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Regulatory T cells (Tregs) suppress effector immune responses and have been implicated in promoting chronic viral infections. Their role during influenza infection and vaccination, however, is still unclear. Influenza is a major public health concern, claiming over 49,000 lives annually in the U.S. alone. Vaccination is the best approach for preventing disease but frequent mutations of immunogenic epitopes requires a new vaccine to be formulated and administered annually. This poses a challenge for vaccine manufacturing and may strain patient compliance. A universal influenza vaccine, which targets the highly conserved extracellular domain of the influenza matrix protein 2 (M2e), may circumvent this problem by generating cross-protective immunity. In this study, we tested the efficacy of the M2e universal vaccine in the ferret, and determined whether vaccination induces a Treg response after influenza infection. We found that vaccination promotes the development of M2e specific IgM and IgG antibodies after boosting. Upon challenge with A/Memphis H1N1, vaccinated ferrets exhibited a lower body temperature and reduced virus titer compared to non-vaccinated animals. Together these findings suggest that the M2e vaccine protects ferrets against influenza infection. In order to determine whether Tregs increase after vaccination in ferrets, we had to first clone and characterize genes involved with Treg phenotype and function including CD25, Foxp3, and IL-10. The reciprocal nature between Tregs and Th17 cells and their involvement during influenza infection prompted us to also clone ferret IL-17F. Using these sequences, we designed a qRT-PCR array to measure the expression of Foxp3, IL-10, and IL-17F in ferret tissue. We also identified cross-reactive antibodies against ferret CD8, CD25, and Foxp3 for use in FACS, western blot, and ICC. Using these tools, we found that vaccination significantly increased the expression of Foxp3 in the spleen. An increased percentage of Foxp3+ lymphocytes was detected in both the PBMCs and splenocytes of immunized animals. In contrast, IL-10 and IL-17F expression decreased significantly in both immunized and non-immunized ferrets compared with naïve animals. These studies suggest that the M2e influenza vaccine induces a regulatory T cell response in ferrets and protects against influenza infection. / acase@tulane.edu

This thesis quantitatively and qualitatively analyzes the economic regulations that govern taxicab firms in Iowa City, Iowa. Based upon a review of the relevant literature, an economic analysis of regulations and market power, and conducted interviews among taxicab owners and drivers, city staff and planners, and members of the general public, this paper will analyze the costs and implications of economic regulations and risks of regulatory capture, and identify improvements to existing ordinances and city codes. Current economic theory argues that economic regulations create both real and perceived entry barriers, and impose costs to producers and consumers. Additionally, these regulations stifles entrepreneurship and innovation, reduces driver pay, and in some instances leads to discrimination and encumbrance for the most vulnerable residents, recent immigrants and the car-less poor. On the question of whether economic regulations causes high concentrations of market power in U.S. cities, regression analysis of medium to large U.S. cities does not reveal a correlation between entry regulation and market power. Additionally, calculations of the Herfindahl (HH) Index for taxi firms in Iowa City yields a HH score of 0.103052, which is considered an un-concentrated market. However, while the quantitative data indicates that economic regulations do not cause an identifiable influence on market power, qualitative data gathered from stakeholder interviews reveal a burden in the form of unavoidable sunk costs for drivers, owners, and riders. These interviews reveal the "true" costs of regulations, as well as the perceived costs by policy makers, regulators, and the general public, who frequently underestimate the burden of regulation. This thesis further highlights how regulations arise in the policy-making process; and to what extend they stem from either anti-competitive interests between established firms, a lack of information among policymakers, or simply planners' failure to integrate taxis into a more comprehensive regional transportation system. Ultimately this thesis argues that some of Iowa City's taxicab regulations, particularly the liability insurance minimums for drivers, the terms of operation for dispatching, and the profiling of immigrant and small firm cabbies by ICPD are burdensome and unnecessary. Loosening of these restrictions would benefit small firm and drivers, the general consumer of taxi services, and compliment larger city planning goals in Iowa City, Iowa. Despite the costs and burdens, this thesis does not justify complete deregulation for Iowa City's transportation policy, particularly when case studies of such efforts have not always yielded positive benefits. Instead, this thesis advocates for "better regulation", to be enforced on a regional level, rather than at a municipal level.

The core focus of this research lies in developing and using intelligent methods to solve biological problems and integrating the knowledge for understanding the complex gene regulatory phenomenon. We have developed an integrative framework and used it to: model molecular interactions from separate case studies on time-series gene expression microarray datasets, molecular sequences and structure data including the functional role of microRNAs; to extract knowledge; and to build reusable models for the central dogma theme. Knowledge was integrated with the use of ontology and it can be reused to facilitate new discoveries as demonstrated on one of our systems – the Brain Gene Ontology (BGO). The central dogma theme states that proteins are produced from the DNA (gene) via an intermediate transcript called RNA. Later these proteins play the role of enzymes to perform the checkpoints as a gene expression control. Also, according to the recently emerged paradigm, sometimes genes do not code for proteins but results in small molecules of microRNAs which in turn controls the gene regulation. The idea is that such a very complicated molecular biology process (central dogma) results in production of a wide variety of data that can be used by computer scientists for modelling and to enable discoveries. We have suggested that this range of data should actually be taken into account for analysis to understand the concept of gene regulation instead of just taking one source of data and applying some standard methods to reveal facts in the system biology. The problem is very complex and, currently, computational algorithms have not been really successful because either existing methods have certain problems or the proven results were obtained for only one domain of the central dogma of molecular biology, so there has always been a lack of knowledge integration. Proper maintenance of diverse sources of data, structures and, in particular, their adaptation to new knowledge is one of the most challenging problems and one of the crucial tasks towards the knowledge integration vision is the efficient encoding of human knowledge in ontologies. More specifically this work has contributed towards the development of novel computational and information science methods and we have promoted the vision of knowledge integration by developing brain gene ontology (BGO) system. With the integrative use of several bioinformatics methods, this research has indeed resulted in modelling of such knowledge that has not been revealed in system biology so far. There are many discoveries made during my study and some of the findings are briefly mentioned as follows: (1) in relation to leukaemia disease we have discovered a new gene “TCF-1” that interacts with the “telomerase” gene. (2) With respect to yeast cell cycle analysis, we hypothesize that exoglucanase gene “exg1” is now implicated to be tied with “MCB cluster regulation” and a “mannosidase” with “histone linked mannoses”. A new quantitative prediction is that the time delay of the interaction between two genes seems to be approximately 30 minutes, or 0.17 cell cycles. Next, Cdc22, Suc22 and Mrc1 genes were discovered that interacts with each other as the potential candidates in controlling the Ribonucleotide reductase (RNR) activity. (3) Upon studying the phenomenon of Long Term Potentiation (LTP) it was found that the transcription factors, responsible for regulation of gene expression, begin to be elevated as soon as 30 min after induction of LTP, and remain elevated up to 2 hours. (4) Human microRNA data investigation resulted in the successful identification of two miRNA families i.e. let-7 and mir-30. (5) When we analysed the CNS cancer data, a set of 10 genes (HMG-I(Y), NBL1, UBPY, Dynein, APC, TARBP2, hPGT, LTC4S, NTRK3, and Gps2) was found to give 85% correct prediction on drug response. (6) Upon studying the AMPA, GABRA and NMDA receptors we hypothesize that phenylalanine (F at position 269) and leucine (L at position 353) in these receptors play the role of a binding centre for their interaction with several other genes/proteins such as c-jun, mGluR3, Jerky, BDNF, FGF-2, IGF-1, GALR1, NOS and S100beta. All the developed methods that we have used to discover above mentioned findings are very generic and can be easily applied on any dataset with some constraints. We believe that this research has established the significant fact that integrative use of various computational intelligence methods is critical to reveal new aspects of the problem and finally knowledge integration is also a must. During this coursework, I have significantly published this research in reputed international journals, presented results in several conferences and also produced book chapters.

Hox genes are regulatory genes encoding small proteins containing a highly conserved 61-amino acid motif, the homeodomain, that enables Hox proteins to bind to DNA at specifically recognised binding sites and transcriptionally activate their target genes. In mammalian species there are 39 Hox genes and they are structural and functional homologs of the Drosophila homeotic complex (Horn-C). During embryogenesis and early development the Hox genes are expressed in a spatiotemporal fashion, where they operate as master transcriptional regulators. Hox genes are further expressed in fully differentiated adult cells, potentially in a tissue-specific manner involving maintenance of the normal phenotype. In selected oncogenic transformations, dysregulated Hox gene expression has been observed, indicating an involvement of these transcriptional regulators in carcinogenesis and metastasis. Utilising quantitative real-time PCR assays, these studies investigated the expression patterns of 20 Hox genes and two wellcharacterised Hox cofactors (Pbx and Meis) in malignant and non-malignant human breast and skin cancer cells. Dysregulated Hox expression was observed for all malignancies tested, of which some misexpressed Hox genes seemed random, whereas other Hox transcripts showed altered levels potentially corresponding with the invasive capacity of the cells. Also, the Hox cofactors Pbx and Meis showed no marked changes in expression levels from the non-malignant to the malignant phenotypes, indicating that it is dysregulated Hox gene expression rather than dysregulated gene expression of Hox cofactors that potentially commit the cell to redifferentiate and undergo oncogenic transformation. Although the Hox proteins are known to be key transcriptional regulators of development, the mechanisms by which they gain their in vivo functional specificity is still largely unknown. They all show strikingly similar transcriptional specificity in vitro, yet show unique specificity in their in vivo environment. This paradox has been the subject of intense scrutiny, however very few direct Hox target genes have been identified, making it a difficult task to decipher the exact manner in which Hox proteins exert their functional potential. Therefore, the studies presented herein were aimed at identifying further Hox target genes in the human system. Utilising differential display approaches, several potential downstream target genes were isolated. Substantiated with real-time PCR assays, one of these potential targets was selected as a likely direct Hox gene target, and as such subjected to further studies. By the combination of bioinformatic analyses, transfection protocols and luciferase assays, a gene encoding the SR-related protein SRrpl3O was shown to be trans-activated in vitro by HOXD4 via a putative Hox binding element within its promoter region. This is the first reported link between Hox transcription factors and the SR and SR-related family of pre-mRNA splicing proteins, offering a new and exciting insight into the complex nature of Hox functional specificity. Finally, this thesis also puts forward new ideas regarding how the Hox proteins gain their transcriptional and functional specificity. Utilising bioinformatic tools in conjunction with performing an extensive review of the disparate catalogue of Hox-related research reports, work herein offers the first comprehensive analysis of the mammalian Hox gene targets in relation to their promoter structures, as well as with respect to the expanded Hox DNA-binding elements. This work reports that identified Hox targets generally contain TATA-less core promoters, many of which have several GC-box elements. The Hox binding elements show no apparent preference regarding their location relative to the transcription start site (TSS), as they are found both upstream and downstream of the TSS, as well as being located close to proximal core promoter elements for some genes and at more distant positions in other gene promoters. Finally, the core Hox binding element TAAT/ATTA contains only part of the necessary recognition sequence involved in Hox-DNA binding, and the notion that flanking base pairs dictate trans-regulatory potential is further explored with the hypothesis that the immediate 3' base pair dictates an activator/repressor-switch of the Hox trans-regulatory effect.

The dual problem space model of writing (Scardamalia, Bereiter and Steinbach, 1984) shows how writers develop their knowledge and understanding of the world by reflecting on problems of substance and problems of presentation in planning a composition. Reflective thought is attributed to a two-way communication between a content problem space and a rhetorical problem space. The content space involves the development of ideas, while the rhetorical space is concerned with achieving various purposes in composition. This thesis reports an instructional experiment comparing alternative approaches to teaching the self-regulatory strategies required for the two-way process of reflection. The experiment compared the dialogue approach of current practice, which relies on the teacher to provide the linking operations between the two problem spaces, with two experimental approaches which promote development of self-regulatory strategies of reflection, so that students are able to sustain such a two-way process independently. The experimental approaches are described as a guided discovery approach proposed by Evans (1991) and an approach described as cognitive apprenticeship developed by Scardamalia, Bereiter and Steinbach (1984). Three instructional programs were prepared by the author to represent the three alternative approaches examined in the present study. The control program utilised the dialogue approach of current practice in which the dialectical process is carried on between teacher and student. The two experimental programs focused on promoting processes of self-questioning rather than questioning by an external agent such as a teacher. The guided discovery program consisted of activities which prompted self-questioning processes. The cognitive apprenticeship program employed scaffolding in the form of procedural facilitation cues to stimulate the self-questioning process. A pre-test and post-test control group design was used involving three groups, two experimental (guided discovery and cognitive apprenticeship) and one control (dialogue), with instructional method as the independent variable and rated reflectiveness of writing as the dependent variable. Instruction was concentrated on teaching the two-way problem formulating and problem solving strategies of the reflective process for opinion essays and factual exposition essays. The experiment compared the effectiveness of programs by measuring changes in overall reflectiveness of writing. Significant improvements were obtained for the experimental teaching methods withrespect to opinion essays. This research provided some support for the hypothesis that instruction which fosters self-regulation of the planning process through processes of reflection results in more reflective writing than instruction in which such regulation is prompted by the teacher.

Since Higgins presented regulatory fit theory in 2000, over 200 studies have demonstrated that regulatory fit processes moderate both information processing and outcome responses substantially. However, scholars characterize regulatory fit diversely and manipulate the definition. Researchers have highlighted this issue and recommended further investigations to clarify regulatory fit heterogeneity, which is derived from two sources: conceptual viewpoints of regulatory fit and methods for regulatory fit induction (Aaker, 2006; Avnet, 2006; Cesario, 2008; Pham, 2009). This study decomposes manipulation heterogeneity for researching regulatory fit from the experimental methodology viewpoint. Rather than providing a summary of existing methods, this study proposes a critical moderator embedded in these techniques, which objectifies the regulatory fit concept using a calculable index. The proposed index can be used to predict the strength of the regulatory fit effect, and explain manipulation heterogeneity when researching regulatory fit.

There is community concern about toxic contaminants in Chinese herbal medicines. The two areas of contamination that attract most attentions are the nephrotoxic chemical, aristolochic acids found to be present in some Chinese herbs and resulting in renal failure of over 200 patients in Belgium, and heavy metals such as lead, arsenic, cadmium, mercury and chromium, which can cause systemic, CNS, neurological and developmental pathologies. Currently there is a lack of systematic information about the aristolochic acid content in Aristolochia species and related genera, nor have there been any studies on metal contamination conducted in Australia which can provide some scientific basis for assessment of potential risk of Chinese herbal medicines posed to consumers in Australia. This research aimed at addressing these concerns by firstly carrying out a systematic measurement of the contents of aristolochic acids in some relevant raw herbal medicines (CHM) and proprietary medicines (CPM)- 27 CHM, and 7 CPM, and secondly analysing the contents of five heavy metals in 100 CHM, 50 CPM, and 5 commonly used Chinese medicinal formulae (CMF) in the form of raw herbs, and finally evaluating the potential systemic metal toxicity caused by routine ingestions of Chinese medicines in the common form of encapsulated concentrated powder extracts formulated for the treatment of seasonal allergic rhinitis by means of measuring the metal concentrations in blood collected from 71 patients in a randomised double-blind control clinical trial (RCT). Results showed that four of the 37 CHM and two of the 7 CPM contained the banned toxic aristolochic acids. Some of these contaminated medicines could still be purchased over-the-counter in Victoria. Quantitative screening of metal contamination in CHM found that metal concentrations were much lower in the aqueous solutions than in the acid-digested samples. Almost all CHM, CPM and the 5 CMF contained the five heavy metals. Contrary to popular perception, their metal concentrations in the clinically ingested form were extremely low. Their prescribed ingested contents calculated as percentages of the universally recognised regulatory safety standards, the WHO provisional tolerable weekly intake (PTWI), would produce only small percentages of the PTWI for the metal concerned. This was true even when the metal intakes from any forms of Chinese medicines were added to the normal Australian daily dietary metal exposure. These new approaches of analysing the aqueous extractions, as well as interpretation with reference to the WHO regulatory standards and in combination with the Australian normal daily diet, are more relevant and realistic. The RCT in vivo study demonstrated no significant metal accumulation in the blood of both the real treatment group and the placebo control group, thus, attesting to the encouraging finding of the herbal medicine analysis. In conclusion, there is still much to improve in Australia in terms of enforcing the regulation of banning the sale of Chinese herbal medicines that might contain the highly nephrotoxic aristolochic acids. On the other hand, all forms of Chinese medicines in Victoria are safe, and do not appear to pose significant health concerns in terms of metal contamination.

Cells of the ventrolateral dermomyotome delaminate and migrate into the limb buds where they give rise to all muscles of the limbs. The migratory cells proliferate and form myoblasts, which withdraw from the cell cycle to become terminally differentiated myocytes. The regulatory mechanisms that control the later steps of this myogenic program are not well understood. The homeodomain transcription factor Pitx2 is expressed specifically in the muscle lineage from the migration of precursors to adult muscle. Ablation of Pitx2 results in distortion, rather than loss, of limb muscle anlagen, suggesting that its function becomes critical during the colonization of, and/or fiber assembly in, the anlagen. Microarrays were used to identify changes in gene expression in flow-sorted migratory muscle precursors from Wild type and Pitx2 null mice. Changes in gene expression were observed in genes encoding cytoskeletal, adhesion and fusion proteins which play a role in cell motility and myoblast fusion. We observed decreased cellular motility, disrupted cytoskeleton organization and focal adhesion distribution, decreased fusion of mononucleated myoblasts into multinucleated myotubes and decreased proliferation in presence of Ptix2. These studies suggest that Pitx2 plays a critical role in regulating the timing of myoblast filling the limb anlagen which may have detrimental consequences for higher order muscle architecture. / Graduation date: 2013

CD4+CD25+FoxP3+ regulatory T cells (Treg) possess the capacity to modulate both adaptive and innate immunity. Due to their suppressive nature, Treg cells have been studied and tested in a variety of scenarios in an attempt to ameliorate undesired immune responses. While graft versus host disease (GVHD) has in fact emerged as the first clinical application for human Treg cells (Riley et al. 2009), equally important are issues concerning hematopoietic engraftment and immune reconstitution. Currently, little is known about the effect(s) that regulatory T cells may exert outside the immune system in this context. Based on cytokine effector molecules they can produce we hypothesized that Treg cells could regulate hematopoietic phenomena. The studies portrayed in this dissertation demonstrate that Treg cells can differentially affect the colony forming activity of myeloid and erythroid progenitor cells. In-vitro as well as in-vivo findings demonstrate the ability of Tregs to inhibit and augment the differentiation of primitive and intermediate myeloid (interleukin (IL)-3 driven) and late erythroid (erythropoietin driven) hematopoietic progenitor cells, respectively. The inhibitory and enhancing affects appeared to be mediated by independent pathways, the former requiring cell-cell contact, major histocompatibility complex (MHC) class II expression on marrow cells and involving transforming growth factor beta (TGF-beta), whereas the latter required interleukin (IL)-9 and was not contact dependent. Strikingly, we observed that in addition to regulating hematopoietic activity in normal primary BM cells, Tregs were also capable of suppressing colony forming activity by the myelogenous leukemia cell line NFS-60. Furthermore, studies involving endogenous Treg manipulations in-situ (i.e. depletion of these cells) resulted in elevated overall myeloid colony activity (CFU-IL3) and diminished colony numbers of erythroid precursors (CFU-E) in recipients following BMT. Consistent with these results, it was found that upon co-transplant with limiting numbers of bone marrow cells, exogenously added Treg cells exert in-vivo regulation of myeloid and erythroid CFU activity during the initial weeks post-transplantation. This regulation of hematopoietic activity by freshly generated Tregs upon transplantation led to the elaboration of a second hypothesis; following lethal total body irradiation (TBI) the host microenvironment facilitates regulatory T cell activation/effector function. Substantial evidence has accumulated in support of this hypothesis, for example we demonstrate up-regulation of surface molecules such as GARP and CD150/SLAM, which have been previously reported as indicators of Treg activation following TCR signaling and co-stimulation, occurs in donor (reporter) Treg populations. Acquisition of an activated phenotype and hence of effector/modulatory function is consistent with the previous in-vivo observations, indicating that both recipient and donor Treg cells can influence hematopoietic progenitor cell activity post-transplant. Finally, the present studies may be of great relevance in the emerging field of Treg cell based immunotherapy for prevention and/or treatment of HSCT complications.