Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 192-197).

The intertidal red algal genus Porphyra is found on rocky shores worldwide. In the Northern Hemisphere the genus is well studied but there is a paucity of data on southern hemisphere Porphyra and even less on New Zealand Porphyra. The species� taxonomy has been undergoing revision since the late 1990�s, when it was discovered that the main species P. columbina and P. lilliputana reported for New Zealand were a combination of several endemic species. These species are found from the low to the high intertidal watermark; hence they are exposed to fluctuating stresses such as desiccation, temperature, high light and UV radiation. Algae have evolved a number of mechanisms to adapt to naturally changing increasing abiotic conditions, such as accumulation of screening pigments and changes in antioxidant metabolism during light stress. For terrestrial plants, polyamines (small aliphatic amines) have been shown to be involved in protecting cells from damage under conditions of stress including UV-B radiation; such mechanisms have yet to be identified in algae. The overall aim of this study was to determine the importance of cellular processes in shaping the community structure of Porphyra on a wave-exposed shore on the east coast of the South Island, New Zealand. Porphyra distribution and community structure was assessed by regular monthly monitoring of presence and absence of Porphyra along four transect lines at the site. Enviromnental information was recorded to determine the effects of temperature, light, UV radiation, humidity and wind on Porphyra�s spatial and temporal distribution. Regular tissue samples were taken for species identification by the application of primers, which were specifically designed during this study. P. cinnamomea and Porphyra spec. "ROS 54" were identified as dominant species present almost throughout the year with a pronounced maximum in presence during late winter and spring, and some weeks of absence during April or May. The two dominant species were recorded from the low to the high intertidal shore, but the mid intertidal was identified as the preferred habitat. Other species that were found were rare and only present for a few months in a very restricted area. It was hypothesised that free radical generation and antioxidant metabolism are associated with desiccation tolerance in Porphyra. An attempt was made to investigate the impact of desiccation stress on Porphyra. The extraction process of antioxidants was problematic and no reproducible results could be obtained. It was attempted to investigate the spatial distribution of spores and conchocelis of different Porphyra species in the field, and determine if those found at Brighton Beach are species-specific in their morphology. This indicated that the two main Porphyra species at Brighton Beach not only prefer to occupy the same habitat but that they also have a morphologically similar conchocelis phase. Mechanisms on a cellular level such as polyamine metabolism affected by environmental (abiotic) stresses are related to the alga�s ability to adapt to stress and therefore can have an effect on Porphyra�s distribution along the shore and its presence throughout the year. The depletion of the ozone layer has become an important issue as the effects of increased UV radiation on the environment, especially the intertidal habitat, are revealed. Marine macrophytes possess the main three. polyamines: putrescine, spermidine and spermine of varying levels. For the few species studied, Rhodophyta generally contain higher levels of polyamines than Chlorophyta, while polyamine levels for the one heterokontophyte analysed were between Chlorophyta and Rhodophyta. Levels of the three most common polyamines (putrescine, spermidine, spermine) were determined in P. cinnamomea under controlled UV exposure. Tissue discs were exposed to visible light (PAR), PAR and UV-A or PAR, UV-A and UV-B radiation. Discs exposed to PAR and PAR and UV-A showed little change in polyamine levels over a six day trial period, while discs exposed to PAR, UV-A and UV-B showed a significant increase in free, bound soluble and bound insoluble polyamines over the same period of time. Correspondingly levels of ADC and ODC, two enzymes involved in polyamine synthesis, were measured. ODC levels changed little while ADC levels increased significantly during UV-B treatment, indicating that under UV-B stress polyamines are mainly synthesized via the ADC pathway. The experimental set-up and process of this study has not been applied in macroalgal polyamine research and results obtained are the first indication that increased levels of polyamines are involved in protection and/or protection mechanisms in macrophytic algae to prevent UV-B damage.

This thesis is concerned with phytoestrogenic isoflavones, which are a group of plant-derived compounds that can be consumed in the diet or as over-the-counter preparations for self-medication, and have been associated with a wide range of health benefits. However, unlike the extract of St John's wort and grapefruit juice, little is known about the potential for phytoestrogenic isoflavones to be involved in pharmacokinetic interactions. This thesis describes a series of experiments that investigate that potential by assessing the effects of the isoflavones on intestinal P-glycoprotein-mediated transport, hepatic metabolism, and hepatic cell membrane transport of conventional drugs.

Thesis (M.SC.) - Carleton University, 2007. / Includes bibliographical references (p. 94-109). Also available in electronic format on the Internet.

Thesis (MSc (Genetics. Institute for Plant Biotechnology))--University of Stellenbosch, 2006. / The main goal of this project was to elucidate the apparent role of neutral invertase (NI) in sucrose accumulation in sugarcane. In the first part of the study putative transgenic cell lines (transformed with antisense NI constructs) were characterised to confirm the stable integration and expression of the transgene. Batch suspension cultures were used to initiate replicate cultures of several of these transgenic lines as well as a control, and the metabolism of the cultures during a 14 day growth cycle was examined. The transgenic lines had substantially reduced levels of NI activity. While the activities of the other invertases remained unchanged, the activity of sucrose synthase (SuSy) was significantly higher in the transgenic suspension cultures relative to the control. Throughout the growth cycle, sucrose concentrations in the transgenic lines were consistently higher, and glucose and fructose concentrations lower, than the control. The transgenic cultures also exhibited a decreased growth rate in comparison to the control. Labelling studies confirmed a decrease in the in vivo rate of invertase-mediated sucrose hydrolysis in the transgenic lines, as well as indicating a decline in the partitioning of carbon to respiratory pathways in these cultures. In the second part of the study, which focussed on greenhouse-grown transgenic plants, similar results were reported. NI activity was significantly decreased, and SuSy activity increased in all of the tissues sampled. The sucrose concentration and purity were also higher in the transgenic tissues, while the in vivo sucrose hydrolysis rate was lower. Allocation of carbon to respiration was lower in the transgenic plants, suggesting that a decrease in sucrose breakdown reduces the availability of hexoses for growth and respiration. Overall, the results suggest that NI plays a key role in the control of sucrose metabolism, and that changes in the activity of this enzyme have far-reaching effects on cellular metabolism. The fact that the trends reported in the whole-plant studies parallel those of the suspension cultures confirms that suspension cultures can be used as a model system in metabolic engineering research in sugarcane. Thus the possibility now exists to analyse large numbers of transgenic lines in a quicker time frame and at a reduced cost in comparison to conventional methods.

As regiões organizadoras de nucléolos (Nucleolar Organizer Regions - NORs) são regiões de cromatina levemente corada em volta da qual, no final da telófase, o nucléolo é novamente formado após a mitose. Um grupo peculiar de proteínas ácidas que têm alta afinidade por prata são localizadas nos mesmos locais que as NORs, o que confere as mesmas a propriedade de serem clara e rapidamente visualizadas por colorações que utilizam nitrato de prata. As NORs, quando coradas por prata são chamadas de AgNORs. O número de AgNORs está estritamente relacionado com a atividade transcricional do RNAr e com a agilidade e rapidez da proliferação celular. A homeostase celular utiliza-se de mecanismos através dos quais os tecidos de um organismo mantêm-se ou renovam-se, e para que isso ocorra, as células dispõe de dois programas genéticos principais: o ciclo celular e a apoptose. Considerando o crescimento da placenta e consequentemente das células trofoblásticas mono e binucleadas, esse trabalho teve dois principais objetivos: 1. determinar a relação quantitativa entre a proliferação celular e o estágio da gestação através da quantificação das AgNORs utilizadas como marcadores em placentas bovinas, evidenciando a intensa atividade proliferativa e metabólica das células binucleadas e estabelecendo o padrão de normalidade para gestações bovinas; e 2. determinar a ploidia das células do placentônio bovino e, através da análise dos estágios do ciclo celular, estabelecer a cinética celular envolvida na formação e diferenciação das células trofoblásticas binucleadas. Houve um aumento da quantidade de AgNORs nas células trofoblásticas mononucleadas a medida em que a gestação avançou, principalmente no terceiro trimestre de gestação. Isso indica um aumento na atividade proliferativa e/ou metabólica dessas células. O número de AgNORs expresso pela células binucleadas apenas aumentou do primeiro para o segundo trimestre, permanecendo, após esse estágio, praticamente constante. Isso nos leva a crer que a atividade das células binucleadas atinge seu ponto máximo no segundo trimestre e permanece com o seu metabolismo alto até o final da gestação. Além disso, esse número foi extremamente maior do que o observado nas células trofoblásticas mononucleadas, evidenciando a intensa atividade metabólica dessa célula. Na análise do ciclo celular, dois tipos celulares diferiam quanto ao seu tamanho (área) e quanto a sua granulosidade no placentônio, caracterizando diferenças quanto sua ploidia, sendo uma população diplóide e a outra tetraplóide. Para a fase G0-G1, a porcentagem obtida para o primeiro trimestre foi significativamente maior quanto comparada com o segundo e com o terceiro trimestre. Para a fase G2-M, encontramos um padrão inverso ao observado na fase G0-G1. Assim, podemos concluir que no primeiro trimestre de gestação as células tetraplóides estão formadas, mas não diferenciadas. A medida em que a gestação avança e a demanda metabólica fetal aumenta, essas células entram em processo de diferenciação para sua completa transformação em células capazes de produzir diversas substâncias e suprir as necessidades gestacionais maternas e fetais. / Nucleolar Organizer Regions (NORs) are weakly stained chromatin regions around which nucleoli reform after mitosis. A group of highly argyrophilic proteins are localized at the same sites as NORs, allowing NORs to be very clearly and rapidly visualized by silver nitrate staining procedures. They are called AgNORs. The number of AgNORs is strictly related to rRNA transcriptional activity and to the rapidity of cell proliferation. Cellular homeostasis uses two mechanisms to maintain or renew all tissues in organisms: the cell cycle and apoptosis. To comprehend the growth and the maturation of several tissues it is important to understand proliferation cellular kinetics and the cell cycle. The aim of this study was to evaluate the functional activity relation between the proliferative and metabolic capacity of trophoblastic mononucleate and binucleate cells from bovine placentomes at different stages of pregnancy. In addition, this study determined the ploidy and established the cellular kinetics involved in cell formation and differentiation through cell cycle analysis. The results demonstrated that: 1. The AgNOR number of mononucleate trophoblastic cells is related to their proliferative activity and stage of pregnancy; 2. The AgNOR number of binucleate trophoblastic cells is related to their level of metabolic activity and stage of pregnancy; 3. The AgNOR number of binucleate cell was just derived metabolic activity, and not proliferative activity, differing to mononucleate cells; 4. The AgNOR number was much greater in binucleate cells than in mononucleate cells, evidencing that the metabolism of binucleate cells was much higher than that of mononucleate cells; 5. Two cell populations form the bovine placentome: one is diploid cells and the other tetraploid; and 6. In the first trimester, the majority of tetraploid cells are not differentiated; with the progress of pregnancy and the increase in metabolic demands of fetus, the tetraploid cells begin a differentiation process and become cells that are able to supply maternal and fetal needs. These parameters could be applied to investigations about placental abnormalities due to pathologies or gestations derived from manipulated embryos.

Based on data from the World Healthcare Organisation, cardiovascular diseases are the primary cause of disease-related death globally, with myocardial infarction (MI) being the most prevalent. If not treated effectively, MI can progress to heart failure (HF). With 70 million prescriptions for HF in 2014 and 515 people in the UK being hospitalised daily with MI, the British Heart Foundation calls for novel robust treatments. Even though cardiac stem cell (CSC) therapy for MI has been under investigation for more than a decade, there still has not been a consensus over the identity of the adult endogenous CSC. Recent clinical trials, using selected Ckit+ cells or the cardiosphere-derived cells (CDCs) have shown moderate results. The aim of this thesis was to develop a digestion-based method for isolation of cardiac progenitor cells (CPCs) from the mouse atria. The resulting "CTs" were isolated by collagenase/trypsin (where their name has resulted from) digestion with a prolonged period step for cell attachment. CTs were compared to isolated CDCs for their marker expression, using RT-PCR and Immunocytochemistry, showing cells with a mesenchymal phenotype which expressed SCA1 and CKIT. The CDCs had more of a fibroblast phenotype with higher Ddr2 and Wt1 expression. Using a TGF-β1 differentiation protocol, the CTs could be differentiated more effectively to a CM lineage than could the CDCs. In addition, Oleic acid (OA) supplementation stimulated the Peroxisome proliferator-activated receptor alpha pathway and led to maturation of the CT cells, both before and after differentiation. The differentiated CTs begin to express Tnnt2, while OA led to Myh7 increase and upregulated their oxidative metabolism. Finally, the CTs were more able to survive under serum-starvation than the CDCs, and transfection with miR-210 could enhance CT survival under these conditions and increased VEGF secretion. By digestion of the whole atria and allowing a prolonged time for attachment, we have developed a novel isolation protocol which generates a cell population containing a range of progenitors. Cells within this population can survive under serum starvation and can be differentiated to a CM lineage, making them a promising therapeutic population.

Os compostos fenólicos pertencem a um grupo tóxico de poluentes ambientais descartados do processo de muitas indústrias tais como refinarias de óleo e indústrias químicas. Embora o fenol possua ação bactericida, alguns micro-organismos adquiriram a habilidade de se adaptar e utilizar este composto como fonte de carbono e energia, através do controle coordenado de vias metabólicas (catabólicas). A expressão destas vias pode ser regulada por: mecanismos de controle globais ou por uma via específica de resposta controlada, porém estes mecanismos ainda não são bem compreendidos. O presente trabalho pretendeu isolar e identificar micro-organismos de um ambiente contaminado para tratamento biológico de efluentes fenólicos, bem como analisar o padrão de proteínas citosólicas expressas em função da exposição à duas diferentes fontes de carbono (glicose ou fenol). As linhagens isoladas de Cubatão-SP foram identificadas pela amplificação e sequenciamento do gene 16S DNAr, resultando em 100 % de similaridade com os gêneros Achromobacter e Pandoraea. Os ensaios de biodegradação em diferentes concentrações de fenol (200 a 600 mg.L-1) mostraram que as duas linhagens foram capazes de degradar 100 % do fenol. As proteínas expressas por Achromobacter sp. em resposta ao fenol foram submetidas a eletroforese 2D e os resultados sugerem que a biodegradação do fenol foi realizada através da meta clivagem do anel aromático, pois três enzimas desta via foram identificadas (proteína degradação do fenol via meta- clivagem, 2- hidroximucônico semialdeído desidrogenase 1 e 4-hidroxi-2-oxovalerate aldolase). Outras enzimas envolvidas no metabolismo celular também foram identificadas, reforçando a hipótese que o fenol altera todo o metabolismo celular, envolvendo as mais diferentes vias metabólicas para que a célula possa superar o estresse celular ocasionado por esta exposição. / Phenolic compounds belong to a group of toxic environmental pollutants discharged from the process in many industries such as oil refineries and chemical plants. Although phenol has bactericidal action, some microorganisms acquired the ability to adapt and use this compound as a source of carbon and energy, through the coordinated control of metabolic pathways (catabolic). The expression of these pathways may be regulated by: control mechanisms via a global or specific response, but these mechanisms are not well understood. This work aims to isolate and identify micro-organisms from a contaminated environment for biological treatment of phenolic wastewaters, as well as analyzing the pattern of cytosolic proteins expressed in terms of exposure to two different carbon sources (glucose or phenol). The strains isolated from Cubatao-SP were identified by amplification and sequencing of 16S rDNA, resulting in 100% similarity with the genera Achromobacter and Pandoraea. The biodegradation assays at different concentrations of phenol (200 to 600 mg.L-1) showed that both strains were able of degrading 100% of the phenol. The proteins expressed by Achromobacter sp. in response to phenol were subjected to 2D electrophoresis and the results suggest that the biodegradation of phenol was performed using the meta cleavage of the aromatic ring, once three enzymes of this pathway have been identified (protein degradation meta-cleavage pathway phenol, 2-hydroxymuconic semialdehyde dehydrogenase 1 and 4-hydroxy-2-oxovalerate aldolase). Other enzymes involved in cellular metabolism were also identified; reinforcing the hypothesis that phenol modifies the entire cellular metabolism, involving very different metabolic pathways for the cell can overcome stress caused by this exposure.

<p>We have provided the significance of Protein Methyltransferase 5 on skeletal muscle function and muscle development.</p>

<p>  </p> <p>Breast cancer is a significant public health concern being the second leading cause of cancer-related death in women, with a projected 43,250 deaths in the US in 2022. However, cancer progression to metastatic sites is the primary cause of death in breast cancer patients. A hallmark of cancer is the dysregulation of cellular metabolism. Cancer cells have the ability to hijack their metabolism and drive cellular processes supporting cancer progression. As cancer cells continue through the metastatic cascade, they are challenged with various bioenergetic processes that can be supported by the influx of glucose. Thus, altering glycogen accumulation, where glucose is stored in cells, may be beneficial in supporting cancer progression. In this study, we aim to determine what drives glycogen accumulation in metastatic cells and if it is utilized to support cancer progression. We employed the non-metastatic MCF10A-<em>ras</em> and the metastatic MCF10CA1a cells for these studies. Our results demonstrate that metastatic MCF10CA1a have 20-fold accumulation of glycogen compared to the MCF10A-<em>ras</em> cells. Utilizing 13C6-glucose flux analysis, surprisingly, most of the glucose incorporated into glycogen of the MCF10CA1a cells was in the M+5 glucose labeling pattern instead of the expected M+6 pattern which occurs when glucose is directly converted to glycogen. We showed that glycogen was accumulated due to increased gluconeogenesis through cataplerosis (PEPCK) utilizing inhibitors of the enzyme. Additionally, in a pulse-chase experiment using 13C6-glucose flux analysis, there was an approximate 50% reduction in labeled glucose in glycogen, 3 hours after removing the label, suggesting that the MCF10CA1a cells also have a rapid turnover of glycogen. Glucose can be released through two mechanisms, glycogenolysis or glycophagy. Utilizing siRNAs to a rate limiting steps in each pathway, results suggest both glycogenolysis (PYGL) and glycophagy (GAA) are necessary to support cell migration, a critical step in metastasis of the MCF10CA1a cells. Thus, glycogen metabolism is dysregulated in the MCF10CA1a breast cancer cells such that they have increased glycogen accumulation and that glycogen is required to support cell migration. Further understanding the mechanism by which glucose is accumulated and released in a specific cancer and in specific steps or stressors in cancer progression may contribute to potential therapeutic targets to help mitigate metastasis, and potentially breast cancer mortality.</p>