Studies on the mode of cytotoxicity of imidazotetraziones

The irnidazotetrazinones are a novel group of anti tumour agents which have demonstrated good activity against a range of murine tumours and human xenografts. They possess a structure activity relationship similar to the anti tumour triazenes, with the chloroethyl (mitozolomide) and methyl (temozolomide) analogues being active antitumour agents, whilst the ethyl (CCRG 82019) and higher homologues are inactive. This thesiS attempts to elucidate the biological mechanisms responsible for the strict structure-activity relationship observed amongst the imidazotetrazinones. Mitozolomide is the only agent chemically capable of cross-linking DNA , which has been suggested to be responsible fo r the cytotoxicity of this group of agents. Only mitozolomide and ternozolornide Exhibit a marked ditferential toxicity towards the 0 -alkylguanine-DNA alkyltransferase deficient GM892A (Mer-) cell line rather than the proficient Raji cell line (Mer+). The rate of uptake of imidazotetrazinones into cells is similar for all three agents in both cell lines, and does not explain the differing sensitivities to these agents. The effect of drug treatment on the incorporation of precursors into macromolecules, and their pool sizes, was examined. Temozolomide administration was found to alter de novo protein synthesis in both GM892A and Raji cells. Flow cytometric analysis revealed that temozolomide and CCRG 82019 block cells in late S/G2/M phase of the cell cycle , similar to that observed with mitozolomide. The extent of reaction of all three drugs with isolated macromolecules and cellular macromolecules was determined, and differences found, with cellular repair processes influencing the number of alkyl lesions remaining bound to macromolecules. The specific bases formed in calf thymus DNA after treatment with either temozolornide and CCRG 82019 was measured, and it was found that the types and relative amounts of lesions formed, differed, as well as the total level of alkylation. Whereas DNA extracted from imidazotetrazinone treated cells is not affected in its ability to support RNA polymerase activity, an effect is observed on the ability to extract DNA polymerase from drug treated cells. This may suggest that the alkylated DNA must be in intact chromatin for the lesion to manifest its effects. Temozolomide and methyl methanesulphonate do got appear to act with a synergistic mode of action. The 0 -position of guanine is suspected to be a critical site for the action of these types of drugs.

Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Studies on DNA methylation and mechanisms of hypomethylation

The methylation of cytosinc residues in DNA is thought to play an important role in the regulation of gene expression, with active genes generally being hypomethylated. With this in mind peptides were synthcsised to mimic the cytosine-5 methylation activity carried out by DNA mcthylase, which however, showed no ability to carry out this function. The imidazotetrazinoncs are a novel group of antitumour agents which have demonstrated good activity against a range of murinc tumours and human tumour xenografts, and hypomethylation of DNA has been implicated in the mechanism of action. Studies have been conducted on the mechanism by which such agents cause hypomethylation, using DNA methylase partially purified from murine L1210 leukaemia cells. Unmodified calf thymus DNA does not inhibit the transfer of methyl groups from SAM to M.lysodeikticus DNA by partially purified DNA methylase. However, if the calf thymus DNA is modified by alkylating agents such as imida-zotetrazinones or nitrosoureas, the treated DNA becomes an inhibitor of the methylation reaction. This has been correlated with the induction of DNA damage, such as single strand breaks, since X-ray treated DNA and deoxyribonuclease treatment produces a similar effect. The mechanism of inhibition by the drug treated or damaged DNA is thought to occur by binding of the enzyme to an increased concentration of non-substrate DNA, presumably by the occurrence of single strand breaks, since neither sonication nor treatment with the restriction enzyme Mspl caused an inhibition. Attempts were made to elucidate the strict structure activity relationship for antitumour activity observed amongst the imidazotctrazinones. The transfection of a murine colon adcnocarcinoma cell line (MAC 13) with DNA extracted from GM892 or Raji cells previously treated with either the methyl (temozolomide) or ethyl (ethazolastone) imidazotetrazinone was performed. X-irradiated DNA did not cause any suppression of cell growth, suggesting that it was not due to physical damage. Transfection of MAC 13 cells with DNA extracted from GM892 cells, was more effective at inhibiting growth than DNA from Raji cells. Temozolomide treated cellular DNA was a more potent growth inhibitor than that from ethazolastone treated cells. For both agents the growth inhibitory effect was most marked with DNA extracted 6h after drug addition, and after 24h no growth suppression was observed. This suggested that the growth inhibitory effect is due to a repairable lesion. .The methylation of M.lysodeikticus DNA by DNA methylase is inhibited potently and specifically by both hereto and homoribo and dcoxyri-bopolynucleotides containing guanine residues. The inhibitory effect is unaffected by chain length or sugar residue, but is abolished when the O-6 residue of guanine is substituted as in poly d(OGG)2o. Potent inhibition is also shown by polyinosinic and polyxanthylic acids but not by polyadenylic acid or by heteropolymers containing adcnine and thymine. These results suggest that the 6 position of the purine nucleus is important in binding of the DNA methylase to particular regions of the DNA and that the hydrogen bonding properties of this group are important in enzyme recognition. This was confirmed using synthetic oligonucleotides as substrates for DNA methylase. Enzymatic methylation of cytosine is completely suppressed, when O6 methylguanine replaces guanine in CG sites.

Biological interactions with synthetic polymers

Anchorage dependent cell culture is a useful model for investigating the interface that becomes established when a synthetic polymer is placed in contact with a biological system. The primary aim of this interdisciplinary study was to systematically investigate a number of properties that were already considered to have an influence on cell behaviour and thereby establish the extent of their importance. It is envisaged that investigations such as these will not only further the understanding of the mechanisms that affect cell adhesion but may ultimately lead to the development of improved biomaterials. In this study, surface analysis of materials was carried out in parallel with culture studies using fibroblast cells. Polarity, in it's ability to undergo hydrogen bonding (eg with water and proteins), had an important affect on cell behaviour, although structural arrangement and crystallinity were not found to exert any marked influence. In addition, the extent of oxidation that had occurred during the process of manufacture of substrates was also important. The treatment of polystyrene with a selected series of acids and gas plasmas confirmed the importance of polarity, structural groups and surface charge and it was shown that this polymer was not unique among `hydrophobic' materials in it's inability to support cell adhesion. The individual water structuring groups within hydrogel polymers were also observed to have controlling effects on cell behaviour. An overall view of the biological response to both hydrogel and non-hydrogel materials highlighted the importance of surface oxidation, polarity, water structuring groups and surface charge. Initial steps were also taken to analyse foetal calf serum, which is widely used to supplement cell culture media. Using an array of analytical techniques, further experiments were carried out to observe any possible differences in the amounts of lipids and calcium that become deposited to tissue culture and bacteriological grade plastic under cell culture conditions.

A new experimental protocol for preferential differentiation of mouse embryonic stem cells into insulin-producing cells

Mouse embiyonic stem (ES) cells have the potential to differentiate into insulin-producing cells, but efficient protocols for in vitro differentiation have not been established. Here we have developed a new optimized four-stage differentiation protocol and compared this with an established reference protocol. The new protocol minimized differentiation towards neuronal progeny, resulting in a population of insulin-producing cells with ß-cell characteristics but lacking neuronal features. The yield of glucagon and somatostatin cells was negligible. Crucial for this improved yield was the removal of a nestin selection step as well as removal of culture supplements that promote differentiation towards the neuronal lineage. Supplementation of the differentiation medium with insulin and fetal calf serum was beneficial for differentiation towards monohor-monal insulin-positive cells. After implantation into diabetic mice these insulin-producing cells produced a time-dependent improvement of the diabetic metabolic state, in contrast to cells differentiated according to the reference protocol. Using a spinner culture instead of an adherent culture of ES cells prevented the differentiation towards insulin-producing cells. Thus, prevention of cell attachment in a spinner culture represents a means to keep ES cells in an undifferentiated state and to inhibit differentiation. In conclusion, this study describes a new optimized four-stage protocol for differentiating ES cells to insulin-producing cells with minimal neuronal cell formation. Copyright © 2008 Cognizant Comm. Corp.

Radiolabelling of antigen and liposomes for vaccine biodistribution studies

A relatively simple and effective method to follow the movement of pharmaceutical preparations such as vaccines in biodistribution studies is to radiolabel the components. Whilst single radiolabelling is common practice, in vaccine systems containing adjuvants the ability to follow both the adjuvant and the antigen is favourable. To this end, we have devised a dual-radiolabelling method whereby the adjuvant (liposomes) is labelled with 3H and the antigen (a subunit protein) with 125I. This model is stable and reproducible; we have shown release of the radiolabels to be negligible over periods of up to 1 week in foetal calf serum at 37° C. In this paper we describe the techniques which enable the radiolabelling of various components, assessing stability and processing of samples which all for their application in biodistribution studies. Furthermore we provide examples derived from our studies using this model in tuberculosis vaccine biodistribution studies. © 2010 by the authors.

In vitro bioactivity of titanium-doped bioglass

Previous studies have suggested that incorporating relatively small quantities of titanium dioxide into bioactive glasses may result in an increase in bioactivity and hydroxyapatite formation. The present work therefore investigated the in vitro bioactivity of a titanium doped bioglass and compared the results with 45S5 bioglass. Apatite formation was evaluated for bioglass and Ti-bioglass in the presence and absence of foetal calf serum. Scanning electron microscopy (SEM) images were used to evaluate the surface development and energy dispersive X-ray measurements provided information on the elemental ratios. X-ray diffraction spectra confirmed the presence of apatite formation. Cell viability was assessed for bone marrow stromal cells under direct and indirect contact conditions and cell adhesion was assessed using SEM. © 2014 Springer Science+Business Media.

Differential effects of chlorinated and oxidized phospholipids in vascular tissue:implications for neointima formation

The presence of inflammatory cells and MPO (myeloperoxidase) in the arterial wall after vascular injury could increase neointima formation by modification of phospholipids. The present study investigates how these phospholipids, in particular oxidized and chlorinated species, are altered within injured vessels and how they affect VSMC (vascular smooth muscle cell) remodelling processes. Vascular injury was induced in C57BL/6 mice and high fat-fed ApoE-/- (apolipoprotein E) mice by wire denudation and ligation of the left carotid artery (LCA). Neointimal and medial composition was assessed using immunohistochemistry and ESI-MS. Primary rabbit aortic SMCs (smooth muscle cells) were utilized to examine the effects of modified lipids on VSMC proliferation, viability and migration at a cellular level. Neointimal area, measured as intima-to-media ratio, was significantly larger in wire-injured ApoE-/- mice (3.62±0.49 compared with 0.83±0.25 in C57BL/6 mice, n=3) and there was increased oxidized low-density lipoprotein (oxLDL) infiltration and elevated plasma MPO levels. Relative increases in lysophosphatidylcholines and unsaturated phosphatidylcholines (PCs) were also observed in wire-injured ApoE-/- carotid arteries. Chlorinated lipids had no effect on VSMC proliferation, viability or migration whereas chronic incubation with oxidized phospholipids stimulated proliferation in the presence of fetal calf serum [154.8±14.2% of viable cells at 1 μM PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) compared with control, n=6]. In conclusion, ApoE-/- mice with an inflammatory phenotype develop more neointima in wire-injured arteries and accumulation of oxidized lipids in the vessel wall may propagate this effect.

A comparison of individual cow versus group concentrate allocation strategies on dry matter intake, milk production, tissue changes, and fertility of Holstein-Friesian cows offered a grass silage diet

A diverse range of concentrate allocation strategies are adopted on dairy farms. The objectives of this study were to examine the effects on cow performance [dry matter (DM) intake (DMI), milk yield and composition, body tissue changes, and fertility] of adopting 2 contrasting concentrate allocation strategies over the first 140 d of lactation. Seventy-seven Holstein-Friesian dairy cows were allocated to 1 of 2 concentrate allocation strategies at calving, namely group or individual cow. Cows on the group strategy were offered a mixed ration comprising grass silage and concentrates in a 50:50 ratio on a DM basis. Cows on the individual cow strategy were offered a basal mixed ration comprising grass silage and concentrates (the latter included in the mix to achieve a mean intake of 6 kg/cow per day), which was formulated to meet the cow’s energy requirements for maintenance plus 24 kg of milk/cow per day. Additional concentrates were offered via an out-of-parlor feeding system, with the amount offered adjusted weekly based on each individual cow’s milk yield during the previous week. In addition, all cows received a small quantity of straw in the mixed ration part of the diet (approximately 0.3 kg/cow per day), plus 0.5 kg of concentrate twice daily in the milking parlor. Mean concentrate intakes over the study period were similar with each of the 2 allocation strategies (11.5 and 11.7 kg of DM/cow per day for group and individual cow, respectively), although the pattern of intake with each treatment differed over time. Concentrate allocation strategy had no effect on either milk yield (39.3 and 38.0 kg/d for group and individual cow, respectively), milk composition, or milk constituent yield. The milk yield response curves with each treatment were largely aligned with the concentrate DMI curves. Cows on the individual cow treatment had a greater range of concentrate DMI and milk yields than those on the group treatment. With the exception of a tendency for cows on the individual cow treatment to lose more body weight to nadir than cows on the group treatment, concentrate allocation strategy had little effect on either body weight or body condition score over the experimental period. Cows on the individual cow treatment had a higher pregnancy rate to first and second service and tended to have a higher 100-d in calf rate than cows on the group treatment. This study demonstrates that concentrate allocation strategy had little effect on overall production performance.

Qual a contribuição das características antropométricas na velocidade de corrida de curta distância ?

v. 19, n. 2, abr./jun. 2016.

Analysis of radiation induced DNA damage by LC-MS/MS in isolated and cellular DNA

Abstract: It is well established that ionizing radiation induces a variety of damage in DNA by direct effects that are mediated by one-electron oxidation and indirect effects that are mediated by the reaction of water radiolysis products, e.g., hydroxyl radicals (•OH). In cellular DNA, direct and indirect effects appear to have about an equal effect toward DNA damage. We have shown that ϒ-(gamma) ray irradiation of aqueous solutions of DNA, during which •OH is the major damaging ROS can lead to the formation several lesions. On the other hand, the methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the gene regulation. The C5 position of cytosine in CG dinucleotides is frequently methylated by DNA methyl transferees (DNMTs) and constitutes 4-5% of the total cytosine. Here, my PhD research work focuses on the analysis of oxidative base modifications of model compounds of methylated and non methylated oligonucleotides, isolated DNA (calf-thymus DNA) and F98 cultured cell by gamma radiation. In addition, we identified a series of modifications of the 2-deoxyribose moiety of DNA arising from the exposure of isolated and cellular DNA to ionizing radiation. We also studied one electron oxidation of cellular DNA in cultured human HeLa cells initiated by intense nanosecond 266 nm laser pulse irradiation, which produces cross-links between guanine and thymine bases (G*-T*). To achieve these goals, we developed several methods based on mass spectrometry to analyze base modifications in isolated DNA and cellular DNA.

Desenvolvimento ponderal de bubalinos da raça Murrah criados em pastagem de Brachiaria brizantha no Centro-Oeste do Estado de São Paulo, Brasil

Foram analisados os dados de desempenho ponderal de bubalinos Murrah do Sistema de Produção de Leite da Universidade Estadual Paulista, Faculdade de Medicina Veterinária e Zootecnia, campus de Botucatu. Os pesos foram corrigidos às diversas idades-padrão e o modelo incluiu os efeitos de sexo (S), mês (M) e ano (A) de nascimento, classe de idade da búfala ao parto (C) e as interações S x M, S x A, S x C e M x A. As médias ajustadas e respectivos erros-padrão estimados para as características estudadas foram: Peso ao Nascer (PN): 37,71 ± 8,25kg; Peso aos 120 dias (P120): 102,08 ± 16,27kg; Peso aos 240 dias (P240): 169,84 ± 22,83kg; Peso aos 365 dias (P365): 250,59 ± 25,12kg; Peso aos 550 dias (P550): 326,13 ± 39,27kg e Peso aos 730 dias (P730): 389,80 ± 31,26kg. O efeito de sexo (S) foi significativo somente para PN e P365, sendo que machos tenderam a nascer mais pesados que fêmeas. O mês de nascimento (M) exerceu efeito sobre o PN, P120 e P730 sendo que animais nascidos em maio foram os mais pesados ao nascer, enquanto os nascidos em janeiro e maio, foram os mais pesados aos 120 e 730 dias, respectivamente. O efeito de ano de foi significativo sobre o PN, P120, P240 e P730. Os filhos de búfalas das classes de idade 1 (3 anos ou menos) e da classe 6 (10,11 e 12 anos) foram os mais leves e mais pesados ao nascer, respectivamente. O fato de a classe de idade da búfala não exercer efeito sobre os P365, P550 e P730 sugere que, em rebanhos comerciais possa ser feita a substituição de búfalas não gestantes por novilhas prenhes, apesar de esta prática reduzir a média de idade do rebanho de cria. Bubalinos da raça Murrah oriundos de rebanhos leiteiros podem ser utilizados para a produção de carne.

Modelagem univariada e regressão múltipla na análise da oferta e do preço do boi gordo no mercado brasileiro

Dissertação (mestrado)—Universidade de Brasília, Faculdade de Agronomia e Medicina Veterinária, Programa de Pós-Graduação em Agronegócios, 2016.

3D Porous Architecture of Stacks of β-TCP Granules Compared with That of Trabecular Bone: A microCT, Vector Analysis, and Compression Study

International audience

Impacto del uso de los sistemas de alimentación a corral como estrategia para el engorde de bovinos para carne

El confinamiento estratégico de los bovinos Argentina es una herramienta muy útil para lograr la terminación de animales destinados al consumo interno y a la exportación. La utilidad de estos encierres consiste permitir un mejor aprovechamiento del forraje y reduciendo la edad a la faena. Para su aplicación es necesario considerar los factores que afectan su resultado económico tales como la característica de las dietas utilizadas, los niveles de consumo y la eficiencia de conversión. Esto interactúa a su vez con el tipo de animal a encerrar. Son dos los sistemas de confinamiento estratégico que van a ser considerados en esta presentación: los corrales de engorde o terminación y los de recría o engorde de los terneros posdete. La actividad del engorde a corral durante los últimos años, ha estado ligada a variaciones operadas en el precio del grano y del tipo de producto logrado. Hay variaciones atribuidas al cambio en las preferencias del consumidor y también al efecto de la concentración de las ventas de carne en determinadas bocas de expendio que exigen uniformidad en la entrega de animales y del tamaño de los cortes. Se comentan entonces algunos aspectos relevantes del engorde a corral americano y de los engordes a corral en Argentina, además de algunos factores que pueden afectar el resultado económico de la actividad. En cuanto al corral de recría, tiene como objetivo la mejora de la ganancia de peso durante esta etapa frente a condiciones de recría a pasto donde pueden existir limitantes en la cantidad y calidad del forraje producido. En esta presentación se hace el comentario y el análisis de los factores a tener en cuenta en los dos tipos de manejo de los animales a corral: los de terminación o engorde final y los de recría o engorde de terneros después del destete. En el primer caso, los efectos del sexo, la edad, la eficiencia de conversión, la raza, el estado nutricional previo, así como factores de la dieta en si mismo. En el segundo caso se analizan sobre todo los distintos tipos de terneros que pueden incorporar al sistema, así como el uso de diferentes dietas. El desarrollo de los encierres de terneros permitió diseñar nuevos sistemas de producción basados en un aumento de la carga, de la ganancia de peso, la modificación del peso final y los posibles cambios en el momento de terminación. En el caso de los corrales de terminación, han sido una herramienta muy útil para lograr la terminación de animales destinados al consumo interno como exportación.