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<p>Exposure to ultraviolet radiation (UVR) has been shown to cause cellular damage in cyanobacteria. In response to UVR exposure, some cyanobacteria produce scytonemin, an indole-alkaloid sunscreen capable of absorbing long-wavelength UVA radiation. Previous genomic and transcriptomic analyses have determined that the production of scytonemin is controlled by a two-component regulatory system (TCRS), encoded by Npun_F1277 and Npun_F1278 in the cyanobacterium <i>Nostoc punctiforme </i>ATCC 29133. This TCRS is thought to not only regulate scytonemin biosynthesis, but also other responses to light and UVR stimuli. To better understand the functionality of the sensor kinase (SK) Npun_F1277 and to determine if it could activate alternative UVR protection pathways, the SK was expressed in <i>Escherichia coli.</i> The first objective of this study was to observe and quantify the level of fitness conferred to <i>E. coli</i> expressing Npun_F1277 from <i>N. punctiforme </i>(strain SKE) when exposed to white light, UVA, and UVB stress. Results from these experiments do not indicate that expression of the <i>N. punctiforme</i> SK conferred an advantage to <i>E. coli</i> under white light, UVA, or UVB stress based on growth alone. Therefore, the second objective was to study the expression of regulatory genes, such as response regulators, in <i>E. coli</i> that are homologs to those associated with the SK Npun_F1277 in <i>N. punctiforme </i>using quantitative-PCR. Expression of the selected genes was measured following exposure to white light and UVA after 30 and 60 minutes as well as UVB after 15 and 30 minutes. Comparison of SKE to empty-vector (EV) control cells exposed to the same stress showed that there were significant changes in the expression of important regulatory genes (e.g. <i>recA, spoT, relA</i>) in the SKE strain. Moreover, when comparing SKE cells exposed to the same conditions above to unstressed SKE cells, a similar result was seen for SKE cells exposed to UVA and UVB as was found in the studies comparing SKE to EV cells. These results suggest that the SK Npun_F1277 may play a role in multiple defense mechanisms of <i>N. punctiforme</i> in addition to initiation of the scytonemin biosynthesis pathway. </p>

<p>As part of development of a human pathogen suppressing in plantae model system within <i>Spinacia oleracea</i>, a known pathogen transmitting produce plants, the secretome of the <i>Stemphylium</i> strain PNW2016-02, an endophytic fungal isolate of spinach plants, was studied. This strain was previously isolated in Purdue University Northwest laboratories in the Biological Sciences Department. As the secondary metabolite secretions of PNW2016-02 were shown to have antibiotic properties against a broad range of bacteria, we sought to improve our understanding of these properties and other characteristics of PNW2016-02 by sequencing and annotating the genome of this strain. Chemical compound characterization was achieved using HPLC-MS, which provided the ability to identify and quantify chemical compounds contained within the PNW2016-02 secretome. Through multi-gene phylogenetic analysis and comparative genomics, we found that PNW2016-02 clusters with a clade representing <i>Stemphylium vesicarium</i>; however, genome annotation also uncovered several genes unique to PNW2016-02. To assist in more fully understanding the secondary metabolites PNW2016-02, a semi-automated bioinformatic pipeline was developed in the R statistical environment in order to reconstruct and characterize the metabolic pathways, especially those pertinent to antibiotic production. Analysis using the bioinformatic pipeline revealed the presence of a number of antimicrobial metabolites, such as flavonoids, and suggests these compounds are produced and transformed in pathways, such as the phenylpropanoid synthesis pathway. These findings also suggest an important vital role of the shikimate pathway for antimicrobial metabolite production within PNW2016-02. Overall, the findings presented here have implications for understanding the antimicrobial strategies employed by <i>Stemphylium</i> PNW2016-02 and its potential for use in the above-mentioned model system.</p>

Type 1 Diabetes (T1D) is a complex autoimmune disorder in which T cells destroy the pancreatic islets, leading to a loss of insulin production and hyperglycemia. The disease incidence has increased globally over the last decades, primarily in individuals with low to moderate genetic risk. There is evidence that environmental factors play a role alongside genetic risk to trigger the disease. An environmental factor that has global influence is adoption of the Western diet, characterized by increased consumption of n-6 fatty acids, including linoleic acid (LA), and decreased consumption of n-3 fatty acids. Increased n-6/n-3 ratios are associated with enhanced susceptibility to autoimmune diseases. We sought to understand how linoleic acid affects the survival and function of T cells from the non-obese diabetic (NOD) mouse, a model for T1D. We found that linoleic acid's presence during in vitro activation of T cells led to an increased expansion of the cells in culture. Additionally, CD4+ and CD8+ T cells activated in linoleic acid's presence produced increased levels of pro-diabetogenic cytokines, including Interleukin-21 (IL-21) and Interferon-gamma (IFN-γ). In contrast, linoleic acid reduced IL-10-producing CD4+ T cells, which are protective in T1D, significantly changing the balance between pro-and anti-inflammatory T cell subsets. Gene expression analysis of T cells exposed to linoleic acid during in vitro activation revealed decreased gene expression of lipid-regulated transcription factors, peroxisome proliferator-activated receptors (PPAR), PPARα and PPARγ. These data suggest a role for these transcription factors and their associated pathways in linoleic acid-mediated T cell functions. Finally, we tested whether the T cell fatty acid response is regulated by the cytokine IL-7, which modulates T cell immunometabolism. However, our data did not reveal a prominent role for IL-7 in regulating the T cell response to linoleic acid. Together, these studies add to evidence that fatty acids present in the microenvironment can directly alter T cell functions and that changes in dietary components may contribute to enhanced T1D susceptibility.

Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Nutrition and Food Science, 1982. / MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE. / Pages 373-383 reprint from Developments in Industrial Microbiology. / Bibliography: p. 243-261. / by Robert James Fleischaker, Jr. / Ph.D.

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.

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.

In this thesis, we develop and apply top-down, quantitative approaches to gain novel insights into various complex biological systems. Beginning at the multicellular level, we study human gut microbiome dynamics from an ecological perspective. We develop computational frameworks to enable a global understanding of the spatiotemporal variability of gut bacterial abundances. We demonstrate the utility of our frameworks to elucidate the ecological processes governing abundance changes of gut microbiota. We then shift our focus to the intracellular level by investigating the metabolic limitations of cancer cell growth. We use coarse-grained mathematical modeling to identify a major growth limitation of cancer cells associated with electron acceptor deficiency, which we then experimentally validate. Collectively, these set of approaches help to decipher the organizing principles of complex biological systems at both the individual and multicellular levels.

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.

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.