DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.

The shift of medical technology from a doctor's application of individualized medicine toward precision medicine has been accelerated by the advent of Next Generation Sequencing. Individualized medicine is where a doctor tries to understand the intricacies of a patient's medical state, where precision medicine uses a wealth of data to understand the individuality of a patient on a biological level to determine treatment course. Next Generation Sequencing allows for the collection of genome wide analyses such as genomic, transcriptomic, and epigenomic sequencing, which provides the backbone of the data driven precision medicine. In order to obtain and use this data, it needs to be produced from minimal amounts of patient tissue, such as the amount from a needle biopsy. In order to perform so many different assays it is paramount that we develop high sensitivity methodologies, such that we can gain an understanding of the patient's physiology without causing much discomfort in gathering large amounts of sample. In pursuit of making more tests, data, and assays available for use in precision medicine, we have developed 3 different microfluidic technologies, which automate and simplify the assays needed for the data collection at a high sensitivity, as well as a versatile platform for therapeutic production. First, we developed a epigenomic assay for chromatin immunoprecipitation, which gives us information on histone modifications across the genome. These histone modifications heavily impact gene expression and how the chromatin is organized, as well promoting or inhibiting transcription of genes. Our technology allowed us to perform multiple parallel assays from as few as 50 cells quickly and reliably using our fluidized bed technology. Next, we developed a library preparation system, which reduces the cost of library preparation by 20x and reduces operator pipetting by 100x. Our system uses a droplet based reactor to quickly and reliably prepare sequencing libraries using the lowest amount of DNA to date, 10 pg. Finally, we designed a therapeutics-on-a-chip platform which is capable of producing clinically relevant proteins on demand from temperature stable components. Using our system, we are capable of producing a number of different therapeutics on demand quickly without rearrangement of the system. / Doctor of Philosophy / Technical advances in the healthcare industry have made a range of new data available to physicians and patients. Home use DNA testing kits have made it possible to examine one’s predisposition to certain genetic diseases. Using these advanced methods, we are able to gain insights into a patient’s disease state where we were previously unable. Unfortunately, some of these new analyses currently require large amounts of patient sample, which make the examinations largely impractical to perform. In order to overcome the sample requirements, which make these analyses impractical, we develop microscale reactor systems capable of reducing the amount of material required for these new analyses. Here I demonstrate our developed technologies to automate 3 different processes aimed at enabling the study of protein-DNA interactions and produce therapeutics at the point of care. First, we developed an analytical system to study protein-DNA interactions (which are important to understanding patient responses to treatment), that allow for parallel analyses which can be done with sample from less than one needle biopsy, where existing methods would require dozens or more (50 vs 10,000,000 cells.) Next, we developed automated system for preparing DNA sequencing libraries using as little as 10 pg DNA (~2 cells of DNA). The device run multiple reactions simultaneously while reducing batch to batch variation and operator hands-on time. Finally, we developed a v Therapeutics-On-a-Chip platform that produces clinically relevant therapeutic proteins in clinically relevant dosages using a cell-free approach, while saving the trouble and cost associated with protein storage and transportation.

Epigenetic alterations are observed in virtually all cancer types, yet there is limited understanding of their role in tumorigenesis and evolution. The role of DNA methylation has been particularly elusive in this context. While this epigenetic mark has been extensively profiled in healthy and cancerous samples, our ability to understand its relationship with underlying biological processes is still limited. Moreover, recent advancements in the profiling of cell-free DNA in circulation have sparked renowned attention toward tissue-specific and cancer-specific DNA methylation patterns. In this thesis, I present results to improve and refine the computational characterization of DNA methylation in cancer, focusing on metastatic castration-resistant prostate cancer. The first contribution is the development and performance assessment of Rockermeth, a computational methodology to leverage large-scale DNA methylation profiling data to nominate robust differentially methylated regions (DMRs). Rocker-meth can retrieve biologically relevant DNA methylation changes, as demonstrated by extensive integrative analyses with gene expression, chromatin states, and genomic annotations. The second contribution is the generation of a map of DNA methylation changes across prostate cancer progression. The application of Rockermeth and other tailored methodologies can be used to trace the critical evolutionary steps of this disease, from the healthy tissue to the most lethal metastatic AR-independent counterpart. The main result is the evidence of the ability of DNA methylation to capture a snapshot of the active transcription factors in each state of the disease, offering orthogonal information compared to standard genomic sequencing. The third contribution is the design and development of NEMO, a tailored liquid biopsy sequencing panel approach to allow non-invasive neuroendocrine castration-resistant prostate cancer detection in patients with metastatic disease. Based on previous results and the comprehensive analysis of multiple datasets, I designed a set of informative genomic regions to estimate disease burden and evidence of neuroendocrine transdifferentiation. The actual implementation of the NEMO panel produced a scalable and cost-effective strategy, which has been extensively benchmarked using both in silico and in vitro approaches. The application of NEMO to patient-derived cfDNA samples demonstrated accurate tumor content estimation and robust detection of neuroendocrine disease, making it a promising instance of liquid biopsy for CRPC.

Previous studies have shown that genes can be switched on or off by age, environmental factors, diseases, and lifestyles. The open or compact structures of chromatin is a crucial factor that affects gene expression. Epigenetics refers to hereditary mechanisms that change gene expression and regulations without changing DNA sequences. Epigenetic modifications, such as DNA methylation, histone modification, and non-coding RNA interaction, play critical roles in cell differentiation and disease processes. The conventional approach requires the use of a few million or more cells as starting material. However, such quantity is not available when samples from patients and small lab animals are examined. Microfluidic technology offers advantages to utilize low-input starting material and for high-throughput. In this thesis, I developed novel microfluidic technologies to study epigenomic regulations, including 1) profiling epigenomic changes associated with LPS-induced murine monocytes for immunotherapy, 2) examining cell-type-specific epigenomic changes associated with BRCA1 mutation in breast tissues for breast cancer treatment, and 3) developing a novel microfluidic oscillatory hybridized ChIRP-seq assay to profile genome-wide lncRNA binding for numerous human diseases. We used 20,000 and 50,000 primary cells to study histone modifications in inflammation and breast cancer of BRCA1 mutation, respectively. In the project of whole-genome lncRNA bindings, our microfluidic ChIRP-seq assay, for the first time, allowed us to probe native lncRNA bindings in mouse tissue samples successfully. The technology is a promising approach for scientists to study lncRNA bindings in primary patients. Our works pave the way for low-input and high-throughput epigenomic profiling for precision medicine development. / Doctor of Philosophy / Traditionally, physicians treat patients with a one-size-fits-all approach, in which disease prevention and treatment are designed for the average person. The one-size-fits-all approach fits many patients, but does not work on some. Precision medicine is launched to improve the low efficiency and diminish side effects, and all of these drawbacks are happening in the traditional approaches. The genomic, transcriptomic, and epigenomic data from patients is a valuable resource for developing precision medicine. Conventional approaches in profiling functional epigenomic regulation use tens to hundreds of millions cells per assay, that is why applications in clinical samples are restricted for several decades. Due to the small volume manipulated in microfluidic devices, microfluidic technology exhibits high efficiency in easy operation, reducing the required number of cells, and improving the sensitivity of assays. In order to examine functional epigenomic regulations, we developed novel microfluidic technologies for applications with the small number of cells. We used 20,000 cells from mice to study the epigenomic changes in monocytes. We also used 50,000 cells from patients and mice to study epigenomic changes associated with BRCA1 mutation in different cell types. We developed a novel microfluidic technology for studying lncRNA bindings. We used 100,000-500,000 cells from cell lines and primary tissues to test several lncRNAs. Traditional approaches require 20-100 million cells per assay, and these cells are infected by virus for over-producing specific lncRNA. However, our technology just needs 100,000 cells (non-over-producing state) to study lncRNA bindings. To the best of our knowledge, this is the first allowed us to study native lncRNA bindings in mouse samples successfully. Our efforts in developing microfluidic technologies and studying epigenomic regulations pave the way for precision medicine development.

Current sequencing technologies allows researchers unprecedented insight into our biology, and how these biological mechanisms can become distorted and lead to disease. These aberrant mechanisms can be brought about by many causes, but some occur as a result of genetic mutations or external factors through the epigenome. Here, we used our microfluidic technology to profile the epigenome and transcriptome to study such aberrant mechanisms in three different diseases and illnesses: breast cancer, chronic inflammation, and mental illness. We profiled the epigenome of breast tissue from healthy women with the BRCA1 mutation to understand how the mutation may facilitate eventual breast cancer. Epigenomic changes in breast cells suggest that cells in the basal compartment may differentiate into a different cell type, and perhaps become the source of breast cancer. Next, we compared the epigenome and genome of murine immune cells under low-grade inflammation and acute inflammation conditions. We found that low-grade inflammation preferentially utilizes different signaling pathways than in acute inflammation, and this may lead to a non-resolving state. Finally, we analyzed the effect of the maternal immune activation on unborn offspring, and how these changes could cause later mental illness. The insights we made into these diseases may lead to future therapies. / Doctor of Philosophy / Despite advances in medical and scientific research, there is still a dearth of information on how diseases affect the expression of our genes, such as breast cancer, chronic inflammation, and influenza. Mutation in the BRCA1 gene is probably the most well-known mutation that can lead to breast cancer. We know the overarching reason that mutation in BRCA1 can lead to cancer, as BRCA1 is responsible for repairing damage in the DNA, so mutations can compound and create cancerous cells. However, we do not know the exact mechanisms by which this actually happens. Another widespread problem is chronic inflammation, which can promote or lead to diseases such as diabetes, cancer, Alzheimer's, Rheumatoid arthritis, and heart disease. In addition, there are many causes of chronic inflammation that many people have experienced at some point in time, including stress, insomnia, being sedentary, poor eating habits, and obesity. Despite this, we still do not fully understand why chronic inflammation differs from normal inflammation, which is a healthy process, or why it does not resolve. There are also other connections that are surprising, and many are not aware of. If a pregnant woman gets the flu during her second trimester, her baby has much higher odds of developing schizophrenia later in its lifetime. Given the prevalence of the flu, there is a very real chance that an expecting mother will be infected during her pregnancy.

INTRODUÇÃO. Os efeitos da poluição atmosférica sobre a saúde humana são cada vez mais reconhecidos. A exposição ao combustível diesel, que contribui significativamente para a poluição em zonas de tráfego veicular intenso, pode ser ao menos parcialmente, responsável por estes efeitos. Estudos epidemiológicos demonstram maior incidência de pré-eclampsia, prematuridade e baixo peso ao nascer. Maiores taxas de falha de implantação e alterações da morfologia placentária são demonstrados em estudos experimentais. OBJETIVO. Análise morfológica do ovário e do útero de camundongos após exposição crônica ao combustível diesel durante a fase intra-uterina e pós-natal. MÉTODO. Estudo experimental prospectivo com crossover. Camundongos foram expostos durante uma hora a doses de combustão do diesel que correspondem a média diária de material particulado fino (MP2.5) recomendado pela Organização Mundial da Saúde (WHO) e a doses acima da média anual recomendada pela mesma. A dose de exposição a MP2.5 foi abaixo da dose recomendada pela Comissão Nacional do Meio Ambiente (CONAMA). Quatro grupos foram formados: animais expostos a ar filtrado no período intra-uterino e no período pós-natal (CC); animais expostos a diesel apenas no período intra-uterino, sendo que no período pós-natal foram expostos a ar filtrado (EC); animais expostos a ar filtrado no período intra-uterino e a diesel no pós-natal (CE), e animais expostos a diesel nos períodos intra-uterino e pós-natal (EE). A análise morfométrica do ovário e do útero foi realizada para definir a área relativa ocupada por cada tipo de folículo, pelo corpo lúteo e pelo estroma, e a proporção de área ocupada por glândulas, estroma e epitélio no endométrio. RESULTADOS. Uma redução significativa de folículos primordiais foi observada em animais expostos a diesel durante o período intra-uterino (p=0,035) e durante o período pós-natal (p=0,015), assim como em animais expostos a diesel nos dois períodos (p=0.004). Houve redução significativa de folículos primários quando os animais foram expostos no período intra-uterino (p=0.04). Não foram demonstradas alterações significativas no útero. CONCLUSÃO. A exposição a níveis considerados aceitáveis de combustão do diesel é prejudicial ao potencial reprodutivo de camundongos fêmeas, diminuindo sua reserva ovariana ao atingir a maturidade sexual, sugerindo o envolvimento de alterações epigenéticas. Tal efeito aumenta o risco de menopausa precoce. Estes resultados sugerem que diretrizes ambientais sejam revisadas, já que a exposição prejudica gerações futuras, diminuindo a janela reprodutiva, já comprometida pelo desejo de grande parte das mulheres de adiar a maternidade / BACKGROUND. There is growing awareness that ambient air pollution compromises human health. Exposure to diesel exhaust, which significantly contributes to pollution in heavy traffic areas, may be at least partially responsible for the observed effects of pollution on human health. Poor reproductive outcomes, such as preeclampsia, preterm deliveries and low birth weight, are described in epidemiological studies. Moreover, experimental evidence shows increased implantation failure rates and placental morphology alterations in exposed mice. OBJECTIVE. The purpose of this study was to analyse ovarian and uterine morphological changes resulting from chronic intrauterine and postnatal exposure to diesel exhaust in mice. METHODS. A experimental prospective crossover study. Mice were exposed to diesel exhaust with doses that correspond to the daily average PM2.5 levels reported by the World Health Organization (WHO) and above the annual average PM2.5 levels. The diesel exhaust exposure doses were also below daily and annual levels recommended by the National Council of Environment (CONAMA). Four groups were examined: intrauterine and postnatal clean, filtered air exposure; intrauterine exposure to diesel only; postnatal exposure to diesel only; and intrauterine and postnatal exposure to diesel. Morphometric analyses of the ovaries and uterus were performed to define the relative area occupied by follicles, corpus luteum and stroma. These analyses also aimed to determine the proportionate area of glands, the epithelial layer and stroma within the uterine endometrium. RESULTS: A significant reduction in primordial follicles was observed in intra-uterine-exposed animals (p=0.035), those exposed during the postnatal period (p=0.015) and in animals exposed during both phases (p=0.004). Primary follicles were reduced in animals exposed during pregnancy (p=0.04). No significant changes were detected in uterine morphology. CONCLUSIONS: Intrauterine exposure to currently acceptable levels of diesel exhaust compromises the reproductive potential of female mice, diminishing ovarian reserve when sexual maturity is achieved suggesting involvement of epigenetic changes. This effect in turn increases the risk of premature menopause. These findings suggest that environmental guidelines should be reviewed because diesel exposure affects future generations. Premature menopause reduces the span of the reproductive window, which is already shortening due to womens desire to bear children later in life

The transcriptional repressor Polycomb Repressive Complex 1 (PRC1) is critical for stem cell maintenance and proper differentiation and as such is involved in the development and progression of cancer. Canonical PRC1, composed of PCGF, PHC, RING and CBX, binds histone H3 lysine 27 trimethylation (H3K27me3) allowing for ubiquitination, chromatin compaction and subsequently transcriptional silencing. In mammals, each subunit has multiple paralogs creating functional and compositional diversity. The greatest diversity is contributed by the CBX targeting subunit with five mutually exclusive paralogs (CBX2/4/6/7/8). The CBX paralogs contain an N-terminal chromodomain for methyllysine binding. There has been interest in the CBX paralogs due to their misregulation in various cancers and the “druggability” of the chromodomain histone interaction. However, the unique biochemical and transcriptional functions of the paralogs are unclear. Expression changes during lineage specification and the context-dependent misregulation of CBX paralogs in cancers suggest the paralogs have paralog-specific functions. However, little has been done to define differences in paralog-mediated chromatin binding and regulation. This work utilizes a variety of approaches to tease apart the biological and biochemical functions of the CBX paralogs in chromatin binding and oncogenesis. In this dissertation, we identify a combinatorial therapeutic strategy using a CBX chromodomain inhibitor to enhance chemotherapeutic response. Further, this work demonstrates a role for CBX8 and its chromodomain in glioblastoma oncogenesis suggesting it may serve as a therapeutic target. Finally, we identify a binding mechanism for the CBX8 chromodomain in which DNA and H3K27me3 binding contribute to full chromatin association.

INTRODUÇÃO. Os efeitos da poluição atmosférica sobre a saúde humana são cada vez mais reconhecidos. A exposição ao combustível diesel, que contribui significativamente para a poluição em zonas de tráfego veicular intenso, pode ser ao menos parcialmente, responsável por estes efeitos. Estudos epidemiológicos demonstram maior incidência de pré-eclampsia, prematuridade e baixo peso ao nascer. Maiores taxas de falha de implantação e alterações da morfologia placentária são demonstrados em estudos experimentais. OBJETIVO. Análise morfológica do ovário e do útero de camundongos após exposição crônica ao combustível diesel durante a fase intra-uterina e pós-natal. MÉTODO. Estudo experimental prospectivo com crossover. Camundongos foram expostos durante uma hora a doses de combustão do diesel que correspondem a média diária de material particulado fino (MP2.5) recomendado pela Organização Mundial da Saúde (WHO) e a doses acima da média anual recomendada pela mesma. A dose de exposição a MP2.5 foi abaixo da dose recomendada pela Comissão Nacional do Meio Ambiente (CONAMA). Quatro grupos foram formados: animais expostos a ar filtrado no período intra-uterino e no período pós-natal (CC); animais expostos a diesel apenas no período intra-uterino, sendo que no período pós-natal foram expostos a ar filtrado (EC); animais expostos a ar filtrado no período intra-uterino e a diesel no pós-natal (CE), e animais expostos a diesel nos períodos intra-uterino e pós-natal (EE). A análise morfométrica do ovário e do útero foi realizada para definir a área relativa ocupada por cada tipo de folículo, pelo corpo lúteo e pelo estroma, e a proporção de área ocupada por glândulas, estroma e epitélio no endométrio. RESULTADOS. Uma redução significativa de folículos primordiais foi observada em animais expostos a diesel durante o período intra-uterino (p=0,035) e durante o período pós-natal (p=0,015), assim como em animais expostos a diesel nos dois períodos (p=0.004). Houve redução significativa de folículos primários quando os animais foram expostos no período intra-uterino (p=0.04). Não foram demonstradas alterações significativas no útero. CONCLUSÃO. A exposição a níveis considerados aceitáveis de combustão do diesel é prejudicial ao potencial reprodutivo de camundongos fêmeas, diminuindo sua reserva ovariana ao atingir a maturidade sexual, sugerindo o envolvimento de alterações epigenéticas. Tal efeito aumenta o risco de menopausa precoce. Estes resultados sugerem que diretrizes ambientais sejam revisadas, já que a exposição prejudica gerações futuras, diminuindo a janela reprodutiva, já comprometida pelo desejo de grande parte das mulheres de adiar a maternidade / BACKGROUND. There is growing awareness that ambient air pollution compromises human health. Exposure to diesel exhaust, which significantly contributes to pollution in heavy traffic areas, may be at least partially responsible for the observed effects of pollution on human health. Poor reproductive outcomes, such as preeclampsia, preterm deliveries and low birth weight, are described in epidemiological studies. Moreover, experimental evidence shows increased implantation failure rates and placental morphology alterations in exposed mice. OBJECTIVE. The purpose of this study was to analyse ovarian and uterine morphological changes resulting from chronic intrauterine and postnatal exposure to diesel exhaust in mice. METHODS. A experimental prospective crossover study. Mice were exposed to diesel exhaust with doses that correspond to the daily average PM2.5 levels reported by the World Health Organization (WHO) and above the annual average PM2.5 levels. The diesel exhaust exposure doses were also below daily and annual levels recommended by the National Council of Environment (CONAMA). Four groups were examined: intrauterine and postnatal clean, filtered air exposure; intrauterine exposure to diesel only; postnatal exposure to diesel only; and intrauterine and postnatal exposure to diesel. Morphometric analyses of the ovaries and uterus were performed to define the relative area occupied by follicles, corpus luteum and stroma. These analyses also aimed to determine the proportionate area of glands, the epithelial layer and stroma within the uterine endometrium. RESULTS: A significant reduction in primordial follicles was observed in intra-uterine-exposed animals (p=0.035), those exposed during the postnatal period (p=0.015) and in animals exposed during both phases (p=0.004). Primary follicles were reduced in animals exposed during pregnancy (p=0.04). No significant changes were detected in uterine morphology. CONCLUSIONS: Intrauterine exposure to currently acceptable levels of diesel exhaust compromises the reproductive potential of female mice, diminishing ovarian reserve when sexual maturity is achieved suggesting involvement of epigenetic changes. This effect in turn increases the risk of premature menopause. These findings suggest that environmental guidelines should be reviewed because diesel exposure affects future generations. Premature menopause reduces the span of the reproductive window, which is already shortening due to womens desire to bear children later in life

Objetivo: El objetivo del estudio fue evaluar si la alimentación complementaria Baby Led Weaning tiene efecto protector de la malnutrición por exceso en preescolares y determinar sus características sociodemográficas y la proporción de preescolares con normopeso y malnutridos por exceso que recibieron este tipo de alimentación. Tuvo un alcance de niños cuyos padres y/o madres pertenecen a grupos de redes sociales de maternidad o paternidad asociados a profesionales de la salud. Metodología: El diseño fue observacional, analítico, retrospectivo tipo caso control, 38 casos y 66 controles con una relación caso control de 1/1.73. La población de estudio estuvo conformada por niños de uno u otro sexo de 24 a 60 meses de edad. Se utilizó una red de contactos de profesionales de la salud para difundir la encuesta virtual. Los datos recabados de duración de lactancia, método de alimentación complementaria y tipo de parto y otros de interés fueron extrapolados a Excel y posteriormente al software SPSS para determinar la asociación y el OR para estudio de casos y controles. Resultados: En el análisis descriptivo se obtuvo mayor participación de los países de Argentina y Perú. En su mayoría, los casos y controles fueron del género femenino con edad media de 34.17 meses. En el análisis inferencial se obtuvo una OR de 0.187 (IC 95% 0.078-0.444), lo que significa que la Alimentación Complementaria BLW reduce significativamente el riesgo de padecer malnutrición por exceso en (1-0.187=0.81) 81%, respecto a los que tuvieron Alimentación Complementaria Convencional. Conclusión: Se concluye que la AC BLW tiene efecto protector de malnutrición por exceso en preescolares, sin embargo, se deben realizar estudios de cohorte para evaluar prospectivamente la evolución del infante desde que se inicia la AC hasta los 60 meses, considerando otras variables biopsicosociales. / Objective: The objective of the study was to evaluate if the Complementary Feeding Baby Led Weaning (AC BLW) has a protective effect against malnutrition due to excess in preschool children and to determine their sociodemographic characteristics and the proportion of preschool children with normal weight and malnourished by excess who received this type of feeding. It had a scope of children whose fathers and / or mothers belong to maternity or paternity social network groups associated with health professionals. Methodology: The design was observational, analytical, retrospective, case-control type, 38 cases and 66 controls with a case-control ratio of 1 / 1.73. The study population consisted of children of either sex between 24 and 60 months of age. A network of contacts of health professionals was used to disseminate the virtual survey. The data collected on the duration of lactation, the method of complementary feeding and the type of delivery, and others of interest, were extrapolated to Excel and later to the SPSS software to determine the association and the OR for a case-control study. Results: In the descriptive analysis, greater participation was obtained from the countries of Argentina and Peru. Most of the cases and controls were female with a mean age of 34.17 months. In the inferential analysis, an OR of 0.187 (95% CI 0.078-0.444) was obtained, which means that the AC BLW significantly reduces the risk of suffering from malnutrition due to excess in (1-0.187 = 0.81) 81%, with respect to the who had Conventional Complementary Food (AC CV). Conclusion: It is concluded that AC BLW has a protective effect against malnutrition due to excess in preschool children, however, cohort studies should be carried out to prospectively evaluate the evolution of the infant from the beginning of the AC to 60 months, considering other biopsychosocial variables. / Tesis

<p>  </p> <p>Aging is characterized by changes in transcriptional outputs that correlate with physiological changes observed as we age, including decreased function, and increased cell death. Importantly, many of these changes are conserved across tissues and organisms . Because one of the molecular hallmarks of aging is epigenetic dysregulation, we are interested in understanding how age-associated changes in chromatin contribute to the aging transcriptome. To accomplish this, we use the <em>Drosophila </em>visual system as a model for aging, with a particular focus on photoreceptor neurons. </p> <p>To perform cell-type specific genomic studies in <em>Drosophila, </em>we previously developed a nuclei immuno-enrichment method that was compatible with RNA-seq. However, due to low nuclei yields, this protocol was not amenable to chromatin-based studies, such as ChIP-seq and ATAC-seq. In Chapter 1, we developed an improved approach to isolate Drosophila melanogaster nuclei tagged with a GFPKASH protein that increased yields without compromising efficiency. We further demonstrate that this protocol is compatible with several chromatin profiling techniques, such as Assay of Transposable-Accessible Chromatin (ATAC)-seq, Chromatin Immunoprecipitation (ChIP-seq), and CUT&Tag. </p> <p>Chromatin accessibility is enriched for transcription factors. Thus, in Chapter 2, we profiled accessible chromatin in aging photoreceptors and integrated this data with RNA-seq to identify transcription factors that showed differential activity in aging Drosophila photoreceptors. Surprisingly, we found that 57 transcription factors had differential binding activity during aging, including two circadian regulators, Clock and Cycle, that showed sustained increased activity during aging. When we disrupted the Clock:Cycle complex by expressing a dominant negative version of Clock (ClkDN) in adult photoreceptors, we observed changes in expression of 15–20% of genes including key components of the phototransduction machinery and many eye-specific transcription factors. Using ATAC-seq, we showed that expression of ClkDN in photoreceptors leads to changes in activity of 37 transcription factors and causes a progressive decrease in global levels of chromatin accessibility in photoreceptors. Supporting a key role for Clock-dependent transcription in the eye, expression of ClkDN in photoreceptors also induced light-dependent retinal degeneration and increased oxidative stress, independent of light exposure. Together, our data suggests that the circadian regulators Clock and Cycle act as neuroprotective factors in the aging eye by directing gene regulatory networks that maintain expression of the phototransduction machinery and counteract oxidative stress.</p> <p>Previous work in the Weake lab found that long, highly expressed genes were more susceptible to be downregulated with age. DNA:RNA hybrids are co-transcriptional structures that form when the nascent RNA hybridizes with the template strand, resulting in a displaced non-template ssDNA. Importantly, accumulation of R-loops is associated with transcriptional inhibition and genomic instability, both hallmarks of aging. In Chapter 3, I characterized R-loop in maintaining proper transcriptional outputs and regulating visual function during aging. Bulk assays to measure R-loop levels revealed a significant increase in nuclear R-loops with age. Further, genome-wide mapping of R-loops revealed that transcribed genes accumulated R-loops over gene bodies during aging, which correlated with decreased expression of long and highly expressed genes. Importantly, while photoreceptor-specific down-regulation of Top3β, a DNA/RNA topoisomerase associated with R-loop resolution, lead to decreased visual function, over-expression of Top3β or nuclear-localized RNase H1, which resolves R-loops, enhanced positive light response during aging. </p> <p>Together, these studies underscore the importance of understanding how age-related changes in genomic processes, such as circadian transcription and maintenance of R-loops, contribute to physiological changes observed during aging. </p>