951 resultados para Specific inhalation challenge test
Resumo:
Expanded understanding of the trends and determinants of food consumption is needed to reduce the ecological impacts of the contemporary agro-food system while also being attentive to broader issues pertaining to health and the environment. Incorporating these additional aspects and formulating meaningful dietary recommendations is a major challenge. This article seeks to highlight differences in ecological footprint (EF) by activity level for various so-cial groups to meet suggested physiological requirements by nutritionists versus actual food consumption. The study is based on a combination of healthy diet requirements (as expressed by national guidelines) and a survey of a repre-sentative sample of 1,013 Hungarian adults using a bottom-up approach for calculating EFs. Students and women with small children have a higher than average food-related EF due to their higher nutritional needs. At the same time, the elderly are characterized by lower footprints. Perhaps most interesting is our finding that people with seden-tary forms of employment have higher food footprints than those with jobs that require physical labor. We offer rec-ommendations for food-policy planning based on encouraging dietary changes for individuals, differentiated by the nature of their work. The research suggests that dietary policy that improves health often has environmental benefits.
Resumo:
A major consequence of contamination at the local level’s population as it relates to environmental health and environmental engineering is childhood lead poisoning. Environmental contamination is one of the pressing environmental concerns facing the world today. Current approaches often focus on large contaminated industrial size sites that are designated by regulatory agencies for site remediation. Prior to this study, there were no known published studies conducted at the local and smaller scale, such as neighborhoods, where often much of the contamination is present to remediate. An environmental health study of local lead-poisoning data in Liberty City, Little Haiti and eastern Little Havana in Miami-Dade County, Florida accounted for a disproportionately high number of the county’s reported childhood lead poisoning cases. An engineering system was developed and designed for a comprehensive risk management methodology that is distinctively applicable to the geographical and environmental conditions of Miami-Dade County, Florida. Furthermore, a scientific approach for interpreting environmental health concerns, while involving detailed environmental engineering control measures and methods for site remediation in contained media was developed for implementation. Test samples were obtained from residents and sites in those specific communities in Miami-Dade County, Florida (Gasana and Chamorro 2002). Currently lead does not have an Oral Assessment, Inhalation Assessment, and Oral Slope Factor; variables that are required to run a quantitative risk assessment. However, various institutional controls from federal agencies’ standards and regulation for contaminated lead in media yield adequate maximum concentration limits (MCLs). For this study an MCL of .0015 (mg/L) was used. A risk management approach concerning contaminated media involving lead demonstrates that the linkage of environmental health and environmental engineering can yield a feasible solution.
Resumo:
The increasing amount of available semistructured data demands efficient mechanisms to store, process, and search an enormous corpus of data to encourage its global adoption. Current techniques to store semistructured documents either map them to relational databases, or use a combination of flat files and indexes. These two approaches result in a mismatch between the tree-structure of semistructured data and the access characteristics of the underlying storage devices. Furthermore, the inefficiency of XML parsing methods has slowed down the large-scale adoption of XML into actual system implementations. The recent development of lazy parsing techniques is a major step towards improving this situation, but lazy parsers still have significant drawbacks that undermine the massive adoption of XML. ^ Once the processing (storage and parsing) issues for semistructured data have been addressed, another key challenge to leverage semistructured data is to perform effective information discovery on such data. Previous works have addressed this problem in a generic (i.e. domain independent) way, but this process can be improved if knowledge about the specific domain is taken into consideration. ^ This dissertation had two general goals: The first goal was to devise novel techniques to efficiently store and process semistructured documents. This goal had two specific aims: We proposed a method for storing semistructured documents that maps the physical characteristics of the documents to the geometrical layout of hard drives. We developed a Double-Lazy Parser for semistructured documents which introduces lazy behavior in both the pre-parsing and progressive parsing phases of the standard Document Object Model’s parsing mechanism. ^ The second goal was to construct a user-friendly and efficient engine for performing Information Discovery over domain-specific semistructured documents. This goal also had two aims: We presented a framework that exploits the domain-specific knowledge to improve the quality of the information discovery process by incorporating domain ontologies. We also proposed meaningful evaluation metrics to compare the results of search systems over semistructured documents. ^
Resumo:
This study investigated the effects of word prediction and text-to-speech on the narrative composition writing skills of 6, fifth-grade Hispanic boys with specific learning disabilities (SLD). A multiple baseline design across subjects was used to explore the efficacy of word prediction and text-to-speech alone and in combination on four dependent variables: writing fluency (words per minute), syntax (T-units), spelling accuracy, and overall organization (holistic scoring rubric). Data were collected and analyzed during baseline, assistive technology interventions, and at 2-, 4-, and 6-week maintenance probes. ^ Participants were equally divided into Cohorts A and B, and two separate but related studies were conducted. Throughout all phases of the study, participants wrote narrative compositions for 15-minute sessions. During baseline, participants used word processing only. During the assistive technology intervention condition, Cohort A participants used word prediction followed by word prediction with text-to-speech. Concurrently, Cohort B participants used text-to-speech followed by text-to-speech with word prediction. ^ The results of this study indicate that word prediction alone or in combination with text-to-speech has a positive effect on the narrative writing compositions of students with SLD. Overall, participants in Cohorts A and B wrote more words, more T-units, and spelled more words correctly. A sign test indicated that these perceived effects were not likely due to chance. Additionally, the quality of writing improved as measured by holistic rubric scores. When participants in Cohort B used text-to-speech alone, with the exception of spelling accuracy, inconsequential results were observed on all dependent variables. ^ This study demonstrated that word prediction alone or in combination assists students with SLD to write longer, improved-quality, narrative compositions. These results suggest that word prediction or word prediction with text-to-speech be considered as a writing support to facilitate the production of a first draft of a narrative composition. However, caution should be given to the use of text-to-speech alone as its effectiveness has not been established. Recommendations for future research include investigating the use of these technologies in other phases of the writing process, with other student populations, and with other writing styles. Further, these technologies should be investigated while integrated into classroom composition instruction. ^
Resumo:
Public schools traditionally have been held accountable for educating the majority of the nation’s school children, and through the years, these schools have been evaluated in a variety of ways. Currently, evaluation measures for accountability purposes consist solely of standardized test scores. In the past, only test scores of general education students were analyzed. Laws governing the education of students with disabilities, however, have extended accountability measures not only to include those students, but to report their scores in a disaggregated form (No Child Left Behind Act, 2001). The recent emphasis on accountability and compliance has resulted in the need for schools to carefully examine how programs, services, and policies impact student achievement (Bowers & Figgers, 2003). ^ Standard-based school reform and accountability systems have raised expectations about student learning outcomes for all students, including those with disabilities and minority students. Yet, overall, racial/ethnic minority students are performing well below their White non-Hispanic peers in most academic areas. Additionally, with respect to special education, there exists an enduring problem of disproportionate representation of racial/ethnic minority students (National Research Council, 2000). ^ This study examined classroom placement (inclusive versus non-inclusive) relative to academic performance of urban, low socioeconomic Hispanic students with and without disabilities in secondary content area classrooms. A mixed method research design was used to investigate this important issue using data from a local school district and results from field observations. The study compared performance levels of four middle school Hispanic student subgroups (students with disabilities in inclusive settings, students without disabilities in inclusive settings, students with disabilities in resource settings, and student without disabilities in general education settings) each in their respective placements for two consecutive years, exploring existing practices within authentic settings. ^ Significant differences were found in the relationship of educational placement and achievement between grade level and disability in the areas of math and reading. Additionally, clear and important differences were observed in student-teacher interactions. Recommendations for further researchers and stakeholders include soliciting responses from teams at the schools composed of general education and special education teachers, administrative personnel, and students as well as broadening the study across grade levels and exceptionalities. ^
Resumo:
Black students, in general, are underserved academically (Darling-Hammond, 2000; Townsend, 2002) and overrepresented in special education (Donovan & Cross, 2002). Black students with disabilities are further overrepresented in more restrictive educational environments (Skiba, Poloni-Staudinger, Gallini, Simmons & Feggins-Azziz, 2006). Although the National Longitudinal Transition Study 2 (NLTS2) revealed that the academic performance of students with learning disabilities is positively related to the percentage of courses taken in the general education setting (Newman, 2006), the research specifically on placement of Black students with disabilities, particularly at the secondary level, as it relates to academic achievement is lacking. While previous studies have sought to determine which placement is better for students with disabilities, no study was found that specifically examined the impact of placement specific to Black students with specific learning disabilities (SLD) in urban settings (Fore, III, Hagan-Burke, Burke, Boon & Smith, 2008; Rea, McLaughlin & Walther-Thomas, 2002). This study examined educational placement, instructional best practices, and achievement gains of Black students with SLD in urban secondary settings using an ex post facto research design. Achievement, placement, and demographic data were collected and analyzed on approximately 314 Black eighth grade students with SLD. The Teacher Instructional Practices Survey was developed and used to collect and analyze data from the teachers of 78 of these students as it relates to instructional best practices. Results indicate no significant difference in reading but a significant difference in math gains of students served in inclusive settings as compared to resource settings with a small effect size. Also, no significant relationship was found between achievement gains and the reported use of instructional best practices. However, there was a relationship between educational placement and the use of instructional best practices. The results implied that there is a need for training with both general and special education teachers on instructional best practices for SWD and that there should be certain IEP team considerations when making placement decisions for this population of students with disabilities. It is recommended that future research in this area include classroom observations and factors other than test scores to measure growth in achievement.
Resumo:
The purpose of the study was to determine the degree of relationships among GRE scores, undergraduate GPA (UGPA), and success in graduate school, as measured by first year graduate GPA (FGPA), cumulative graduate GPA, and degree attainment status. A second aim of the study was to determine whether the relationships between the composite predictor (GRE scores and UGPA) and the three success measures differed by race/ethnicity and sex. A total of 7,367 graduate student records (masters, 5,990; doctoral: 1,377) from 2000 to 2010 were used to evaluate the relationships among GRE scores, UGPA and the three success measures. Pearson's correlation, multiple linear and logistic regression, and hierarchical multiple linear and logistic regression analyses were performed to answer the research questions. The results of the correlational analyses differed by degree level. For master's students, the ETS proposed prediction that GRE scores are valid predictors of first year graduate GPA was supported by the findings from the present study; however, for doctoral students, the proposed prediction was only partially supported. Regression and correlational analyses indicated that UGPA was the variable that consistently predicted all three success measures for both degree levels. The hierarchical multiple linear and logistic regression analyses indicated that at master's degree level, White students with higher GRE Quantitative Reasoning Test scores were more likely to attain a degree than Asian Americans, while International students with higher UGPA were more likely to attain a degree than White students. The relationships between the three predictors and the three success measures were not significantly different between men and women for either degree level. Findings have implications both for practice and research. They will provide graduate school administrators with institution-specific validity data for UGPA and the GRE scores, which can be referenced in making admission decisions, while they will provide empirical and professionally defensible evidence to support the current practice of using UGPA and GRE scores for admission considerations. In addition, new evidence relating to differential predictions will be useful as a resource reference for future GRE validation researchers.
Resumo:
Persistence of HIV-1 reservoirs within the Central Nervous System (CNS) remains a significant challenge to the efficacy of potent anti-HIV-1 drugs. The primary human Brain Microvascular Endothelial Cells (HBMVEC) constitutes the Blood Brain Barrier (BBB) which interferes with anti-HIV drug delivery into the CNS. The ATP binding cassette (ABC) transporters expressed on HBMVEC can efflux HIV-1 protease inhibitors (HPI), enabling the persistence of HIV-1 in CNS. Constitutive low level expression of several ABC-transporters, such as MDR1 (a.k.a. P-gp) and MRPs are documented in HBMVEC. Although it is recognized that inflammatory cytokines and exposure to xenobiotic drug substrates (e.g HPI) can augment the expression of these transporters, it is not known whether concomitant exposure to virus and anti-retroviral drugs can increase drug-efflux functions in HBMVEC. Our in vitro studies showed that exposure of HBMVEC to HIV-1 significantly up-regulates both MDR1 gene expression and protein levels; however, no significant increases in either MRP-1 or MRP-2 were observed. Furthermore, calcein-AM dye-efflux assays using HBMVEC showed that, compared to virus exposure alone, the MDR1 mediated drug-efflux function was significantly induced following concomitant exposure to both HIV-1 and saquinavir (SQV). This increase in MDR1 mediated drug-efflux was further substantiated via increased intracellular retention of radiolabeled [3H-] SQV. The crucial role of MDR1 in 3H-SQV efflux from HBMVEC was further confirmed by using both a MDR1 specific blocker (PSC-833) and MDR1 specific siRNAs. Therefore, MDR1 specific drug-efflux function increases in HBMVEC following co-exposure to HIV-1 and SQV which can reduce the penetration of HPIs into the infected brain reservoirs of HIV-1. A targeted suppression of MDR1 in the BBB may thus provide a novel strategy to suppress residual viral replication in the CNS, by augmenting the therapeutic efficacy of HAART drugs.
Resumo:
ackground Following incomplete spinal cord injury (iSCI), descending drive is impaired, possibly leading to a decrease in the complexity of gait. To test the hypothesis that iSCI impairs gait coordination and decreases locomotor complexity, we collected 3D joint angle kinematics and muscle parameters of rats with a sham or an incomplete spinal cord injury. Methods 12 adult, female, Long-Evans rats, 6 sham and 6 mild-moderate T8 iSCI, were tested 4 weeks following injury. The Basso Beattie Bresnahan locomotor score was used to verify injury severity. Animals had reflective markers placed on the bony prominences of their limb joints and were filmed in 3D while walking on a treadmill. Joint angles and segment motion were analyzed quantitatively, and complexity of joint angle trajectory and overall gait were calculated using permutation entropy and principal component analysis, respectively. Following treadmill testing, the animals were euthanized and hindlimb muscles removed. Excised muscles were tested for mass, density, fiber length, pennation angle, and relaxed sarcomere length. Results Muscle parameters were similar between groups with no evidence of muscle atrophy. The animals showed overextension of the ankle, which was compensated for by a decreased range of motion at the knee. Left-right coordination was altered, leading to left and right knee movements that are entirely out of phase, with one joint moving while the other is stationary. Movement patterns remained symmetric. Permutation entropy measures indicated changes in complexity on a joint specific basis, with the largest changes at the ankle. No significant difference was seen using principal component analysis. Rats were able to achieve stable weight bearing locomotion at reasonable speeds on the treadmill despite these deficiencies. Conclusions Decrease in supraspinal control following iSCI causes a loss of complexity of ankle kinematics. This loss can be entirely due to loss of supraspinal control in the absence of muscle atrophy and may be quantified using permutation entropy. Joint-specific differences in kinematic complexity may be attributed to different sources of motor control. This work indicates the importance of the ankle for rehabilitation interventions following spinal cord injury.
Resumo:
In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050°C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1μg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.
Resumo:
There is a national need to increase the STEM-related workforce. Among factors leading towards STEM careers include the number of advanced high school mathematics and science courses students complete. Florida's enrollment patterns in STEM-related Advanced Placement (AP) courses, however, reveal that only a small percentage of students enroll into these classes. Therefore, screening tools are needed to find more students for these courses, who are academically ready, yet have not been identified. The purpose of this study was to investigate the extent to which scores from a national standardized test, Preliminary Scholastic Assessment Test/ National Merit Qualifying Test (PSAT/NMSQT), in conjunction with and compared to a state-mandated standardized test, Florida Comprehensive Assessment Test (FCAT), are related to selected AP exam performance in Seminole County Public Schools. An ex post facto correlational study was conducted using 6,189 student records from the 2010 - 2012 academic years. Multiple regression analyses using simultaneous Full Model testing showed differential moderate to strong relationships between scores in eight of the nine AP courses (i.e., Biology, Environmental Science, Chemistry, Physics B, Physics C Electrical, Physics C Mechanical, Statistics, Calculus AB and BC) examined. For example, the significant unique contribution to overall variance in AP scores was a linear combination of PSAT Math (M), Critical Reading (CR) and FCAT Reading (R) for Biology and Environmental Science. Moderate relationships for Chemistry included a linear combination of PSAT M, W (Writing) and FCAT M; a combination of FCAT M and PSAT M was most significantly associated with Calculus AB performance. These findings have implications for both research and practice. FCAT scores, in conjunction with PSAT scores, can potentially be used for specific STEM-related AP courses, as part of a systematic approach towards AP course identification and placement. For courses with moderate to strong relationships, validation studies and development of expectancy tables, which estimate the probability of successful performance on these AP exams, are recommended. Also, findings established a need to examine other related research issues including, but not limited to, extensive longitudinal studies and analyses of other available or prospective standardized test scores.
Resumo:
The increasing amount of available semistructured data demands efficient mechanisms to store, process, and search an enormous corpus of data to encourage its global adoption. Current techniques to store semistructured documents either map them to relational databases, or use a combination of flat files and indexes. These two approaches result in a mismatch between the tree-structure of semistructured data and the access characteristics of the underlying storage devices. Furthermore, the inefficiency of XML parsing methods has slowed down the large-scale adoption of XML into actual system implementations. The recent development of lazy parsing techniques is a major step towards improving this situation, but lazy parsers still have significant drawbacks that undermine the massive adoption of XML. Once the processing (storage and parsing) issues for semistructured data have been addressed, another key challenge to leverage semistructured data is to perform effective information discovery on such data. Previous works have addressed this problem in a generic (i.e. domain independent) way, but this process can be improved if knowledge about the specific domain is taken into consideration. This dissertation had two general goals: The first goal was to devise novel techniques to efficiently store and process semistructured documents. This goal had two specific aims: We proposed a method for storing semistructured documents that maps the physical characteristics of the documents to the geometrical layout of hard drives. We developed a Double-Lazy Parser for semistructured documents which introduces lazy behavior in both the pre-parsing and progressive parsing phases of the standard Document Object Model's parsing mechanism. The second goal was to construct a user-friendly and efficient engine for performing Information Discovery over domain-specific semistructured documents. This goal also had two aims: We presented a framework that exploits the domain-specific knowledge to improve the quality of the information discovery process by incorporating domain ontologies. We also proposed meaningful evaluation metrics to compare the results of search systems over semistructured documents.
Resumo:
Brazil is a country in development, rich in natural resources. In order to grow sustainably, it is necessary to Brazil to preserve its environment, which is an expressive challenge, especially to industries, such as those producing ceramic materials. This study was developed using Porcelain Tile Polishing Residue (RPP) in blends with soil to build compacted fills. This residue is a slurry generated during the polishing process of porcelain tiles and contains powdery material from the polished tile, the abrasives used during the process and cooling water. The RPP was collected from a private company located in Conde/PB and it was mixed with a sandy-clayey soil, to build the fills. Laboratorial tests were conducted with pure soil, pure RPP and blends in proportions of 5%, 10%, 15% and 20% of RPP in addition to the dry mass of pure soil. The Chemical and Physical Characterization tests performed were: specific solid weight, grain size distribution, laser analysis of grain size distribution, Atterberg limits, X ray fluorescence, X ray diffraction, scanning electron microscopy and soil compaction,. The materials and blends were also compacted and direct shear tests and plate load tests were performed. Plate load tests were conducted using a circular plate with 30 cm diameter, on specimens of pure soil and 5% blend, compacted in a metallic box inside the Soil Mechanics Laboratory of the Federal University of Rio Grande do Norte, Brazil. Both mechanical tests performed were conducted under inundated conditions, willing to reduce the influence of soil suction. An evaluation of the results of the tests performed shows that RPP is a fine material, with grain size distribution smaller than 0,015mm, composed mainly of silica and alumina, and particles in angular shape. The soil was characterized as a clayey sand, geologically known as a lateritic soil, with high percentages of alumina and iron oxide, and particles with rounded shape. Both the Soil and the blends presented low plasticity, while the residue showed a medium plasticity. Direct shear tests showed that the addition of RPP did not cause major changes into blends’ friction angle data, however, it was possible to note that, for the proportions studied, that is a tendency of obtain lower shear stresses for higher percentages of RPP in the blends. Both pure soil and 5% mixture showed a punching disruption for the Plate load test. For this same test, the allowable stress for 5% mixture was 44% higher than the pure soil, and smaller vertical settlement results for all stresses.
Resumo:
Brazil is a country in development, rich in natural resources. In order to grow sustainably, it is necessary to Brazil to preserve its environment, which is an expressive challenge, especially to industries, such as those producing ceramic materials. This study was developed using Porcelain Tile Polishing Residue (RPP) in blends with soil to build compacted fills. This residue is a slurry generated during the polishing process of porcelain tiles and contains powdery material from the polished tile, the abrasives used during the process and cooling water. The RPP was collected from a private company located in Conde/PB and it was mixed with a sandy-clayey soil, to build the fills. Laboratorial tests were conducted with pure soil, pure RPP and blends in proportions of 5%, 10%, 15% and 20% of RPP in addition to the dry mass of pure soil. The Chemical and Physical Characterization tests performed were: specific solid weight, grain size distribution, laser analysis of grain size distribution, Atterberg limits, X ray fluorescence, X ray diffraction, scanning electron microscopy and soil compaction,. The materials and blends were also compacted and direct shear tests and plate load tests were performed. Plate load tests were conducted using a circular plate with 30 cm diameter, on specimens of pure soil and 5% blend, compacted in a metallic box inside the Soil Mechanics Laboratory of the Federal University of Rio Grande do Norte, Brazil. Both mechanical tests performed were conducted under inundated conditions, willing to reduce the influence of soil suction. An evaluation of the results of the tests performed shows that RPP is a fine material, with grain size distribution smaller than 0,015mm, composed mainly of silica and alumina, and particles in angular shape. The soil was characterized as a clayey sand, geologically known as a lateritic soil, with high percentages of alumina and iron oxide, and particles with rounded shape. Both the Soil and the blends presented low plasticity, while the residue showed a medium plasticity. Direct shear tests showed that the addition of RPP did not cause major changes into blends’ friction angle data, however, it was possible to note that, for the proportions studied, that is a tendency of obtain lower shear stresses for higher percentages of RPP in the blends. Both pure soil and 5% mixture showed a punching disruption for the Plate load test. For this same test, the allowable stress for 5% mixture was 44% higher than the pure soil, and smaller vertical settlement results for all stresses.
Resumo:
Cryptococcus neoformans is an opportunistic fungal pathogen that causes significant disease worldwide. Even though this fungus has not evolved specifically to cause human disease, it has a remarkable ability to adapt to many different environments within its infected host. C. neoformans adapts by utilizing conserved eukaryotic and fungal-specific signaling pathways to sense and respond to stresses within the host. Upon infection, two of the most significant environmental changes this organism experiences are elevated temperature and high pH.
Conserved Rho and Ras family GTPases are central regulators of thermotolerance in C. neoformans. Many GTPases require prenylation to associate with cellular membranes and function properly. Using molecular genetic techniques, microscopy, and infection models, I demonstrated that the prenyltransferase, geranylgeranyl transferase I (GGTase I) is required for thermotolerance and pathogenesis. Using fluorescence microscopy, I found that only a subset of conserved GGTase I substrates requires this enzyme for membrane localization. Therefore, the C. neoformans GGTase I may recognize its substrate in a slightly different manner than other eukaryotic organisms.
The alkaline response transcription factor, Rim101, is a central regulator of stress-response genes important for adapting to the host environment. In particular, Rim101 regulates cell surface alterations involved in immune avoidance. In other fungi, Rim101 is activated by alkaline pH through a conserved signaling pathway, but this pathway had yet been characterized in C. neoformans. Using molecular genetic techniques, I identified and analyzed the conserved members of the Rim pathway. I found that it was only partially conserved in C. neoformans, missing the components that sense pH and initiate pathway activation. Using a genetic screen, I identified a novel Rim pathway component named Rra1. Structural prediction and genetic epistasis experiments suggest that Rra1 may serve as the Rim pathway pH sensor in C. neoformans and other related basidiomycete fungi.
To explore the relevance of Rim pathway signaling in the interaction of C neoformans with its host, I characterized the Rim101-regulated cell wall changes that prevent immune detection. Using HPLC, enzymatic degradation, and cell wall stains, I found that the rim101Δ mutation resulted in increased cell wall chitin exposure. In vitro co-culture assays demonstrated that increased chitin exposure is associated with enhanced activation of macrophages and dendritic cells. To further test this association, I demonstrated that other mutant strains with increased chitin exposure induce macrophage and dendritic cell responses similar to rim101Δ. We used primary macrophages from mutant mouse lines to demonstrate that members of both the Toll-like receptor and C-type lectin receptor families are involved in detecting strains with increased chitin exposure. Finally, in vivo immunological experiments demonstrated that the rim101Δ strain induced a global inflammatory immune response in infected mouse lungs, expanding upon our previous in vivo rim101Δ studies. These results demonstrate that cell wall organization largely determines how fungal cells are detected by the immune system.