Improving Molecular Diagnosis Of Suspected Mendelian Disorders With Rna Splicing Analysis

Exome sequencing is the most advanced standard-of-care genetic test for people with suspected Mendelian disorders. Yet, the diagnostic rate of exome sequencing is only 31%. RNA sequencing (RNA-seq) is a promising molecular test for detecting potentially pathogenic changes in RNA splicing as part of obtaining a molecular diagnosis. In this dissertation, I develop new computational tools and perform analyses towards improving how we detect these potentially pathogenic changes in RNA splicing with the goal of improving the molecular diagnostic rate. First, in Chapter 1, I review background on how we diagnose patients and how RNA splicing and RNA-seq could be used to improve this process. Then, in Chapter 2, I describe my contributions to MAJIQ v2 as methodology to study RNA splicing from large and heterogeneous RNA-seq datasets. Afterwards, I use MAJIQ v2 in Chapter 3 to evaluate how tissue-specific expression and splicing affects what clinically-relevant splicing changes we can identify from clinically-accessible tissues. Then, in Chapter 4, I describe the limitations of MAJIQ v2 for our approach to detect splicing aberrations and the development and evaluation of MAJIQ v3 to address these challenges. With MAJIQ v3, I develop MAJIQ-CLIN in Chapter 5 to identify and prioritize splicing aberrations in patient RNA-seq data and compare our method to previous approaches. Finally, in Chapter 6, I discuss overall conclusions for the work and exciting areas for future work. Together, the work in this dissertation pushes forward how we can study and use RNA-seq to improve the diagnostic rate of patients with suspected Mendelian disorders

How sexism leads to intentions to leave an organization among coaches of women's teams in Division I intercollegiate athletics

Since the passage of Title IX, there has been a steady decline of women head coaches in intercollegiate athletics. Previous research indicated that perceived treatment and access discrimination may be a plausible cause of the decline; however, research has not identified the antecedents of discrimination. Research indicates that sexism levels are associated with hiring intentions, ascription of attributes to managers, and performance appraisals. This study attempted to identify sexism as one possible antecedent of discrimination. A dyadic study between head and assistant collegiate coaches was utilized to determine the relationship between hostile and benevolent sexism, treatment and access discrimination, and intentions to leave the profession. I sampled 364 head coaches and 163 assistant coaches, creating 71 dyads. Each of the measures was validated in previous research. Results indicated that men possessed higher levels of hostile (M = 2.02, SD = 1.02) and benevolent (M = 2.33, SD = 1.01) sexists beliefs than females (M = 2.00, SD = .97, M = 1.62, SD = 1.00). Additionally, females coaches indicated significantly higher levels of perceived access discrimination (F [1, 384] = 38.05, p < .01), treatment discrimination (F [1,384] = 7.353, p < .01) and intentions to leave (F [1, 384] = 13.146, p < .01) than men. Results indicate that there is a correlation between benevolent sexism and access discrimination (r = .322, p < .001) within the coach dyads. Further, to support previous literature, this study found that 17% of the variance in intentions to leave the profession was explained by perceived treatment and access discrimination. Though the results of this study show only one relationship between sexism and perceived discrimination, the results that sexism is present in intercollegiate athletics and that females perceive higher levels of discrimination and intentions to leave the profession are an interesting finding. Sexism may have an effect on perceived discrimination; however, the relationship may be mediated through other variables such as group identity or organizational citizenship behavior. Ultimately, this study has indicated that sexist beliefs are present in intercollegiate athletics and has negative implications that should be further researched

The Impact a Diversity Culture Has on the "Think Manager, Think Male" Stereotype: A Social Identity Theory of Leadership Perspective on Gender Stereotypes in Sport Organizations

Women in intercollegiate athletics have faced numerous challenges in breaking through the "glass ceiling." This issue has received a plethora of attention in the literature; however, the impact of culture on leadership stereotypes has yet to be evaluated. Therefore, the purpose of this study was to determine the impact a diversity culture may or may not have on gendered leadership stereotypes. Utilizing the social identity theory of leadership and the expectations of gender stereotypes, I predicted men would be considered more prototypical of a sport organization than would women. Moving forward, I argued culture would moderate this relationship. Specifically, women would be considered more prototypical in a proactive culture (diversity viewed as an asset), whereas men would be perceived as more prototypical in compliant cultures (diversity viewed as a liability). Finally, when a leader was determined as prototypical, then (s)he would be rated as more effective than nonprototypical leaders. A 2 (culture: compliant, proactive) by 2 (leader's sex: male, female) design was employed to determine the relationship between culture, sex and leadership prototypicality. Respondents to this research experiment included students participating in activity classes at a major Southwest University (N = 278). Respondents were first asked to read through two scenarios: one describing culture and the other manipulating the leader. Next, they were asked to complete a series of items to measure prototypicality and leadership effectiveness. Results indicated the manipulation in the scenarios was successful. A majority of the respondents correctly identified the leader?s sex (N = 241), and a proactive culture was viewed as supporting diversity when compared to a compliant culture (F [1, 274] = 120.83, p .05, n2 =.001), and culture did not affect prototypicality ratings (b = -.04, p greater than .05). However, culture did have a significant positive relationship with leadership effectiveness (b = 21, p less than .01). Prototypicality was significantly positively related to leadership effectiveness (b = .54, p less than .001), thus supporting the third hypothesis

Accumulation of total nutrients, dry matter and phytochrome content in Boesenbergia stenophylla R.M Smith as affected by different light conditions

A study was conducted to determine the best agronomic practices for the cultivation of jerangau merah as a new medicinal crop. Jerangau merah is scientifically known as Boesenbergia stenophylla is a ginger plants that is highly endemic to the Borneo Highland. This understorey herb grows in cluster and under the heavy shades of forest canopies and perceived only the sunflecks that pass through the forest canopies. Jerangau Merah has been consumed by the locals for its medicinal values since decades ago. Generally, B. stenophylla is well known for its function as antidote for alcohol. Moreover, the B. stenophylla is also beneficial for rheumatic pains, remedies for stomach ache and toothache and as antiseptic wash as well. This herb is not propagate by the locals due to its sensitivity to sunlight and lack of agronomic information for B. stenophylla. Harvesting it from their natural habitat, however, seen the decline it its population size hence cultivation is essential. This paper aimed to determine the total nutrients, dry matter yield and phytochrome content of Bosenbergia stenophylla R.M. Smith under different light regimes. A study for determining suitable planting media for B. stenophylla was conducted at Universiti Putra Malaysia (UPM). The treatments are: i) (M1) with the ratio of 3:2:1 top soil: sand: organic matter, media ii) (M2) consists of soil mixture 3:2:1 placed in water-logged polyethylene bags and media iii) (M3) contained only leaf litters. The results showed that the herbs showed significantly higher number of shoots and leaves in the common soil mixture. But, better root development in media containing leaf litters. The study proceeded with field experiment at Ba’Kelalan to determine the effect of different shade levels and fertilizing regimes on growth. B. stenophylla was cultivated under two different levels of shade cloths: 70% and 90% level of shade and different fertilizing regimes (T1 as control, no fertilizer applied to the plants; T2, chicken dung; T3, NPKMg and T4, mixed of chicken dung and NPKMg). The study for determining the effects of different shade level and fertilizing regimes on seedling’s growth which conducted at Ba’Kelalan in factorial randomized completely blocked design (RCBD). The data collected for 9 weeks of planting which include nutrient content in soil, leaf, PAR and phytochrome content and growth parameters. Regarding the nutrient uptake, the results showed no interactions between fertilizing regimes and shade levels. There were no significant different in nutrient elements except for magnesium and potassium. Magnesium is essential for the formation of chlorophyll which ensure efficiency of photosynthesis when under higher light intensity. Among all the fertilizer treatment, it was showed that chicken dung amendment has higher nutrient uptake. Thus, it is recommended chicken dung should be added into the NPKMg for better nutrient uptake. Further study on suitable fertilizer rates apply to B. stenophylla should be taken into consideration. Moreover, plants cultivated under 70% have been higher and have higher dry matter yield than those plants cultivated under 90%. The result also revealed that there was significant different in chlorophyll content of B. stenophylla cultivated under 70% which treated either with chicken dung and NPKMG respectively. However, plants under 90% have higher chlorophyll content than those under 70%. Based on the results, plants under 70% shade was taller than those under 90% and there were significant difference in height among treatments under 70%.&nbsp; It was observed plant treated with NPKMg was taller. This study showed that shade and fertilizers significantly affected the dry matter yield of B. stenophylla. Moreover, adding NPKMg to the treatments yields more dry matter content of jerangau merah. As for phytochrome content, there was no significant effect of fertilizer on phytochrome content. However, there was significant difference among the shade levels. 90% shade showed higher phytochrome content than those under 70%. In overall, both shade and fertilizer is important in cultivation of Jerangau Merah. 70% shade was observed to have significant effects on growth of jerangau merah and also more economical compared to 90%. Moreover, combination of organic matter and NPKMg also promote the growth of jerangau merah enhance the nutrient uptake efficiency of jerangau merah. However, further investigation of suitable fertilizer and application rate are required to determine suitable fertilizer for jerangau merah and application rate for optimum growth of jerangau merah

ChromoShake: a chromosome dynamics simulator reveals that chromatin loops stiffen centromeric chromatin

A novel chromosome simulator recapitulates the position and dynamics of centromeric chromatin in a model composed of cross-linked intramolecular loops. Simulations reveal that chromatin loops stiffen the centromere and dictate the distribution of pericentric cohesin.ChromoShake is a three-dimensional simulator designed to find the thermodynamically favored states for given chromosome geometries. The simulator has been applied to a geometric model based on experimentally determined positions and fluctuations of DNA and the distribution of cohesin and condensin in the budding yeast centromere. Simulations of chromatin in differing initial configurations reveal novel principles for understanding the structure and function of a eukaryotic centromere. The entropic position of DNA loops mirrors their experimental position, consistent with their radial displacement from the spindle axis. The barrel-like distribution of cohesin complexes surrounding the central spindle in metaphase is a consequence of the size of the DNA loops within the pericentromere to which cohesin is bound. Linkage between DNA loops of different centromeres is requisite to recapitulate experimentally determined correlations in DNA motion. The consequences of radial loops and cohesin and condensin binding are to stiffen the DNA along the spindle axis, imparting an active function to the centromere in mitosis

Conservation strategies for jerangau merah (Boesenbergia stenophylla) using DNA profiling and micropropagation

Jerangau merah (Boesenbergia stenophylla) is highly endemic to the highland of Borneo. Their medicinal value attracts many plant collectors which raise up to the concern on their population size. A study was carried out to establish the conservation approaches for this species. The objectives of this study are to determine the genetic variations among accession from Bario and to develop the in vitro culturing protocol for productions of seedlings. Genetic variation studies were done using simple sequence repeats (SSR) and random amplified polymorphic DNA markers (RAPD). Micropropagation of shoot tips was carried out using BAP and NAA plant growth regulator supplemented in MS media. The genetic variation studies using SSR and RAPD marker show no variations among accession and three sub populations. Two steps protocol was recommended for the tissue culture of B. stenophylla. But it start with culturing using shoot tips in MS media containing 0.2 mg/L NAA for shoot induction followed by sub–culturing to MS media with 2 mg/L BAP +0.4 mg/L NAA for rapid shoot elongation. This study suggests that their conservation should remain as in situ and seedling production under optimum nursery conditions should be carried out near to their natural populations

Molecular and micropropagation approaches for ex-situ conservation of boesenbergia stenophylla R. M. SM.

Jerangau Merah (Boesenbergia stenophylla) is highly endemic to the highland of Borneo. Due to its medicinal properties, it is highly sought which made their population declining. Rapid development and anthropogenic disturbance near the habitat may give negative impact on the Jerangau Merah population in their natural habitat. To date, there are no conservation strategies prepared for this species. Therefore a study was conducted to develop strategies to conserve this species which includes DNA profiling, DNA barcoding, micropropagation and nursery planting. A total of three natural locations of B. stenophylla were found in Bario, Ba’kelalan and Long Semadoh forest. Based on the DNA profiling with RAPD and SSRs markers there was no genetic variations found among sub populations from Bario. This finding implied that no variations may be caused to low hybridization because flowering is rare and propagation through rhizome is more dominant. DNA analysis using CpDNA and NrDNA markers showed that this species clades under the Borneo-Boesenbergia section. This proves further that this species is endemic and could not be found outside of Borneo. A micropropagation protocol was achieved for the in vitro cultures of B. stenophylla. It includes initial culturing using shoot tips in MS media containing 0.2 mg/L NAA for shoot induction followed by sub-culturing to MS media with 2 mg/L BAP + 0.4 mg/L NAA for rapid shoot elongation. B. stenophylla is highly endemic, having small populations, possibly lacks genetic variations and difficult to grow in in vitro cultures. The nursery studies show that the plant has it unique sympodial growth that proved that cannot be confined in the small polybag, so bedded planting was suggested for improving the growth in the nursery conditions. Therefore, this study suggests that their conservation should remain as in situ, seedlings production under optimum nursery conditions should be carried out near to their natural populations and further monitoring of new population occurrence for documentations

Improving Molecular Diagnosis of Suspected Mendelian Disorders with RNA Splicing Analysis

Exome sequencing is the most advanced standard-of-care genetic test for people with suspected Mendelian disorders. Yet, the diagnostic rate of exome sequencing is only 31%. RNA sequencing (RNA-seq) is a promising molecular test for detecting potentially pathogenic changes in RNA splicing as part of obtaining a molecular diagnosis. In this dissertation, I develop new computational tools and perform analyses towards improving how we detect these potentially pathogenic changes in RNA splicing with the goal of improving the molecular diagnostic rate. First, in Chapter 1, I review background on how we diagnose patients and how RNA splicing and RNA-seq could be used to improve this process. Then, in Chapter 2, I describe my contributions to MAJIQ v2 as methodology to study RNA splicing from large and heterogeneous RNA-seq datasets. Afterwards, I use MAJIQ v2 in Chapter 3 to evaluate how tissue-specific expression and splicing affects what clinically-relevant splicing changes we can identify from clinically-accessible tissues. Then, in Chapter 4, I describe the limitations of MAJIQ v2 for our approach to detect splicing aberrations and the development and evaluation of MAJIQ v3 to address these challenges. With MAJIQ v3, I develop MAJIQ-CLIN in Chapter 5 to identify and prioritize splicing aberrations in patient RNA-seq data and compare our method to previous approaches. Finally, in Chapter 6, I discuss overall conclusions for the work and exciting areas for future work. Together, the work in this dissertation pushes forward how we can study and use RNA-seq to improve the diagnostic rate of patients with suspected Mendelian disorders