From molecules to organs : Microscopy and multi-scale nature of development

Morphogenesis is a phenomenon of intricate balance and dynamic interplay between processes occurring at a wide range of scales (spatial, temporal and energetic). During development, a variety of physical mechanisms are employed by tissues to simultaneously pattern, move, and differentiate based on information exchange between constituent cells, perhaps more than at any other time during an organism's life. To fully understand such events, a combined theoretical and experimental framework is required to assist in deciphering the correlations at both structural and functional levels at scales that include the intracellular and tissue levels as well as organs and organ systems. Microscopy, especially diffraction-limited light microscopy, has emerged as a central tool to capture the spatio-temporal context of life processes. Imaging has the unique advantage of watching biological events as they unfold over time at single-cell resolution in the intact animal. In this work I present a range of problems in morphogenesis, each unique in its requirements for novel quantitative imaging both in terms of the technique and analysis. Understanding the molecular basis for a developmental process involves investigating how genes and their products- mRNA and proteins-function in the context of a cell. Structural information holds the key to insights into mechanisms and imaging fixed specimens paves the first step towards deciphering gene function. The work presented in this thesis starts with the demonstration that the fluorescent signal from the challenging environment of whole-mount imaging, obtained by in situ hybridization chain reaction (HCR), scales linearly with the number of copies of target mRNA to provide quantitative sub-cellular mapping of mRNA expression within intact vertebrate embryos. The work then progresses to address aspects of imaging live embryonic development in a number of species. While processes such as avian cartilage growth require high spatial resolution and lower time resolution, dynamic events during zebrafish somitogenesis require higher time resolution to capture the protein localization as the somites mature. The requirements on imaging are even more stringent in case of the embryonic zebrafish heart that beats with a frequency of ~ 2-2.5 Hz, thereby requiring very fast imaging techniques based on two-photon light sheet microscope to capture its dynamics. In each of the hitherto-mentioned cases, ranging from the level of molecules to organs, an imaging framework is developed, both in terms of technique and analysis to allow quantitative assessment of the process in vivo. Overall the work presented in this thesis combines new quantitative tools with novel microscopy for the precise understanding of processes in embryonic development.

Is inbreeding avoidance driving female mate choice in Verreaux's sifaka lemurs?

Females of different species might exert female mate choice for different reasons, one of them the aim of avoiding inbreeding. In this study I examine the implication of inbreeding avoidance as a mechanism driving female mate choice in Verreaux’s sifaka lemurs (Propithecus verreauxi). In fact, in this species females are dominant and appear to be able to choose certain males to mate with, while observations indicate that rank, body size, canine size and proportions of fights won are not factors influencing female mate choice. So I hypothesized that females mate choice is driven by inbreeding avoidance in Verreaux’s sifaka lemurs. Tissue and fecal samples were collected in the Kirindy Mitea National Park in western Madagascar as a source of DNA. Parentage was assigned for a sample of the population and relatedness coefficients between dams and sires were estimated and compared to those of between random female and male pairs, dams and other candidate sires within the population and within the groups were the offspring were conceived. I found that there were no significant differences in none of the comparisons which means that Verreaux’s sifaka females do not mate more with males that are more distantly related to them. I concluded that inbreeding avoidance does not appear to be the main force driving female mate choice in Verreaux’s sifaka lemurs and I addressed explanations for these findings. With this study I contribute to our knowledge of female mate choice in lemurs.

Female slave narratives: consistency and permanence: a study of two texts from the XIXth and XXth centuries

Esta dissertação tem por objetivo investigar o papel das slave narratives como poderoso gênero literário na denúncia da escravidão africana e na representação do homem negro e da mulher negra nos séculos dezoito e dezenove. Este trabalho também se propõe a investigar o papel das neo-slave narratives no estudo do passado e a representação da identidade negra no século vinte. Ambos os gêneros desafiam seus tempos presentes ao discutirem questões de etnia e subjugação humana, em uma abordagem crítica. Em Incidents in the Life of a Slave Girl (1861), Harriet Jacobs narra sua experiência na escravidão, deixando um importante legado não somente para a História mas também para a Literatura Afro-Americana. Em Dessa Rose (1986), Sherley Anne Williams, revisa o passado para resgatar a memória da escravidão e reescrever a história para examinar seu tempo presente. Além disso, as duas autoras apresentam questões de gênero, levantando questões feministas em suas obras

Diasporization and family relations in Nella Larsens Quicksand and Jamaica Kincaids Lucy: the construction of female identity

O principal objetivo desta dissertação é investigar e analisar como os movimentos diaspóricos e as relações familiares exercem influência na construção das identidades das mulheres em Quicksand, de Nella Larsen, e Lucy, de Jamaica Kincaid. As questões a que pretendo responder são: Como as personagens principais, Helga Crane e Lucy, lidam com as diferentes culturas que encontram nas suas trajetórias ? ; Como essas diferentes culturas lidam com essas mesmas personagens?; e Como o encontro entre essas diferentes culturas e as relações familiares são descritos e influenciam na construção da identidade feminina nessas obras?. Minha hipótese é que encontraremos nas obras selecionadas duas jornadas, portadoras tanto de aspectos comuns quanto distintos, que começam com o desejo das personagens principais de escapar da opressão patriarcal. Isto é, Helga Crane e Lucy passam por um período de muitas descobertas sobre elas mesmas e as sociedades com que têm que lidar, o que determina dois produtos diferentes: a construção das identidades híbridas de Lucy, em meio a sua solidão, na obra de Kincaid, e a construção e o sufocamento das identidades de Helga pela religião, o patriarcado e as relações familiares, na obra de Larsen

Aspects of mineral composition of male and female Heterobranchus bidorsalis adults exposed to different concentrations of Bonny-light crude oil

Studies were carried out to assess some macro and trace elements of mineral composition of the male and female Heterobranchus bidorsalis adults exposed to graded concentrations (1.00-8.00m/L-1) of Bonny-light crude oil (BLCO). The experiment was monitored for 4 days (toxicity) and 42days (recovery) periods. Significant decreases (P < 0.05)in the sodium (Na), potassium (K), magnesium (Mg), calcium (Ca), phosphorus (P), zinc (Zn), iron (Fe), vanadium (Va), lead (Pb) and manganese (Mn) contents of the male H. bidorsalis corresponded with the increasing concentrations of BLCO. In contrast, the female fishes recorded significant increases (P < 0.05) in the values of the above elements in their tissues as the concentrations of BLCO increased. Furthermore, the values of Na, K, Mg, Ca, P, Zn, Fe, Va, Pb and Mn recorded in the male fishes where generally lower than those of their female counterparts and the control fish. Increased values of these elements were also recorded during the recovery periods (days 14, 28 and 42) of this study in the magnitudes of 15% at day 14, 20% at day 28 and 20% at day 42. This implied that the removal of crude oil stress during this period improved the quantity of these minerals deposited in the fish tissues. The highest percent proportion of Zn and the lowest proportion of Pb recorded in both male and female H. bidorsalis adults agreed with the report of other workers for other fish species. KEYWORDS: Heterobranchus bidorsalis, Mineral composition, Bonny-light crude oil, Toxicity, Recovery.

Ações educativas em contracepção: teoria e prática dos profissionais de saúde.

Esta pesquisa objetivou analisar as repercussões do Curso de PAISM/Contracepção, nas práticas, conhecimentos e percepções dos profissionais que desenvolvem atividades educativas nas ações de contracepção, no que diz respeito à saúde e aos direitos na esfera da sexualidade, da reprodução e do gênero; e identificar através dos relatos dos profissionais, os conhecimentos sobre a história do PAISM e do planejamento familiar, o quadro jurídico e normativo, as temáticas e a metodologia do trabalho educativo. Foi um estudo descritivo, com abordagem etnográfica. O corpus de análise foi composto pelo registro da observação participante, entrevistas e análise documental. O universo empírico contou com três cenários no âmbito da Secretaria Municipal de Saúde do Rio de Janeiro: o Espaço Mulher, grupos educativos de contracepção de duas Unidades Básicas de Saúde, e grupos coordenados por profissionais treinadas no referido curso e como informantes, sete enfermeiras e quatro assistentes sociais. Segundo os relatos das informantes, a mudança de visão sobre alguns temas abordados e a aquisição de novos conhecimentos como possibilidade para a mudança de suas práticas, foram as principais contribuições do curso. Esta tese comprovou parcial conhecimento a respeito dos direitos sexuais e reprodutivos; do marco histórico, quadro jurídico e normativo do PAISM. No que se refere ao aborto, o discurso predominante foi no sentido contrário a sua prática, em geral por argumentos de natureza religiosa. A sexualidade para a maioria das informantes é relacional e para além do sexo, uma expressão de marca típica do gênero feminino. O tom dominante nos discursos das informantes restringiu-se ao domínio de ações informativas no âmbito da prevenção de doenças e gravidez e à esfera humanitária, numa retórica próxima ao do discurso da moral religiosa cristã. A ética cristã de acolhimento e tolerância à liberdade individual independe do pertencimento religioso, e de certo modo, pode-se articular esse ethos ao discurso dos direitos sexuais e reprodutivos. Embora apresentando um discurso fundado em valores cristãos (tolerância, compreensão e acolhimento) e mesmo não tendo uma posição política e/ou acadêmica na esfera dos direitos, ou até mesmo não (re) conhecendo alguns deles, as informantes parecem ter uma prática que, de certa forma, expressa uma postura de respeito. Mesmo assim, a abordagem dos direitos sexuais e reprodutivos precisa ser mais explícita e melhor discutida nos cursos de capacitação. Os profissionais que atuam nos grupos educativos em contracepção são atores sociais que podem/devem contribuir à garantia dos direitos sexuais e reprodutivos, para que se alcance a tão proclamada noção de integralidade em saúde. No entanto, para isto, é preciso que as práticas sejam estruturadas segundo o marco cognitivo emancipatório (BONAN, 2005), no sentido de evitar a manutenção de desigualdades sociais e de gênero, principalmente no que tange às questões da sexualidade e da reprodução. Este desafio está posto aos gestores de atenção à saúde da mulher e aos órgãos de formação em saúde.

Desenvolvimento do sistema reprodutivo de Echinostoma paraensei (Trematoda: Digenea) de hamsters (Mesocricetus auratus) experimentalmente infectados, analisado por microscopia de luz de campo e microscopia laser confocal

O conhecimento da morfologia e ultraestrutura dos helmintos permite a correta classificação destes organismos, bem como fornece subsídios que poderão ser utilizados para diagnóstico e controle. A microscopia laser confocal é uma ferramenta para estudar a organização estrutural de várias espécies de helmintos, possibilitando acesso a detalhes morfológicos não evidenciados pela microscopia óptica. Echinostoma paraensei é um trematódeo, digenético, hermafrodita parasito de numerosos hospedeiros vertebrados. Neste trabalho foi investigado o desenvolvimento dos órgãos reprodutivo e a morfometria de E. paraensei, desde a fase jovem até a adulta, como contribuição ao conhecimento do desenvolvimento reprodutivo desta espécie. Os trematódeos foram recuperados aos 3, 4, 5, 6, 7, 10, 14 e 21 dias posterior à infecção (dpi) experimental em hamsters. Estes foram corados em carmim clorídrico, desidratados em série alcoólica e montados em lâmina permanente em bálsamo do Canadá, fotografados e medidos usando microscopia de luz de campo claro (MCC) e microscopia de varredura laser confocal (MVLC). Entre 3 e 4 dpi, os primórdios genitais estavam presentes e nenhuma organização do sistema reprodutivo foi visualizada por MCC e MVLC. Os primórdio do ovário, dos testículos e da bolsa do cirro foram visualizados por MCC aos 5 e 6 dpi, no entanto, MVLC dos helmintos aos 5dpi mostra que estes primórdios, o ootipo e o útero estavam presentes, como estruturas individualizadas. A bolsa do cirro apresenta metratermo e o ovário com primórdio do oótipo adjacente aos 7dpi por MVLC. A vesícula seminal, receptáculo seminal, células diferenciadas nos testículos, ducto e reservatório vitelínico e oviducto foram visualizados após 10 dias, enquanto os espermatozóides na vesícula seminal, ovos e oócitos, células vitelínicas, poro e canal de Laurer aos 14 dias. A morfometria evidencia um acelerado crescimento dos órgãos reprodutores a partir do 7 dia. Os testículos apresentam aumento significativo no comprimento do 7 ao 21 dia e o ovário durante o período de 7 à 10 dpi. Aos 21 dpi, todos os helmintos apresentaram glândulas vitelínicas, útero contendo ovos e espermatozóides no oviducto enquanto outros ovos estão sendo formados. As mudanças morfológicas acentuadas durante a gametogênese consistem no aumento do comprimento do helminto, maturação das gônadas, desenvolvimento e maturação das glândulas vitelínicas. O desenvolvimento do helminto como um todo está relacionado à maturação dos órgãos reprodutivo masculino e feminino indicando o investimento deste trematódeo em garantir a produção e eliminação dos ovos ao meio exterior.

Analysis of the Response Speed of Musculature of the Knee in Professional Male and Female Volleyball Players

The aim of this study was to evaluate the normalized response speed (Vrn) of the knee musculature (flexor and extensor) in high competitive level volleyball players using tensiomyography (TMG) and to analyze the muscular response of the vastus medialis (VM), rectus femoris (RF), vastus lateralis (VL), and biceps femoris (BF) in accordance with the specific position they play in their teams. One hundred and sixty-six players (83 women and 83 men) were evaluated. They belonged to eight teams in the Spanish women's superleague and eight in the Spanish men's superleague. The use of Vrn allows avoiding possible sample imbalances due to anatomical and functional differences and demands. We found differences between Vrn in each of the muscles responsible for extension (VM, RF, and VL) and flexion (BF) regardless of the sex. Normalized response speed differences seem to be larger in setters, liberos and outside players compared to middle blockers and larger in males when compared to females. These results of Vrn might respond to the differences in the physical and technical demands of each specific position, showing an improved balance response of the knee extensor and flexor musculature in male professional volleyball players.

Maturity and growth of female dusky rockfish (Sebastes variabilis) in the central Gulf of Alaska

The dusky rockfish (Sebastes variabilis) has recently been resurrected as a distinct species in the genus Sebastes. Reproductive biology and growth were examined for this redescribed species in the central Gulf of Alaska. Age and length at 50% maturity were 9.2 years and 365 mm fork length, respectively, which are lower than previously reported. Fertilized ova and eyed embryos were observed in April and evidence of postparturition was not observed until May. The gonadosomatic index decreased with the onset of postparturition in May. Von Bertalanffy growth parameters for female dusky rockfish, estimated from the maturity samples, were significantly different from growth parameters derived from Gulf of Alaska fishery-independent survey data.

Age- and length-at-maturity of female arrowtooth flounder (Atheresthes stomias) in the Gulf of Alaska

Arrowtooth flounder (Atheresthes stomias) has had the highest abundance of any groundfish species in the Gulf of Alaska since the 1970s (Matarese et al., 2003; Turnock et al., 2005; Blood et al., 2007); however, commercial catches have been restricted because Pacific halibut (Hippoglossus stenolepis) are caught as bycatch in the fishery. Arrowtooth flounder plays a key role in the ecosystem because it is a dominant organism within the food web, both as an apex predator of fish and invertebrates, as well as an important prey for walleye pollock (Theragra chalcogramma; Aydin et al., 2002). Walleye pollock is the dominant groundfish in the Bering Sea, a principal groundfish in the Gulf of Alaska, and the primary prey for marine mammals. The distribution of arrowtooth flounder extends from Cape Navarin and the eastern Sea of Okhotsk in Russia, across the Bering Sea, Aleutian Islands, Gulf of Alaska, and south to the coast of central California (Shuntov, 1964; Britt and Martin, 2001; Chetvergov, 2001; Weinberg et al., 2002; Zenger, 2004). Because of the importance of arrowtooth flounder in the marine ecosystem of A laska, a maturity study of this species was undertaken to determine age-at-maturity, which is essential for age-based stock management models. Before these results, management has had to rely upon a length-at-maturity-based estimate (Zimmermann, 1997) to manage stocks in the Gulf of Alaska (GOA), Bering Sea, and Aleutian Islands. The central GOA was selected as the location for this maturity study Age- and length-at-maturity of female arrowtooth flounder (Atheresthes stomias) in the Gulf of Alaska because it contains approximately 70% of the total Gulf of Alaska arrowtooth flounder biomass (1.9×106 t, age 3 and older)— the highest percentage in the world (Shuntov, 1964; Britt and Martin, 2001; Weinberg et al., 2002; Wilderbuer and Nichol, 2006).

Geographic and seasonal variations in maturation and growth of female Pacific cod (Gadus macrocephalus) in the Gulf of Alaska and Bering Sea

This study investigates the temporal stability of length- and age-at-maturity estimates for female Pacific cod (Gadus macrocephalus) in the Gulf of Alaska and eastern Bering Sea. Females reached 50% maturity (A50) at 4.4 years in the Gulf of Alaska and at 4.9 years in the eastern Bering Sea. Total body length at 50% maturity (LT50) was significantly smaller (503 mm) in the Gulf of Alaska than in the eastern Bering Sea (580 mm). The estimated length- and age-at-maturity did not differ significantly between winter and spring in either the Gulf of Alaska (1999) or Bering Sea (2003) areas. The results of this study raised the spawning biomass estimate of female Alaskan Pacific cod from 298×103 t for 2005 to 499×103 t for 2006. The increased spawning biomass estimate resulted in an increased over-fishing limit for Pacific cod.

Reproductive biology, spawning season, and growth of female rex sole (Glyptocephalus zachirus) in the Gulf of Alaska

Rex sole (Glyptocephalus zachirus) have a wide distribution throughout the North Pacific, ranging from central Baja California to the western Bering Sea. Although rex sole are an important species in the commercial trawl fisheries off the U.S. West Coast, knowledge of their reproductive biology is limited to one study off the Oregon coast where ovaries were analyzed with gross anatomical methods. This study was initiated to determine reproductive and growth parameters specific to rex sole in the Gulf of Alaska (GOA) stock. Female rex sole (n=594) ranging in total length from 166 to 552 mm were collected opportunistically around Kodiak Island, Alaska, from February 2000 to October 2001. All ovaries were analyzed by using standard histological criteria to determine the maturity stage. Year-round sampling of rex sole ovaries confirmed that rex sole are batch spawners and have a protracted spawning season in the GOA that lasts at least eight months, from October to May; the duration of the spawning season and the months of spawning activity are different from those previously estimated. Female rex sole in the GOA had an estimated length at 50% maturity (ML50) of 352 mm, which is greater than the previously estimated ML50 at southern latitudes. The maximum age of collected female rex sole was 29 years, and the estimated age at 50% maturity (MA50) in the GOA was 5.1 years. The von Bertalanffy growth model for rex sole in the GOA was significantly different from the previously estimated model for rex sole off the Oregon coast. This study indicated that there are higher growth rates for rex sole in the GOA than off the Oregon coast and that there are differences in length at maturity and similarity in age at maturity between the two regions.