Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development

Programmed cell death is an essential process during animal development. One type of cell death—apoptosis—is well understood at the molecular level, in large part due to genetic studies in the nematode Caenorhabditis elegans. The central apoptotic pathway in C. elegans consists of the four conserved genes egl-1 (BH3-only), ced-9 (Bcl-2), ced-4 (Apaf-1), and ced-3 (caspase), which act in a linear pathway. It is crucial that the activity of this pathway only triggers apoptosis in those cells programmed to die, and this requires regulation at multiple levels of gene expression. Although several studies have examined this regulation, post-transcriptional control of the pathway is still not well characterized. Here, I investigate regulation of the central apoptotic pathway by two prominent post- transcriptional mechanisms: microRNAs (miRNAs) and RNA-binding proteins (RBPs). First, I present evidence that the miR-35 family and the miR-58 bantam family of miRNAs directly target conserved elements in the 3ʹUTR of egl-1 mRNA and act cooperatively to repress its expression. This repression is crucial during embryogenesis, as loss of the mir-35 family leads to the inappropriate death of cells that are not programmed to die, and this phenotype is enhanced by the additional loss of the mir-58 family. These inappropriately dying cells are exclusively mothers and sisters of cells that are programmed to die, and their precocious and collateral deaths result in the formation of abnormally large cell corpses. Using single-molecule RNA FISH, I show that egl-1 is already transcribed in mother cells, and that both miR-35- and miR-58-family miRNAs function to maintain the copy number of egl-1 mRNA below a critical threshold—failure to do so results in precocious death of the mother cell. Furthermore, it seems that these two miRNA families are not required for the turnover of egl-1 mRNA over time in the daughter that survives. Considering that egl-1 transcription is controlled by numerous factors with varying modes of activity, the cooperative activity of miR-35- and miR-58- family miRNAs may buffer any lineage-specific differences in egl-1 transcription, thereby ensuring EGL-1 activity only reaches a level sufficient to trigger death in daughter cells that are programmed to die. Next, I describe a novel cell-death role for the gene puf-8, which encodes a conserved member of the Pumilio/FBF (PUF) family of RBPs. I show that animals lacking puf-8 exhibit two contrasting cell-death abnormalities. First, some cells die inappropriately, and these cells belong to both cell-death and non-cell-death lineages. Second, programmed cell death is delayed during the first wave of embryonic cell death. These abnormalities are not present upon nor enhanced by the loss of closely related puf-9, nor is the phenotype present upon knockdown of another member of the PUF family, fbf; however, fbf knockdown does suppress the large-cell-corpse phenotype in mir-35-family mutants. All four genes of the central apoptotic pathway harbor PUF-8-binding elements (PBEs) and/or FBF-binding elements (FBEs) in their 3ʹUTRs, and two FBEs in egl-1 can mediate the repression of a transgenic reporter. Therefore, puf-8 and fbf exhibit activity that both promotes and suppresses the cell-death pathway, and their prevailing activity might be regulated in a tissue- or cell-specific manner. Taken together, these findings show complex regulation of the central apoptotic pathway by both miRNAs and RBPs, and mounting evidence in the field suggests these two mechanisms can function cooperatively in the regulation of common targets. Evidence of cell-death genes being targeted by miRNAs and RBPs in mammals supports the possibility of this regulation being conserved in higher animals, which could have implications for the medical field. Our understanding of programmed cell death and its regulation has already led to the development of drugs that trigger apoptosis in cancer cells, and furthering this understanding could aid in the development of novel disease treatments

Post-transcriptional regulation of the central apoptotic pathway by microRNAs and RNA-binding proteins during C. elegans development

Programmed cell death is an essential process during animal development. One type of cell death—apoptosis—is well understood at the molecular level, in large part due to genetic studies in the nematode Caenorhabditis elegans. The central apoptotic pathway in C. elegans consists of the four conserved genes egl-1 (BH3-only), ced-9 (Bcl-2), ced-4 (Apaf-1), and ced-3 (caspase), which act in a linear pathway. It is crucial that the activity of this pathway only triggers apoptosis in those cells programmed to die, and this requires regulation at multiple levels of gene expression. Although several studies have examined this regulation, post-transcriptional control of the pathway is still not well characterized. Here, I investigate regulation of the central apoptotic pathway by two prominent post- transcriptional mechanisms: microRNAs (miRNAs) and RNA-binding proteins (RBPs). First, I present evidence that the miR-35 family and the miR-58 bantam family of miRNAs directly target conserved elements in the 3ʹUTR of egl-1 mRNA and act cooperatively to repress its expression. This repression is crucial during embryogenesis, as loss of the mir-35 family leads to the inappropriate death of cells that are not programmed to die, and this phenotype is enhanced by the additional loss of the mir-58 family. These inappropriately dying cells are exclusively mothers and sisters of cells that are programmed to die, and their precocious and collateral deaths result in the formation of abnormally large cell corpses. Using single-molecule RNA FISH, I show that egl-1 is already transcribed in mother cells, and that both miR-35- and miR-58-family miRNAs function to maintain the copy number of egl-1 mRNA below a critical threshold—failure to do so results in precocious death of the mother cell. Furthermore, it seems that these two miRNA families are not required for the turnover of egl-1 mRNA over time in the daughter that survives. Considering that egl-1 transcription is controlled by numerous factors with varying modes of activity, the cooperative activity of miR-35- and miR-58- family miRNAs may buffer any lineage-specific differences in egl-1 transcription, thereby ensuring EGL-1 activity only reaches a level sufficient to trigger death in daughter cells that are programmed to die. Next, I describe a novel cell-death role for the gene puf-8, which encodes a conserved member of the Pumilio/FBF (PUF) family of RBPs. I show that animals lacking puf-8 exhibit two contrasting cell-death abnormalities. First, some cells die inappropriately, and these cells belong to both cell-death and non-cell-death lineages. Second, programmed cell death is delayed during the first wave of embryonic cell death. These abnormalities are not present upon nor enhanced by the loss of closely related puf-9, nor is the phenotype present upon knockdown of another member of the PUF family, fbf; however, fbf knockdown does suppress the large-cell-corpse phenotype in mir-35-family mutants. All four genes of the central apoptotic pathway harbor PUF-8-binding elements (PBEs) and/or FBF-binding elements (FBEs) in their 3ʹUTRs, and two FBEs in egl-1 can mediate the repression of a transgenic reporter. Therefore, puf-8 and fbf exhibit activity that both promotes and suppresses the cell-death pathway, and their prevailing activity might be regulated in a tissue- or cell-specific manner. Taken together, these findings show complex regulation of the central apoptotic pathway by both miRNAs and RBPs, and mounting evidence in the field suggests these two mechanisms can function cooperatively in the regulation of common targets. Evidence of cell-death genes being targeted by miRNAs and RBPs in mammals supports the possibility of this regulation being conserved in higher animals, which could have implications for the medical field. Our understanding of programmed cell death and its regulation has already led to the development of drugs that trigger apoptosis in cancer cells, and furthering this understanding could aid in the development of novel disease treatments

The C. elegans PUM1, 2-like RNA binding protein PUF-8 is required for robustness of the cell death fate

During C. elegans development, 1090 somatic cells are generated of which 959 survive and 131 die, many through apoptosis. We present evidence that PUF-8, a C. elegans ortholog of the mammalian RNA binding proteins PUM1 and PUM2, is required for the robustness of this 'survival and death' pattern. We found that PUF-8 prevents the inappropriate death of cells that normally survive, and we present evidence that this anti-apoptotic activity of PUF-8 is dependent on PUF-8's ability to interact with ced-3caspase mRNA thereby repressing the activity of the pro-apoptotic ced-3caspase gene. PUF-8 also promotes the death of cells that are programmed to die, and we propose that this pro-apoptotic activity of PUF-8 may depend on PUF-8's ability to repress the expression of the anti-apoptotic ced-9Bcl-2 gene. Our results suggest that stochastic differences in the expression of genes within the apoptosis pathway can disrupt the highly reproducible and robust survival and death pattern during C. elegans development, and that PUF-8PUM1, 2 acts at the post-transcriptional level to level out these differences, thereby ensuring proper cell number homeostasis

miRNAs cooperate in apoptosis regulation during C. elegans development

Programmed cell death occurs in a highly reproducible manner during Caenorhabditis elegans development. We demonstrate that, during embryogenesis, miR-35 and miR-58 bantam family microRNAs (miRNAs) cooperate to prevent the precocious death of mothers of cells programmed to die by repressing the gene egl-1, which encodes a proapoptotic BH3-only protein. In addition, we present evidence that repression of egl-1 is dependent on binding sites for miR-35 and miR-58 family miRNAs within the egl-1 3\u27 untranslated region (UTR), which affect both mRNA copy number and translation. Furthermore, using single-molecule RNA fluorescent in situ hybridization (smRNA FISH), we show that egl-1 is transcribed in the mother of a cell programmed to die and that miR-35 and miR-58 family miRNAs prevent this mother from dying by keeping the copy number of egl-1 mRNA below a critical threshold. Finally, miR-35 and miR-58 family miRNAs can also dampen the transcriptional boost of egl-1 that occurs specifically in a daughter cell that is programmed to die. We propose that miRNAs compensate for lineage-specific differences in egl-1 transcriptional activation, thus ensuring that EGL-1 activity reaches the threshold necessary to trigger death only in daughter cells that are programmed to die

An evaluation of the effectiveness of annual health checks and quality of health care for adults with intellectual disability: an observational study using a primary care database

Background People with intellectual disability (ID) have poorer health than the general population; however, there is a lack of comprehensive national data describing their health-care needs and utilisation. Annual health checks for adults with ID have been incentivised through primary care since 2009, but only half of those eligible for such a health check receive one. It is unclear what impact health checks have had on important health outcomes, such as emergency hospitalisation. Objectives To evaluate whether or not annual health checks for adults with ID have reduced emergency hospitalisation, and to describe health, health care and mortality for adults with ID. Design A retrospective matched cohort study using primary care data linked to national hospital admissions and mortality data sets. Setting A total of 451 English general practices contributing data to Clinical Practice Research Datalink (CPRD). Participants A total of 21,859 adults with ID compared with 152,846 age-, gender- and practice-matched controls without ID registered during 2009–13. Interventions None. Main outcome measures Emergency hospital admissions. Other outcomes – preventable admissions for ambulatory care sensitive conditions, and mortality. Data sources CPRD, Hospital Episodes Statistics and Office for National Statistics. Results Compared with the general population, adults with ID had higher levels of recorded comorbidity and were more likely to consult in primary care. However, they were less likely to have long doctor consultations, and had lower continuity of care. They had higher mortality rates [hazard ratio (HR) 3.6, 95% confidence interval (CI) 3.3 to 3.9], with 37.0% of deaths classified as being amenable to health-care intervention (HR 5.9, 95% CI 5.1 to 6.8). They were more likely to have emergency hospital admissions [incidence rate ratio (IRR) 2.82, 95% CI 2.66 to 2.98], with 33.7% deemed preventable compared with 17.3% in controls (IRR 5.62, 95% CI 5.14 to 6.13). Health checks for adults with ID had no effect on overall emergency admissions compared with controls (IRR 0.96, 95% CI 0.87 to 1.07), although there was a relative reduction in emergency admissions for ambulatory care-sensitive conditions (IRR 0.82, 95% CI 0.69 to 0.99). Practices with high health check participation also showed a relative fall in preventable emergency admissions for their patients with ID, compared with practices with minimal participation (IRR 0.73, 95% CI 0.57 to 0.95). There were large variations in the health check-related content that was recorded on electronic records. Limitations Patients with milder ID not known to health services were not identified. We could not comment on the quality of health checks. Conclusions Compared with the general population, adults with ID have more chronic diseases and greater primary and secondary care utilisation. With more than one-third of deaths potentially amenable to health-care interventions, improvements in access to, and quality of, health care are required. In primary care, better continuity of care and longer appointment times are important examples that we identified. Although annual health checks can also improve access, not every eligible adult with ID receives one, and health check content varies by practice. Health checks had no impact on overall emergency admissions, but they appeared influential in reducing preventable emergency admissions. Future work No formal cost-effectiveness analysis of annual health checks was performed, but this could be attempted in relation to our estimates of a reduction in preventable emergency admissions. Funding The National Institute for Health Research Health Services and Delivery Research programme

Trichomonas vaginalis: Clinical relevance, pathogenicity and diagnosis

Trichomonas vaginalis is the etiological agent of trichomoniasis, the most prevalent non-viral sexually transmitted disease worldwide. Trichomoniasis is a widespread, global health concern and occurring at an increasing rate. Infections of the female genital tract can cause a range of symptoms, including vaginitis and cervicitis, while infections in males are generally asymptomatic. The relatively mild symptoms, and lack of evidence for any serious sequelae, have historically led to this disease being under diagnosed, and under researched. However, growing evidence that T. vaginalis infection is associated with other disease states with high morbidity in both men and women has increased the efforts to diagnose and treat patients harboring this parasite. The pathology of trichomoniasis results from damage to the host epithelia, caused by a variety of processes during infection and recent work has highlighted the complex interactions between the parasite and host, commensal microbiome and accompanying symbionts. The commercial release of a number of nucleic acid amplification tests (NAATs) has added to the available diagnostic options. Immunoassay based Point of Care testing is currently available, and a recent initial evaluation of a NAAT Point of Care system has given promising results, which would enable testing and treatment in a single visit

Pollution in the Río Santiago: A Qualitative Analysis

67 pages. A thesis presented to the Latin American Studies Program and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2016.The Rio Grande de Santiago-colloquially known as the Rio Santiago-has become one of the most polluted rivers in North America. The river, which spans more than 430 kilometers, directly borders numerous towns whose citizens have been afflicted with several pollution related health issues. Much of the pollution stems from the industrial center of Guadalajara where over a hundred corporations, many of which are international, set up plants and factories in the wake of NAFTA. Although the extent of the pollution and its repercussions for public health have been known for some time, the Mexican government has been slow make meaningful action towards preventing the pollution. This study performs a qualitative analysis on the economics of pollution in the Rio Santiago, employing a systematic review of relevant literature in order to explore the culpability ofNAFTA in promoting pollution, the mechanisms which encourage environmentally unsustainable behavior, as well as the sources and long-run economic effects of the pollution

Essays in the Economics of Crime

This dissertation consists of three essays that each explore the United States criminal justice system. In the first chapter I evaluate the impact of "Ban the Box" Laws on rates of criminal recidivism. Despite their goal of increasing ex-offender employment and reducing recidivism, several recent studies of "Ban the Box" (BTB) policies have cast doubt on BTB's efficacy at improving ex-offender employment outcomes. Evidence of BTB's effect on criminal recidivism, however, remains limited. Using administrative prison data, this chapter examines the direct effect of BTB policies on rates of criminal recidivism. I find that, while BTB policies don't appear to reduce criminal recidivism in the aggregate, they may be exacerbating racial disparities. In particular, I show that being released into a labor market with a BTB policy is associated with higher rates of recidivism for black ex-offenders, with young black ex-offenders being particularly affected. In contrast, older white ex-offenders seem to benefit from the policies. In the second chapter I estimate the effect of electoral pressure on the sentencing behavior of prosecutors in California. Prosecutors in the United States wield immense discretionary power over the outcome of criminal cases. Despite this, there has been relatively little research concerning the effect that electoral cycles might have on their sentencing behavior. Conventional wisdom dictates that prosecutors will likely pursue harsher sentences on average, in an attempt to appear "tough on crime". To test this, I construct a novel dataset of California prosecutors and electoral outcomes. Using criminal sentencing data, I then estimate the impact of electoral pressure, as measured by electoral proximity and competition, on criminal sentencing. I find that electoral pressure is associated with a decrease in the average severity of criminal sentences for serious violent crimes. Then, using data from the San Francisco District Attorney's Office, I provide evidence that this effect can be explained, in part, by prosecutors engaging in charge bargaining. Finally, in the third chapter I estimate the impact of changes in local television broadcast news on criminal sentencing. The local television broadcasting industry in the United States has undergone significant consolidation over the past two decades, leading to the rise of large national media conglomerates. It is unclear, however, what impact consolidation will have on the quantity, quality, and content of local news broadcasts. This chapter uses the rapid expansion of one of the largest media conglomerates, Sinclair Broadcast Group, on the sentencing behavior of local criminal justice officials. I find that Sinclair's entrance into a media market is associated with 2.55% decrease in the average sentence length for serious violent crimes. Heterogeneity analyses show that this effect is concentrated among black defendants, and is primarily driven by sentences for robberies and aggravated assaults. Similarly, I show that this effect is almost entirely driven by counties that select their judges via elections rather than appointments. Taken together, these results suggest that a reduction in local crime coverage associated with Sinclair's acquisition limits voter information concerning local judges, reducing their incentive to appeal to voters by imposing harsher sentences

Photosystem II and Pigment Dynamics among Ecotypes of the Green Alga Ostreococcus1

We investigated the photophysiological responses of three ecotypes of the picophytoplankter Ostreococcus and a larger prasinophyte Pyramimonas obovata to a sudden increase in light irradiance. The deepwater Ostreococcus sp. RCC809 showed very high susceptibility to primary photoinactivation, likely a consequence of high oxidative stress, which may relate to the recently noted plastid terminal oxidase activity in this strain. The three Ostreococcus ecotypes were all capable of deploying modulation of the photosystem II repair cycle in order to cope with the light increase, but the effective clearance of photoinactivated D1 protein appeared to be slower in the deepwater Ostreococcus sp. RCC809, suggesting that this step is rate limiting in the photosystem II repair cycle in this strain. Moreover, the deepwater Ostreococcus accumulated lutein and showed substantial use of the xanthophyll cycle under light stress, demonstrating its high sensitivity to light fluctuations. The sustained component of the nonphotochemical quenching of fluorescence correlated well with the xanthophyll deepoxidation activity. Comparisons with the larger prasinophyte P. obovata suggest that the photophysiology of Ostreococcus ecotypes requires high photosystem II repair rates to counter a high susceptibility to photoinactivation, consistent with low pigment package effects in their minute-sized cells