Thesis (M.A.)--University of Wisconsin--Madison, 1965. / eContent provider-neutral record in process. Description based on print version record. Bibliography: l. 83-89.

Rhodopsin-linked retinitis pigmentosa (RP) is the most common form of autosomal dominant RP, an inherited retinal degeneration, in which rod degeneration is followed by secondary cone loss leading to loss of vision and blindness. The overall objective of this work was to develop an optimized gene replacement therapy, delivering the rhodopsin gene for rhodopsin- linked RP and establish whether secondary cone loss can be delayed. A fast-acting single mutant serotype 8 self-complementary adeno-associated virus vector was produced containing the human rhodopsin promoter and the human rhodopsin coding sequence. In vivo studies in rhodopsin knockout mouse showed that the vector administration led to widespread and robust expression of the transgene. Subretinal injection of the vector into three-week pups of rhodopsin knockout mice with cones expressing green fluorescent protein showed restoration of rod-derived electroretinogram (ERG) responses, and preservation of cone- driven ERG responses three months after injection. Similarly, the longitudinal follow-up with confocal scanning ophthalmoscopy found preservation of fluorescent cones up to three months after injection. Overall, these data provided evidence that the designed vector resulted in significant benefit to rod photoreceptors as well as in delay in secondary cone degeneration and built a basis for future use of this vector on dominant models of RP.

Résumé Les protéines XMAP215/ch-TOG appartiennent à une famille de protéines associées aux microtubules (MAPs), bien conservée tout au long de l'évolution, la famille XMAP215/Dis1. Cette famille joue un rôle dans la régulation du cytosquelette des microtubules (MT), en particulier pendant la division cellulaire. Chez l'humain, ch-TOG est la protéine surexprimée dans les tumeurs du colon et du foie, une protéine qui provient de cellules blastiques et de plusieurs formes de cancer. Certaines protéines XMAP215/ch-TOG ont été retrouvées dans différentes localisations cellulaires, toujours reliées aux MTs, donnant origine à une activité spécifique. Cependant, la localisation exacte de XMAP215/ch-TOG ainsi que son activité restait à être déterminées. Dans ce contexte scientifique, nous avons développé une série d'anticorps monoclonaux (mcAB) qui nous ont permis d'identifier deux populations différentes de la famille des protéines XMAP215/Dis1. Les images de microscopie confocale des cellules fixées ont montré une première localisation, la colocalisation bien connue XMAP215-microtubulaire (MT-XMAP215) qui s'observe pendant l'interphase et pendant la mitose (fuseau mitotique). Une deuxième localisation a été identifiée sur le bout plus des MTs, donnant XMAP215/ch-TOG comme faisant parti de la famille des protéines de bout plus (+TIPs). Cette deuxième colocalisation a été identifiée comme +TIP XMAP215/ch-TOG. La +TIP XMAP215 est la protéine la plus distale du bout des MTs. La hiérarchie a été établie en faisant la comparaison de la localisation de XMAP215/ch-TOG avec les protéines les plus connues du bout plus, telles qu'EB1, CLIP170 et p150Glued. Dans l'extrait mitotique de Xenopus laevis, les images obtenues in vivo par la microscopie de fluorescence par réflexion totale interne (TIRF) ont permis d'identifier une +TIP XMAP215 présente au bout des MTs qui polymérisent et dépolymérisent. Les images de microscopie cryo-électronique (Cryo-EM) ont montré une activité spécifique de la population +TIP XMAP215. Dans les solutions de tubuline pure, XMAP215 induit la formation de structures au bout des MTs, cette activité est compatible avec les mécanismes de croissance des MTs. Sur la base de nos résultats, nous proposons un modèle où XMAP215 se charge des dimères de tubuline en devenant une structure de type protofilament. Cette structure se lie au bout du MT en utilisant son domaine C-terminal, en rajoutant les dimères de tubuline et aussi certainement en participant à la fermeture de la structure microtubulaire même. La protéine interviendrait donc dans la dépolymérisation et aurait un rôle dans le mécanisme de dépolymérisation contrôlée. Une fois que l'addition de tubuline a eu lieu, la +TIP XMAP215 pourrait évoluer en MT-XMAP215, la forme la plus connue de la protéine qui a été associée au trafic des granules d'ARN. Mots cles : XMAP215, ch-TOG, microtubules, anticorps monoclonaux. / Summary XMAP215/ch-TOG are members of an evolutionary conserved family of microtubule-associated proteins (MAPs), the XMAP215/Dis1 family. This family of proteins plays a key role in the regulation of the microtubule (MT) cytoskeleton, particularly during the cell division. In humans, ch-TOG is the colon-hepatic tumor overexpresed gene, a protein whose sequence was originally reported from blastic cells and from several forms of cancer. A few members of the XMAP215/ch-TOG family have been found to be present in different cell localizations, always MT-related, perhaps providing in this way a selected activity. However, the XMAP215/ch-TOG exact localization and activity has remained as a theory to be probed. In this scientifical context, we developed a series of monoclonal antibodies (mcABs) that allow us to identify two different populations of the XMAP215/ Dis1 family of proteins. Confocal images of fixed cells revealed a first and well known XMAP215/ch-TOG population, a microtubular-XMAP215 (MT-XMAP215), co-localizing with microtubules (MTs) in interphase and mitotic spindle. A second localization was identified at the tips of growing MTs, placing XMAP215/ch-TOG as one more member of the known microtubule plus end tracking proteins (+TIPs). This second population was identified as the +TIP XMAP215/ch-TOG. The + TIP XMAP215 is the most distal +TIP protein at the tip of MTs. The + TIPs hierarchy was established comparing the XMAP215/ch-TOG localization with other well known + TIPs proteins such as EB1, CLIP170 and p150Glued. In the Xenopus laevis mitotic egg extract, the Total Internal Reflection Fluorescence (TIRF) In vivo images, identified a +TIP XMAP215 that is present at the tip of polymerizing and depolymerising MTs. Cryo-electron microscopy (Cryo-EM) images probed a selective activity for the +TIP XMAP215 population. In pure tubulin solutions XMAP215 shown to promote the formation of outwardly sheet structures at the tip of MTs, compatible with growing mechanisms in MTs. Based in our results we propose a model where free XMAP215 is previously loaded with tubulin dimers to become a protofilament-like structure. This structure joins the MT tip using its C-terminal domain, not only adding the tubulin dimmers but also perhaps participating in the MT sheet closure. The possibility that the protein participates in the MT depolymerization could be associated to a “controlled” depolymerization mechanism. Once the tubulin addition has taken place, the +TIP XMAP215 protein could evolve to a MT-XMAP215, the well know form of the protein that has been related to the RNA granules traffic. Key words: XMAP215, ch-TOG, microtubules, monoclonal antibodies.

Les plantes adaptent leur croissance en fonction des facteurs environnementaux dont la gravité qui est un facteur constant. Une modification de l’orientation de la plante par rapport à l’axe de la gravité, i.e. une inclinaison induit une réponse de redressement : le gravitropisme. Pour les parties aériennes le gravitropisme est négatif, c’est-à-dire que les plantes vont adapter leur croissance dans la direction opposée à la gravité. Chez les arbres, le redressement est assuré par à la formation asymétrique d’un bois aux propriétés physico-chimiques particulières appelé le bois de réaction. Des récepteurs kinases pourraient participer à la perception et à la réponse précoce au stimulus gravitropique qu’est l’inclinaison. Parmi ces familles protéiques, les kinases associées à la paroi (WAK) sont des candidats intéressants. La liaison de ces protéines à la paroi permettrait de percevoir les déformations qui sont supposées se produire par l’inclinaison de la tige. Nous avons alors identifié et caractérisé pour la première fois la famille WAKs chez une espèce ligneuse, le peuplier, qui est composée de 175 membres. L’étude d’accumulation des transcrits WAKs a permis d’identifier les gènes WAKs qui s’expriment dans la tige puis l’expression de ces gènes a été suivie lors d’une cinétique après inclinaison. Il s’avère que les gènes WAKs sont faiblement exprimés et que 25% des gènes présentent une expression différentielle après inclinaison. Ces données transcriptomiques suggèrent que les WAKs participeraient aux événements cellulaires précoces après l’inclinaison de tiges chez le peuplier. Enfin, une étude plus approfondie a été initiée sur PtWAK70 qui est localisée dans le jeune xylème et le phloème secondaire. Nous avons également généré des outils moléculaires dont l’objectif est d’identifier les interacteurs potentiels de la partie apoplastique de PtWAK70. / Plants adapt their growth to environmental factors whose gravity is a continuous one. A modification of plant orientation by tilting leads to a straightening response: gravitropism. For aerial parts, plants will adapt their growth upward (negative gravitropism). In trees, straightening is accomplished through asymmetric formation of reaction wood which exhibits modifications in its physicochemical properties. Receptors-like kinases could play a role in both perception and early response to a gravitropic stimulus. Among them, Wall-Associated Kinases (WAKs) are interesting candidates because they are bind to the cell wall. They could detect wall deformations that are supposed to occur after tilting of the stem. We identified and characterized for the first time in a woody species (poplar) the largest WAKs family with 175 members. An extensive study of WAKs transcripts accumulation was carried out to identify genes expressed in woody stem. These genes expressions were analyzed during a kinetic after tilting. WAKs genes were overall weakly expressed but 25% of analyzed genes showed a modulation in their transcript accumulation after tilting. This suggests they could play a role in early events after tilting of poplar stems. Lastly, a deeper study was initiated on PtWAK70 which is localized in young xylem and secondary phloem. We generated molecular tools to identify potentials interactors of PtWAK70 apoplastic part.

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Severe infections, trauma or major surgery can each cause a state of systemic inflammation. These causes for systemic inflammation often coexist and complicate each other. Mechanical ventilation is commonly used during major surgical procedures and when respiratory functions are failing in the intensive care setting. Although necessary, the use of mechanical ventilation can cause injury to the lungs and other organs especially under states of systemic inflammation. Moreover, a course of mechanical ventilator therapy can be complicated by ventilator-associated pneumonia, a factor greatly influencing mortality. The efforts to avoid additional ventilator-induced injury to patients are embodied in the expression ‘protective ventilation’. With the use of pig models we have examined the impact of protective ventilation on systemic inflammation, on organ-specific inflammation and on bacterial growth during pneumonia. Additionally, with a 30-hour ventilator-associated pneumonia model we examined the influence of mechanical ventilation and systemic inflammation on bacterial growth. Systemic inflammation was initiated with surgery and enhanced with endotoxin. The bacterium used was Pseudomonas aeruginosa. We found that protective ventilation during systemic inflammation attenuated the systemic inflammatory cytokine responses and reduced secondary organ damage. Moreover, the attenuated inflammatory responses were seen on the organ specific level, most clearly as reduced counts of inflammatory cytokines from the liver. Protective ventilation entailed lower bacterial counts in lung tissue after 6 hours of pneumonia. Mechanical ventilation for 24 h, before a bacterial challenge into the lungs, increased bacterial counts in lung tissue after 6 h. The addition of systemic inflammation by endotoxin during 24 h increased the bacterial counts even more. For comparison, these experiments used control groups with clinically common ventilator settings. Summarily, these results support the use of protective ventilation as a means to reduce systemic inflammation and organ injury, and to optimize bacterial clearance in states of systemic inflammation and pneumonia.

<p> </p><p>In previous studies the carboxyl-terminus of microtubule-associated protein 1S was shown to interact with the <em>N</em>-methyl-D-aspartate receptor subunit NR3A (Eriksson <em>et. al.</em>)¹.  In this study, interactions between three truncations of the microtubule-associated proteins 1B and one truncation of the microtubule-associated protein 1S carboxyl-terminus and the <em>N</em>-methyl-D-aspartate receptor subunits NR1 and NR3A were examined. The study showed that an interaction occurred between amino acids 2167 to 2365 of the microtubule-associated protein 1B and NR3A.  That region of microtubule associated protein 1B corresponds to a microtubule-binding region in the light chain. It has been shown in earlier studies (Reviewed in Halpain S. <em>et a1²</em>, Riederer, BM<em>.  et.al³</em>.) that the light chain is a active part of the protein that have been post translational cleaved. The MAP 1 proteins are present in all tissue but has higher concentrations in the Post Synaptic Density of neurons in the central nervous system.  The <em>N</em>-methyl-D-aspartate receptors are present in glial cells and in the dendritic shafts of the central nervous system neurons (Eriksson <em>et. al.</em>)¹. The diseases were these proteins may play a part is mainly memory destructive diseases such as Alzheimers disease and in muscular dystrophy, but these assumptions are still being speculated.</p><p> </p>

Viruses have evolved a multitude of mechanisms, which allow immune evasion in both initial and persistent infection. Understanding the intricacies of these pathways is essential to our future ability to combat primary and reactive viral infections. The murine gamma-2 herpesvirus 68 (γHV68) encodes a protein mK3, which targets Major Histocompatibility Complex (MHC) class I heavy chains for ubiquitin-dependent proteasome degradation. MK3 is able to target and ubiquitinate MHC class I by binding to Endoplasmic Reticulum (ER) resident proteins tapasin, Transporter associated with antigen processing (TAP) 1 and TAP2 that are subunits in the complex known as the peptide-loading complex (PLC). The aforementioned characteristics of mK3 make this novel protein an excellent vehicle to study MHC class I assembly, immune evasion, and ER associated degradation (ERAD). Deepening our understanding of class I assembly and viral immune evasion will impact both the fields of immunology and virology. The homology between γHV68 and many of the human γ-herpesviruses makes this an indispensable model to clarify mechanisms that can then be applied to a broader spectrum of viruses. ERAD, an emerging field of study, is known to play a key role in numerous cellular housekeeping pathways as well as a number of disease states. Illuminating the mechanisms implicated in the mK3-mediated ubiquitination of MHC class I, specifically requirements for substrate recognition and degradation, will yield an increased understanding of cellular pathways involved in ERAD. The studies in this dissertation aim to expand our understanding of the relationship between mK3 and adapter proteins TAP/tapasin as well as mK3 and mK3-targeted substrates. The results show that TAP/tapasin act as adapter proteins by recruiting substrates for mK3. Further, mK3 ubiquitinates TAP/tapasin-associated substrates as long as the substrates have a tail greater than 6aa in length and the tail possesses an ubiquitin acceptor residue (lysine, serine or threonine). These studies also confirm that location of a protein within the PLC will determine the substrate’s susceptibility to mK3-mediated degradation. In the field of ubiquitin ligases and ERAD, these studies lend support to the concept of adapter mediated substrate recruitment.

Ventilator-associated pneumonia (VAP) is a healthcare-associated infection (HAI), or more specifically, a healthcare-associated pneumonia (HAP) that can lead to significant morbidity and mortality in hospitalized patients that are being mechanically ventilated. There are established evidence-based guidelines in existence designed to reduce or eliminate VAP from occurring and when properly maintained have been shown to reduce the incidence of VAP. Nurses are at the frontline adhering to the VAP protocol through its integration into their workflow. It is yet unknown what elements of the protocol and workflow contribute to a successful VAP reduction in occurrence and increased patient safety. This program evaluation project, guided by an adapted Systems Engineering Initiative for Patient Safety (SEIPS) model, takes a human-factors approach towards answering these questions. It specifically examines the VAP protocol in a large urban southwestern teaching hospital to evaluate program effectiveness using a human factors approach. Building on the work of Carayon, et al. (2006) and Jansson et al. (2013), I present the findings from this program evaluation project using an adapted SEIPS model that sought to evaluate the VAP prevention program from a human factors perspective addressing the following aims: Aim 1. Determine the effectiveness of using the adapted SEIPS model to evaluate a VAP quality improvement (QI) project; Aim 2. Evaluate a VAP QI program taking a human factors approach; and Aim 3. Using the adapted SEIPS model, identify elements of the VAP bundle that nurses perceive as strength and weaknesses. The project was completed with the following findings: Based on this work the adapted SEIPS model demonstrates usefulness for evaluating QI projects. It would be interesting to continue this work with QI projects to see how well it performs.