Orientador: Ana Silvia Alves Meira Tavares Moura / Resumo: O objetivo do presente trabalho foi avaliar o efeito da adição de óleo de linhaça (OL) ou farelo de linhaça (FL) na dieta de ovelhas da raça Bergamácia em lactação, sobre o teor de aminas biogênicas e polifenóis no leite. Foram utilizadas 70 ovelhas distribuídas aleatoriamente entre os tratamentos. As dietas experimentais foram: Controle (CT) – concentrado sem adição de suplementação lipídica; Óleo de linhaça (OL) – concentrado com adição de 3% de OL (%MS); e Farelo de linhaça (FL) – concentrado com adição de 15% de FL (%MS). Durante todo o período experimental, os animais foram mantidos confinados em baias coletivas e recebiam dieta composta por 60% de silagem de milho e 40% de concentrado referente a cada tratamento. As ovelhas foram ordenhadas uma vez ao dia, no período da manhã, e tiveram suas produções controladas diariamente. As amostras de leite para a avaliação das aminas biogênicas foram colhidas a cada 14 dias a partir do primeiro dia de ordenha e as amostras de leite para análise dos polifenóis foram colhidas a cada 14 dias a partir do quinto dia pós-parto. O experimento foi conduzido no delineamento inteiramente casualizado e os dados avaliados por análise de variância (P< 0,05). Não foi encontrada a presença de cadaverina no leite e a serotonina foi a amina predominante no leite de ovelhas em todos os tratamentos. O tratamento controle apresentou maiores teores de espermina (0,300 mg/100g) entretanto, o tratamento com óleo de linhaça apresentou menores teores de ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim in this study was to determine the effects of feeding diets with linseed oil (OL) or flaxseed meal (FM) of Bergamasca ewes on to the content of biogenic amines and phenols in milk.Seventy ewes were distributed in three groups: Control (CT) - no lipids; Linseed (L) – with addition of 3% of OL (DM basis); and Flaxseed meal (F) – with addition of 15% of FM (DM basis). Throughout the experiment, the ewes remained confined in collective pens and were fed a diet containing 60% corn silage and 40% concentrate according to each treatment. They were mechanically milked once a day and had their productions controlled. The milk samples for biogenic amines analysis were collected every 14 days from the first milking day and the milk samples for phenols analysis were collected every 14 days from the 5th day postpartum until de end of the lactation. The experiment was conducted in a random customized design and the data evaluated by analysis of variance (P< 0,05). Cadaverine was not found and the serotonine was the predominant amine in the milk from ewes in all treatments. The control treatment showed high contents of espermine (0,300 mg/100g), however the OL had lower contents of histamine (0,0049 mg/100g). The espermidine was the biogenic amine more correlated with greater numbers of amines. There was no difference between treatments for polyphenols contents in milk, however the treatment with oil it was more efficient. / Doutor

Colorectal cancer (CRC) has a 5-year survival rate of 68% yet it still has a mortality rate of 50,000 per year. While CRC has a host of causes, one that stands out is TGFβ deficient signaling, which is disrupted in a majority of high-microsatellite-instability or inflammation-associated CRCs. Since TGFβ is a multifunctional cytokine, it has been elusive to determine whether its effect on cancer development is operating through inflammation, differentiation or developmental pathways. Additionally, it is now becoming apparent that a great number of CRC cases can be associated with and possibly caused by gut bacteria dysbiosis. Here, I present a metagenomic and metatranscriptomic study of the interactions between TGFβ deficient signaling, inflammatory signaling, and the microbiome in a CRC mouse model. TGFβ deficient mice have reduced amounts of Firmicutes as well as mRNA counts of a key butyrate enzyme. Lack of butyrate, as shown by previous literature, could be inhibiting apoptosis and promoting growth. Also, TGFβ deficient mice have increased mRNA counts of polyamine producing genes, which could act synergistically with butyrate reduction. I find that H. hepaticus inoculation, as a source of inflammatory signaling, affects another species, M. schaedleri, to produce pro- inflammatory lipopolysaccharides. Additionally, H. hepaticus itself has increased oxidative phosphorylation; reactive oxygen species from this process could be adding to cancer-promoting DNA damage. Taken together, TGFβ deficient signaling and H. hepaticus inoculation, disrupt enough pathways to cross the threshold of carcinogenicity in 40% of the mice in our study. The results of this study emphasize the importance of microbiome function and represent possible new avenues of treatment.

Abstract Fetal alcohol syndrome (FAS) is a pattern of anomalies in affected children due to maternal alcohol administration at vulnerable stages of fetal development. Intrauterine growth restriction and facial malformation are the presenting phenotypes of FAS. In this investigation, novel pathomechanisms of intrauterine growth restriction and facial malformation were the primary aims. We found by a FAS mouse model that AceCS1 gene expression and polyamines are the immediate targets of fetal alcohol exposure. The AceCS1 product is a precursor for lipid synthesis and protein acetylation and possibly, for polycation acetylation. We cloned the Mus musculus nuclear-cytosolic AceCS1 gene, and showed that its expression is developmentally regulated with a dynamic localization in the cytosolic and nuclear compartment. The enzyme plays an essential role in de novo synthesis of acetyl Coenzyme A. Fetal alcohol administration targets nutrient supplying networks, which are localized at critical barriers. The main findings were reduced surface of the labyrinthine zone, destruction of gap junctions in the hemotrichorial placenta, reduced syncytiotrophoblastic cell layers and loosening of interaction between cell layers and embryo endothelial cells, reduced Reichert’s membrane thickness with discontinued Reichert’s trophoblast and loss of interaction by Reichert’s-parietal cells, reduction of capillary network and reduced vascularization in the brain area, and perturbed neural crest migration and formation of neural tube defect. Alteration of angiogenesis -regulating proteins such as VEGF, PlGF, PECAM was detected in FAS, with no significant changes in placental angiogenesis of the labyrinthine zone, but up-regulation of VEGF/PlGF caused permeability changes in the placenta and yolk sac. On the other hand, the PECAM pool in embryos’ brain was reduced, which in turn led to decreased angiogenesis and vascularization. / Tiivistelmä Sikiön alkoholisyndrooma (engl. Fetal alcohol syndrome, FAS) on joukko muutoksia, joita esiintyy äidin raskaudenaikaisen alkoholin käytön seurauksena, kun käyttö osuu sikiökehityksen kannalta kriittiseen vaiheeseen. Kohdunsisäisen kasvun rajoittuminen ja kasvojen epämuodostumat ovat FAS:n tyypillisimpiä ilmentymiä. Tässä tutkimuksessa pyrittiin löytämään uusia patomekanismeja kohdunsisäisen kasvun rajoittumiselle ja kasvojen epämuodostumille. Hiiren FAS-mallin avulla selvisi, että sikiön altistuminen alkoholille vaikuttaa suoraan AceCS1-geenin ilmentymiseen ja polyamiinien pitoisuuteen. AceCS1-geenin tuote on esiaste lipidien synteesissä ja proteiinien asetylaatiossa sekä mahdollisesti myös polykationien asetylaatiossa. Työssä myös kloonattiin hiiren (Mus musculus) AceCS1-geeni, jonka tuotetta esiintyy sekä tumassa että solulimassa. Lisäksi osoitettiin, että geenin ekspressio oli kehityksen aikana säädelty tuottamaan entsyymiä dynaamisesti eri paikkoihin solussa. Entsyymillä on lisäksi merkittävä osuus asetyyli-koentsyymi-A:n de novo–synteesissä. Sikiön altistuminen alkoholille kohdistuu sellaisten ravintoaineiden saatavuuteen, jotka sijaitsevat kriittisesti tärkeissä kudosrajapinnoissa. Päälöydöksinä olivat vähentynyt labyrinttikudoksen pinta-ala, gap-liitosten tuhoutuminen istukan veriesteessä (hemotrichorial?), ohentunut trofoblastisolujen kerros ja Reichertin kalvon paksuus, harventunut hiusverisuonten verkosto sekä verisuonitus aivojen alueella sekä hermopienan solujen siirtymishäiriö ja hermostoputken sulkeutumishäiriö. Verisuonten muodostumista (angiogeneesiä) säätelevien proteiinien (kuten VEGF, PlGF, PECAM) muutoksia todettiin FAS:ssa, mutta merkittäviä muutoksia ei havaittu istukan verisuonten muodostumisessa. VEGF/PlGF-suhteen suureneminen muutti istukan ja ruskuaispussin verisuonten läpäisevyyttä. Toisaalta sikiöiden aivojen PECAM-määrä pieneni, mikä johti verisuonten ja verisuoniverkoston muodostumisen vähenemiseen.

Plasmodium falciparum causes the most severe form of human malaria, and the continual development of resistance of this parasite to current anti-malarial drugs underpins a pressing need for the discovery of novel chemotherapeutic approaches. Polyamines and their biosynthetic enzymes are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of the malaria parasite’s polyamine biosynthesis pathway causes cytostatic arrest in the trophozoite stage, but does not cure infections in vivo. This may be due to the salvage of exogenous polyamines from the host, replenishing the intracellular polyamine pool; however the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study the uptake of the polyamines putrescine and spermidine into P. falciparum-infected erythrocytes (iRBC) well as into P. falciparum parasites functionally isolated from their host cell by saponin-permeabilisation of the erythrocyte membrane was investigated using radioisotope flux techniques. While the characteristics of transport of putrescine into infected erythrocytes were similar to those of transport into uninfected erythrocytes, spermidine entered iRBC in part via the ‘new permeation pathways’ induced by the parasite in the erythrocyte membrane. Both putrescine and spermidine were taken up across the plasma membrane of isolated parasites via a saturable, temperature-dependent process that showed competition between different polyamines as well as the polyamine precursor ornithine and basic amino acids. Inhibition of polyamine biosynthesis led to increased total uptake of both putrescine and spermidine. The influx of putrescine and spermidine into isolated parasites was independent of Na+ but increased with increasing pH and showed a marked dependence on the membrane potential, decreasing with membrane depolarisation and increasing with membrane hyperpolarisation. Both anthracene and polyamine derivatives have been shown to have anti-malarial activity. Anthracene-polyamine conjugates have been developed with the aim of utilising the polyamine uptake mechanisms of cancer cells to deliver the cytotoxic anthracene moieties to these cells. Here, several anthracene-polyamine conjugates showed promising anti-malarial activity. These compounds inhibited parasite proliferation with IC50 values in the nM range, and caused an arrest in the cell cycle, as well as a decrease in the mitochondrial membrane potential. Cytotoxicity could not be reversed by the addition of exogenous polyamines, nor did the conjugates have an effect on intracellular polyamine levels. This doctoral study showed that P. falciparum parasites not only synthesise polyamines, but can also acquire putrescine and spermidine from the extracellular environment and paves the way for interfering with polyamine metabolism as an anti-parasitic strategy. / Thesis (PhD)--University of Pretoria, 2012. / Biochemistry / unrestricted

Orientador: Sonia Maria Alves Bueno / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-27T12:23:49Z (GMT). No. of bitstreams: 1 Carmignotto_GabrielaPannunzio_M.pdf: 6143089 bytes, checksum: aa61b4762764a38220fc51226e410a80 (MD5) Previous issue date: 2015 / Resumo: Os fragmentos Fab de imunoglobulina G são utilizados principalmente em testes diagnósticos e terapias. Por apresentarem tamanho reduzido, os fragmentos Fab possuem maior facilidade de penetração em tecidos e maior velocidade de circulação no plasma quando comparados à IgG intacta. A purificação dos fragmentos Fab é realizada geralmente por cromatografia de afinidade utilizando os ligantes bioespecíficos proteína A, proteína G e proteína L. Entretanto, esses ligantes apresentam algumas desvantagens, entre elas seu elevado custo e a necessidade de eluição em condições drásticas. Como alternativa, muitos estudos têm sido realizados empregando ligantes pseudobioespecíficos e de interação mista visando o desenvolvimento de novas estratégias que proporcionem a purificação dos fragmentos Fab. Neste contexto, este trabalho tem por objetivo avaliar a seletividade das poliaminas ?-aminohexil e poli(etilenoimina) imobilizadas em gel de agarose na separação de fragmentos Fab humano a partir de uma solução de IgG humana clivada enzimaticamente, empregando diferentes sistemas tamponantes. A recuperação seletiva dos fragmentos Fab foi avaliada por eletroforese SDS-PAGE, Western blot e imunodifusão radial. Os resultados indicaram que nas cromatografias realizadas em gel ?-aminohexil-agarose em Tris-HCl a pH 8,0, os fragmentos Fab foram recuperados nas frações não retidas, com valores calculados de pureza de 97% e recuperação de 100%. Nas cromatografias realizadas em gel PEI-agarose empregando o tampão Tris-HCl nos valores de pH de 7,5 e 8,0, a pureza obtida foi de 96% e a recuperação foi de 94% e 93%, respectivamente. As curvas de ruptura para os adsorventes foram determinadas e os resultados obtidos indicaram melhor recuperação do fragmento Fab com alta pureza no flowthrough para o ligante ?-aminohexil (2,7 mg) quando comparado ao ligante PEI (0,8 mg). A isoterma de adsorção de fragmentos Fc para o adsorvente ?-aminohexil-agarose foi determinada e os parâmetros capacidade de adsorção de 44,32 mg.mL-1 de gel e constante de dissociação de 1,11.10-5 mol.L-1 foram obtidos por ajuste destes parâmetros segundo o modelo de Langmuir-Freundlich. O valor de n obtido foi igual a 1,68, indicando cooperatividade positiva na adsorção dos fragmentos Fc e IgG não clivada. Os resultados mostraram que as poliaminas ?-aminohexil e PEI apresentaram potencial como ligantes para a purificação de fragmentos Fab / Abstract: Fab fragments from immunoglobulin G are considered an important tool for the therapy and diagnose of diseases. Their reduced molecular size is an advantage over the intact IgG allowing faster tissue penetration and circulation in the plasm. Fab fragments are often purified by affinity chromatography using biospecifics ligands, such as protein A, protein G, and protein L. However, the use of those ligands is hindered by their high costs and elution in drastic conditions. Several studies have been performed for the development of new techniques to improve the chromatographic purification of Fab fragment, e.g., using pseudobiospecifics or mix-mode ligands. In this context, we have evaluated the efficiencies of polyamines ?-aminohexyl and poly(ethyleneimine) (PEI) ligands immobilized in agarose gel aiming the purification of human Fab fragments. Our chromatographic studies were conducted feeding cleaved human IgG solution into the aforementioned adsorbents using different adsorption buffers and conditions. The selectivity towards Fab fragments was evaluated using SDS-PAGE, Western blot, and radial immunodiffusion. The chromatography performed with ?-aminohexyl-agarose using Tris-HCl buffer pH 8.0 recovered 100% of the Fab fragments with 97% of purity. Studies done with adsorbent PEI-agarose using Tris-HCl pH 7.5 and 8.0 as adsorption buffers recovered 94% and 93% of Fab fragments, respectively, with 97% of purity. Breakthrough curves experiments showed that the ?-aminohexyl ligand was superior than PEI ligand in terms of recovering Fab fragment with high purity in flowthrough (2.7 mg against 0.8 mg, respectively). The adsorption isotherm of ?-aminohexil-agarose adsorbent was determined adjusted by the Langmuir-Freundlich model as well (Qm: 44.32 mg.mL-1 of gel; Kd: 1.11.10-5 mol.L-1). Additionally, Fc fragments and IgG binding showed positive cooperativity (n=1.68). All these results indicate that ?-aminohexyl and PEI polyamines are potential ligands for the purification of Fab fragments and can possibly improve the existing techniques employed for this purpose / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestra em Engenharia Química

Malaria is considered the most prevailing human parasitic disease. Despite various chemotherapeutic interventions being available, the parasite responsible for the most lethal form of malaria, Plasmodium falciparum, is continuously developing resistance towards drugs targeted against it. This, therefore, necessitates the need for validation of new antimalarial development. Polyamine biosynthetic enzymes, particularly S-adenosylmethionine-L-decarboxylase (PfAdoMetDC), has been identified as a suitable drug target for protozoan parasitic diseases due to its essential role in cell proliferation. Furthermore, in Plasmodium polyamine biosynthesis, PfAdoMetDC is organised into a unique bifunctional complex with ornithine decarboxylase (PfAdoMetDC/ODC) covalently linked by a hinge region, distinguishing this enzyme as unique a drug target. However, inhibitors targeting this pathway have not been successful in clinical assessment, creating the need for further research in identifying novel inhibitors. This study focused on the structural and functional characterisation of protein-specific properties of the AdoMetDC domain in P. falciparum parasites, as well as identifying novel inhibitors targeting this enzyme as a potential antimalarial therapeutic intervention. In order to develop novel inhibitors specifically targeting PfAdoMetDC through a structure-based drug discovery approach, the three-dimensional structure is required. However, due to a lack of structural and functional characterisation, determination of the crystal structure has been challenging. Heterologous expression of monofunctional PfAdoMetDC was achieved from a wild-type construct of the PfAdoMetDC domain including the covalently linked hinge region. In chapter 2, deletion of a large non-homologous, low-complexity parasite-specific insert (A3) in monofunctional PfAdoMetDC resulted in an increased yield, purity and sample homogeneity, whilst maintaining protein functionality and structural integrity. However, truncation of the proposed non-essential hinge region resulted in low-level expression of insoluble protein aggregates and a complete loss of protein activity, indicating that the hinge region is essential for monofunctional PfAdoMetDC. However, in the absence of the three-dimensional PfAdoMetDC crystal structure, novel derivatives of a well-known AdoMetDC inhibitor, MDL73811, were tested for their activity against heterologous PfAdoMetDC, as well as their potency against P. falciparum parasites, in chapter 3. The compound Genz-644131 was identified as a lead inhibitor of PfAdoMetDC, however, the poor membrane permeability of the compound resulted in low in vitro activity. Drug permeability of Genz-644131 into P. falciparum infected erythrocytes and its potency was significantly improved by its encapsulation into a novel immunoliposome based drug delivery system. The results presented here provide essential information for development of a unique strategy in obtaining suffiecient levels of fully active recombinant PfAdoMetDC of sufficient purity for crystallisation studies and subsequent structure-based drug design efforts. The combination of Genz-644131 with the novel drug delivery system, which markedly improved its potency against PfAdoMetDC may proof to be a viable antimalarial chemotherapeutic strategy for future investigations. / Thesis (PhD)--University of Pretoria, 2014. / tm2015 / Biochemistry / PhD / Unrestricted

The ability of the accumulation of thorium and study of the stress responses on his presence was tested on a selected cultivar of tobacco, La Burley 21. Plants were cultivated in Hoagland's hydroponic medium under artificial light. Except to the ability of accumulation and distribution of thorium in the all parts of plant was investigated the effect of selected organic and inorganic additions on accumulation. Among organic substances included citric acid, tartaric and oxalic acid in their presence was observed the increase of thorium in all parts of the plant. Were also tested products from the diamine and polyamines (putrescine, cadaverine, spermine and spermidin). These substances, also known for their antioxidant activity in plants, had an impact on reducing the accumulation of thorium, especially in the root system of plants. The most important factor influencing the accumulation of thorium was the absence of phosphate ions in a hydroponic medium, which caused the rise of the concentration of thorium about several levels in all parts of the plants. The initial decrease of pH after additions of organic acids or addition of high concentrations of thorium and the gradual increase of pH during cultivation had proved significant. It was also compared the uptake of accumulation and distribution of...

Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

A rapid method was developed for the quantitative analysis of N, N'hexamethylenebisacetamide (HMBA) and its deacetylated metabolites, N-acetyl-1, 6- diaminohexane (NADAH) and 1, 6-diaminohexane (DAH). The method used flowinjection liquid chromatography (LC)-atmospheric pressure chemical ionization (APCI)selected ion monitoring at low/high resolving power (LR/HR-SIM)-mass spectrometry (MS). Samples were extracted from murine erythroleukemia (MEL) cell culture and were analyzed without chemical derivatization. The limits of detection (LOD) for HMBA, NADAH and DAH measured by HR-SIM were 0.05, 0.1 and 0.2 picomole, respectively. Calibration curves were constructed by using tetradeuterated DAH as an internal standard for both SIM methods. The ratio of the analyte concentration in the extract of a MEL cell culture treated with HMBA for 120 hours was found to be 1. 7, 27 and 17 times that of the control for DAH, NADAH and HMBA, respectively. The HRSIM that used a lock-mass technique helped eliminate chemical interferences that had mlz values similar to the one of interest. Although this technique was developed using HMBA, NADAH and DAH, it is expected to work for natural polyamines and their acetylated metabolites. Qualitative analysis was performed by both positive electrospray (ES)- and APCIMS under full mass range scanning. The mass spectra of polyamine standards were characterized. HMBA exhibited an ability to form mono- and bi-molecular adducts with metal ions, itself and with other polyamine molecules in ES-MS. The LODs estimated by ES-MS were consistently higher than the APCI-SIM methods that is described here.

Putrescine, spermidine and spermine are classified as polyamines and are found in virtually all living tissues. Although polyamines have been proposed to influence cell proliferation and differentiation, the mechanism by which they do so is not well understood. Previous studies in our laboratory have been focused on the interconversion of spermidine and N8-acetylspermidine (N8-AcSpd) and the enzyme N8-AcSpd deacetylase. In the present study, a N8-AcSpd deacetylase inhibitor, 7-[N-(3-aminopropyl) amino] heptan-2-one (APAH) was employed to study the effect of N8-AcSpd deacetylase inhibition on proliferation of cultured cells. This study for the first time shows the pharmacological effects of elevated N8-AcSpd levels and inhibition of N8-AcSpd deacetylation. Based on the fact that inhibition of N8-AcSpd deacetylation stimulates the growth of cells in culture, we propose to search for a new class of antitumor agents among the inhibitors of spermidine N8-acetyltransferase.