Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

Despite the availability of contraception in Canada, almost half of pregnancies are unintended and account for approximately 95% of elective, induced abortions. Nearly one-third of Canadian women will access an elective abortion in their lifetime and 30-40% of these women will access at least one further termination. Although contraceptive counselling is generally provided as part of abortion care, there is a paucity of research about the effectiveness of peri-abortive counselling methods or women’s experiences with this care. This research was designed to better understand peri-abortive contraceptive decision-making through the lens of the Ottawa Decision Support Framework and addressed gaps in previous research. The thesis was completed in two parts, a literature review and the first Canadian qualitative study on this topic. Unlike most of the available research that focused on typical outcomes such as contraceptive uptake or continuation, the data from the interpretive descriptive study described women’s informational needs, desired supports, and preferences for peri-abortive contraceptive care. The literature review described the current research and the findings from the qualitative study demonstrated that Canadian women’s experiences with peri-abortive contraceptive care are similar to those in studies published in other countries. This thesis contributes to the limited body of knowledge in understanding what women need and want when receiving contraceptive care as part of abortion services.

Single-molecule Förster resonance energy transfer (smFRET) has emerged as an important tool for studying biological reactions. This thesis describes smFRET investigations into the mechanism of bacterial transcription initiation. We developed protocols to immobilize RNAP-DNA initiation complexes using vesicles and antibodies. We used these techniques to show that the transcription bubble conformation in immobilized complexes exhibits inter-molecular heterogeneity. We observed large FRET changes that we attribute to transcription bubble opening and closing dynamics. We found that σ⁷⁰ region 3.2 (σR3.2) influences the kinetics of the bubble dynamics, which supports proposals that σR3.2 interacts with the transcription bubble template strand. We extended our investigations to RNA synthesis and were able to observe abortive initiation cycles directly. We observed RNAP pausing and backtracking for the first time in transcription initiation. We obtained data suggesting that σR3.2 stabilises short RNAs at the active centre and forms a barrier to the extension of RNAs longer than 5-nt in length. We extended our abortive initiation assay to observe signal changes that we attribute to promoter escape. Our data revealed the number of abortive cycles that occur prior to escape, the kinetics of promoter escape, and pausing events that may have some regulatory function. We investigated the conformational dynamics of the RNAP β clamp and observed dynamic conformational changes between clamp-open and clamp-closed states. Our work confirms proposals that the clamp remains stably closed once the open complex (RPO) is formed. We investigated what affect the antibiotics Myxopyronin and Lipiarmycin have on the clamp conformation. Our results revealed that Myxopyronin traps the clamp in a closed conformation, while Lipiarmycin traps it in an open conformation. Overall, we made a number of novel observations that we believe advance our understanding of the mechanism of transcription. We hope that the discoveries reported here will direct future research efforts into RNAP function.

A boldina é o principal alcaloide encontrado no Peumus boldus, muito utilizado na medicina tradicional principalmente pelos seus efeitos gastrointestinais e hepáticos. Estudos indicam que apresenta diversas atividades farmacológicas como coletérica, hipnótica, citoprotetora, antitumoral, antiinflamatória, antipirética, antiplaquetária, antiplasmódica, antidiabética, antihipertensiva, inibidora da tirosinase e da acetilcolinesterase, antagonista dopaminérgico (receptores D1 e D2 like), adrenérgico (receptores α1 e α2) e serotoninérgico (receptores 5-HT3), entre outras. O chá de boldo (Peumus boldus) é muito utilizado por gestantes para aliviar os efeitos negativos da gestação como, constipação, cólicas uterinas, cefaleia e náuseas; controverso a esse uso ele também é muito utilizado como abortivo. Este estudo procurou identificar qual sua ação em ratas Wistar tratadas durante o período gestacional, GD1 ao dia anterior ao parto, nas doses de 1 mg/kg, 10 mg/kg e 100 mg/kg, uma vez ao dia, no ciclo claro, por meio gavagem. Conclui-se que o uso da boldina e do chá de P. boldus deve ser evitado durante o período gestacional pois seu uso na gestação causou alterações no desempenho reprodutiva das fêmeas, perda pré e pós implantação e mortes ao nascimento e pós-natais, além da modificação do comportamento materno (latência para lamber); foram encontradas também alterações no desenvolvimento da proles de fêmeas tratadas com boldina, em seu desempenho nos testes de reflexos (teste de endireitamento, geotaxia e agarrar), físico (abertura de olhos) e reprodutivo (abertura vaginal, descida de testículos e separação prepucial), assim como alteração no peso de órgãos de machos e fêmeas, na produção de espermatozoides e no percentual de espermatozoides com alteração (anormais). / Boldine is the main alkaloid found in Peumus boldus, widely used in traditional medicine mainly for its gastrointestinal and hepatic effects. Studies indicate that it presents several pharmacological activities as a choleretic, hypnotic, cytoprotective, anti-inflammatory, anti-plasmodic, antipyretic, antiplatelet, antidiabetic, antihypertensive, tyrosinase and acetylcholinesterase inhibitor, dopaminergic antagonist (D1 and D2 like receptors), α-adrenergic receptors antagonist (α 1 and α 2 receptors) and serotonergic receptors antagonist (5-HT 3 receptors), among others. Boldo’s tea (Peumus boldus) is widely used by pregnant women to alleviate the negative effects of pregnancy such as constipation, uterine cramps, headache and nausea; Controversely it is also widely used as an abortive substance The aim of this study was to identify the effect of boldine on Wistar rats treated during the gestational period, day 1 of gestation to the day before birth, at doses of 1mg/kg, 10mg/kg and 100mg/kg, daily, light cycle, by gavage. We concluded that the use of boldine and P. boldus tea should be avoided during the gestational period because its use in the gestacional period caused alterations in the reproductive performance of females, loss of pre and post implantation and deaths at birth and postnatal, besides modification of maternal behavior (latence to pup licking); alterations in the development of the offspring which the females were treated with boldine were observed, in their performance in the reflex tests (straightening, geotaxy and grab), physical development (eye opening) and reproductive development (vaginal opening, testicle descent and preputial separation), as well as changes in the weight of male and female organs, in the production of spermatozoa and in the percentage of abnormal spermatozoa.

Fontes de poluição atmosférica têm aumentado em países em desenvolvimento, tornando extremamente difícil o acesso aos efeitos da poluição atmosférica na saúde pública. Bioensaios com plantas têm sido extensivamente utilizados para monitorar a poluição atmosférica em áreas desprovidas de sistemas de monitoramento da qualidade do ar convencionais pela sua simplicidade, baixo custo e alta sensibilidade. O presente estudo visa caracterizar a distribuição espacial da concentração de poluentes no entorno de indústrias através de evidências de toxicidade celular e acúmulo foliar utilizando planta local (Catharanthus roseus (L.) G. Don). O abortamento polínico foi quantificado em microscópio óptico (400x). O acúmulo de elementos traços foi avaliado em EDXRF (Energy Dispersive X-Ray Fluorescence). Os resultados obtidos para a área de estudo 1 mostram que a taxa de abortamento polínico é maior quanto mais próximo da indústria (p=0,047). Para o acúmulo foliar, foram encontrados os elementos S, Fe e Na que se relacionam com produtos de combustão; processo industrial; e aerossol marinho. Para a área de estudo 2, a taxa de abortamento polínico aumenta com a proximidade da área de alto tráfego veicular assim como a concentração dos elementos Al, Cu, S e Zn nas folhas pela Análise de Regressão Linear. Este estudo mostra que as áreas no entorno de fontes emissoras de poluição têm alta concentração de elementos - traço e dano celular o que indica riscos à saúde da população local. Nossos resultados reforçam que o uso de metodologias alternativas e simples como testes de abortamento polínico e bioacumulação em folhas são apropriados para caracterizar gradientes de concentração em áreas desprovidas de sistema de monitoramento da qualidade do ar, em escalas maiores do que as já estudadas. A combinação de técnicas de biomonitoramento e geoprocessamento pode ser utilizada para monitoramento ambiental em áreas com tecnologia limitada / Developing countries are experiencing increases in air pollution sources, making assessment of air pollution impact on human health extremely difficult. Plant bioassays have been widely employed to monitor air pollution in areas devoid of conventional air pollution monitoring systems because of their simplicity, low cost and high sensitivity. The aim of this study was to determine the area of influence of industries using local flora (Catharanthus roseus (L.) G. Don). Abortive pollen tests and the accumulation of toxic elements in leaves were used to detect possible risks to health of the local population. Abortive grains were evaluated through the use of a microscope under 400-fold magnification. The trace elements accumulation was evaluated by using EDXRF (Energy Dispersive X-Ray Fluorescence). Results for study site 1 shown that the closer the distance from the industry, the higher the pollen abortion rate (p=0,047). For bioaccumulation test, S, Fe and Na were associated to combustion of fossil fuels, industrial process and marine aerosol. For study site 2, the closer the distance from the major source of traffic, the higher the pollen abortion rate. The same behavior was observed for Al, Cu, S and Zn concentrations in Linear Regression Analyses. The distribution of element concentrations was elaborated in maps with the obtained results. This work showed that the areas surrounding the emission sources had a higher concentration of toxic elements, leading to greater biological damage, which indicates health risks to the local population. Our results reinforce that the use of alternative and simple methods such as pollen abortion rates and bioaccumulation in leaves are appropriate for characterizing air quality gradients in areas devoid of conventional air pollution monitoring systems, even at greater scales than have been tested before. The association of this technique and geographic information system can be used for environmental screening in areas with limited technology

Fontes de poluição atmosférica têm aumentado em países em desenvolvimento, tornando extremamente difícil o acesso aos efeitos da poluição atmosférica na saúde pública. Bioensaios com plantas têm sido extensivamente utilizados para monitorar a poluição atmosférica em áreas desprovidas de sistemas de monitoramento da qualidade do ar convencionais pela sua simplicidade, baixo custo e alta sensibilidade. O presente estudo visa caracterizar a distribuição espacial da concentração de poluentes no entorno de indústrias através de evidências de toxicidade celular e acúmulo foliar utilizando planta local (Catharanthus roseus (L.) G. Don). O abortamento polínico foi quantificado em microscópio óptico (400x). O acúmulo de elementos traços foi avaliado em EDXRF (Energy Dispersive X-Ray Fluorescence). Os resultados obtidos para a área de estudo 1 mostram que a taxa de abortamento polínico é maior quanto mais próximo da indústria (p=0,047). Para o acúmulo foliar, foram encontrados os elementos S, Fe e Na que se relacionam com produtos de combustão; processo industrial; e aerossol marinho. Para a área de estudo 2, a taxa de abortamento polínico aumenta com a proximidade da área de alto tráfego veicular assim como a concentração dos elementos Al, Cu, S e Zn nas folhas pela Análise de Regressão Linear. Este estudo mostra que as áreas no entorno de fontes emissoras de poluição têm alta concentração de elementos - traço e dano celular o que indica riscos à saúde da população local. Nossos resultados reforçam que o uso de metodologias alternativas e simples como testes de abortamento polínico e bioacumulação em folhas são apropriados para caracterizar gradientes de concentração em áreas desprovidas de sistema de monitoramento da qualidade do ar, em escalas maiores do que as já estudadas. A combinação de técnicas de biomonitoramento e geoprocessamento pode ser utilizada para monitoramento ambiental em áreas com tecnologia limitada / Developing countries are experiencing increases in air pollution sources, making assessment of air pollution impact on human health extremely difficult. Plant bioassays have been widely employed to monitor air pollution in areas devoid of conventional air pollution monitoring systems because of their simplicity, low cost and high sensitivity. The aim of this study was to determine the area of influence of industries using local flora (Catharanthus roseus (L.) G. Don). Abortive pollen tests and the accumulation of toxic elements in leaves were used to detect possible risks to health of the local population. Abortive grains were evaluated through the use of a microscope under 400-fold magnification. The trace elements accumulation was evaluated by using EDXRF (Energy Dispersive X-Ray Fluorescence). Results for study site 1 shown that the closer the distance from the industry, the higher the pollen abortion rate (p=0,047). For bioaccumulation test, S, Fe and Na were associated to combustion of fossil fuels, industrial process and marine aerosol. For study site 2, the closer the distance from the major source of traffic, the higher the pollen abortion rate. The same behavior was observed for Al, Cu, S and Zn concentrations in Linear Regression Analyses. The distribution of element concentrations was elaborated in maps with the obtained results. This work showed that the areas surrounding the emission sources had a higher concentration of toxic elements, leading to greater biological damage, which indicates health risks to the local population. Our results reinforce that the use of alternative and simple methods such as pollen abortion rates and bioaccumulation in leaves are appropriate for characterizing air quality gradients in areas devoid of conventional air pollution monitoring systems, even at greater scales than have been tested before. The association of this technique and geographic information system can be used for environmental screening in areas with limited technology

<p>In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in <i>Escherichia coli</i>.</p><p>The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon.</p><p>The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR.</p><p>The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in <i>Escherichia coli</i>.</p>

In prokaryotes, several mRNA sequences surrounding the initiation codon have been found to influence the translation process; these include the downstream region and its codon context, the Shine-Dalgarno sequence and the S1 ribosomal protein-binding site. In this thesis, the purpose has been to study the role of the downstream region and Shine-Dalgarno-like sequences on early translation elongation and gene expression in Escherichia coli. The downstream region (DR) after the initiation codon (around five to seven codons), has an important role in the initiation of translation. We find that most of the codons which give very low gene expression at +2 (considering AUG as +1), reach 5 to 10 fold higher expression when those codons are positioned posteriori to +2, with the exception of the NGG codons. The NGG codons abort the translation process if located within the first five codons of the DR, due to peptidyl-tRNA drop-off. However, when the NGG codons are situated further down from the DR, the protein expression was increased at the same level of expression as in the presence of any other codon. The Shine-Dalgarno (SD) is an important region of initiation in translation of bacteria. In spite of this, it has been found that Gram-negative bacteria could translate mRNAs with weak or non-functional SD, while the DR carries out a main role in the efficiency of translation. In addition, positions of SD and SD-like sequences are very important to direct initiation of translation in the choice between two possible initiation codons. A strong SD between two initiation sites will favor the second initiation site if it consists of a canonical start codon followed by a good DR. The results suggest that the mRNA sequences surrounding the initiation codon: the downstream region and the Shine-Dalgarno and SD-like sequences, are very important contributors to the translation level and gene expression in Escherichia coli.

The overall objective of this study was to investigate how NS0 cell proliferation is regulated in protein-free media. The hypothesis was that during the adaptation to growth factor-free media, animal cell lines start to produce their own autocrine growth factors to support proliferation, and after some time in a culture the effects of these factors are lost which results in cessation of proliferation. A chemically defined, protein-free and animal component-free medium was developed for the NS0 cells. This medium was comprised of a basal hybridoma medium to which phosphatidyl¬choline, cholesterol, β-cyclodextrin, ferric citrate and amino acids were added. A fed-batch process was then developed in this medium. The feed profile was optimised in a step-wise manner with a final feed solution containing glucose, glutamine, lipids, amino acids, vitamins, sodium selenite and ethanolamine. Specifically, supplementation with lipids (cholesterol) had a drastic effect on cell growth. Calcium, magnesium and potassium were not depleted during culture and a feed containing also iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield further. More than 8 x 106 viable cells mL-1 and 600 mg antibody L-1 was obtained in the final fed-batch. This corresponded to a 4.3-fold increase in viable cell yield and an 11.4-fold increase in product yield compared to bioreactor batch culture when the dilution of the fed-batch culture was also accounted for. The presence of autocrine growth factors in NS0 cell cultures was initially investigated by studying the effects of conditioned medium (CM). Concentrated CM had a significant positive effect on cell growth and part of this effect could be attributed to factor(s) eluting from a gel-filtration column at 20-25 kDa. In the search for cell-derived factors affecting cell growth the following proteins were identified as released/secreted by the NS0 cells; cyclophilin A, cyclophilin B, cystatin C, D-dopachrome tautomerase, IL-25, isopentenyl-diphosphate delta-isomerise, macrophage migration inhibitory factor (MIF), β2-microglobulin, niemann pick type C2, secretory leukocyte protease inhibitor (SLPI), thioredoxin-1, TNF-α, tumour protein translationally controlled-1 and ubiquitin. Zymogram electrophoresis further identified aspartic acid, papain-like cysteine (including cathepsin L) and serine protease activity in the CM. Pro/cathepsin L, CypB, EGF, IFN-α/β/γ, IGF-I/II, leukaemia inhibitory factor, IL-6, IL-11, IL-25, MIF, oncostatin M, TGF-β and TNF-α were excluded as involved in autocrine regulation of NS0 cell proliferation. The serine protease activity was suggested to affect the cells negatively and since the serine protease inhibitor SLPI is also present in NS0 CM, a balance in serine protease activity may be crucial for optimal cell growth. Further, the receptor gp130, known to be associated with myeloma cell growth, was shown to be essential for NS0 cell proliferation as demonstrated by siRNA gene silencing. The results suggested that autocrine regulation of proliferation in NS0 cell cultures involves the receptor subunit gp130. / QC 20100920

Orientador: Ione Pellegatti Lemonica / Banca: João Lauro Viana de Camargo / Banca: Márcia Guimarães da Silva / Banca: Silvana Lima Górniak / Banca: Regiane Kawakami / Tese não possui um resumo geral, possue um resumo para cada capítulo / Resumo : O objetivo do presente estudo foi verificar, experimentalmente, o possível mecanismo pelo qual o extrato aquoso de Plectranthus barbatus (boldo-brasileiro), planta utilizada popularmente como abortiva, atua sobre o organismo materno ou sobre o desenvolvimento do concepto durante o período de pré-implantação, correlacionando sua ingestão com possíveis alterações no transporte e desenvolvimento embrionário ou com alterações hormonais maternas...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract : The present study was conducted to determine the possible mechanism by which the aqueous extract of Plectranthus barbatus (brazilian-boldo), a plant used popularly as abortive agent, can lead to early loss of pregnancy, correlating this possible effect with morphological alterations in the embryo, oviductal motility dysfunctions or maternal hormonal level modifications...(Complete abstract, access undermentioned electronic address) / Doutor