Use of Galleria mellonella as a model organism to study Legionella pneumophila infection.

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires' disease, is an important human pathogen that infects and replicates within alveolar macrophages. Its virulence depends on the Dot/Icm type IV secretion system (T4SS), which is essential to establish a replication permissive vacuole known as the Legionella containing vacuole (LCV). L. pneumophila infection can be modeled in mice however most mouse strains are not permissive, leading to the search for novel infection models. We have recently shown that the larvae of the wax moth Galleria mellonella are suitable for investigation of L. pneumophila infection. G. mellonella is increasingly used as an infection model for human pathogens and a good correlation exists between virulence of several bacterial species in the insect and in mammalian models. A key component of the larvae's immune defenses are hemocytes, professional phagocytes, which take up and destroy invaders. L. pneumophila is able to infect, form a LCV and replicate within these cells. Here we demonstrate protocols for analyzing L. pneumophila virulence in the G. mellonella model, including how to grow infectious L. pneumophila, pretreat the larvae with inhibitors, infect the larvae and how to extract infected cells for quantification and immunofluorescence microscopy. We also describe how to quantify bacterial replication and fitness in competition assays. These approaches allow for the rapid screening of mutants to determine factors important in L. pneumophila virulence, describing a new tool to aid our understanding of this complex pathogen.

LtpD is a novel legionella pneumophila effector that binds phosphatidylinositol 3-phosphate and inositol monophosphatase IMPA1

The Dot/Icm type IV secretion system (T4SS) of Legionella pneumophila is crucial for the pathogen to survive in protozoa and cause human disease. Although more than 275 effector proteins are delivered into the host cell by the T4SS, the function of the majority is unknown. Here we have characterized the Dot/Icm effector LtpD. During infection, LtpD localized to the cytoplasmic face of the membrane of the Legionella-containing vacuole (LCV). In A549 lung epithelial cells, ectopically expressed LtpD localized to large vesicular structures that contained markers of endosomal compartments. Systematic analysis of LtpD fragments identified an internal 17-kDa fragment, LtpD_471-626, which was essential for targeting ectopically expressed LtpD to vesicular structures and for the association of translocated LtpD with the LCV. LtpD_471-626 bound directly to phosphatidylinositol 3-phosphate [PtdIns(3)P] in vitro and colocalized with the PtdIns(3)P markers FYVE and SetA in cotransfected cells. LtpD was also found to bind the host cell enzyme inositol (myo)-1 (or 4)-monophosphatase 1, an important phosphatase involved in phosphoinositide production. Analysis of the role of LtpD in infection showed that LtpD is involved in bacterial replication in THP-1 macrophages, the larvae of Galleria mellonella, and mouse lungs. Together, these data suggest that LtpD is a novel phosphoinositide- binding L. pneumophila effector that has a role in intracellular bacterial replication. © 2013, American Society for Microbiology.

Effectiveness of Community versus Hospital Eye Service follow-up for patients with neovascular age-related macular degeneration with quiescent disease (ECHoES): a virtual non-inferiority trial

OBJECTIVES: To compare the ability of ophthalmologists versus optometrists to correctly classify retinal lesions due to neovascular age-related macular degeneration (nAMD).

DESIGN: Randomised balanced incomplete block trial. Optometrists in the community and ophthalmologists in the Hospital Eye Service classified lesions from vignettes comprising clinical information, colour fundus photographs and optical coherence tomographic images. Participants' classifications were validated against experts' classifications (reference standard).

SETTING: Internet-based application.

PARTICIPANTS: Ophthalmologists with experience in the age-related macular degeneration service; fully qualified optometrists not participating in nAMD shared care.

INTERVENTIONS: The trial emulated a conventional trial comparing optometrists' and ophthalmologists' decision-making, but vignettes, not patients, were assessed. Therefore, there were no interventions and the trial was virtual. Participants received training before assessing vignettes.

MAIN OUTCOME MEASURES: Primary outcome-correct classification of the activity status of a lesion based on a vignette, compared with a reference standard. Secondary outcomes-potentially sight-threatening errors, judgements about specific lesion components and participants' confidence in their decisions.

RESULTS: In total, 155 participants registered for the trial; 96 (48 in each group) completed all assessments and formed the analysis population. Optometrists and ophthalmologists achieved 1702/2016 (84.4%) and 1722/2016 (85.4%) correct classifications, respectively (OR 0.91, 95% CI 0.66 to 1.25; p=0.543). Optometrists' decision-making was non-inferior to ophthalmologists' with respect to the prespecified limit of 10% absolute difference (0.298 on the odds scale). Optometrists and ophthalmologists made similar numbers of sight-threatening errors (57/994 (5.7%) vs 62/994 (6.2%), OR 0.93, 95% CI 0.55 to 1.57; p=0.789). Ophthalmologists assessed lesion components as present less often than optometrists and were more confident about their classifications than optometrists.

CONCLUSIONS: Optometrists' ability to make nAMD retreatment decisions from vignettes is not inferior to ophthalmologists' ability. Shared care with optometrists monitoring quiescent nAMD lesions has the potential to reduce workload in hospitals.

TRIAL REGISTRATION NUMBER: ISRCTN07479761; pre-results registration.

Manutenção da Heterozigosidade em populações de plantas autógamas

Neste trabalho objetivou-se verificar, teoricamente , a possibilidade de manutenção de heterozigotos, em populações de plantas autógamas, na presença e na ausência de seleção dependente de frequência (SDF). Considerou-se apenas um loco com dois alelos. Utilizou-se a dedução algébrica de formulas referentes a alguns modelos de populações e um programa para simular a sucessão das gerações em uma calculadora científica avançada. Comparou-se os resultados do modelo com os dados obtidos experimentalmente por Allard e Workman (1963) e por Harding, Allard e Smeltzer (1966). Os coeficientes de determinação foram 0,9653 e 0,9166 para a primeira e a segunda comparação, respectivamente. Estes coeficientes indicam que o modelo D representa de modo bastante fidedigno as variações observadas em populações experimentais de plantas predominantemente autógamas

Sex steroid feedback regulation of pituitary gonadotropins during early secondary oocyte growth in coho salmon (Oncorhynchus kisutch) – a potential target of endocrine disruption.

Thesis (Ph.D.)--University of Washington, 2016-06

Jung and Goddess: the significance of Jungian and post-Jungian theory to the development of the Western Goddess Movement

This study is concerned with the significance of Jungian and post-Jungian theory to the development of the contemporary Western Goddess Movement, which includes the various self-identified nature-based, Pagan, Goddess Feminism, Goddess Consciousness, Goddess Spirituality, Wicca, and Goddess-centred faith traditions that have seen a combined increase in Western adherents over the past five decades and share a common goal to claim Goddess as an active part of Western consciousness and faith traditions. The Western Goddess Movement has been strongly influenced by Jung’s thought, and by feminist revisions of Jungian Theory, sometimes interpreted idiosyncratically, but presented as a route to personal and spiritual transformation. The analysis examines ways in which women encounter Goddess through a process of Jungian Individuation and traces the development of Jungian and post-Jungian theories by identifying the key thinkers and central ideas that helped to shape the development of the Western Goddess Movement. It does so through a close reading and analysis of five biographical ‘rebirth’ memoirs published between 1981 and 1998: Christine Downing’s (1981) The Goddess: Mythological Images of the Feminine; Jean Shinoda Bolen’s (1994) Crossing to Avalon: A Woman’s Midlife Pilgrimage; Sue Monk Kidd’s (1996) The Dance of the Dissident Daughter: A Woman’s Journey from Christian Tradition to the Sacred Feminine; Margaret Starbird’s (1998) The Goddess in the Gospels: Reclaiming the Sacred Feminine; and Phyllis Curott’s (1998) Book of Shadows: A Modern Woman’s Journey into the Wisdom of Witchcraft and the Magic of the Goddess. These five memoirs reflect the diversity of the faith traditions in the Western Goddess Movement. The enquiry centres upon two parallel and complementary research threads: 1) critically examining the content of the memoirs in order to determine their contribution to the development of the Goddess Movement and 2) charting and sourcing the development of the major Jungian and post-Jungian theories championed in the memoirs in order to evaluate the significance of Jungian and post-Jungian thought in the Movement. The aim of this study was to gain a better understanding of the original research question: what is the significance of Jungian and post-Jungian theory for the development of the Western Goddess Movement? Each memoir is subjected to critical review of its intended audiences, its achievements, its functions and strengths, and its theoretical frameworks. Research results offered more than the experiences of five Western women, it also provided evidence to analyse the significance of Jungian and post-Jungian theory to the development of the Western Goddess Movement. The findings demonstrate the vital contributions of the analytical psychology of Carl Jung, and post-Jungians M Esther Harding, Erich Neumann, Christine Downing, E.C. Whitmont, and Jean Shinoda Bolen; the additional contributions of Sue Monk Kidd, Margaret Starbird, and Phyllis Curott, and exhibit Jungian and post-Jungian pathways to Goddess. Through a variety of approaches to Jungian categories, these memoirs constitute a literature of Individuation for the Western Goddess Movement.

Manutenção da Heterozigosidade em populações de plantas autógamas

Neste trabalho objetivou-se verificar, teoricamente , a possibilidade de manutenção de heterozigotos, em populações de plantas autógamas, na presença e na ausência de seleção dependente de frequência (SDF). Considerou-se apenas um loco com dois alelos. Utilizou-se a dedução algébrica de formulas referentes a alguns modelos de populações e um programa para simular a sucessão das gerações em uma calculadora científica avançada. Comparou-se os resultados do modelo com os dados obtidos experimentalmente por Allard e Workman (1963) e por Harding, Allard e Smeltzer (1966). Os coeficientes de determinação foram 0,9653 e 0,9166 para a primeira e a segunda comparação, respectivamente. Estes coeficientes indicam que o modelo D representa de modo bastante fidedigno as variações observadas em populações experimentais de plantas predominantemente autógamas

Ciclo de los negocios en Colombia: el papel de la política de estabilización

Este documento mide la sincronización entre las políticas de estabilización y el ciclo de los negocios en Colombia en el periodo comprendido entre marzo de 1990 y junio de 2013. Para la construcción de los ciclos se utiliza la metodología clásica y se construye un ciclo de referencia a partir de ciclos individuales de tres índices de actividad económica real. Con el fin de medir la sincronización entre el ciclo de nego­cios y los ciclos de las políticas fiscal y monetaria se utiliza el estadístico de Harding y Pagan (2006). Se concluye que durante el periodo de estudio las políticas de estabilización tienden a ser procíclicas (por el lado fiscal) y acíclicas (por el lado monetario). En la literatura se conoce a este fenómeno como when it rains, it pours y es común que se presente en economías emergentes como la colombiana

LOOKING FOR DIONYSUS: HEDWIG RAABE AND THE HAUNTING OF THE BIRTH OF TRAGEDY

This thesis explores the relationship of the actress Hedwig Raabe’s 1866 performance in Charlotte Birch-Pfeiffer’s play Die Grille to the philosopher Friedrich Nietzsche’s 1872 book The Birth of Tragedy. This exploration is structured by theatre scholar Marvin Carlson’s concept of haunting. I conclude that the haunting of Nietzsche’s text by Raabe’s performance destabilizes the former and points towards new ways of understanding The Birth of Tragedy in the fields of theatre and performance studies.

Effects of advanced glycation end-products (AGEs) on retinal pigment epithelial (RPE) cells lysosomal function:implications for age-related macular degeneration (AMD)

Purpose: RPE lysosomal dysfunction is a major contributor to AMD pathogenesis. Controlled activity of a major class of RPE proteinases, the cathepsins, is crucial in maintaining correct lysosomal function. Advanced glycation end-products (AGEs) accumulate in the Bruch’s membrane (BM) with age, impacting critical RPE functions and in turn, contributing to the development of AMD. The aim of this study was to assess the effect of AGEs on lysosomal function by analysing the expression, processing and activity of the cysteine proteinases cathepsins B, L and S, and the aspartic proteinase cathepsin D. Methods: ARPE-19 cells were cultured on AGE-containing BM mimics (matrigel) for 14 days and compared to untreated substrate. Expression levels and intracellular processing of cathepsins B, D, L and S, were assessed by qPCR and immunoblotting of cell lysates. Lysosomal activity was investigated using multiple activity assays specific to each of the analysed cathepsins. Statistical analysis was performed using the Student’s independent T-test. Results: AGE exposure produced a 36% decrease in cathepsin L activity when compared to non-treated controls (p=0.02, n= 3) although no significant changes were observed in protein expression/processing under these conditions. Both the pro and active forms of cathepsin S decreased by 40% (p=0.04) and 74% (p=0.004), respectively (n=3). In contrast, the active form of the cathepsin D increased by 125% (p=0.005, n= 4). However, no changes were observed in the activity levels of both cathepsins S and D. In addition, cathepsin B expression, processing and activity also remained unaltered following AGE exposure. Conclusions: AGEs accumulation in the extracellular matrix, a phenomenon associated with the natural aging process of the BM, attenuates the expression, intracellular processing and activity of specific lysosomal effectors. Altered enzymatic function may impair important lysosomal processes such as endocytosis, autophagy and phagocytosis of photoreceptor outer segments, each of which may influence the age-related dysfunction of the RPE and subsequently, AMD pathogenesis.