Studies of the serotonin type 3A receptor and the chemical preparation of tRNA

This thesis describes studies surrounding a ligand-gated ion channel (LGIC): the serotonin type 3A receptor (5-HT₃AR). Structure-function experiments using unnatural amino acid mutagenesis are described, as well as experiments on the methodology of unnatural amino acid mutagenesis. Chapter 1 introduces LGICs, experimental methods, and an overview of the unnatural amino acid mutagenesis.

In Chapter 2, the binding orientation of the clinically available drugs ondansetron and granisetron within 5-HT₃A is determined through a combination of unnatural amino acid mutagenesis and an inhibition based assay. A cation-π interaction is found for both ondansetron and granisetron with a specific tryptophan residue (Trp183, TrpB) of the mouse 5-HT₃AR, which establishes a binding orientation for these drugs.

In Chapter 3, further studies were performed with ondansetron and granisetron with 5-HT₃A. The primary determinant of binding for these drugs was determined to not include interactions with a specific tyrosine residue (Tyr234, TyrC2). In completing these studies, evidence supporting a cation-π interaction of a synthetic agonist, meta-chlorophenylbiguanide, was found with TyrC2.

In Chapter 4, a direct chemical acylation strategy was implemented to prepare full-length suppressor tRNA mediated by lanthanum(III) and amino acid phosphate esters. The derived aminoacyl-tRNA is shown to be translationally competent in Xenopus oocytes.

Appendix A.1 gives details of a pharmacological method for determining the equilibrium dissociation constant, K_B, of a competitive antagonist with a receptor, known as Schild analysis. Appendix A.2 describes an examination of the inhibitory activity of new chemical analogs of the 5-HT₃A antagonist ondansetron. Appendix A.3 reports an organic synthesis of an intermediate for a new unnatural amino acid. Appendix A.4 covers an additional methodological examination for the preparation of amino-acyl tRNA.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Reconstructing Past Depositional and Diagenetic Processes through Quantitative Stratigraphic Analysis of the Martian Sedimentary Rock Record

High-resolution orbital and in situ observations acquired of the Martian surface during the past two decades provide the opportunity to study the rock record of Mars at an unprecedented level of detail. This dissertation consists of four studies whose common goal is to establish new standards for the quantitative analysis of visible and near-infrared data from the surface of Mars. Through the compilation of global image inventories, application of stratigraphic and sedimentologic statistical methods, and use of laboratory analogs, this dissertation provides insight into the history of past depositional and diagenetic processes on Mars. The first study presents a global inventory of stratified deposits observed in images from the High Resolution Image Science Experiment (HiRISE) camera on-board the Mars Reconnaissance Orbiter. This work uses the widespread coverage of high-resolution orbital images to make global-scale observations about the processes controlling sediment transport and deposition on Mars. The next chapter presents a study of bed thickness distributions in Martian sedimentary deposits, showing how statistical methods can be used to establish quantitative criteria for evaluating the depositional history of stratified deposits observed in orbital images. The third study tests the ability of spectral mixing models to obtain quantitative mineral abundances from near-infrared reflectance spectra of clay and sulfate mixtures in the laboratory for application to the analysis of orbital spectra of sedimentary deposits on Mars. The final study employs a statistical analysis of the size, shape, and distribution of nodules observed by the Mars Science Laboratory Curiosity rover team in the Sheepbed mudstone at Yellowknife Bay in Gale crater. This analysis is used to evaluate hypotheses for nodule formation and to gain insight into the diagenetic history of an ancient habitable environment on Mars.

Smooth sets for borel equivalence relations and the covering property for σ-ideals of compact sets

This thesis is divided into three chapters. In the first chapter we study the smooth sets with respect to a Borel equivalence realtion E on a Polish space X. The collection of smooth sets forms σ-ideal. We think of smooth sets as analogs of countable sets and we show that an analog of the perfect set theorem for Σ¹₁ sets holds in the context of smooth sets. We also show that the collection of Σ¹₁ smooth sets is ∏¹₁ on the codes. The analogs of thin sets are called sparse sets. We prove that there is a largest ∏¹₁ sparse set and we give a characterization of it. We show that in L there is a ∏¹₁ sparse set which is not smooth. These results are analogs of the results known for the ideal of countable sets, but it remains open to determine if large cardinal axioms imply that ∏¹₁ sparse sets are smooth. Some more specific results are proved for the case of a countable Borel equivalence relation. We also study I(E), the σ-ideal of closed E-smooth sets. Among other things we prove that E is smooth iff I(E) is Borel.

In chapter 2 we study σ-ideals of compact sets. We are interested in the relationship between some descriptive set theoretic properties like thinness, strong calibration and the covering property. We also study products of σ-ideals from the same point of view. In chapter 3 we show that if a σ-ideal I has the covering property (which is an abstract version of the perfect set theorem for Σ¹₁ sets), then there is a largest ∏¹₁ set in I^int (i.e., every closed subset of it is in I). For σ-ideals on 2^ω we present a characterization of this set in a similar way as for C₁, the largest thin ∏¹₁ set. As a corollary we get that if there are only countable many reals in L, then the covering property holds for Σ¹₂ sets.

Nuclear Energy Levels of ^31S and ^33Cl

The reaction ³²S(³He, α) ³¹S has been used to locate 42 levels in ³¹S. For 11 of the first 17 levels ℓ_n-values have been determined. The first 6 excited states of ³¹S have been studied by applying the particle-gamma correlation method of Litherland and Ferguson (their Method II) to the reaction ³²S(³He, αγ) ³¹S. The resulting spins and parities are: E_X, J^π = 1.25 MeV, 3/2⁺; 2.23 MeV, 5/2⁺; 3.08 MeV, 1/2⁺; 3.29 MeV, 5/2⁺, 3/2⁺; 3.35 MeV, 7/2, 3/2; 3.44 MeV, 3/2⁺. Mixing and branching ratios have also been determined. The ground state Q-value for the reaction ³²S(³He, α)³¹S has been measured to be 5.538 ± 0.006 MeV. Analysis of the spectra of the reaction ³²S(³He, α)³³Cl which were obtained as a by-product of the spectra of the reaction ³²S(³He, α) ³¹S located levels in ³³Cl at the following excitation energies: 0, 810 ± 9, (1978 ± 14), 2351 ± 9, 2686 ± 8, 2848 ± 9 (a known doublet), 2980 ± 9, and 4119 ± 10 keV. The 2.0 MeV level was only weakly populated, and to confirm its existence the reaction ³⁶Ar(p, α)³³Cl has been studied. In this reaction the 2.0 MeV level was strongly populated and the measured excitation energy was 1999 ± 20 keV. The experimental results for ³¹S and ³³Cl are compared with their analogs and with nuclear model predictions.

Some constructions, related to noncommutative tori; Fredholm modules and the Beilinson-Bloch regulator

A noncommutative 2-torus is one of the main toy models of noncommutative geometry, and a noncommutative n-torus is a straightforward generalization of it. In 1980, Pimsner and Voiculescu in [17] described a 6-term exact sequence, which allows for the computation of the K-theory of noncommutative tori. It follows that both even and odd K-groups of n-dimensional noncommutative tori are free abelian groups on 2^n-1 generators. In 1981, the Powers-Rieffel projector was described [19], which, together with the class of identity, generates the even K-theory of noncommutative 2-tori. In 1984, Elliott [10] computed trace and Chern character on these K-groups. According to Rieffel [20], the odd K-theory of a noncommutative n-torus coincides with the group of connected components of the elements of the algebra. In particular, generators of K-theory can be chosen to be invertible elements of the algebra. In Chapter 1, we derive an explicit formula for the First nontrivial generator of the odd K-theory of noncommutative tori. This gives the full set of generators for the odd K-theory of noncommutative 3-tori and 4-tori.

In Chapter 2, we apply the graded-commutative framework of differential geometry to the polynomial subalgebra of the noncommutative torus algebra. We use the framework of differential geometry described in [27], [14], [25], [26]. In order to apply this framework to noncommutative torus, the notion of the graded-commutative algebra has to be generalized: the "signs" should be allowed to take values in U(1), rather than just {-1,1}. Such generalization is well-known (see, e.g., [8] in the context of linear algebra). We reformulate relevant results of [27], [14], [25], [26] using this extended notion of sign. We show how this framework can be used to construct differential operators, differential forms, and jet spaces on noncommutative tori. Then, we compare the constructed differential forms to the ones, obtained from the spectral triple of the noncommutative torus. Sections 2.1-2.3 recall the basic notions from [27], [14], [25], [26], with the required change of the notion of "sign". In Section 2.4, we apply these notions to the polynomial subalgebra of the noncommutative torus algebra. This polynomial subalgebra is similar to a free graded-commutative algebra. We show that, when restricted to the polynomial subalgebra, Connes construction of differential forms gives the same answer as the one obtained from the graded-commutative differential geometry. One may try to extend these notions to the smooth noncommutative torus algebra, but this was not done in this work.

A reconstruction of the Beilinson-Bloch regulator (for curves) via Fredholm modules was given by Eugene Ha in [12]. However, the proof in [12] contains a critical gap; in Chapter 3, we close this gap. More specifically, we do this by obtaining some technical results, and by proving Property 4 of Section 3.7 (see Theorem 3.9.4), which implies that such reformulation is, indeed, possible. The main motivation for this reformulation is the longer-term goal of finding possible analogs of the second K-group (in the context of algebraic geometry and K-theory of rings) and of the regulators for noncommutative spaces. This work should be seen as a necessary preliminary step for that purpose.

For the convenience of the reader, we also give a short description of the results from [12], as well as some background material on central extensions and Connes-Karoubi character.

Analog methods of nonlinear vibration analysis

This thesis presents methods by which electrical analogies can be obtained for nonlinear systems. The accuracy of these methods is investigated and several specific types of nonlinear equations are studied in detail.

In Part I a general method is given for obtaining electrical analogs of nonlinear systems with one degree of freedom. Loop and node methods are compared and the stability of the loop analogy is briefly considered.

Parts II and III give a description of the equipment and a discussion of its accuracy. Comparisons are made between experimental and analytic solutions of linear systems.

Part IV is concerned with systems having a nonlinear restoring force. In particular, solutions of Duffing's equation are obtained, both by using the electrical analogy and also by approximate analytical methods.

Systems with nonlinear damping are considered in Part V. Two specific examples are chosen: (1) forced oscillations and (2) self-excited oscillations (van der Pol’s equation). Comparisons are made with approximate analytic solutions.

Part VI gives experimental data for a system obeying Mathieu's equation. Regions of stability are obtained. Examples of subharmonic, ultraharmonic, and ultrasubharmonic oscillat1ons are shown.

Modulação da ativação de monócitos por lipoxinas

Lipoxinas (LXs) são metabólitos do ácido araquidônico com reconhecidas atividades antiinflamatórias e pró-resolução. Apesar do grande número de trabalhos publicados descrevendo o papel das LXs e seus análogos em leucócitos e outros tipos celulares envolvidos em doenças inflamatórias, pouco é sabido a respeito dos mecanismos de ação que desencadeiam estas respostas. Neste trabalho investigamos o papel do 15-epi-16-(para-flúor)-fenoxi-lipoxina A4 (ATL-1), um análogo sintético da 15-epi-lipoxina A4, sobre diversos processos de ativação de monócitos. Caracterizamos, pela primeira vez, o receptor da lipoxina A4 (ALX) na linhagem monocítica U937, através da avaliação de sua expressão gênica e protéica e de sua funcionalidade analisando a ativação de ERK-2, o que torna esta célula uma ferramenta apta para estudo dos mecanismos de ação das LXs e seus análogos sobre os monócitos. Além disso, demonstramos que o ATL-1 aumenta a expressão da enzima heme oxigenase (HO) -1 em células U937 via ativação da p38 MAP quinase (MAPK) e diminui a secreção da Monocyte chemoattractant protein-1 (MCP-1), uma quimiocina envolvida com o recrutamento de monócitos para o foco inflamatório, em células U937 estimuladas com LPS. A inibição da secreção de MCP-1 foi revertida pela utilização do SB203580, sugerindo que este efeito é dependente da ativação da via p38 MAPK. O presente estudo elucida alguns dos mecanismos envolvidos na ativação de monócitos pelas lipoxinas que podem levar a novas abordagens para o controle de diversas doenças nas quais o componente inflamatório é importante

Humoral Immunity Links Candida albicans Infection and Celiac Disease

Objective The protein Hwp1, expressed on the pathogenic phase of Candida albicans, presents sequence analogy with the gluten protein gliadin and is also a substrate for transglutaminase. This had led to the suggestion that C. albicans infection (CI) may be a triggering factor for Celiac disease (CeD) onset. We investigated cross-immune reactivity between CeD and CI. Methods Serum IgG levels against recombinant Hwp1 and serological markers of CeD were measured in 87 CeD patients, 41 CI patients, and 98 healthy controls (HC). IgA and IgG were also measured in 20 individuals from each of these groups using microchips sensitized with 38 peptides designed from the N-terminal of Hwp1. Results CI and CeD patients had higher levels of anti-Hwp1 (p= 0.0005 and p= 0.004) and anti-gliadin (p= 0.002 and p= 0.0009) antibodies than HC but there was no significant difference between CeD and CI patients. CeD and CI patients had higher levels of anti-transglutaminase IgA than HC (p= 0.0001 and p= 0.0039). During CI, the increase in anti-Hwp1 paralleled the increase in anti-gliadin antibodies. Microchip analysis showed that CeD patients were more reactive against some Hwp1 peptides than CI patients, and that some deamidated peptides were more reactive than their native analogs. Binding of IgG from CeD patients to Hwp1 peptides was inhibited by gamma III gliadin peptides. Conclusions Humoral cross-reactivity between Hwp1 and gliadin was observed during CeD and CI. Increased reactivity to Hwp1 deamidated peptide suggests that transglutaminase is involved in this interplay. These results support the hypothesis that CI may trigger CeD onset in genetically-susceptible individuals.

A influência do metabolismo redox na transmissão da doença de Chagas

As formas epimastigotas de Trypanosoma cruzi proliferam e se diferenciam no interior de diferentes compartimentos do trato digestivo dos triatomíneos. Esses ambientes antagônicos, no que diz respeito à concentração de nutrientes, pH e status redox, constituem um desafio para o protozoário por conterem moléculas e fatores capazes de deflagrar diferentes sinalizações e respostas no parasito. Por isso, testamos a influência de produtos abundantes do metabolismo do vetor e de status redox distintos, frente aos processos de proliferação e diferenciação in vivo e in vitro. Como exemplo temos o heme e a hemozoína, subprodutos da digestão da hemoglobina, e o urato, rico na urina dos insetos. O heme é uma importante molécula em todos os seres vivos. Nosso grupo mostrou seu papel na proliferação in vitro de T. cruzi e que esse sinal é governado pela enzima redox-sensível CaMKII (Lara et al., 2007; Souza et al., 2009). Esse efeito parece depender de uma sinalização redox, onde o heme e não seus análigos induz a formação de EROs, modulando a atividade da CaMKII (Nogueira et al, 2011). Apesar de gerar espécies reativas de oxigênio (EROs) em formas epimastigotas, o heme não alterou a ultraestrutura desses parasitos mostrando uma adaptação a ambientes oxidantes. Além disso, a adição de FCCP inibiu a formação de EROs mitocondrial, diminuindo a proliferação dos parasitos. Em contrapartida, a AA aumentou drasticamente a produção de EROs mitocondrial levando à morte dos epimastigotas. Estes resultados confirmam a hipótese de regulação redox do crescimento de epimastigotas. A formação de β- hematina (hemozoína) constitui uma elegante estratégia para minimizar o efeito tóxico do heme nos insetos hematófagos. Contudo, a β-hematina não influenciou a proliferação ou a metaciclogênese in vitro. Já o urato, e outros antioxidantes clássicos como o GSH e o NAC prejudicaram a proliferação in vitro de epimastigotas. Estes efeitos foram parcialmente revertidos quando os antioxidantes foram incubados juntamente com o heme. Durante a metaciclogênese in vitro, o NAC e o urato induziram um aumento significativo das formas tripomastigotas e levaram a diminuição da porcentagem de formas epimastigotas. Em contrapartida, o heme e a β-hematina apresentaram o efeito oposto, diminuindo a porcentagem de formas tripomastigotas e aumentando a de epimastigotas. No intuito de confirmar a influencia do status redox na biologia do parasito in vivo, nós quantificamos a carga parasitária nas porções anterior e posterior e no reto do triatomíneo alimentado na presença ou na ausência de NAC e urato por qPCR. O tratamento com os antioxidantes aumentou a carga parasitária em todas as partes do intestino analisadas. Posteriormente, para diferenciar as formas evolutivas responsáveis pelo incremento da carga parasitária, foram realizadas contagens diferenciais nas mesmas porções do intestino do inseto vetor. Cinco dias após a infecção foi observado aumento significativo de formas tripomastigotas e diminuição de formas epimastigotas in vivo. Em conjunto, estes dados sugerem que, assim como a concentração de nutrientes e o pH, o status redox também pode influenciar a biologia do T. cruzi no interior do inseto vetor. Neste cenário, moléculas oxidantes agiriam a favor da proliferação, e em contraste, antioxidantes parecem favorecer a metaciclogênese.

Avaliação citotóxica, mutagênica e genotóxica dos análogos do Megazol

A doença de Chagas é uma doença tropical infecciosa e negligenciada responsável por um grande número de pessoas infectadas e em risco de infecção, principalmente nas regiões pobres da América Latina. No momento, apenas duas drogas, Benzonidazol e Nifurtimox, estão disponíveis para o tratamento da doença de Chagas, mas são ineficazes por apresentarem baixa taxa de cura. O Megazol é um importante representante da classe dos nitroimidazóis e é uma alternativa promissora devido ao seu potencial tripanocida com um perfil superior de ação quando comparado ao tratamento disponível. No entanto, o Megazol não é utilizado clinicamente uma vez que possui atividade mutagênica e carcinogênica relatada. O Instituto de Tecnologia em Fármacos (Farmanguinhos) desenvolveu três análogos do Megazol: PTAL 05-02 (3-amino-5-(1-metil-5-nitro-1H-imidazol-2-il)-1H-1,2,4-triazol), PAMT 09 (2-amino-N-(1-metil-4-nitro-1H-imidazol-5-il)-5-(trifluorometil)-1H-1,2,4-triazol) e PTAL 04-09 (1-(1-metil-4-nitro-1H-imidazol-5-il)-1H-pirazol). O objetivo deste trabalho é apresentar novas moléculas análogas do Megazol com atividade tripanocida, desenvolvidas a partir de estratégias racionais de desenvolvimento de substâncias bioativas ao manter o perfil farmacodinâmico do Megazol enquanto tenta diminuir ou remover o efeito genotóxico. Testes genotóxicos na avaliação segura de novas substâncias bioativas foram utilizados, de acordo com as diretrizes da OECD. O teste da Salmonella/microssoma foi utilizado na avaliação mutagênica e citotóxica, utilizando linhagens de Salmonella enterica sorovar Typhimurium, deficientes e supercompetentes na síntese de enzimas nitroredutase e acetiltransferase. O análogo PAMT 09 não foi mutagênico em nenhuma concentração e linhagem utilizada. Os análogos PTAL 05-02 e PTAL 04-09 foram mutagênicos, na ausência de S9 mix, para a linhagem TA98/1,8-DNP6. Na avaliação de citotoxicidade, os três análogos foram citotóxicos, independente de metabolização exógena S9 mix. O teste do micronúcleo, utilizando células de macrófago de rato, foi realizado para a avaliação genotóxica dos análogos do Megazol. Os três análogos foram capazes de induzir a formação de micronúcleos e apresentaram efeito citotóxico.

Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 μg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

A modern pollen record from the central Gulf of California

As one facet of an effort to tie the pollen record of central Gulf of California deep cores to modern analogs, pollen was analyzed in the uppermost 150-200 years of varved core 7807-1410 taken nearby. Sampling at 2- to 8-year resolution yielded a noncomplacent record, suggesting pollen in these sediments may be a potential high resolution proxy record of short-term climatic events. The pollen spectrum as a whole matches that of upper-most DSDP Site 480 (means of all samples). Lack of a ratio or influx shift following damming of local rivers and a surplus of low-spine Compositae pollen relative to mainland sites support Baumgartner's theory that terrigenous influx to the site is largely aeolian and also suggest that a significant fraction of the pollen influx may come from Baja California.

Stable isotopes in paleosols and origins of the Asian monsoon

The stable isotopic composition of buried soil carbonate and organic matter from northern Pakistan and Nepal can be used to reconstruct aspects of the paleoecology of riverine floodplain ecosystems over the past 17 Myr. Probable dry woodland dominated the floodplain biomass of large rivers ancestral to the modern Indus and Ganges up to 7.3 Myr. Between 7.3 and about 6 Myr, tropical grasses gradually displaced woodland and have dominated floodplain biomasses to the present. The paleovegetational transition beginning about 7.3 Myr likely signals the onset of the strongly seasonal precipitation pattern that typifies the monsoonal climate of the region today. One possible analog to the dry woodland soils of the Miocene are found under the Sal woodlands of the northern Indian subcontinent, while undisturbed modern analogs to the Plio-Pleistocene floodplain grasslands can still be found in the Chitwan area of southern Nepal.

Anti HIV-1 agents 5: Synthesis and anti-HIV-1 activity of some N-arylsulfonyl-3-acetylindoles in vitro

In continuation of our program aimed at the discovery and development of compounds with superior anti-human immunodeficiency virus type 1 (HIV-1) activity, 21N-arylsulfonyl-3-acetylindole analogs (2a-u) were synthesized and preliminarily evaluated as HIV-1 inhibitors in vitro. Among of all the analogs, several compounds exhibited significant anti-HIV-1 activity, especially N-phenylsulfonyl-3-acetyl-6-methylindole (2j) and N-(p-ethyl)phenylsulfonyl-3-acetyl-6-methylindole (2n) showed the most potent anti-HIV-1 activity with EC50 values of 0.36 and 0.13 mu g/mL, and TI values of >555.55 and 791.85, respectively. It demonstrated that introduction of the acetyl group at the 3-position of N-arylsulfonyl-6-methylindoles could generally lead to the more potent analogs. (C) 2010 Elsevier Ltd. All rights reserved.