Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), which is one of the most important diseases impacting the US catfish industry. Though this disease has been very common, progress has been slow to find an economical and practical treatment method. Our long-term goal is to determine the mechanisms of E. ictaluri virulence in ESC. The overall objective of this study was to identify E. ictaluri genes required for host encounter and serum resistance and to determine their roles in pathogenesis. The central hypothesis is that E. ictaluri must differentially regulate its genes to invade fish and evade host defenses, thus, mutation of these differentially expressed genes (DEG) should cause attenuation of E. ictaluri virulence. To test this hypothesis, we first determined the global gene expression patterns of the wild type (wt) E. ictaluri 93-146 and EiAKMut02 mutant during catfish encounter and serum exposure using microarray analysis. Results indicated that in E. ictaluri wt, 377 and 16 DEGs were identified during host encounter and serum exposure, respectively. In EiAKMut02, 82 and 296 DEGs were identified during host encounter and serum experiment. Through functional analysis using Blast2GO, PSORTb, Host Pathogen Interaction Database (HPIDB), and Microbe Virulence Database (MVirDB), 38 DEGs in 9 KEGG pathways have been identified as potential virulence factors. The KEGG pathways represented were 1) bacterial secretion system including T3SS and T6SS, 2) ABC transporters including cystine transport system, iron complex transport system, d-methionine transport system, arginine transport system, thiamine transport system, and molybdate transport system, 3) protein export, 4) flagellar assembly, 5) two-component system, 6) bacterial chemotaxis, 7) ascorbate and aldarate metabolism, 8) phosphotransferase system, and 9) metabolic pathways. In order to understand their role in the E. ictaluri virulence, selected DEGs were inrame deleted by allelic exchange, and their virulence and efficacy were characterized in channel catfish fingerlings. Our results showed that the virulence of E. ictaluri ssaV and yscR mutants was completely attenuated while their efficacies were moderate in catfish fingerlings. These results support that the T3SS and T6SS, ABC transporters, protein export, and flagella seem to be important in E. ictaluri virulence.

22q11.2 deletion syndrome (22q), also known as Velocardiofacial Syndrome or DiGeorge Syndrome, is one of the most common genetic syndromes with an incidence of 1 in 2500 to 1 in 4000 (Wang et al., 2009). It is the most identified human chromosomal microdeletion syndrome to date (Wang et al., 2009). 22q is associated with a wide spectrum of clinical features including various palate, cardiac, and immunological abnormalities (Lynch et al., 1995; Wang et al., 2009). 22q is also the most common genetic cause of velopharyngeal dysfunction (VPD). Posterior cranial fossa (PCF) and cervical spine variations may influence velopharyngeal (VP) port closure. Although some studies have analyzed PCF size in individuals with 22q, there has not been extensive analysis of skull base anomalies and their correlation to velopharyngeal depth. The purpose of this study was to examine PCF measures and their effects on VP dimensions in children with 22q using a non-sedated imaging protocol. 34 participants, 17 with 22q and 17 with normal VP anatomy (age range: 4-12 years) completed the study. Participants were imaged using a 3D anatomical scan. MRIs were transferred into Amira 6.4 Visualization Volume Modeling software. Linear and angular measures were obtained in the sagittal image plane on the 3D MRI scans. Measures included: distance from the palatal plane to C1, pharyngeal depth, anterior cranial base angle, posterior cranial base angle, length of the clivus, McRae line and supraocciput of the PCF, angle of clivus, and the PCF angle formed by the McRae line and the supraocciput. It is hypothesized that shorter clivus length and smaller PCF angle between McRae line and supraocciput noted in individuals with 22q DS could be related to larger pharyngeal depth, which contributes to hypernasality typically seen in 22q. Results from this study indicate that children with 22q demonstrate larger pharyngeal depth, a more obtuse anterior cranial base angle (NSB angle), a more acute posterior cranial base angle (SBO angle), shorter length of the clivus, longer supraocciput length, and a more obtuse angle of clivus. The NSB angle was positively correlated with pharyngeal depth while the SBO angle was negatively correlated with pharyngeal depth. The angle of clivus was positively correlated with both pharyngeal depth and resonance severity.

Product deletion is the end of a product’s life cycle, when it ceases to be produced and is removed from the portfolios of those companies which used to sell them. This project seeks to depict how product deletion is done by manufacturers in Sweden. Product deletion is a scarcely studied subject, which leads to problems such as shortage of existing literature and disconnectedteachings from different perspectives, making the formation of a complete picture problematic.What seems to unify the literature is that product deletion is a valuable tool that is not well enough explored. Through qualitative, semi-structured interviews with representatives from three different companies, a depiction of the relevant aspects of product deletion is formed as it is today. Some aspects were consistent between sources, while others highlighted differences. The similarities were many and considered to strengthen one another as well as the existing literature. The differences were seen as an opportunity for discussion and further studies. The sources agreed that product deletion is of high priority to the operation of a manufacturer. Each participating company therefore spent significant resources to ensure an effective product deletion process. Relevant experience, combined with monetary calculations are highly prized as the backbone of the process. The companies, as well as existing literature, also describe a divide between faster and slower decisions for product deletion. Products which were deemed more important took longer time and more careful consideration before a decision was made than product which occupied less of the company’s focus. Intersections between the groups were rare, showing a clear polarization.Product deletion regularly occurs in tandem with, or as a consequence of, product development. When a new product is made it often replaces an old one, meaning the total product range rarely grows or shrinks in size. Other possible driving forces for product deletion are legal regulations, which might prohibit certain products. Product deletion typically starts as a relatively informal process initiated by monetary calculations and are later applied practically in a more structuredand systematic manner. Preserving investments made in the deleted product and using them for future operations is the primary focus. Considerable contrast was seen in the relationship with product deletion, the level of standardization and how the process has changed historically for each company. Two companies viewed product deletion mainly as a mellow subject and a symptom of error, while the third considered it a great tool for improving their work. This third company also had the most standardized process for product deletion of the three. However, historically, each company had an entirely different development of the process. The clearest possible conclusion would be that each industry, company, and product is unique and needs to be treated as such when considering product deletion. It also seems that companies are not allocating enough resources to product deletion in general. To further our understanding of product deletion a more comprehensive consensus is needed, as well as a clear distinction between meaningful parameters which makes up the holistic understanding of the subject. Through a combination of quantitative and qualitative methodology, each such parameter should be examined in relation to one another. Examples of such parameters could be the different stages of product deletion, quantitative parameters of the product or manufacturer or the culture, structure or relationships of the companies involved. In short, a tighter focus with a wider sample range could give experts on the subject the details they need to construct and refine it.

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The process of xylem tracheary element differentiation involves the coordination of vascular cambium activity, cell fate determination, cell expansion/elongation, secondary wall synthesis, programmed cell death, and cellular autolysis. The end result of tracheary element differentiation is a cellular corpse lacking a protoplast and consisting of a thickened cell wall composed mostly of lignin and cellulose. Little is known about the genetic mechanisms regulating the process of tracheary element differentiation. XCP2 expression localizes to tracheary elements according to two independent methods of analysis: promoter reporter experiments and immunogold localization by electron microscopy. XCP2 may be involved in catalyzing the degeneration of the protoplast during the final autolytic stages of tracheary element differentiation. To this date XCP2 function has not been directly demonstrated. In principle, any tracheary element-specific markers can be linked to upstream regulatory genes with roles in tracheary element differentiation. To develop the XCP2 promoter as a tool for identification of transacting factors, a promoter deletion analysis was carried out. Utilizing information from 5â and 3â deletion constructs, a 70-bp region upstream of the XCP2 translational start site is both necessary and sufficient for TE-specific expression of the UidA reporter gene. Mutational analysis of the ACTTTA element at position -113-bp strongly suggests it is a cis element required for XCP2 expression. In silico analysis of an 18-bp promoter region located within 200-bp of the translation start site and including the ACTTTA element revealed high indentity shared between xylem-specific XCP2 homologs from Zinnia elegans, Populus trichocarpa, and XCP1 from Arabidopsis thaliana. / Master of Science

Intellectual disability (ID) affects approximately 1-3% of the population and is defined as having an IQ below 70 as well as a significant limitation in adaptive behavior. The implementation of chromosomal microarrays (CMA) into the field of clinical genetics has revolutionized the ability to find genetic aberrations responsible for different genetic disorders. Importantly. these technologies have allowed several new microdeletion and microduplication aberrations to be identified that otherwise would have escaped detection using more conventional methods. Finding the genetic etiology of a syndrome and its association to the phenotype is paramount to better health care, provision of tailored therapy, presymptomatic screening, accurate prognosis, recurrence risk evaluation and in some cases prenatal testing. Despite the plethora of new information available, there are still a number of clinical and genetic features we do not fully understand. The aim of this work was to identify regions and syndromes associated with ID by CMA analysis and to make a detailed clinical description of the affected patients’ phenotype. In paper I we studied the 22q11.2 duplication syndrome and presented two familial cases with a description of both their genotype and phenotype. Additionally, 36 cases harboring the duplication were reviewed to further delineate the phenotype of the syndrome. In paper II, we revealed two unrelated patients with a deletion at 6q14.1-q15 and a distinct phenotype. Together with one previously reported patient our study suggests that a novel, clinically recognizable microdeletion syndrome exists in these patients. In paper III the phenotype and genotype of six unrelated patients with partially overlapping microdeletions at 10p12.31-p11.21 were described. Taken together with a previously reported patient we propose that these findings represent a new contiguous gene syndrome. In paper IV, two sisters; one presenting with two tandem interstitial duplications and the other a large deletion over the same region (6q13-q16) were reported. The reason for the CNVs was a maternal de novo translocation. This is the first case describing the genotype and phenotype of this duplicated region at 6q13-q16. In conclusion, four different genetic aberrations involved in the etiology of ID and their corresponding phenotypes and candidate genes have been characterized.

Smith-Magenis syndrome (SMS) is a multiple congenital abnormalities intellectual disability syndrome that results from a deletion of chromosome 17p11.2 or mutation of the retinoic acid inducted one gene (RAI1). SMS is characterized by a multitude of phenotypic features including craniofacial defects, short stature, obesity, intellectual disability, self-abusive behavior, sleep disturbance and behavioral abnormalities. Interestingly, although SMS is a clearly defined syndrome with a known molecular change at its foundation, ~40% of all candidate cases sent to the Elsea lab for evaluation do not have a mutation or deletion of RAI1. We hypothesize that at least one other locus must be responsible for this Smith-Magenis-like (SMS-like) phenotype. To address this hypothesis, we first compiled a cohort of 52 subjects who had been referred to the Elsea lab for a clinical diagnosis of SMS. Once these individuals were confirmed to not have an RAI1 mutation or deletion, their phenotypes were compiled and statically analyzed to distinguish whether SMS and SMS-like cohorts are different in the prevalence of the core phenotypes of SMS such as, but not limited to, sleep disturbance, self-abusive behavior and developmental delay. SMS-like and SMS cohorts are not different in prevalence for these core features. Next, all SMS-like subjects were sent for whole genome array comparative genomic hybridization (aCGH) to identify duplications or deletions of each individual’s genome which contribute to the phenotype observed. We identified 6 pathogenic copy number variants (CNVs) in six individuals which contribute directly to the clinical phenotype, including two del(2)(q37). This study enabled us to draw relationships between SMS and other syndromes that had never been appreciated before and helped to identify pathways in which RAI1 may function. Using the data from our SMS-like study we were able to further characterize two known syndromes; Deletion 2q37 syndrome (brachydactyly mental retardation syndrome) and deletion 2q23 syndrome. With regard to deletion 2q37, syndrome we used genomic data from known and new deletion 2q37 subjects to refine the critical region to one gene: the histone deacetylase 4 gene (HDAC4). Using both clinical and molecular clues, we were able to identify one subject from our SMS-like cohort who has an insertion in HDAC4 which results in a premature stop codon. We conclude from this study that mutation of HDAC4 results in brachydactyly mental retardation syndrome. With regard to deletion 2q23 syndrome there were only five known cases in the published literature to which we were able to add two more. Using as similar approach to our del2q37 study we refined the critical region for this syndrome to one gene, the methyl binding domain 5 gene (MBD5). Using a molecular and clinical approach we were able to conclude that haploinsufficiency of MBD5 results in the core phenotypes seen in del2q23 syndrome including microcephaly, intellectual disabilities, severe speech impairment, and seizures. Using all the data generated from the three previous studies we set out to characterize the molecular function of RAI1. We hypothesize that RAI1 is a transcription factor that regulates gene expression of core genes involved in development, neurological function, and circadian rhythm. Using a ChIP-chip based approach we identified 257 transcripts we believe RAI1 regulates. Following up on these transcripts, using in vitro and in vivo methods, we have been able to conclude that RAI1 is a positive regulator of CLOCK, the master regulator of the central circadian cycle. Taken together, these studies have given us insight into the specific molecular changes that contribute to SMS and SMS-like syndromes. We have unveiled pathways and genes which are important to normal human development and behavior and identified novel functions of RAI1. These studies will provide the foundation for the future discovery of the pathways affected.

No description available.