New Oncogenic Drivers in Hepatocellular Carcinoma: Role of the RNA Binding Protein Hu antigen R

156 p. : graf.

1-42 beta-Amyloid peptide requires PDK1/nPKC/Rac 1 pathway to induce neuronal death

1-42 beta-Amyloid (A beta(1-42)) peptide is a key molecule involved in the development of Alzheimer's disease. Some of its effects are manifested at the neuronal morphological level. These morphological changes involve loss of neurites due to cytoskeleton alterations. However, the mechanism of A beta(1-42) peptide activation of the neurodegenerative program is still poorly understood. Here, A beta(1-42) peptide-induced transduction of cellular death signals through the phosphatidylinositol 3-kinase (PI3K)/phosphoinositol- dependent kinase (PDK)/novel protein kinase C (nPKC)/Rac 1 axis is described. Furthermore, pharmacological inhibition of PDK1 and nPKC activities blocks Rac 1 activation and neuronal cell death. Our results provide insights into an unsuspected connection between PDK1, nPKCs and Rac 1 in the same signal-transduction pathway and points out nPKCs and Rac 1 as potential therapeutic targets to block the toxic effects of A beta(1-42) peptide in neurons.

Early astrocytic atrophy in the entorhinal cortex of a triple transgenic animal model of Alzheimer's disease

The EC (entorhinal cortex) is fundamental for cognitive and mnesic functions. Thus damage to this area appears as a key element in the progression of AD (Alzheimer's disease), resulting in memory deficits arising from neuronal and synaptic alterations as well as glial malfunction. In this paper, we have performed an in-depth analysis of astroglial morphology in the EC by measuring the surface and volume of the GFAP (glial fibrillary acidic protein) profiles in a triple transgenic mouse model of AD [3xTg-AD (triple transgenic mice of AD)]. We found significant reduction in both the surface and volume of GFAP-labelled profiles in 3xTg-AD animals from very early ages (1 month) when compared with non-Tg (non-transgenic) controls (48 and 54%, reduction respectively), which was sustained for up to 12 months (33 and 45% reduction respectively). The appearance of Lambda beta (amyloid beta-peptide) depositions at 12 months of age did not trigger astroglial hypertrophy; nor did it result in the close association of astrocytes with senile plaques. Our results suggest that the AD progressive cognitive deterioration can be associated with an early reduction of astrocytic arborization and shrinkage of the astroglial domain, which may affect synaptic connectivity within the EC and between the EC and other brain regions. In addition, the EC seems to be particularly vulnerable to AD pathology because of the absence of evident astrogliosis in response to A beta accumulation. Thus we can consider that targeting astroglial atrophy may represent a therapeutic strategy which might slow down the progression of AD.

The BACOMA cod-end. History and recent developments in BACOMA cod-end regulations and a proposal for abetter specification of the BACOMA cod-end for Baltic Sea cod fishery

To improve the cod stocks in the Baltic Sea, a number of regulations have recently been established by the International Baltic Sea Fisheries Commission (IBSFC) and the European Commission. According to these, fishermen are obliged to use nets with escape windows (BACOMA nets) with a mesh size of the escape window of 120 mm until end of September 2003. These nets however, retain only fish much larger than the legal minimum landing size would al-low. Due to the present stock structure only few of such large fish are however existent. As a consequence fishermen use a legal alternative net. This is a conventional trawl with a cod-end of 130 mm diamond-shaped meshes (IBSFC-rules of 1st April 2002), to be increased to 140 mm on 1st September 2003, according to the mentioned IBSFC-rule. Due legal alterations of the net by the fishermen (e.g. use of extra stiff net material) these nets have acquired extremely low selective properties, i. e. they catch very small fish and produce great amounts of discards. Due to the increase of the minimum landing size from 35 to 38 cm for cod in the Baltic, the amount of discards has even increased since the beginning of 2003. Experiments have now been carried out with the BACOMAnet on German and Swedish commercial and research vessels since arguments were brought forward that the BACOMA net was not yet sufficiently tested on commercial vessels. The results of all experiments conducted so far, are compiled and evaluated here. As a result of the Swedish, Danish and German initiative and research the European Commission reacted upon this in June 2003 and rejected the increase of the diamond-meshed non-BACOMA net from 130 mm to 140mm in September 2003. To protect the cod stocks in the Baltic Sea more effectively the use of traditional diamond meshed cod-ends with-out escape window are prohibited in community waters without derogation, becoming effective 1st of September 2003. To enable more effective and simplified control of the bottom trawl fishery in the Baltic Sea the principle of a ”One-Net-Rule“ is enforced. This is going to be the BACOMA net, with the meshes of the escape window being 110 mm for the time being. The description of the BACOMA net as given in the IBSFC-rules no.10 (revision of the 28th session, Berlin 2002) concentrates on the cod-end and the escape window but only to a less extent on the design and mesh-composition of the remaining parts of the net, such as belly and funnel and many details. Thus, the present description is not complete and leaves, according to fishermen, ample opportunity for manipulation. An initiative has been started in Germany with joint effort from scientists and the fishery to better describe the entire net and to produce a proposal for a more comprehensive description, leaving less space for manipulation. A proposal in this direction is given here and shall be seen as a starting point for a discussion and development towards an internationally uniform net, which is agreed amongst the fishery, scientists and politicians. The Baltic Sea fishery is invited to comment on this proposal, and recommendations for further improvement and specifications are welcomed. Once the design is agreed by the Baltic Fishermen Association, it shall be proposed to the IBSFC and European Commission via the Baltic Fishermen Association.

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca2+ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca2+ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP(3)Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP(3)Rs. Blockade of Ca2+-induced Ca2+ release by TMB-8 following alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca2+ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca2+ overload whereas IP3R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by alpha subunit of the eukaryotic initiation factor 2 alpha phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca2+ release through RyRs contributes to cytosolic Ca2+ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes. Cell Death and Disease (2010) 1, e54; doi:10.1038/cddis.2010.31; published online 15 July 2010

Änderung wissenschaftlicher Gattungsnamen der Echten Rochen (Raja spp.), Familie Rajidae

Based on a comprehensive compilation and evaluation of hitherto published revisions by international rajid systematists, the U.S. authors McEachran and Dunn (1998) published a summarizing revision on phylogenetic interrelationships within the family Rajidae. The family has been subdivided into the two subfamilies Rajinae and Arhynchobatinae comprising 15 and 11 rajid genera, respectively. Within the Rajinae, seven former subgenera of Raja and Gurgesiella have been newly elevated to generic rank, likewise two former subgenera of Raja within the Arhynchobatinae. As a consequence, generic names have changed for quite a number of mainly former Raja species not only in European and North Atlantic waters. Table 1 presents for comparison a selection of European skate species on the shelf with the former and new generic assignment, as well as their German names. As Fishery Conventions and Societies, as well as regional (e.g., ICES) and international organizations (e.g., FAO) are increasingly using the new scientific name combinations for fishery statistics and in relevant publications, all involved in fisheries should be aware of these nomenclatorial alterations.

TUNPOP: A simulation of the dynamics and structure of the yellowfin tuna stock and surface fishery of the Eastern Pacific Ocean

ENGLISH: Three distinct versions of TUNP0P, an age-structured computer simulation model of the eastern Pacific yellowfin tuna, Thunnus albacores, stock and surface tuna fishery, are used to reveal mechanisms which appear to have a significant effect on the fishery dynamics. Real data on this fishery are used to make deductions on the distribution of the fish and to show how that distribution might influence events in the fishery. The most important result of the paper is that the concept of the eastern Pacific yellowfin tuna stock as a homogeneous unit is inadequate to represent the recent history of the fishery. Inferences are made on the size and distribution of the underlying stock as well as its potential yield to the surface fishery as a result of alterations in the level and distribution of the effort. SPANISH: Se han empleado tres versiones diferentes de TUNP0P, un modelo de simulación de la computadora (basado en la estructura de la edad) de la población y la pesca epipelágica del atún aleta amarilla, Tbunnus albacares, del Pacífico oriental, para revelar los mecanismos que parecen tener un efecto importante en la dinámica pesquera. Se emplean los datos verdaderos de esta pesca para hacer deducciones sobre la distribución de los peces y para mostrar cómo puede influir esta distribución en los eventos de pesca. La conclusión más importante de este estudio es que el concepto de que la población del aleta amarilla del Pacífico oriental es una unidad homogénea, es inadecuado para representar la historia reciente de pesca. Se teoriza sobre la talla y distribución de la población subyacente como también sobre su producción potencial en la pesca epipelágica al cambiar el nivel y distribución del esfuerzo.

Fisheries statistics of Kainji Lake, northern Nigeria, Nov. 1994 - Dec. 1998

Results are presented for the first 4 years data (1994-1998) of the Kainji Lake catch assessment survey, collected and analysed by the Nigerian-German Kainji Lake Fisheries Promotion Project. The following areas are covered: Methodology and alterations of the original sampling concept; Frame survey results - factors relating to the CAS; Catch assessment survey results - general; Gill net fishery; Drift net fishery; Beach seine fishery; Cast net fishery; Longline fishery; Trap fishery; and, Catch statistics from fisherwomen. (PDF contains 143 pages)

Dynamics and scaling of self-excited passive vortex generators for underwater propulsion

A series of experiments was conducted on the use of a device to passively generate vortex rings, henceforth a passive vortex generator (PVG). The device is intended as a means of propulsion for underwater vehicles, as the use of vortex rings has been shown to decrease the fuel consumption of a vehicle by up to 40% Ruiz (2010).

The PVG was constructed out of a collapsible tube encased in a rigid, airtight box. By adjusting the pressure within the airtight box while fluid was flowing through the tube, it was possible to create a pulsed jet with vortex rings via self-excited oscillations of the collapsible tube.

A study of PVG integration into an existing autonomous underwater vehicle (AUV) system was conducted. A small AUV was used to retrofit a PVG with limited alterations to the original vehicle. The PVG-integrated AUV was used for self-propelled testing to measure the hydrodynamic (Froude) efficiency of the system. The results show that the PVG-integrated AUV had a 22% increase in the Froude efficiency using a pulsed jet over a steady jet. The maximum increase in the Froude efficiency was realized when the formation time of the pulsed jet, a nondimensional time to characterize vortex ring formation, was coincident with vortex ring pinch-off. This is consistent with previous studies that indicate that the maximization of efficiency for a pulsed jet vehicle is realized when the formation of vortex rings maximizes the vortex ring energy and size.

The other study was a parameter study of the physical dimensions of a PVG. This study was conducted to determine the effect of the tube diameter and length on the oscillation characteristics such as the frequency. By changing the tube diameter and length by factors of 3, the frequency of self-excited oscillations was found to scale as f~D_0^{-1/2} L_0^0, where D_0 is the tube diameter and L_0 the tube length. The mechanism of operation is suggested to rely on traveling waves between the tube throat and the end of the tube. A model based on this mechanism yields oscillation frequencies that are within the range observed by the experiment.

Avaliação da função endotelial microvascular, da rigidez arterial e da resposta inflamatória sistêmica em pacientes submetidos a cirurgia cardíaca com circulação extracorpórea

A Cirurgia de Revascularização do Miocárdio, realizada com o auxílio da Circulação Extracorpórea, está associada a alterações importantes na microcirculação e na produção e circulação de citocinas e marcadores inflamatórios. No presente estudo, foram avaliados 23 pacientes com indicação de Revascularização do Miocárdio, no dia do procedimento e 7 e 28 dias após a cirurgia. A microcirculação cutânea, enquanto reflexo da microcirculação coronariana, foi estudada através da hiperemia térmica e/ ou reativa pós oclusiva e da iontoforese de substâncias vasoativas por mecanismos dependentes e independentes do endotélio. A rigidez arterial foi aferida através da análise da onda de pulso digital. Foi avaliado ainda o impacto da doença e do procedimento cirúrgico sobre a produção e circulação sérica de citocinas e marcadores inflamatórios, tais como: PCR-HS, nitrito/ nitrato, IL-6, Il-7, IL-8, IL-10, IFN-γ, TNF-α e G-CSF. Foi observada uma tendência à redução da vasodilatação da microcirculação cutânea após a administração de doses acumulativas de acetilcolina (endotélio dependente) através da iontoforese de 7 e 28 dias após o procedimento cirúrgico. A hiperemia térmica foi mais pronunciada na avaliação basal do que aos 7 e 28 dias. A hiperemia reativa pós oclusiva não demonstrou alterações 7 dias após o procedimento. Aos 28 dias, houve um aumento da condutância microvascular cutânea. Quando avaliada a vasodilatação endotélio-independente (nitroprussiato de sódio), observamos aumento do fluxo microvascular cutâneo diretamente proporcional à carga/ dose aplicada, sem diferenças nos valores obtidos no basal e 7 e 28 dias após o procedimento. A rigidez arterial não apresentou alterações. A análise dos fatores inflamatórios e das citocinas demonstrou aumento marcante da IL-6 e da IL-8 após 7 dias do procedimento cirúrgico, com retorno parcial aos níveis basais da IL-6 e total da IL-8 após 28 dias. O IFN-γ, TNF-α e G-CSF apenas apresentaram níveis detectáveis na avaliação basal e IL-7 e IL-10 não demonstraram alterações significativas nos tempos avaliados. A PCR-HS demonstrou níveis mais elevados após 7 dias e retorno parcial aos níveis basais após 28 dias. O nitrito/ nitrato, após 7 dias, apresentou leve queda em sua concentração plasmática. Concluímos que a pequena diferença entre o valores obtidos entre o basal e após 7 dias do procedimento cirúrgico com a iontoforese de acetilcolina resulta em minimização do impacto endotelial e um valor constante deste dado após 28 dias, sugere recomposição fisiológica completa. Este resultado foi semelhante com a análise da hiperemia térmica e reativa pós oclusiva. As interleucinas IL-6 e IL-8, bem como a PCR-HS apresentaram comportamento correlacionável, refletindo a cinética inflamatória. A rigidez arterial não demonstrou alterações.

O agravamento do risco no contrato de seguro

Este trabalho objetivou demonstrar o que é e quais os requisitos adotados pela doutrina e pela jurisprudência para que seja aplicado o instituto do agravamento do risco (previsto nos artigos 768 e 769 do Código Civil) nos contratos de seguro, e quais os seus efeitos jurídicos. Para tanto, examinou-se o contrato de seguro buscando revelar a dimensão coletiva que este tipo negocial possui por excelência, em detrimento de parte da doutrina ainda restrita a uma leitura atomística e individualista deste contrato. Partiu-se, ainda, da premissa de que a boa-fé (seja na sua acepção objetiva ou subjetiva) é qualificada no contrato de seguro, eis que este tipo contratual é todo sob ela estruturado. O princípio da boa-fé é uma via de mão dupla que cria deveres para ambas as partes, cujas declarações e comportamentos serão fundamentais para a delimitação do objeto do seguro e para o alcance da função social desse tipo contratual. A boa-fé estará, ainda, incisivamente presente e modelando a relação obrigacional do seguro em todas as fases contratuais: antes da conclusão do contrato, na apresentação da proposta do contrato; durante a relação obrigacional, nas declarações necessárias sobre eventuais alterações no risco (tais como o seu agravamento), e, ainda, na fase pós-contratual, sempre considerando a natureza comunitária do seguro. Passou-se, também, por algumas questões polêmicas envolvendo a utilização de determinadas cláusulas no seguro, tidas como contrárias à boa-fé, a exemplo da cláusula perfil, cuja validade deve ser avaliada no caso concreto, e possui íntima relação com o agravamento do risco.

Avaliação prospectiva longitudinal da qualidade de vida de adolescentes submetidos a tratamento ortodôntico

A literatura científica ainda não é consistente em relação aos benefícios psicossociais proporcionados pelo tratamento ortodôntico. Os objetivos deste estudo foram conhecer as alterações na qualidade de vida relacionada com a saúde bucal (OHRQoL) e com a autopercepção estética de adolescentes brasileiros de 12 a 15 anos de idade tratados ortodonticamente, durante dois anos de avaliação prospectiva longitudinal. A amostra foi constituída de 318 jovens: 92 que iniciaram tratamento ortodôntico em uma instituição de ensino (grupo orto), e 226 indivíduos não tratados: 124 que procuraram avaliação ortodôntica na mesma instituição e não receberam tratamento durante os dois anos em que foram acompanhados na pesquisa, pois estavam aguardando uma vaga na lista de espera para iniciar o tratamento (grupo de espera), e 102 que nunca buscaram tratamento ortodôntico e que estudam em uma escola vizinha à instituição (grupo escola). A qualidade de vida foi mensurada utilizando o OHIP-14. A necessidade normativa e estética de tratamento ortodôntico foi avaliada com o índice IOTN, o nível social com o Critério de Classificação Econômica Brasil e a saúde dental com o índice CPO-D. As avaliações foram repetidas em três momentos: no exame inicial (T1); um ano depois do início do tratamento ortodôntico, para o grupo orto, e um ano após o exame inicial, para os grupos de espera e escola (T2); e dois anos depois do início do tratamento para o grupo orto, e dois anos depois do exame inicial para os grupos de espera e escola (T3). O tratamento ortodôntico reduziu significativamente os escores de OHRQoL: as médias do escores tiveram uma redução de 10,4 para 9,2 e para 1,6 entre a primeira, segunda e terceira avaliações (p<0,001). A autopercepção estética se comportou de maneira similar, com uma redução progressiva e significativa (p<0,001) nos pacientes tratados, que também tiveram melhora significativa na gravidade da má oclusão (p<0,001). Porém, os indivíduos que removeram o aparelho tiveram OHRQoL e autopercepção estética significativamente melhores em relação aos pacientes que não finalizaram o tratamento no período de dois anos. Os adolescentes do grupo de espera tiveram significativa piora na OHRQoL, que sofreu um aumento de 10,8 para 12,0 da primeira para a terceira avaliações (p<0,001), o que também ocorreu na autoavaliação estética, que sofreu um aumento significativo (p<0,001). Por outro lado, os adolescentes do grupo escola não tiveram nenhuma alteração desses índices nos períodos de avaliação, apresentando uma tendência estacionária para OHRQoL (p=0,34) e para a autopercepção estética (p=0,09). A gravidade da má oclusão não foi alterada nos grupos não tratados durante os dois anos de avaliação e o CPO-D não teve alteração significativa para nenhum dos três grupos. Foi possível concluir que o tratamento ortodôntico melhorou significativamente a qualidade de vida relacionada com a saúde bucal e a autopercepção estética dos adolescentes brasileiros submetidos a tratamento.

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.

Dieta hiperlipídica com ou sem adição de sal modula diferentemente a produção de peptídeo natriurético atrial e a expressão de renina em camundongos

Estudo dos efeitos de uma dieta rica em sal e / ou gordura saturada em grânulos de peptídeo natriurético atrial (ANP), hipertensão, expressão da renina e ultraestrutura cardíaca em camundongos C57Bl / 6. Camundongos machos adultos jovens foram separados em quatro grupos (n = 12) e alimentados com uma das seguintes dietas por 9 semanas: dieta padrão para roedores (Grupo P), dieta hiperlipídica (Grupo HL), dieta hipersódica (Grupo HS) e dieta hiperlipídica e hipersódica simultaneamente (HL-HS). Foram examinados: alterações no ANP sérico, ultra-estrutura dos cardiomiócitos produtores de ANP, estrutura do ventrículo esquerdo, pressão arterial sanguínea, expressão da renina no rim, taxa de filtração glomerular (TFG), eficiência alimentar, parâmetros lipídicos e glicídicos. Os animais alimentados com dieta hiperlipídica mostraram um pequeno aumento na produção de ANP, discreta hipertrofia ventricular esquerda, aumento da eficiência alimentar, dislipidemia e hiperglicemia. Animais alimentados com dieta hipersódica tiveram um grande aumento na produção de grânulos de ANP e correspondente elevação do seu nível sérico, hipertrofia ventricular esquerda, hipertensão arterial, diminuição dos níveis de renina e aumento da TFG. A combinação das duas dietas (HL-HS) teve um efeito aditivo. A ingestão de uma dieta com alto teor de sal e lipídeos induz alterações ultraestruturais dos cardiomiócitos, aumento da produção de ANP e elevação de seu nível sérico e reduz a quantidade de renina no rim.

Relação entre a doença de Parkinson e o gene LRRK2: um estudo na população brasileira

A doença de Parkinson (DP) é a segunda doença neurodegenerativa mais frequente depois da Doença de Alzheimer, afetando aproximadamente 1% da população com idade superior a 65 anos. Clinicamente, esta doença caracteriza-se pela presença de tremor em repouso, bradicinesia, rigidez muscular e instabilidade postural, os quais podem ser controlados com a administração do levodopa. As características patológicas da DP incluem a despigmentação da substância nigra devido à perda dos neurônios dopaminérgicos e a presença de inclusões proteicas denominadas corpos de Lewy nos neurônios sobreviventes. As vias moleculares envolvidas com esta patologia ainda são obscuras, porém a DP é uma doença complexa, resultante da interação entre fatores ambientais e causas genéticas. Mutações no gene leucine-rich repeat kinase 2 (LRRK2; OMIM 609007) constituem a forma mais comum de DP. Este gene codifica uma proteína, membro da família de proteínas ROCO, que possui, entre outros domínios, dois domínios funcionais GTPase (ROC) e quinase (MAPKKK). Neste estudo, os principais domínios do gene LRRK2 foram analisados em 204 pacientes brasileiros com DP por meio de sequenciamento dos produtos da PCR. Através da análise de 14 exons correspondentes aos domínios ROC, COR e MAPKKK foram identificadas 31 variantes. As alterações novas, p.C1770R e p.C2139S, possuem um potencial papel na etiologia da DP. Três alterações exônicas (p.R1398R, p.T1410M e p.Y2189C) e nove intrônicas (c.4317+16C>T, c.5317+59A>C, c.5509+20A>C, c.5509+52T>C, c.5509+122A>G, c.5657-46C>T, c.6382-36G>A, c.6382-37C>T e c.6576+44T>C) são potencialmente não patogênicas. Ao todo, dezessete variantes exônicas e intrônicas constituem polimorfismos já relatados na literatura (p.R1398H, p.K1423K, p.R1514Q, p.P1542S, c.4828-31T>C, p.G1624G, p.K1637K, p.M1646T, p.S1647T, c.5015+32A>G, c.5170+23T>A, c.5317+32C>T, p.G1819G, c.5948+48C>T, p.N2081D, p.E2108E e c.6381+30A>G). A frequência total de alterações potencialmente patogênicas ou patogênicas detectadas em nossa amostra foi de 3,4% (incluindo a mutação p.G2019S, anteriormente descrita em 2 artigos publicados por nosso grupo: Pimentel et al., 2008; Abdalla-Carvalho et al., 2010), sendo a frequência de mutações nos casos familiares (11,1%) cerca de seis vezes maior do que a encontrada nos casos isolados da DP (1,8%). Os resultados alcançados neste estudo revelam que mutações no gene LRRK2 desempenham um papel significativo como fator genético para o desenvolvimento da DP em pacientes brasileiros.