Multiscale Modeling of Familial Cardiomyopathy-linked Tropomyosin Mutations

Mutations in proteins of the cardiac sarcomere can alter muscle function, leading to a hypercontractile or a hypocontractile state of the heart. These mutational insults can also lead to disease states over time, such as hypertrophic cardiomyopathy (HCM) or dilated cardiomyopathy (DCM). One such gene, TPM1, which encodes the sarcomeric regulatory protein α-tropomyosin, has been linked to cases of both familial HCM and DCM. However, it remains unclear how different mutations to the same gene can cause different pathogenic phenotypes. Without clear predictive genotype-phenotype relationships, the value of clinical genetic testing in screening and treating families is inherently limited. This work focuses on the development of two tools to improve understanding of the connection between genotype and phenotype for sarcomeric genes: 1) a computational model of the cardiac thin filament and 2) an engineered tissue model capable of expressing arbitrary mutations through use of adenoviral transduction. By pairing these tools with other complementary methodologies (molecular dynamics and in vitro motility assays [IVMA]) we seek to demonstrate that they can form the basis of accurate classification of TPM1 variants of unknown significance (VUS) into HCM or DCM phenotypes. We first designed and implemented a Markov chain-Monte Carlo model for simulating thin filament activation. We wanted to produce a detailed model that was capable of predicting both steady-state and dynamic force production while incorporating detailed mechanisms of regulation. To do this, we investigated the regulatory mechanism of the sarcomeric protein troponin I (TnI). It was long thought the inhibitory peptide domain (IP) of TnI acted as the sole actin-binding region that holds tropomyosin in the myosin-blocking position. More recently, evidence has arisen that the C-terminal mobile domain (MD) of TnI also binds actin and may also contribute to this inhibition. To properly incorporate these findings, we created both a 16-state model with TnI-IP as the sole regulatory domain and a 24-state TnI-IP+MD version. Comparison of these models showed that assumption of a second actin-binding site allows the individual domains to have a lower affinity for actin than with IP acting alone. We also tested the 24-state model’s ability to represent steady-state experimental data in the case of disruption of either the IP or MD and we were able to capture qualitative changes in several properties as seen in experimental data. Overall, our analyses support a paradigm in which two domains of TnI bind with moderate affinity to actin, working in tandem to regulation the thin filament. To begin the characterization of mutations to TPM1, molecular dynamics simulations were used to predict important structural and mechanical changes. We applied this to two mutants: the DCM-linked M8R and the HCM-linked S215L. M8R increased flexibility of the tropomyosin chain and enhanced affinity for the blocked or inactive state of tropomyosin on actin. S215L also increased flexibility of the tropomyosin chain while enhancing affinity for the closed state of tropomyosin on actin in which myosin binding sites are revealed. Applying these molecular effects to the 24-state Markov model reproduced the shifts in calcium sensitivity, maximum force, and cooperativity that were also observed in IVMA experiments. The model was then used to simulate the impact of M8R or S215L expression on twitch behavior. These dynamic simulations predicted that M8R would reduce peak force and duration of contraction in a dose-dependent manner. To evaluate this prediction, TPM1 M8R was expressed via adenovirus in engineered heart tissues and isometric twitch force was observed. The mutant tissues showed depressed contractility and twitch duration that agreed in detail with model predictions. For S215L, simulations predicted a hypercontractile twitch phenotype. Mechanical testing of genetically engineered tissues homozygous for mutant S215L TPM1 also showed an increase in peak force and slowed relaxation when compared to isogenic WT tissues. In the final study, we characterized four TPM1 VUS using a combination of molecular modeling, IVMA, and engineered tissue. First, 20 candidate VUS were analyzed computationally using molecular dynamics and energy minimization calculations to predict each variant’s effects on TPM1 structure and association with thin filament proteins. From this analysis, four variants representing a spectrum from most to least predicted pathogenicity (A102D, D258E, K233N, and A239T) were selected for further study. Predictions were tested for each variant via engineered heart tissues. Mechanical testing of the tissues revealed an HCM phenotype for A102D and D258E, but a DCM phenotype for A239T and K233N. The pathogenic phenotypes of these selected variants reveals robust progress toward our long-term goal of computational prediction of disease risk for novel TPM1 variants

Sound effects: the effects of sound-producing toys on the level of social and cognitive play in 3, 4, and 5-year-olds

Master of ScienceDepartment of Family Studies and Human ServicesAnn D. MurrayThis study investigated the effects of sound-producing toys on the social and cognitive levels of play in young children. Thirty-four pairs of children were observed (N=68), ages 3, 4, and 5 years, during 15-minute play sessions. The play conditions were: 1) farm set with sound, 2) farm set with no sound, 3) doctor set with sound, and 4) doctor set with no sound. Independent variables included age, gender, and the presence or absence of sound. Dependent variables included the percentages of time that children engaged in levels of social play (solitary, parallel, and group), levels of cognitive play (functional, constructive, and dramatic), and non-play. Overall, sound was marginally associated with more time spent in play. For group play there was a marginally significant sound by gender interaction, suggesting that sound doubled female group play. There was a significant sound by age interaction suggesting that 5-year olds engaged in group play more with sound than with no sound. A significant sound by gender interaction indicated that the presence of sound increased dramatic play in both males and females, but more so in females. A sound by age interaction indicated that 5-year-olds engaged in more dramatic play with sound than without. Three-year-olds also engaged in more dramatic play when sound was present. There was a significant sound by gender interaction indicating males engaged in more functional play than girls when sound was present. There was a significant sound by age interaction, indicating that 5-year-olds engaged in less constructive play when sound was present. In conclusion, young children were more likely to engage in play when sound was present. Sound enhanced the play of 5-year-olds who engaged in more group and dramatic play, but less constructive play, with sound-producing toys. Sound also enhanced the play quality of 3-year-olds, who engaged in more dramatic play when sound was present. Furthermore, sound enhanced the play of girls who engaged in more group and dramatic play with sound, and boys, who engaged in more dramatic play with sound. Limitations of the study along with implications for future research are discussed

The Constitutionality of Federal Climate Change Legislation

Over the last decade, the Canadian government has not managed to produce a comprehensive climate change statute and has failed to adequately consider the constitutional implications of doing so. The Clean Air Bill, an unsuccessful 2006 amendment to the Canadian Environmental Protection Act (CEPA), focused on carbon credit trading and a technology fund credit mechanism to permit certain emissions-heavy industries to mitigate their production of greenhouse gases. However, this bill would likely have infringed on provincial jurisdiction over electricity generation facilities, trumping any attempt to address greenhouse gas emissions beneath the rubric of criminal law. The present federal government’s proposed coal-fired electricity generation regulations are similarly flawed. This paper thoroughly analyzes both pieces of legislation from a constitutional standpoint, using a rich body of case law to offer policymakers invaluable guidance in properly framing legally sound emission reduction statutes

Quantification of the impacts of water hyacinth on riparian communities in Cameroon and assessment of an appropriate method of control : the case of the Wouri River Basin

Some plants, when transported to non-endemic areas undergo reproduction and rapid growth and will out-compete native plants and become weeds. These weeds then become troublesome giving rise to socioeconomic and environmental impacts. Water hyacinth (Eicchornia crassipes), one of such weeds is often considered the world’s worst invasive aquatic species of weed, indigenous to the Amazon Basin of South America and has spread over the years to various areas in Africa and Asia. This dissertation analyses the problem of water hyacinth infestation in the Wouri River Basin, done through the quantification of its socio-economic and environmental impacts on riparian communities. It aims at providing information to decision-makers and other stakeholders to assist them invest in projects geared towards preventing its spread thereby reducing the impacts on the riparian communities. The riparian communities are identified and data on the socioeconomic and environmental impacts of water hyacinth are collected through focus group discussions and interviews. The data are then presented and analysed. Furthermore, the various available methods of control are examined taking into account the benefits and setbacks of each method. Finally, a suitable strategy for the control of water hyacinth in the Wouri River Basin is proposed. The method shows that the total lost benefits of the communities due to water hyacinth infestations, outweigh the cost of implementing the appropriate control measures; hence justifies the need for urgent measures to be taken to manage water hyacinth in the Wouri River Basin

Prison, power, and people with learning disabilities: the complexities of curtailed lives

Very little is known about how people with learning disabilities symbolically engage with imprisonment and discursively account for it within their wider self-narratives. Although there are no cohesive incarceration figures for people with a learning disability, prevalence studies suggest that they are over-represented among prison populations worldwide. This research addresses a major gap in literature as people with learning disabilities’ accounts are largely absent from prison sociologies, and offending and imprisonment experiences are missing from learning disability studies literature. By bringing together empirical, theoretical, and methodological knowledge from criminology and disability studies, the research fills this void and heralds the value of drawing on two distinct fields of study. This thesis provides a platform for the experiences of 25 men and women with learning disabilities who were serving a custodial sentence or who had been recently liberated from custody at the time of research. Through a critical realist lens, the study used innovative qualitative research methods - multiple and semi-structured interviews - in order to preserve the ethical and moral integrity of researching inclusively with people with learning disabilities while being sensitive to the challenges of researching within prison. The research found that people with learning disabilities are disadvantaged and marginalised in unique ways as a result of the increasing psychological demands associated with late modern imprisonment. In prison, they negotiate distinct barriers to their full social participation with and through their punishment, sentence, and the demands of daily prison life. As a result, they face intersectional forms of oppression and are further socially disenfranchised through institutional process which render them vulnerable and dependent on the structures, supports, and regime of prison which are often absent from their lives in the community. Participants internalised their exclusion, and characterised their lives through experiences of labelling, governance, and curtailment

The domestic architecture of William Street-Wilson, 1889-1913.

Thesis (M.Arch.)-University of Natal, Durban, 1996.The topic of this thesis is the Domestic Architecture of the Natal Architect, William Street-Wilson (1856-1928) and spans the period from 1889 to 1913. It is based on the original drawing collection which can be found in the Barrie Bierman Architectural Library, University of Natal. Street-Wilson carried out his architectural training in England, and the study sets out to identify the stylistic changes which took place within the Arts and Crafts Movement in England, to establish any English precedent in Street-Wilson domestic architecture in Natal. In order to contexturalise Street-Wilson's domestic architecture in Natal, an overview of the political, social, economic and architectural background is given prior to, and during the year of Street-Wilson's arrival in 1887. The drawing collection is set chronologically during the period of 1889-1913, against the political, social and economic background, in order to study the effect of such influences on the Street-Wilson's houses. The study defines and identifies the different components which compose the plan and then carries out an analysis of the plan, form and style of the different categories of house types, namely the typical single storey, villa/double storey and semi-detached house, to highlight the evolution of the house type from the late nineteenth century to the early twentieth century. Since the verandah is the common thread throughout the drawing collection, a special study explores its possible origin and application in a colonial context. Examples of detail drawings have been extracted from the drawing collection to illustrate construction techniques and typical detailing employed by Street-Wilson and his partners, accompanied by a brief history of building materials to contexturalise materials within the studied period. The evolution of services, pertaining to comfort and sanitation, have been examined and applied to the houses designed by Street-Wilson

Fostering home-school relationships to nurture children\u27s literacy

The purpose of this paper is to explore ways teachers can promote a relationship between the home and school to nurture the literacy of primary-age children. A review of professional literature will address the home environment\u27s influence on children\u27s emerging literacy, ways parents can support their children\u27s school program, and avenues teachers can use in communicating with parents. From this information base, the writer will plan ways to foster school-home communication

Development and application of liquid-observed vapor exchange NMR to study dehydrated protein structure and protection at the residue-level

Life on Earth evolved in the oceans, and therefore the stability, dynamics, and function of proteins – the molecules that carry out the majority of life’s processes – are intricately linked to the properties of liquid water. However, despite decades of research, our understanding of the fundamental interaction between proteins and water remains surprisingly limited, in part because we have been unable to study, at adequate resolution, what happens to proteins when water is removed. Such technological boundaries have also hindered our understanding of how molecules known as excipients protect proteins from the destructive effects of dehydration, which is important in the context of protein-based drugs such as insulin and vaccines. For my dissertation work, I sought to help fill these knowledge gaps by developing Liquid-Observed Vapor Exchange (LOVE) NMR, a solution NMR technique that provides residue-level information on the structure and interactions of dehydrated proteins. My dissertation begins with a brief overview and contextualization of what we do and do not know about dehydrated protein structure and mechanisms of dehydration protection (Chapter 1), and then goes into the initial proof-of-concept experiments for LOVE NMR, which show that LOVE NMR reports on the fraction of dried protein population for which a given residue is protected from exchange with D2O vapor, and that this fraction is related to the amount of local structure and/or inter-molecular interactions in the dry state (Chapter 2). I then applied LOVE NMR to uncover water’s variable role in the mutation-induced (de)stabilization of two different proteins (Chapter 3) and to probe the importance of surface-area, charge-patterning, and electrostatic interactions in protein dehydration protection by two distinct disordered proteins from desiccation-tolerant animals (Chapter 4). Finally, in Chapter 5 I suggest strategies to increase the accuracy and precision of LOVE NMR and deepen our understanding of the results it produces. Overall, the results presented in this dissertation demonstrate the potential of LOVE NMR to provide new insights into protein-water and protein-excipient interactions.Doctor of Philosoph

The presence of developmental role models in Iowa Teen Award fiction

The purpose of this study was to determine if the choice of winning fiction titles by Iowa teens in the Iowa Teen Award Competition reflect developmental role models. This study was a content analysis using the Havighurst Tasks of Adolescence as categories to record the frequency of the occurrence of developmental tasks by main characters of the Iowa Teen Award Winners and Runnersup, 1984-1994. The research findings of this study showed that today\u27s Iowa Teen Award winners and contenders reflected all of Havighurst\u27 s Developmental Tasks. It fmther showed that the developmental tasks attempted or accomplished by the main characters in the Iowa Teen Award books were directly related to the themes of the books