The role of splicing factors and small nuclear RNAs in spliceosomal formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatiotemporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Indirect and direct controls of macroinvertebrates and small fish by abiotic factors and trophic interactions in the Florida Everglades

1. The roles of nutrients, disturbance and predation in regulating consumer densities have long been of interest, but their indirect effects have rarely been quantified in wetland ecosystems. The Florida Everglades contains gradients of hydrological disturbance (marsh drying) and nutrient enrichment (phosphorus), often correlated with densities of macroinvertebrate infauna (macroinvertebrates inhabiting periphyton), small fish and larger invertebrates, such as snails, grass shrimp, insects and crayfish. However, most causal relationships have yet to be quantified. 2.  We sampled periphyton (content and community structure) and consumer (small omnivores, carnivores and herbivores, and infaunal macroinvertebrates inhabiting periphyton) density at 28 sites spanning a range of hydrological and nutrient conditions and compared our data to seven a priori structural equation models. 3.  The best model included bottom-up and top-down effects among trophic groups and supported top-down control of infauna by omnivores and predators that cascaded to periphyton biomass. The next best model included bottom-up paths only and allowed direct effects of periphyton on omnivore density. Both models suggested a positive relationship between small herbivores and small omnivores, indicating that predation was unable to limit herbivore numbers. Total effects of time following flooding were negative for all three consumer groups even when both preferred models suggested positive direct effects for some groups. Total effects of nutrient levels (phosphorus) were positive for consumers and generally larger than those of hydrological disturbance and were mediated by changes in periphyton content. 4.  Our findings provide quantitative support for indirect effects of nutrient enrichment on consumers, and the importance of both algal community structure and periphyton biomass to Everglades food webs. Evidence for top-down control of infauna by omnivores was noted, though without substantially greater support than a competing bottom-up-only model.

The Effect of Natural Products and Small Gtpases in Adipogenesis

Insulin signaling is one of the main initiators of adipogenesis, the conversion from pre-adipocyte to adipocyte or lipid droplet. Rab proteins are the master regulator of intracellular trafficking and endosome fusion in endocytosis, making them potential regulators of insulin signaling in adipogenesis. Pre-adipocytes 3T3-Ll cells expressing several Rab5 constructs were used to examine the effect of dehydroleucodine (DhL ), a sesquiterpene lactone isolated from aerial parts of Artemisia douglasiana Besser. The results obtained identify Rab5 deactivation as a key step for adipogenesis by forming signaling endosomes. The addition of DhL significantly inhibited the lipid droplet accumulation in a dose-dependent manner and dramatically attenuated the synthesis of adipogenic transcriptional factors, C/EBPa and PPARy. Activation of AMPKa, Erk and Akt during adipocytic differentiation was not inhibited by treatment with DhL. This data suggest that DhL has an important role in Rab5 dependent adipogenesis by regulating several transcriptional factors including PP ARy expression, which is known to play an essential role during fat formation.

The Role of Splicing Factors and Small Nuclear RNAS in Spliceosomal Formation

Protein coding genes are comprised of protein-coding exons and non-protein-coding introns. The process of splicing involves removal of the introns and joining of the exons to form a mature messenger RNA, which subsequently undergoes translation into polypeptide. The spliceosome is a large, RNA/protein assembly of five small nuclear RNAs as well as over 300 proteins, which catalyzes intron removal and exon ligation. The selection of specific exons for inclusion in the mature messenger RNA is spatio-temporally regulated and results in production of an enormous diversity of polypeptides from a single gene locus. This phenomenon, known as alternative splicing, is regulated, in part, by protein splicing factors, which target the spliceosome to exon/intron boundaries. The first part of my dissertation (Chapters II and III) focuses on the discovery and characterization of the 45 kilodalton FK506 binding protein (FKBP45), which I discovered in the silk moth, Bombyx mori, as a U1 small nuclear RNA binding protein. This protein family binds the immunosuppressants FK506 and rapamycin and contains peptidyl-prolyl cis-trans isomerase activity, which converts polypeptides from cis to trans about a proline residue. This is the first time that an FKBP has been identified in the spliceosome. The second section of my dissertation (Chapters IV, V, VI and VII) is an investigation of the potential role of small nuclear RNA sequence variants in the control of splicing. I identified 46 copies of small nuclear RNAs in the 6X whole genome shotgun of the Bombyx mori p50T strain. These variants may play a role in differential binding of specific proteins that mediate alternative splicing. Along these lines, further investigation of U2 snRNA sequence variants in Bombyx mori demonstrated that some U2 snRNAs preferentially assemble into high molecular weight spliceosomal complexes over others. Expression of snRNA variants may represent another mechanism by which the cell is able to fine tune the splicing process.

Development and implementation of a tool to assess the micro/macro elements in an organization

This study examines the congruency of planning between organizational structure and process, through an evaluation and planning model known as the Micro/Macro Dynamic Planning Grid. The model compares day-to-day planning within an organization to planning imposed by organizational administration and accrediting agencies. A survey instrument was developed to assess the micro and macro sociological analysis elements utilized by an organization.^ The Micro/Macro Dynamic Planning Grid consists of four quadrants. Each quadrant contains characteristics that reflect the interaction between the micro and macro elements of planning, objectives and goals within an organization. The Over Macro/Over Micro, Quadrant 1, contains attributes that reflect a tremendous amount of action and ongoing adjustments, typical of an organization undergoing significant changes in either leadership, program and/or structure. Over Macro/Under Micro, Quadrant 2, reflects planning characteristics found in large, bureaucratic systems with little regard given to the workings of their component parts. Under Macro/Under Micro, Quadrant 3, reflects the uncooperative, uncoordinated organization, one that contains a multiplicity of viewpoints, language, objectives and goals. Under Macro/Under Micro, Quadrant 4 represents the worst case scenario for any organization. The attributes of this quadrant are very reactive, chaotic, non-productive and redundant.^ There were three phases to the study: development of the initial instrument, pilot testing the initial instrument and item revision, and administration and assessment of the refined instrument. The survey instrument was found to be valid and reliable for the purposes and audiences herein described.^ In order to expand the applicability of the instrument to other organizational settings, the survey was administered to three professional colleges within a university.^ The first three specific research questions collectively answered, in the affirmative, the basic research question: Can the Micro/Macro Dynamic Planning Grid be applied to an organization through an organizational development tool? The first specific question: Can an instrument be constructed that applies the Micro/Macro Dynamic Planning Grid? The second specific research question: Is the constructed instrument valid and reliable? The third specific research question: Does an instrument that applies the Micro/Macro Dynamic Planning Grid assess congruency of micro and macro planning, goals and objectives within an organization? The fourth specific research question: What are the differences in the responses based on roles and responsibilities within an organization? involved statistical analysis of the response data and comparisons obtained with the demographic data. (Abstract shortened by UMI.) ^

Latin America and the Caribbean in 2011 and Beyond: A conversation among Latin America and Caribbean Specialists.

For the first time in more than fifty years, the domestic and external conflicts in Latin America and the Caribbean (LAC) are not primarily ideological in nature. Democracy continues to thrive and its promise still inspires hope. In contrast, the illegal production, consumption, and trading of drugs – and its links to criminal gangs and organizations – represent major challenges to the region, undermining several States’ already weak capacity to govern. While LAC macroeconomic stability has remained resilient, illegal economies fill the region, often offering what some States have not historically been able to provide – elements of human security, opportunities for social mobility, and basic survival. Areas controlled by drug trafficking organizations (DTOs) are now found in Central America, Mexico, and the favelas of Rio de Janeiro and São Paulo, reflecting their competition for land routes and production areas. Cartels such as La Familia, Los Zetas, and Primeiro Comando da Capital (PCC-Brazil), among others, operate like trade and financial enterprises that manage millions of dollars and resources, demonstrating significant business skills in adapting to changing circumstances. They are also merciless in their application of violence to preserve their lucrative enterprises. The El Salvador-Guatemala-Honduras triangle in Central America is now the most violent region in the world, surpassing regions in Africa that have been torn by civil strife for years. In Brazil’s favelas and Guatemala’s Petén region, the military is leaving the barracks again; not to rule, however, but to supplement and even replace the law enforcement capacity of weak and discredited police forces. This will challenge the military to apply lessons learned during the course of their experience in government, or from the civil wars that plagued the region for nearly 50 years during the Cold War. Will they be able to conduct themselves according to the professional ethics that have been inculcated over the past 20 years without incurring violations of human rights? Belief in their potential to do good is high according to many polls as the Armed Forces still enjoy a favorable perception in most societies, despite frequent involvement in corruption. Calling them to fight DTOs, however, may bring them too close to the illegal activities they are being asked to resist, or even rekindle the view that only a “strong hand” can resolve national troubles. The challenge of governance is occurring as contrasts within the region are becoming sharper. There is an increasing gap between nations positioned to surpass their “developing nation” status and those that are practically imploding as the judicial, political and enforcement institutions fall further into the quagmire of illicit activities. Several South American nations are advancing their political and economic development. Brazil in particular has realized macro-economic stability, made impressive gains in poverty reduction, and is on track to potentially become a significant oil producer. It is also an increasingly influential power, much closer to the heralded “emerging power” category that it aspired to for most of the 20th century. In contrast, several Central American States have become so structurally deficient, and have garnered such limited legitimacy, that their countries have devolved into patches of State controlled and non-State-controlled territory, becoming increasingly vulnerable to DTO entrenchment. In the Caribbean, the drug and human trafficking business also thrives. Small and larger countries are experiencing the growing impact of illicit economies and accompanying crime and violence. Among these, Guyana and Suriname face greater uncertainty, as they juggle both their internal affairs and their relations with Brazil and Venezuela. Cuba also faces new challenges as it continues focusing on internal rather than external affairs and attempts to ensure a stable leadership succession while simultaneously trying to reform its economy. Loosening the regime’s tight grip on the economy while continuing to curtail citizen’s civil rights will test the leadership’s ability to manage change and prevent a potential socio-economic crisis from turning into an existential threat. Cuba’s past ideological zest is now in the hands of Venezuela’s President Hugo Chavez, who continues his attempts to bring the region together under Venezuelan leadership ideologically based on a “Bolivarian” anti-U.S. banner, without much success. The environment and natural disasters will merit more attention in the coming years. Natural events will produce increasing scales of destruction as the States in the region fail to maintain and expand existing infrastructure to withstand such calamities and respond to their effects. Prospects for earthquakes, tsunamis, and hurricanes are high, particularly in the Caribbean. In addition, there are growing rates of deforestation in nearly every country, along with a potential increase in cross-sector competition for resources. The losers might be small farmers, due to their inability to produce quantities commensurate to larger conglomerates. Regulations that could mitigate these types of situations are lacking or openly violated with near impunity. Indigenous and other vulnerable populations, including African descendants, in several Andean countries, are particularly affected by the increasing extraction of natural resources taking place amongst their terrain. This has led to protests against extraction activities that negatively affect their livelihoods, and in the process, these historically underprivileged groups have transitioned from agenda-based organization to one that is bringing its claims and grievances to the national political agenda, becoming more politically engaged. Symptomatic of these social issues is the region’s chronically poor quality of education that has consistently failed to reduce inequality and prepare new generations for jobs in the competitive global economy, particularly the more vulnerable populations. Simultaneously, the educational deficit is also exacerbated by the erosion of access to information and freedom of the press. The international panorama is also in flux. New security entities are challenging the old establishment. The Union of South American Nations, The South American Defense Council, the socialist Bolivarian Alliance, and other entities seem to be defying the Organization of American States and its own defense mechanisms, and excluding the U.S. And the U.S.’s attention to areas in conflict, namely Iraq, Afghanistan, and Pakistan – rather than to the more stable Latin America and Caribbean – has left ample room for other actors to elbow in. China is now the top trading partner for Brazil. Russian and Iran are also finding new partnerships in the region, yet their links appear more politically inclined than those of China. Finally, the aforementioned increasing commercial ties by LAC States with China have accelerated a return to the preponderance of commodities as sources of income for their economies. The increased extraction of raw material for export will produce greater concern over the environmental impact that is created by the exploitation of natural resources. These expanded trade opportunities may prove counterproductive economically for countries in the region, particularly for Brazil and Chile, two countries whose economic policies have long sought diversification from dependence on commodities to the development of service and technology based industries.