Academic literature on the topic 'Differential accumulation'

碩士<br>高雄醫學大學<br>生物醫學暨環境生物學研究所<br>97<br>Non-alcoholic fatty liver disease (NAFLD) is defined histologically by the accumulation of lipid droplet, mostly in the form of triglyceride, in hepatocytes. Recently, NAFLD becomes a common cause of chronic liver disease. Especially, NAFLD is cross-linked with metabolic syndrome. The prevalence of NAFLD in Taiwan is estimated to be one-third in general adult population. In order to study the pathogenesis of NAFLD in hepatocyte level, we induced human hepatoma cell line HepG2 into steatotic state with 200 ?嵱 palmitic acid for 2 to 6 days. Subsequentially, we surveyed the RNA expression profile with microarray gene chips. The differential expressed genes at day 2 and day 6 were clustered into 10 functional groups, including cell cycle regulation, anti-apoptotic regulation, inflammatory response, carbohydrate and lipid metabolism, cytoskeleton and intracellular trafficking, muscle contraction and cysteine protease inhibitors. The up-regulation of lymphocyte antigen 96 (LY96), glycerol kinase, interleukin-8 were validated with RT-PCR. In high fat-diet induced NAFLD mouse model, we observed the increase of LY96 RNA expression in the mouse liver. Because palmitate treatment increased LY96 expression but inhibit NF?羠 activity, it suggested that LY96 may play unknown roles during lipid influx. We shall silence the endogenous LY96 to study the roles of LY96 in the development of steatosis.

This thesis is the first report linking differential distribution of cadmium (Cd) among tissues of hydroponically-grown soybean with Cd amendments – lower seed Cd-accumulating OAC Champion and higher seed Cd-accumulating OAC Bayfield – and the mechanisms responsible for these differences. OAC Champion retains 94% (64% for OAC Bayfield) of accumulated Cd in the root stock and Cd in its xylem sap is eleven-fold less concentrated than OAC Bayfield by seed fill. Though the movement of Cd to shoots is more restricted in OAC Champion, the concentration in some seed still approximates or exceeds 0.1 mg Cd • kg-1, indicating that in soils with elevated available Cd, there is potential for many cultivars of soybean to exceed the new suggested maximum for soybean, under an amendment to regulation (EC) No 1881/2006. Phytic acid was also assayed in seed tissue and OAC Bayfield was found to contain the highest concentrations.

Microglia are a unique type of immune cell found within the brain, spinal cord and retina. In the healthy brain, their job is to support neurons, defend against infectious microbes, clear extracellular debris and remove dead or dying cells through phagocytosis. This diverse array of functions presents the possibility of unique subsets of microglia existing in the healthy brain, yet none have been described thus far. By utilizing cellular autofluorescence as a discriminating characteristic, we identified two novel subsets of microglia present in the healthy brains of mice and non-human primates. Approximately 70% of microglia displayed autofluorescence (AF+) while the remaining 30% did not (AF–). While the proportion of AF+ and AF– microglia remained constant throughout most of adult life, the autofluorescence intensity increased exclusively in the AF+ subset at an almost linear rate with age. This gain in autofluorescence correlated with equivalent increases in the size and complexity of storage bodies, as detected by transmission electron microscopy and increases in LAMP1 levels, a key component of the lysosomal compartment. As the brain ages, lysosomal storage material builds up inside AF+ microglia, further increasing the accumulation of autofluorescence as a result. The analysis of protein content in autofluorescent subsets revealed that AF+ microglia produced more proteins and enzymes involved in the storage and degradation of waste material, as well as more proteins involved in the regulation of mTOR, a key cellular pathway governing nutrient availability and energy production. Interestingly, the disruption of lysosomal function in microglia through genetic mutations accelerated the accumulation of storage material in AF+ cells, which led to impaired microglia physiology and increased cell death, mimicking the effects observed during advanced aging. Increasing evidence suggests that the accumulation of waste materials inside the brain contributes to diseases of aging and these data are suggestive of a mechanistic convergence between aging and lysosomal storage disorders.

The dissertation develops the concept of repurposing as a means for thinking with activists and the issues they confront. It moves alongside pirate television collective insu^tv as they draw on a variety of histories, traditions and technological resources for their practices. Repurposing functions on multiple levels and at multiple scales, from the recycling of materials and spaces to the harnessing and relaying of encounters and events within an ever-expanding field of social relations. When seen as a way of connecting activist groups and communities, the repurposing of media contributes to strengthening an often fragmented and conflicted activist field. Indeed, insu^tv’s use of information and technology brings to the fore the value of media activism for the creation of social assemblages in which the “media” literally mediates between individuals and among individuals and their environment, instituting and developing an ontogenetic relation (Simondon, 1989). Yet, rather than simply making sense of insu^tv’s practices, the concept of repurposing also provokes a discussion regarding the ethics of connection. For insu^tv, this connective ethics can be understood as a set of rules and principles that facilitate the evaluation of actions, communication, and thought according to an immanent mode of collective existence (Deleuze, 1988; Simondon, 1989). For the author, herself a member of insu^tv and an academic researcher, this immanent position helps challenge traditional models of knowing and envisioning social change and instead proposes alternatives that attend to the singularity and relation among new political movements, and to the political potential of research methods that focus on process and fold activism into academia. The methodology is inspired by the militant research methods of the Italian Autonomia movement (conricerca or inchiesta), as developed and performed by activists themselves. While attending to the complexity of social struggles, the concept of repurposing enables an approach to research and experimentation as modes of sociability, where these modes are themselves repurposed through an ethics of connection. This line informs the relation between ethics and subjectivation, as well as between ethics and micropolitics, facilitating the emergence of new modes of political action through the repurposing of the social field itself.

Freshwater planarians are animals that possess a set of features that has made them pivotal for research areas such as stem cell research, neurophar-macology, or ageing research. The appeal of these animals has stemmed from features such as the existence of adult stem cells distributed over their bodies, the presence of a simple brain sharing similarities with the vertebrate nervous system, or their apparent lack of ageing. The interest in freshwater planarians as experimental animals has spread to ecotoxicity testing, given features such as easy maintenance in laboratory setting, their sensitivity to environmental contaminants, and also the range of effects that can be evaluated in response to contaminants, such as teratogenicity, carcinogenicity, reproductive and be-havioural toxicity or neurotoxicity. Their predatory nature offers yet another interesting opportunity to include them in multi-species tests as epibenthic in-vertebrate predators. In the context of exploring the sensitivity of freshwater planarian endpoints to environmental contaminants, the present work used polycyclic aromatic hydrocarbons (PAHs) as model compounds. PAHs are ubiquitous contaminants in the environment, given the multitude of natural and anthropogenic sources and their ability to be transported over long distances through the air. As a major contaminant class in the environment, the risk as-sessment of PAHs has been challenging, given the array of metabolizing abili-ties among invertebrates, as well as the multitude of effects these compounds can elicit. For some invertebrate groups, such as freshwater planarians, there is virtually no (eco)toxicological information. With the ultimate goal of exploring freshwater planarians in the context of ecotoxicological research, this work used the freshwater Girardia tigrina as experimental animal and three PAHs with distinct number of aromatic rings, the 3-, 4- and 5-ringed phenanthrene, pyrene and benzo[a]pyrene, respectively. Several specific objectives were ad-dressed: development of novel planarian bioassays, evaluation of PAH toxicity potential accumulation in G. tigrina under different exposure scenarios. Along 6 experimental chapters, experimental protocols were refined and, more specifi-cally, a feeding protocol was developed in accordance with the specific features of G. tigrina. This freshwater planarian was also shown to be sensitive to PAHs, with mortality, disintegration of tissues, regeneration delays, behavioural impairments, biochemical changes, decreases in reproductive output and de-creased newborn fitness being observed: The severity of effects was well relat-ed with the concentration-dependent increase of PAH-type compounds in pla-narian tissues. However, despite chemical similarities, each PAH elicited differ-ent effects in planarians. The evaluation of feeding and locomotion in response to chemical stress was evidenced to be sensitive at short exposure periods, showcasing the potentialities of planarian behavioural endpoints for ecotoxicity testing. With longer exposure periods, fecundity was shown to be a sensitive endpoint, while increased behavioural anomalies in unexposed newborns re-sulting from B[a]P-exposed parents, evidenced the importance of newborn condition as a reproduction-related endpoint in planarians and for transgenera-tional studies. The evaluation of internal concentrations of PAH-type com-pounds in the head and tail portions of exposed planarians revealed that these compounds accumulated more in the heads of planarians, providing a hint on the potential neurotoxicity of these compounds, as suggested by the observed behavioural effects. Moreover, the accumulation of PAH-type compounds in G. tigrina exposed to B[a]P-contaminated sediments or to contaminated prey, evidenced their potential for ecotoxicological experiments with contaminated sediments and to evaluate trophic transfer of contaminants. The results ob-tained in the present work indicate that planarians are sensitive to chemical stress, with a multitude of available endpoints, while showing potential for in-clusion as invertebrate predators in multispecies studies as invertebrate preda-tors. Therefore, their usage for ecotoxicological research is strongly recom-mended.<br>As planárias de água doce são animais que possuem uma variedade de carac-terísticas que as tornou fundamentais para várias áreas do conhecimento, tais como a investigação em células estaminais, a neurofarmacologia ou a investi-gação sobre envelhecimento. O interesse por estes animais deve-se a atributos únicos como a existência de células estaminais distribuídas pelos seus corpos, a presença de um cérebro simples, mas com semelhanças ao sistema nervoso dos vertebrados, ou a sua aparente ausência de envelhecimento. O interesse pelas planárias como animais experimentais difundiu-se à área da investigação ecotoxicológica, não só devido à sua fácil manutenção em contexto laboratorial e sensibilidade a contaminantes ambientais, mas também à vasta gama de respostas que podem ser usadas para avaliar a teratogénese, a carcinogénese, a toxicidade comportamental e reprodutiva ou a neurotoxicidade. Para além disto, as planárias podem ser usadas em testes com múltiplas espécies como invertebrados epibentónicos predadores. Neste contexto, o presente trabalho pretendeu explorar os parâmetros ecotoxicológicos das planárias de água doce em resposta a contaminantes ambientais, através da utilização de hidrocarbo-netos aromáticos policíclicos (PAHs) como compostos modelo. Os PAHs são contaminantes ubíquos no ambiente, devido à variedade de fontes emissoras, tanto naturais, como antropogénicas, e à sua capacidade de serem transporta-dos através do ar por longas distâncias. Contudo, a avaliação de risco ambien-tal dos PAHs tem-se mostrado difícil, devido à variabilidade das capacidades de metabolização destes compostos nos invertebrados e à grande variedade de efeitos que os PAHs podem provocar nos organismos. Para alguns grupos de invertebrados, como é o caso das planárias, os efeitos destes compostos são (quase) desconhecidos. No sentido de explorar a potencial utilização das planá-rias de água doce no contexto da ecotoxicologia, este trabalho utilizou a espé-cie Girardia tigrina como organismo modelo e três PAHs com diferentes núme-ros de anéis aromáticos, o fenantreno, o pireno e o benzo[a]pireno (B[a]P), de 3, 4 e 5 anéis aromáticos, respetivamente. Foram delineados vários objetivos específicos: o desenvolvimento de ensaios para a avaliação dos parâmetros ecotoxicológicos nas planárias de água doce, a avaliação da toxicidade dos PAHs e a potencial acumulação dos PAHs em diferentes cenários de exposi-ção. Ao longo de 6 capítulos experimentais, os protocolos para a avaliação dos parâmetros ecotoxicológicos foram refinados e, mais especificamente, foi de-senvolvido um protocolo para avaliar a inibição alimentar de acordo com as características específicas da planária G. tigrina. Também se verificou que esta espécie de planária é sensível aos PAHs, tendo sido observada mortalidade, desintegração de tecidos, atrasos na regeneração, alterações comportamen-tais, alterações bioquímicas, diminuição da taxa reprodutiva e efeitos em planá-rias recém-nascidas. A severidade dos efeitos observados aumentou de acordo com as concentrações internas de PAHs nos tecidos das planárias. No entanto, apesar das semelhanças químicas entre os compostos, cada um dos PAHs provocou diferentes efeitos nas planárias. A avaliação da taxa de alimentação e locomoção mostrou ser sensível em resposta aos contaminantes em exposi-ções de curta duração, e evidenciou o potencial destes parâmetros para a ava-liação ecotoxicológica. Em períodos de exposição mais longos, a fecundidade mostrou ser um parâmetro sensível. Para além disso, através da observação dos recém-nascidos resultantes de progenitores expostos ao B[a]P, foi verifica-do que estes apresentavam anomalias comportamentais, evidenciando, assim, a importância da avaliação da condição dos recém-nascidos como um parâme-tro reprodutivo e em exposições multigeracionais. Constatou-se ainda que, nos tecidos das planárias, os PAHs se acumularam em maior quantidade na zona cefálica, potencialmente indicando que estes compostos poderão ser neurotóxi-cos para as planárias, como sugerem também as alterações comportamentais. Para além disto, a bioacumulação de PAHs em planárias expostas a sedimen-tos contaminados ou que consumiram presas contaminadas com B[a]P, de-monstrou o potencial destes animais para testes de avaliação ecotoxicológica de sedimentos e na avaliação da transferência trófica de contaminantes. Os resultados obtidos no presente trabalho indicam que as planárias são sensíveis a contaminantes, permitem a avaliação de uma grande variedade de parâme-tros e podem também ser usadas como predadores em testes com múltiplas espécies. Assim, a utilização das planárias de água doce para a investigação ecotoxicológica é fortemente recomendada.<br>Programa Doutoral em Biologia e Ecologia das Alterações Globais

Initially, protoplasts were isolated to detect various parameters using flow cytometric analysis. The most efficient ratio of cells to enzyme solution, for digestion of cell walls, needed to be established. To detect whether the time of incubation with the enzyme solution influenced the state or viability of the protoplasts, they were observed periodically under the light microscope during digestion at different concentrations of enzyme solution. After 2 h digestion with light swirling every 20 min, the protoplasts were still intact (Figure 1), and viable as detected with Trypan blue staining (results not shown). Increasing the digestion period led to a decrease in cell membrane integrity. The size of the protoplasts varied between 60 mm and 90 mm. Figure 1 shows the difference between cells before digestion with an enzyme solution and protoplasts after digestion. Size determination of protoplasts was important since the flow tip of the flow cytometer is limited to 100 mm and if the protoplasts exceeded this size, could lead to blockages in the flow tip of the flow cytometer, with ineffective readings and a time consuming clean-up process.<br>Dr. Marianne J. Cronje

This thesis presents the analysis, design and characterization of an integrated high-frequency phase-locked loop (PLL) frequency multiplier. The frequency multiplier is novel in its use of a low multiplication factor of 4 and a fully differential topology for rejection of common mode interference signals. The PLL is composed of a voltage controlled oscillator (VCO), injection-locked frequency divider (ILFD) for the first divide-by-two stage, a static master-slave flip-flop (MSFF) divider for the second divide-by-two stage and a Gilbert cell mixer phase detector (PD). The circuit has been fabricated using a standard CMOS 0.18-um process based on its relatively low cost and ready availability. The PLL frequency multiplier generates an output signal at 26 GHz and is the highest operational frequency PLL in the technology node reported to date. Time domain phase plane analysis is used for prediction of PLL locking range based on initial conditions of phase and frequency offsets. Tracking range of the PLL is limited by the inherent narrow locking range of the ILFD, and is confirmed via experimental results. The performance benefits of the fully differential PLL are experimentally confirmed by the injection of differential- and common-mode interfering signals at the VCO control lines. A comparison of the common- and differential-mode modulation indices reveals that a common mode rejection ratio (CMRR) of greater than 20 dB is possible for carrier offset frequencies of less than 1 MHz. Closed-loop frequency domain transfer functions are used for prediction of the PLL phase noise response, with the PLL being dominated by the reference and VCO phase noise contributions. Regions of dominant phase noise contributions are presented and correlated to the overall PLL phase noise performance. Experimental verifications display good agreement and confirm the usefulness of the techniques for PLL performance prediction. The PLL clock multiplier has an operational output frequency of 26.204 to 26.796 GHz and a maximum output frequency step of 16 MHz. Measured phase noise at 1 MHz offset from the carrier is -103.9 dBc/Hz. The PLL clock multiplier core circuit (VCO/ILFD/MSFF Divider/PD) consumes 186 mW of combined power from 2.8 and 4.3 V DC rails.<br>Thesis (Ph.D, Electrical & Computer Engineering) -- Queen's University, 2009-04-24 11:31:35.384