utilizing the techniques of a) the intracellular measurement of [Ca2+]i by fluorescence videomicroscopy, b) the photorelease of second messengers IP3 and Ca2+from their photolabile carriers, c) the loading of specific drugs by electroporation into defined zones of glial cultures, and d) identification of cell types by immunocytochemistry. The results of the Specific Aim 1 demonstrated the following: Mechanically-induced intercellular Ca2+ waves reequired PLC activation, the subsequent production of IP3 within the stimulated cell, and release of Ca2+ from intracellular calcium stores. Propagation of Ca2+waves depended on the presence of gap junctions. The release of Ca2+ via IP3 receptor/channels (IP3Rs) was necessary for Ca2+ wave propagation. In contrast, release of Ca2+ from ryanodine receptor/channels (RyRs) occurred in the mechanically-stimulated cell as well as in cells propagating a Ca2+ wave, but was not required for Ca2+ wave initiation and propagation. The propagation of Ca2+ waves through cells that contained heparin to block IP3Rs, or additional [Ca2+]i buffers, demonstrated that the regeneration of IP3 in the non-stimulated cells was not necessary for the propagation of the Ca2+ wave. Ca2+ waves were not mediated by extracellular signals, since Ca2+ waves were not affected by the extracellular perfusion or the inhibition of G proteins. Ca2+ was found to be a poor propagating signal of Ca2+ waves, since intercellular Ca2+ diffusion was not detected during Ca2+ wave propagation. These results are consistent with the hypothesis that Ca2+ waves propagate by diffusion of IP3molecules between neighboring cells via GIs. The [Ca2+]i increase in the stimulated cell occurred due to a Ca2+ influx from extracellular environment, and a release of Ca2+ from intracellular Ca2+ stores, and appeared to contribute to the activation of PLC and the generation of IP3. Ca2+ influx however, was not a necessary event in Ca2+ wave initiation or propagation, because Ca2+ waves occurred in the absence of extracellular Ca2+. By contrast, a [Ca2+]i increase in the absence of [IP3]i increase did not generate intercellular Ca2+waves. The results of the Specific Aim 2 demonstrated the following: An intercellular Ca2+ wave induced intracellular Ca2+ oscillations in a zone of cells at a specific distance from the stimulated cell. The initiation, frequency and duration of Ca2+ oscillations depended on the cells' distance from the Ca2+ wave origin, and not on the cell type or the magnitude of the Ca2+ wave. Modulation of the [IP3]i achieved by acetylcholine (ACh), a neurotransmitter that initiates IP3 production, or by intracellular photorelease of IP3 altered the oscillatory activity of individual cells and shifted the zone of oscillating cells away from the stimulated cell. Ca2+ oscillations spread through individual cells as an intracellular Ca2+ wave that was initiated from a specific site within the cell, independent of the orientation of the initial intercellular Ca2+ wave. These results are consistent with the hypothesis that an intercellular Ca2+ wave initiates Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The findings of this study support the hypothesis that intercellular diffusion of IP3 is the dominant mechanism of Ca2+ wave propagation and initiation of Ca2+ wave-induced Ca2+ oscillations. The significance of these results is that the glial syncytium may utilize specific intracellular Ca2+ oscillations to decode the position and strength of stimuli that induce intercellular Ca2+ waves, and thus integrate and coordinate multicellular functions of glia in the CNS.

The homodimeric hemoglobin (HbI) from the blood clam Scapharca inaequivalvis binds oxygen cooperatively and thus offers a simple model system for studying communication between two chemically identical sites. Although the individual subunits of HbI have the same myoglobin-fold as mammalian hemoglobins, the quaternary assemblage is radically different. Upon oxygen binding by HbI, only small tertiary changes are seen at the subunit interface in contrast to the relatively large quaternary changes observed with mammalian hemoglobins. Analysis of structures of this hemoglobin in the liganded (02or CO) and unliganded states has provided a framework for understanding the role of individual amino acid side-chains in mediating cooperativity. The work presented in this dissertation has directly tested the central tenets of the proposed structural mechanism for cooperativity in HbI, illuminating the key roles played by residue Phe 97 and interface water molecules in intersubunit communication. Heterologous expression of Scapharca dimeric hemoglobin: A synthetic gene has been utilized to express recombinant RbI in Escherichia coli. The HbI apoprotein constitutes 5-10% of the total bacterial protein in this system. Addition of the heme precursor δ-aminolevulinic acid to the expression culture results in a ~3-fold increase in the production of soluble hemoglobin. Recombinant HbI has been successfully purified to homogeneity, resulting in a final yield of 80-100 mg of pure holoprotein from a 12 L expression culture. Analysis of recombinant HbI reveals its oxygen binding properties to be indistinguishable from native HbI. It was necessary to correct a protein sequence error by mutating residue Asn 56 to aspartate in order to obtain diffraction quality crystals, that are isomorphous to native HbI crystals. These recombinant HbI crystals diffract to high resolution, permitting the functional effects of mutant HbI proteins to be correlated with detailed structural analysis.

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.

Orientadores: Afonso Celso Vigorito, Maria Elvira Pizzigatti Corrêa / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-27T23:05:57Z (GMT). No. of bitstreams: 1 Boer_CamilaCominato_D.pdf: 4791570 bytes, checksum: ef421fd26cc41a5841f0e57323bce220 (MD5) Previous issue date: 2015 / Resumo: Recentemente, a quantificação do fluxo salivar e dos componentes bioquímicos da saliva, foram considerados possíveis biomarcadores para a patogênese da doença do enxerto contra o hospedeiro crônica (DECHc) oral em pacientes submetidos ao Transplante de Células Tronco Hematopoiéticas (TCTH). O objetivo deste estudo foi avaliar prospectivamente a composição inorgânica salivar em diferentes períodos do transplante alogênico e no diagnóstico da DECHc e correlacioná-los com as manifestações clínicas orais. A saliva não estimulada foi coletada para determinação de fluxo salivar e das concentrações de cálcio (Ca), fosfato (Pi), cloro (Cl), magnésio (Mg), potássio (K) e sódio (Na), utilizando reações colorimétricas e absorção atómica. Os Índices de saúde bucal, mucosite e DECHc orais foram avaliados por exame clínico odontológico. A hiposalivação foi avaliada por parâmetros visuais e os pacientes responderam a um questionário sobre xerostomia, dor oral e sensibilidade. Foram incluídos no estudo, consecutivamente, 55 pacientes submetidos ao primeiro TCTH alogênico. Destes 55 pacientes, 49 (89%) foram reavaliados entre os dias D+ 8 e D+10 e 6 (11%) pacientes foram a óbito antes desse período. Após a avaliação entre os dias D+ 8 e 10, 2/ 49 (4%) pacientes recaíram, 13/49 (27%) foram a óbito 34/49 (69%) pacientes permaneceram vivos até o final deste estudo. Dos 34 pacientes 28 (82%) foram avaliados entre os dias D+ 80 e D+ 100, sem o diagnóstico de DECHc, e 12/ 34 (35%) pacientes avaliados no momento do diagnóstico da DECHc, com mediana de dias do diagnóstico de 110 (68-412). A avaliação da saúde bucal entre os dias 8 e 10 apresentou mediana do índice gengival (IG) maior quando comparada com os dados do pré-TCTH (p=0,03). Os índices Dentes Cariados, Perdidos e Obturados (CPOD), Índice de Placa (IP) e o (IG) não apresentaram qualquer alterações nos períodos do estudo. De 49 pacientes, 32 (65%) apresentaram mucosite grau 2 a 4 e a frequência foi maior ao condicionamento de altas doses (p=0,03) e nos pacientes mais jovens (p=0,02). Entre os dias 8 e 10 a queixa de dor oral foi maior nos pacientes mais jovens e no condicionamento de altas doses, (p=0,03) e (p=0,02), respectivamente. O fluxo salivar foi significativamente maior entre os dias 8 e 10 (p=0,05) em comparação ao pré-TCTH. O Pi diminuiu entre os dias 8 e 10 dias (p=0,007) e as concentrações de Na e Cl aumentaram entre os dias 8 e 10 (p=0,001 para ambos). O fluxo salivar no mesmo período demonstrou correlação negativa com a concentração de Pi (p=0,02) e positiva com as concentrações de Na e Cl, (p=0.003) e (p=0.001), respectivamente. Pacientes submetidos ao condicionamento de altas doses apresentarem menores concentrações de Pi (p=0,05). Entre os dias 80 e 100, a xerostomia foi mais frequente nos pacientes submetidos ao condicionamento com altas doses, comparada com de baixa dose (p=0,05). O fluxo salivar foi significativamente menor entre os dias 80-100 (p=0,02) em comparação com o pré-TCTH e as concentrações de Na, Cl e K aumentaram entre 80-100 dias (p=0,03), (0,02) e (p=0,003), respectivamente. O fluxo salivar no mesmo período demonstrou correlação negativa com as concentrações de Na e Cl, (p=0.01) e (p=0.013), respectivamente. Pacientes submetidos ao condicionamento de altas doses apresentaram menor concentração de Na (p=0.05), enquanto que os pacientes que receberam condicionamento de baixas doses apresentaram maiores concentrações de Na e Pi (p=0.05),( p=0,04), respectivamente. No momento do diagnostico da DECHc, o fluxo salivar não mostrou diferença estatística significativa entre os períodos do estudo. Foi observada um tendência de maior concentração de Na no diagnóstico da DECHc (p=0,06) e diminuição significativa da concentração de Pi (p=0.004) em comparação com o pré-TCTH. As concentrações de cálcio e magnésio não apresentaram qualquer alteração durante o mesmo período avaliado. O presente estudo mostrou alterações nos componentes salivares inorgânicos em períodos pós-TCTH, principalmente durante a inflamação da cavidade oral, como mucosite e DECHc. Nós podemos especular que o sódio, cloreto, fosfato e, na saliva, pode ser utilizado como um biomarcador potencial de atividade inflamatória oral em estudos posteriores / Abstract: Recent studies have considered the qualitative and quantitative assessment of salivary flow, as well the biochemical components of saliva, as possible biomarkers that might contribute to the pathogenesis of chronic Graft-Versus-host-Disease (cGHVD) in HSCT patients. The aim of this study was to evaluate the inorganic salivary status at different periods of allogeneic HSCT and cGVHD onset, prospectively. Patients were evaluated in pre-HSCT, between the days 8 and 10, 80 and 100 days without cGVHD, and at the onset of oral cGVHD. Oral health indexes, mucositis, and oral cGVHD were prospectively evaluated by oral clinical examination. Hyosalivation was clinically evaluated by visual parameters, and patients answered a questionnaire concerning xerostomia, oral pain and sensitivity. Unstimulated saliva was collected to determine the salivary flow rate and the concentrations of Calcium (Ca), Phosphate (Pi), Chloride (Cl), Magnesium (Mg), Potassium (K), and Sodium (Na) using colorimetric reactions and atomic absorption. Fifty-five consecutive patients undergoing first allogeneic HSCT were included in this study. Forty-nine out of 55 (89%) patients were evaluated between the days 8 and 10, and 6 (11%) had died before the evaluation. After the evaluation between 8 and 10 days, 2 out of 49 (4%) patients relapsed, 13 out of 49 (27%) patients died and thirty-four out of 49 (69%) patients remained alive until the end of the study. Twenty-eight out of 34 (82%) patients were evaluated between days 80 and 100 without cGVHD, and twelve out of 34 (35%) were evaluated at oral cGVHD onset, at a median time of 110 days (68-412). Oral health evaluation between the days 8 and 10 showed a higher median of Gingival Index (GI) compared with baseline data (p=0.03), and the Decayed, Missing and Filled Teeth Index (DMFT) and Plaque Index (PI) presented no statistical difference. Thirty-two out of 49 (65%) patients developed grade 2-4 oral mucositis associated with a high dose conditioning regimen (p=0.03) and younger patients (p=0.02). Between the days 8 and 10 younger patients and those who received high dose conditioning had more oral pain (p=0.03) and (p=0.02), respectively. The salivary flow rate was significant higher compared with the baseline between days 8 and 10 (p=0.05), Pi concentration was decreased, (p=0.007) and Na and Cl were increased (p=0.001), (p=0.001), respectively. The analysis of the salivary flow rate during the same period showed a negative correlation with Pi concentration (p=0.02) and a positive correlation with Na and Cl concentration, (p=0.003) and (p=0.001), respectively. Patients who received a high dose conditioning regimen presented a lower Pi concentration (p=0.05), and regardless of the conditioning regimen, no other biochemical component showed statistical difference. Xerostomia was more frequent between the days 80 and 100 in high dose, compared with reduced dose conditioning (p=0.05). The salivary flow rate was decreased between days 80 and 100 (p=0.02) and Na, Cl and K concentration was increased, (p=0.03), (p=0.02) and (p=0.003), respectively. The analysis of the salivary flow rate during the same period showed a negative correlation with Na and Cl, (p=0.01) and (p=0.013), respectively. Patients who received high dose conditioning regimen presented a lower Na concentration (p=0.05), whereas those who underwent a reduced dose conditioning regimen presented higher Na and Pi concentrations (p=0.05),( p=0,04), respectively. No other statistical difference was detected in the other biochemical components regardless of the conditioning regimen. At cGVHD onset, the salivary flow rate showed no statistical difference compared with the other periods. A trend was observed in the higher Na concentration compared with the baseline (p=0.06) and Pi concentration presented a significant decrease (p=0.004). Ca and Mg concentrations showed no changes during all evaluation periods. The present study showed changes in inorganic salivary components in post-HSCT periods, mainly during oral cavity inflammation, such as mucositis and cGVHD. We speculate that Na, Cl, and Pi in saliva could be used as a potential biomarker for oral inflammatory activity in further studies / Doutorado / Clinica Medica / Doutora em Clínica Médica

In the 1960s, Rosenberg and his colleagues confirmed the anti-cancer activity of cisplatin. Although cisplatin was capable of killing testicular cancer cells there were also serious side effects. It was necessary to find alternate ways of overcoming side effects, and soon many researchers have discovered novel platinum compounds that show similar reactivity. Recently, replacing one chloride group to a heterocyclic amine group showed significant cytotoxicity with a different binding activity than cisplatin. Previously in our lab, [Pt(Me5dien)(NO3)]+ and [Pt(Et2dien)Cl]+ have been synthesized and reacted with NAcetyl- L-methionine (N-AcMet) and Guanosine 5’-monophosphate (5’-GMP) showed unusual reactivity. Unlike most previously studied platinum triamine compounds, Me5dien compound was reacting faster with 5’-GMP than N-AcMet, due to the bulkiness of the triamine ligand. When both N-AcMet and 5’-GMP were reacted with Et2dien, 5’- GMP displaced one amine group of the triamine ligand and replaced that spot to form a bis-adducts, when the pH was kept below 4. Here a new novel platinum compound has been synthesized with a seven-membered chelate ring triamine ligand, Chloro[2-(4- methyl-1,4-diazepan-1-yl)ethanamine]platinum(II) chloride ([Pt(L)Cl]+). The unusual binding activity of [Pt(L)Cl]+ showed a unique pair of products under 1H NMR, 195Pt NMR and LC/MS spectrometry.

Monofunctional platinum(II) complexes, such as phenanthriplatin and pyriplatin, have notably different characteristics from the bifunctional anticancer complexes, such as cisplatin and oxaliplatin, which have detrimental toxicities and resistance associated with them. The unique properties of the monofunctional complexes may be exploited to target cancer cells without producing the toxic side effects associated with the current FDA-approved platinum-based anticancer drugs. To advance the understanding of these monofunctional platinum(II) complexes, this study replaced the chloride leaving ligand with an acetate group, which should increase solubility and alter the rate of reactivity with key amino acid and nucleotide targets. Phenanthriplatin and pyriplatin compounds were reacted with silver acetate to form insoluble silver chloride and the desired complex. Proton nuclear magnetic resonance (1H NMR) spectroscopy was used to characterize the new complexes and conduct kinetic assays with guanosine 5'-monophosphate (5’-GMP). A rate constant of 2.9 (± 0.7) x 10-2 M-1s-1 was determined for the reaction between pyriplatin and 5’-GMP, previously. A preliminary rate constant of 1.8 (± 0.1) x 10-2 M-1s-1 was determined for the newly synthesized cis-[Pt(NH3)2(py)OAc]+ complex with 5’-GMP. Ligand exchange kinetics directly influences the anticancer activity and toxicity of platinum drugs. Initial results indicate that the solubility is increased, and the rate of reaction is decreased by the acetate ligand.

A dentina esclerosada é um substrato comumente encontrado em pacientes idosos. No entanto, existem poucos estudos comparativos entre dentina humana e bovina esclerosadas. O objetivo deste estudo foi comparar os componentes inorgânicos e orgânicos da dentina saudável e esclerosada humana e bovina, através de cinco parâmetros: nanodureza, módulo de elasticidade, análise quantitativa da concentração de Cálcio (Ca) e Fósforo (P), densidade tubular e morfologia do colágeno. Trinta dentes humanos e 30 bovinos foram distribuídos em 4 grupos experimentais (n=15 por grupo): dentina humana saudável (DHS), esclerosada humana (DEH), bovina saudável (DBS), e bovina esclerosada (DBE). Os dentes saudáveis foram preparados na mesma altura e inclinação dos dentes esclerosados expondo níveis similares da dentina e obtendo fragmentos com 2mm de espessura. Foram realizadas 3 medições por espécime em 3 áreas pré determinadas de dentina intertubular com a utilização do Nanoindentador (carga de 500N por 5 s). Cinco espécimes de cada grupo foram preparados para Microscopia Eletrônica de Varredura (MEV). Com o auxílio da Energia Dispersiva por Raios-X EDX foram obtidos os valores (em percentagem) das concentrações de Ca e P e calculada a relação Ca:P. A contagem dos túbulos por área foi realizada em todas as eletromicrografias. Após descalcificação e preparo, o restante dos espécimes foi analisado em Microscopia Eletrônica de Transmissão (MET). DBS obteve maiores valores de nanodureza comparada à DBE e DHS. DHE sem diferenças com DHS e DBE (p=0,0008). DBS exibiu maiores valores de módulo de elasticidade somente comparada à DHS (p=0,000). A análise estatística não demonstrou diferenças estatisticamente significantes (p=0,71) entre as concentrações de Ca e P. Quanto à densidade tubular (número de túbulos/mm²), os grupos saudáveis foram maiores que os esclerosados e os humanos maiores que os bovinos. As fibras colágenas da DBS mostraram-se mais compactadas e mais desorganizadas que as demais. Pôde se concluir que apenas as concentrações de Ca e P foram similares e que as dentinas esclerosadas humana e bovina mostraram similaridade. Esta pesquisa teve suporte da Fapesp sob o número 2008/10290-8. / The sclerotic dentin has been commonly found in elderly patients. However, there are scarce reports in the literature comparing on the use of human and bovine sclerotic dentins. The objective of this study was to compare inorganic and organic components of healthy and sclerotic dentins from human and bovine. Five parameters were analyzed: nanohardness, elastic modulus, quantitative analysis of Calcium (Ca) and Phosphorous (P) concentrations, tubular density and ultrastructural morphology. Thirty human teeth plus 30 bovine teeth were distributed in 4 experimental groups (n=15 per group): human healthy dentin (HHD), human sclerotic dentin (HSD), bovine healthy dentin (BHD) and bovine sclerotic dentin (BSD). Healthy teeth were cut in the same level and inclination of the sclerotic superficial dentins. The nanohardness and elastic modulus (GPa) of three pre determined areas of each exposed dentin was measured using a nanoindenter (500N for 5s). Five samples of each group were prepared for scanning electron microscopy (SEM) examination. Energy Dispersive X-ray (EDX) was used for obtaining the Ca/P ratio. The tubular density was obtained by counting the tubules in scanning electron micrographs taken in the same magnification and work distance. Data were statistically analyzed by ANOVA complemented by the Tukeys test (p0.05). The ultrastructure of the dentins was observed in specimens processed for transmission electron microscopy (TEM). BHD exhibited significant higher nanohardness than BSD and HHD. HSD nanohardness was similar to those of HHD and BSD (p=0,0008). BHD exhibited significant higher elastic modulus than HHD (p=0,000). The Ca:P ratios were similar amongst all groups (p=0.71). The tubular densities were higher in the healthy dentins than in the sclerotic for both human and bovine. The human dentins presented higher tubular densities than bovine dentins (p=0.000). The intertubular dentin of BHD showed short collagen fibers distributed in a condensed fashion; whereas the other dentins exhibited well-organized long bundles of collagen fibers. It was concluded that sclerotic dentins of human and bovine share most morphological and structural characteristics. This research was supported by Fapesp grants number 2008/10290-8.