Small populations, specifically those that are isolated from others, are more prone to extinction than larger inter-connected populations. The risks that these small isolated populations face include loss of genetic diversity due to founder effects and inbreeding due to population bottlenecks, as well as demographic uncertainty due to fluctuating fecundity and mortality rates and impacts of external environmental factors. Ex situ populations, including those managed as conservation breeding programs with species recovery aims, as well as those that do not have reintroduction goals but are managed for long term population sustainability, suffer from the same extinction risks as small and isolated natural populations. Using three separate avian species which have different life histories and population structures, I investigated impacts of multiple genetic and demographic management strategies on these ex situ populations. I examined the use of molecular genetic datasets including microsatellites and single nucleotide polymorphisms (SNPs) to determine their utility for reconstructing pedigrees, examining individual relatedness within populations, and compared results of measuring genetic diversity through theoretical methods verses those obtained from a molecular dataset. These methods can then ultimately be applied to improve future management including improving studbook datasets and to measure actual loss of genetic diversity. I also used analytical strategies including population viability analysis to determine how management practices influence demographic parameters and determine the future probability of population extinction. The genetic and demographic analyses of both the historic management of an ex situ population, and its current status, are a first step in hypothesizing the potential directions for future management and understanding the likelihood of survival of an ex situ population.

Yes / Integrating a desired DNA sequence into the yeast genomes is a widely-used genetic manipulation in the budding yeast Saccharomyces cerevisiae. The conventional integration method is to use an integrative plasmid such as pRS or YIplac series as the target DNA carrier. The nature of this method risks multiple integrations of the target DNA and the potential loss of integrated DNA during cell proliferation. In this study, we developed a novel yeast integration strategy based on the widely used CRISPR-Cas9 system and created a set of plasmids for this purpose. In this system, a plasmid bearing Cas9 and gRNA expression cassettes will induce a double-strand break (DSB) inside a biosynthesis gene such as Met15 or Lys2. Repair of the DSB will be mediated by another plasmid bearing upstream and downstream sequences of the DSB and an integration sequence in between. As a result of this repair the sequence is integrated into genome by replacing the biosynthesis gene, the disruption of which leads to a new auxotrophic genotype. The newly-generated auxotroph can serve as a traceable marker for the integration. In this study, we demonstrated that a DNA fragment up to 6.3 kb can be efficiently integrated into the Met15 or Lys2 locus using this system. This novel integration strategy can be applied to various yeasts, including natural yeast isolated from wild environments or different yeast species such as Candida albicans. / This work was supported by the Wellcome Trust [202062 to B.H. and 207127 to A.M.] This work was also supported by a SURE studentship from the University of Sheffield [325537] awarded to P.W.D

The Y-chromosome and mitochondria (containing mtDNA) appear entirely dissimilar, the former being a sex chromosome found in the nucleus and the latter an organelle with thousands of copies in the cytoplasm. However, both experience uniparental inheritance, with Y-chromosomes being patrilineal and mitochondria matrilineal. This leads to various other similarities including haploidy, high mutation rates, and limited or lack of genetic recombination. Moreover, the low number of protein-coding genes contained on both led to the belief that neither contributes significantly to the adaptive evolution of complex traits. However, recent studies show the Y-chromosome and mitochondria both influence roughly 10% of male transcripts, greatly increasing their evolutionary reach. The nature of the mitochondria's influence is clear – maternal inheritance leads to the accumulation of male-deleterious mutations that negatively impact autosomal and X-linked transcripts (termed mother's curse). However, the nature of the Y-linked variation is less clear, but may impact the manifestation of mother's curse, the evolution of sexual dimorphism, or even act as a constraint on adaptive evolution. In Chapter 2, we explore the potential for Y-chromosomes to influence sexual dimorphism by examining the nature of Y-linked genetic variance. If this variance is additive, it may facilitate the evolution of sexual dimorphism by allowing male traits to be shaped independently of female traits despite males sharing a genome with females. However, if this variance is epistatic, it may slow population level responses to strong selective pressures such as climate change or novel diseases. To date, previous works suggest that Y-linked variation is predominately epistatic. To address our objective, we compare fruit fly populations with and without Y-linked variance and measure the heritability of male and female morphological traits. Surprisingly, we find more heritable variance in populations with Y-linked variance, suggestive of Y-linked additivity. In Chapter 3, we examine whether Y-linked variation can counteract mother's curse. Theoretically, compensatory mutations could evolve on the autosomes, X-chromosome, or Y-chromosome. However, Ys appear best suited to host these mutations as there can be no counter-selection from females due to the Y's uniparental inheritance. To test this, we placed coevolved and non-coevolved Ys and mitochondria into an isogenic background that shared no coevolutionary history with either genetic element. We find that the Y-chromosome has the potential to compensate for mother's curse for at least one trait, male longevity. In Chapter 4, we address a potential limitation in our previous test for Y-linked compensation in Chapter 3: the non-coevolved nuclear background. Theoretically, Ys may compensate for mitochondrial mutations by interacting with the autosomes. Thus, having a coevolved Y and autosomal background may be critical to compensation. To this end, we repeated our experiments from Chapter 3 with the inclusion of a coevolved nuclear genome. We again find Y-chromosomal compensation for mother's curse for longevity, but only when coevolved autosomes are used. This suggests that using non-coevolved backgrounds (as all previous studies have done) may be unreliable and create misleading results.

Triple negative breast cancer (TNBC's) is a highly aggressive and invasive subtype of breast cancer, typically characterised by the lack of estrogen receptor (ER), progesterone receptor (PR) and Human epidermal growth factor receptor 2 (HER2) with an inexplicable partiality towards African women. The acute heterogenicity and complexity of TNBC tumours, together with a lack of well-defined molecular targets, complicates prognosis of the diseases resulting in patient reliance on traditional therapies, like chemotherapy, radiotherapy, and surgery, which are associated with elevated incidence of adverse effects and relapse. A major contributor to the heterogenicity of TNBCs is the tumour microenvironment which is composed of tumour infiltrating lymphocytes (TILs), tumour cells, healthy cells, and tumour vasculature. TILs have commonly been used as a prognostic marker and show robust predictive value for TNBC. In-depth analysis of the TIL composition within TNBC tumours may provide greatly beneficial information for the development of newer tumour microenvironment changing therapies and could assist doctors in understanding what therapies a particular patient maybe susceptible to. Thus, the diagnosis and therapy of this disease may greatly benefit from improved molecular profiling and patient stratification. Precision medicine seeks to provide such a solution, by dividing patients into subpopulations based on disease-specific profiles. The identification of new molecular targets would provide the basis for development of novel therapies. To this end, one of the major aims of this thesis was to develop a phage display based screening technique which could be utilised to isolate novel TNBC specific cancer antibodies. Once selected these antibodies could be used to generate TNBC specific therapies. Specific monoclonal antibodies (mAbs) and derivatives thereof, have already been established as a revolutionary tool for drug delivery to cancerous cells. Such antibodies have been conjugated to cytotoxic drugs to form antibody-drug conjugates, which may exhibit multiple advantages over their unconjugated counterparts, but their general use in clinical application has been restricted due to developmental deliberations. Historical conjugation strategies used for the generation of ADCs commonly resulted in heterogeneous mixtures of ADC species, with varying drug-to-antibody ratios resulting in unpredictable pharmacologic characteristics and safety profiles. In more recent time, self-labelling tags such as Snaptag have provided a means of developing homogenised recombinant immunotherapeutics. Snaptag is a modified version of a human DNA repair enzyme, O6 - alkylguanine-DNA-alkyltransferase (AGT) which naturally removes alkyl residues from damaged DNA. The enzyme reacts specifically with benzylguanine (BG) derivatives via irreversible transfer of alkyl groups to cysteine residues forming stable end products. In this thesis, Snaptag technology, together with other antibody discovery and manipulation tools was used to develop a methodology allowing for the generation of disease specific fusion proteins. Specifically, these fusion proteins consist of single-chain antibody fragments genetically fused to snaptag, allowing for the generation of recombinant ADCs that could be used as a drug delivery system carrying any BG-modified drug to a disease specific targets. In addition, snaptag interacts with BG in a 1:1 stoichiometry giving rise to homogenised combination products which when fused to a scFv provides a fail-safe target-specific therapeutic option. In addition to antibody conjugates, one of the most promising of all mAb based therapies currently used, are checkpoint inhibitors. In a balanced immune response, immune activation is counteracted with immunoregulatory pathways such as checkpoint inhibition. These negative regulatory pathways are necessary for maintaining tolerance and preventing hyperactivation, and are governed by cell surface, inhibitory receptors known as ‘'checkpoint inhibitors''. Blocking of checkpoint pathways during chronic infections and cancer has been shown to improve T-cell functions leading to reduced viral load and tumour burden. These findings have been translated into clinical application where checkpoint inhibitors, which are monoclonal antibodies targeting CTLA-4, PD1, PD-L1 or other inhibitory ligands, have been used to block these inhibitory interactions. The main intention of this research was to develop a methodology which could be used to generate snaptag based recombinant fusion proteins with potential diagnostic and therapeutic applications. Several snaptag based fusion proteins were developed using the recommended methodology these included fusion proteins targeting breast cancer specific antigen BCK1, checkpoint inhibitors PDL1, B7.1/CD80 (interacts with CTLA-4),and TIL characterising markers CD3, CD4, CD8, CD19 and CD20. In addition, to demonstrate the versatility and robustness of this methodology we sought to develop a snaptag based fusion protein not targeting breast cancer related antigens. Zika virus, an emerging infectious disease, currently lacking specific therapies was chosen for this purpose. An scFv derived from antibodies targeting the the Zika-DIII envelop protein, which is essential to the viral infection cycle was used in the snap fusion protein. The resulting ZIKA-DII-snap fusion protein demonstrated specific binding to zika virus membrane fractions. This research demonstrates the feasibility of using snaptag technology as a state-of-the-art conjugation strategy capable of bypassing the challenges previously associated with using antibodies as an effective delivery system for therapeutic drugs. By combining the applicability of snaptag technology with other antibody isolation and manipulation tools we were able to generate several functional snaptag based recombinant fusion proteins. Establishment of this methodology represents an important first step in generating medically necessary, pharmaceutically acceptable immunoconjugates that is instrumental in shifting general therapy towards a more personalized precision medicine approach.

Plusieurs systématiques se côtoient aujourd'hui : la systématique typologique, d'inspiration linnéenne, qui repose sur l'examen morphologique de types porte-nom. Depuis plusieurs années, une systématique phylogénétique conduit également à la création de taxons nouveaux, de niveau spécifique, infra-spécifique et supra-spécifique. Dans ce travail, nous avons cherché une approche globale, qualifiée de taxonomie intégrative couplant les approches morphologiques traditionnelles, la systématique moléculaire et la geomorphométrie alaire, dans le but d'éclairer la notion d'espèce chez les Culicoides avec ses corollaires épidémiologiques, étant donnée l'importance de ces insectes dans des maladies majeures d'intérêt vétérinaire telles que la fièvre catarrhale ovine (FCO) et la maladie causée par le virus de Schmallenberg (SBV). Cette approche nous a permis de mettre en lumière la complexité systématique des espèces affines (C.clastrieri/C.festivipennis, les groupes Obsoletus, Pulicaris et Vexans). De plus, nous nous sommes intéressés aux préférences trophiques des Culicoides de la région Champagne-Ardenne. / Several systematic coexist today: typological systematic of Linnaean spirit, based on morphological examination of type specimens. For several years, a phylogenetic systematics also led to the creation of new taxa, at specific, sub-specific and supra-specific level.In this study, we have sought a comprehensive approach i.e. an integrative taxonomy that coupling traditional morphological approaches, molecular systematic and wing geometry morphometric in order to clarify the concept of species in the Culicoides with epidemiological corollaries, given the importance of these insects in major diseases of veterinary interest such as bluetongue (BT) and the disease caused by the Schmallenberg virus (SBV). This approach allowed us to highlight the systematic complexity of related species (C.clastrieri / C.festivipennis, the Obsoletus, Pulicaris and Vexans groups). In addition, we investigated the host preference of Culicoides of the Champagne-Ardenne region.

Preparing students to be life-long learners that are career and college ready is a goal of agricultural education. Changing expectations of education have pointed to agriculture educators as potential leaders in the STEM education movement. Literature related to STEM education in agricultural education is lacking in guidance for teachers, administrators, and curriculum developers in integrating academic content related to STEM content areas. A review of STEM education literature coupled with the framework of quantitative reasoning, lead to a conceptualization of a framework for integrative agricultural education. This framework was implemented through a case study to investigate collaborative efforts in curriculum development in agricultural education with a specific focus on integrating mathematics to develop students' quantitative reasoning skills. Teacher characteristics were identified that seemed to support the implementation of integrative agricultural education practices. Teaching and planning strategies were also identified in the case study. Recommendations suggest support of collaboration between agriculture and mathematics teachers would best support curriculum design and aid in the quality of instruction that follows. / Ph. D.

Class of 2007 Abstract / Objectives: The objective of this study was to determine the need for an integrative medicine continuing education (CE) program for pharmacists and what pharmacists are looking for in such a CE program. Methods: A focus group was conducted with four pharmacists. They were led on a discussion about their experiences with alternative/herbal medicines and desire to learn more, opinions on CE for pharmacists, and compensation for CE credits. The members of the focus group were also given a demographics questionnaire about their gender, age, years in practice, and practice setting. Results: The focus group thought that most pharmacists would want to learn more about integrative medicine, including alternative and herbal products. They would like to have a CE program that is structured around disease states and presents evidence based medicine. It was agreed that inclusion of law CE credits would make the program more desirable. The length of the program could be between 2 hours (if presented on-line) to an entire day (if conducted with presentations). The major limitation in this study was the generalizability of the results due to the small size of the focus group and its members being form the same practice site. Conclusions: It was determined that there is a need for an integrative medicine CE program for pharmacists. If remuneration is sought for the program, then pharmacists will expect certification upon completion. More studies should be conducted to determine the ideal content in herbal/alternative medicine CE programs.

This thesis examines an integrative health care (IHC) clinic set within a tertiary hospital located in large city in Canada. The enquiry began by exploring how biomedical and CAM practitioners, artists, hospital administrators, and the Artists’ Health Centre Foundation (AHCF) members interacted, communicated, and collaborated with one another for integrative patient/client care at the Artists’ Health Centre (AHC). Individual stakeholders’ knowledge and attitudes toward IHC are explored, to understand how these affected the everyday interactions among stakeholders. The thesis also examines the organizational structures of the hospital and the AHCF. The use of qualitative research provided useful in-depth accounts of respondents’ experiences of IHC. Semi-structured focus groups with artists, health-care practitioners, hospital administrators, and AHCF members, and ten in-depth interviews with the health-care practitioners were conducted between June, 2006, and February, 2007. Steps were taken to ensure the trustworthiness of the collected data. Qualitative research software, NVIVO, was used to manage the data. The findings suggest that despite a perceived lack of scientific evidence, attitudes towards IHC were positively influenced when biomedical practitioners had a personal experience with CAM therapies and when practitioners developed more confidence in their own work. There remained questions about the need for and presence of IHC at the AHC, as neither the hospital nor the AHCF worked in consultation with the practitioners and artists to develop a shared vision of IHC. The majority of respondents described the level of communication as “sporadic” and one-sided and thus not optimal for creating a communicative environment. Most respondents perceived communication among the stakeholders as one-sided and thus not optimal for creating a communicative environment. There was a lack of understanding regarding scope of practice and how to integrate the various practitioners, particularly CAM. This led to a lack of referrals and had a direct effect on practitioners’ level of confidence. Mechanisms of communication were informal and there were no formalized structures in place to facilitate communication or integration with one another. Additionally, there was no systematic way of charting patient information at the AHC. Respondents all agreed that IHC was an ideal to strive for and many noted how financial limitations impeded the evolution of integrative health care at AHC, truly forming a barrier to IHC. Strategies for sustainability and management of AHC funds included using a business model or an insurance model. Important implications of this research include enhancing the current knowledge of teamwork, collaboration, and integration among practitioners in general, and biomedical and CAM practitioners in particular. This research used existing IHC models, interdisciplinary teamwork models, and educational and organizational theories for building a theoretical and conceptual framework of IHC at the AHC. Combining these models with organizational theory shed light on relationship dynamics among CAM and biomedical practitioners while taking into consideration the several structural and process dimensions of integration.

This thesis examines an integrative health care (IHC) clinic set within a tertiary hospital located in large city in Canada. The enquiry began by exploring how biomedical and CAM practitioners, artists, hospital administrators, and the Artists’ Health Centre Foundation (AHCF) members interacted, communicated, and collaborated with one another for integrative patient/client care at the Artists’ Health Centre (AHC). Individual stakeholders’ knowledge and attitudes toward IHC are explored, to understand how these affected the everyday interactions among stakeholders. The thesis also examines the organizational structures of the hospital and the AHCF. The use of qualitative research provided useful in-depth accounts of respondents’ experiences of IHC. Semi-structured focus groups with artists, health-care practitioners, hospital administrators, and AHCF members, and ten in-depth interviews with the health-care practitioners were conducted between June, 2006, and February, 2007. Steps were taken to ensure the trustworthiness of the collected data. Qualitative research software, NVIVO, was used to manage the data. The findings suggest that despite a perceived lack of scientific evidence, attitudes towards IHC were positively influenced when biomedical practitioners had a personal experience with CAM therapies and when practitioners developed more confidence in their own work. There remained questions about the need for and presence of IHC at the AHC, as neither the hospital nor the AHCF worked in consultation with the practitioners and artists to develop a shared vision of IHC. The majority of respondents described the level of communication as “sporadic” and one-sided and thus not optimal for creating a communicative environment. Most respondents perceived communication among the stakeholders as one-sided and thus not optimal for creating a communicative environment. There was a lack of understanding regarding scope of practice and how to integrate the various practitioners, particularly CAM. This led to a lack of referrals and had a direct effect on practitioners’ level of confidence. Mechanisms of communication were informal and there were no formalized structures in place to facilitate communication or integration with one another. Additionally, there was no systematic way of charting patient information at the AHC. Respondents all agreed that IHC was an ideal to strive for and many noted how financial limitations impeded the evolution of integrative health care at AHC, truly forming a barrier to IHC. Strategies for sustainability and management of AHC funds included using a business model or an insurance model. Important implications of this research include enhancing the current knowledge of teamwork, collaboration, and integration among practitioners in general, and biomedical and CAM practitioners in particular. This research used existing IHC models, interdisciplinary teamwork models, and educational and organizational theories for building a theoretical and conceptual framework of IHC at the AHC. Combining these models with organizational theory shed light on relationship dynamics among CAM and biomedical practitioners while taking into consideration the several structural and process dimensions of integration.

Thesis (Ph. D.)--Rutgers University, 2009. / "Graduate Program in Integrative Neuroscience." Includes bibliographical references (p. 124-148).