O estudo do díptero Rhynchosciara americana forneceu informações importantes sobre a biologia cromossômica, por apresentar cromossomos politênicos e amplificação gênica em diferentes órgãos. Dentre os órgãos que apresentam especializações do ciclo celular, o ovário é um dos mais interessantes, possuindo características únicas, como o desenvolvimento sincrônico dos folículos ovarianos e o fato de apresentar uma única e gigante célula nutridora conectada ao ovócito através de um canal citoplasmático. Une em si: meiose no ovócito, célula nutridora com poliploidia seguido de politenia e células foliculares com mitose. Células poliplóides e politênicas possuem cópias extras de DNA genômico através de repetidos ciclos de fase S sem que ocorra a divisão celular, um processo denominado endoreplicação. Os ciclos celulares, são dirigidos pela oscilação da ativação de complexos ciclina/Cdk. Estudos mostraram que a ciclina A atua em células endoreplicativas em Drosophila e a ciclina B inibe ciclos endoreplicativos induzindo a divisão celular. Torna-se relevante investigar a associação dessas moléculas reguladoras do ciclo celular com os ciclos endoreplicativos que ocorrem na ovogênese de R. americana. Perfis de expressão das ciclinas A e B foram detectados nos ovários por RT-PCR ao longo do desenvolvimento. Ensaios de incorporação do nucleosídeo timidina mostraram elevada atividade proliferativa das células foliculares para formar o folículo e o fim da atividade endoreplicativa nas células nutridoras em pupas de 4 dias. Preparações de imunolocalização proteica em ovários na fase de pupa revelaram acúmulo de ciclina A no citoplasma das células nutridoras e dos ovócitos, e acúmulo de ciclina B no citoplasma e na vesícula germinal do ovócito, atuando nos mecanismos meióticos. O estudo de proteínas relacionadas ao ciclo celular nesse modelo é importante para um melhor entendimento dos ciclos celulares incomuns presentes em diferentes órgãos de insetos. / Study of the diptera Rhynchosciara americana has provided important information about chromosome biology, for it displays polytene chromosomes and gene amplification in different organs. Among the organs that possess cell cycle specializations, the ovary is one of the most interesting ones, showing unique characteristics, such as the synchronous development of follicles and the presence of a single giant nurse cell connected to the oocyte through a cytoplasmic channel. This organ gathers: meiosis in the oocyte, nurse cell with polyploidy followed by polyteny, and follicle cells in mitosis. Polyploid and polytene cells have extra copies of genomic DNA obtained via sequential cycles of S phase not followed by cell division, a process called endoreplication. Cell cycles are driven by the oscilation in the activation of different cyclin/CDK complexes. Studies have shown that Cyclin A acts in endoreplicating cells in Drosophila and Cyclin B inhibits endoreplicative cycles, inducing cell division. Thus, it is relevant to investigate the association between these cell cycle-regulating proteins and the endoreplication cycles that occur during the oogenesis of R. americana. Expression profiles of cyclins A and B were evaluated in the ovary via RT-PCR throughout the development. Thymidine nucleoside incorporation assays showed high proliferative activity in follicle cells to build the follicle and the end of endoreplicative activity in nurse cells of 4-day-old pupae. Protein immunolocalization in ovary at the stage of pupa has shown accumulation of Cyclin A in the cytoplasm of nurse cells and oocytes, and accumulation of Cyclin B in the cytoplasm and germinal vesicle of the oocyte, acting on meiosis mechanisms. The study of proteins related to cell cycle in this model is important for a better understanding of uncommon cell cycle in different insect organ.

More than 300 billion cells die in the human body every day. Although there are over a dozen different death paradigms, they all produce the same result - dead and dying cells. As they are no longer actively maintained, persistent corpses can proceed to a secondary necrotic state in which its cell membrane ruptures thus releasing its contents to the extracellular milieu. As many of the intracellular contents act as damage associated molecular patterns (DAMPs), they pose a potential danger to the rest of the surrounding tissue and organism. Excessive cell death has been correlated with diseases such as atherosclerosis, Alzheimer’s, and autoimmune disorders. To avoid damage and disease associated with cell corpses, two classes of cells evolved to clear them away – professional and nonprofessional phagocytes. A professional phagocyte's primary function is to clear away dying cells and other debris. Nonprofessional phagocytes, however, have a primary role other than clearance. When nonprofessional phagocytes encounter a cell corpse, their phagocytic machinery is engaged to clear it away. Interestingly, a recent study demonstrated that most, if not all, tissues contain nonprofessional phagocytes. To investigate nonprofessional phagocytes, the model organism Drosophila melanogaster is ideal. Drosophila is a useful model system as they have orthologs for 70% of human disease genes, a simplified immune system, and a host of genetic tools. Their ovaries have three morphologically distinct cell types – 15 nurse cells and an oocyte all surrounded by an epithelial follicle cell layer. As the ovaries are immunoprivileged, the follicle cell layer acts as the ovaries’ sole phagocytes. During late stage oogenesis, a small subsection of the follicle cell layer – the stretch follicle cells – murder the nurse cells in order to produce a fully developed oocyte. As past studies of cell corpse clearance have predominantly concentrated on the professional phagocytosis in the context of apoptotic cell corpses, there are still many gaps in our knowledge of nonprofessional phagocytosis and non-apoptotic death. This dissertation focuses on the molecular mechanisms that regulate the transition of nonprofessional phagocytes from their primary role as epithelial cells to their phagocytic role in the context of a newly characterized form of non-autonomous cell death known as phagoptosis. To gain a global view of these changes, two large scale experiments were performed – a classic genetic screen of kinases using RNAi and a high-throughput translatome study. The kinase screen identified dozens of kinase genes required for proper clearance. Of the 27 kinase genes that demonstrated a severe phenotype when knocked down, two were previously uncharacterized and six produced an “undead” phenotype, a phenotype that had only been previously witnessed when genes were perturbed in the germline. A follow up study was performed on Gprk2, one of the genes that induced a severe phenotype. By comparing the phenotypes of Gprk2 knockdowns and those of the two canonical clearance pathways, a third clearance pathway was discovered. The translatome study identified over 400 genes that were statistically significantly differentially expressed between primary state and phagocytic state follicle cells, including groups affecting calcium signaling and muscle contraction. This dissertation further describes the expansion of the molecular network of nonprofessional phagocytes driven by these large-scale experiments and their follow up studies.