Together with the advancements in animal cloning, the ability to efficiently target specific genes in somatic cells would greatly enhance several areas of research. While it has been possible for quite some time to target specific genes in the germ cells of mice, the advancements in somatic cell gene targeting has been slowed for two main reasons. First, the finite lifespan of somatic cells, due mainly to the inability of the somatic cells to regenerate or maintain their telomeres, poses a major problem given the lengthy selection process needed to identify a targeting event. The second problem is the overall inefficiency of homologous recombination. A double strand break or introduction of foreign DNA into a cell can be processed either through the homologous recombination or non-homologous end joining pathways. Of these two, non-homologous end joining is dominant in somatic cells. A two plasmid recombination system was used to study the effects of the manipulation of several non-homologous end joining and homologous recombination pathway molecules on the rates of homologous recombination in porcine fetal fibroblasts. In addition, the effect of telomerase expression, cell synchrony, and DNA nuclear delivery was examined. Results indicate a strong positive relationship between inactivation of p53, cell synchronization, and efficient DNA nuclear delivery in enhancing the rate of homologous recombination. These findings were then applied to an actual locus in the pig, the α1,3 galactosyltransferase gene. Results from these transfections are compared to published accounts of successful targeting at this locus and possibilities for the differences found are discussed.

Bone metabolism is regulated by endocrine and paracrine signalling molecules influencing bone cells in the continuously remodelling bone tissue. These molecules include a variety of osteotropic stimulatory and inhibitory cytokines. Degradation of alveolar bone in periodontal disease is believed to be a result of local release of such osteotropic cytokines, although the relative importance of particular cytokines and their cellular origin is currently unknown. The aim of the present project was to study if, and how, pro-inflammatory cytokines in the interleukin-6 (IL-6) family of cytokines, and anti-inflammatory IL-4 and IL-13 type of cytokines, can affect osteoclast differentiation and bone resorption. Additionally, the objective was to study if gingival fibroblasts may influence alveolar bone resorption through secretion of IL-6 type cytokine release and if the secretion is regulated by pro-inflammatory as well as anti-inflammatory mediators such as IL-4 and IL-13. IL-6 in combination with its soluble receptor (sIL-6R) was found to stimulate mouse calvarial bone resorption. Similarly, two other IL-6 family members, leukemia inhibitory factor (LIF) and oncostatin M (OSM) were found to stimulate bone resorption. The stimulatory effect on bone resorption induced by the three cytokines was associated with increased expression of receptor activator of NF- κB ligand (RANKL), a cytokine which is essential in osteoclast formation and activation through binding to receptor activator of NF- κB (RANK) on osteoclastic cells. The interaction between RANKL and RANK can be inhibited by binding of the decoy receptor osteoprotegerin (OPG) to RANKL, and the expression of OPG was also regulated by IL-6, LIF and OSM (Paper I). The two related cytokines IL-4 and IL-13 were found to inhibit osteoclastogenesis and mouse calvarial bone resorption by mechanisms involving a decreased RANKL/OPG ratio in osteoblasts and decreased RANK expression in osteoclastic cells. The results further demonstrated that IL-4 and IL-13 exert their effects on both osteoblasts and osteoclasts by a mechanism involving the transcription factor signal transducer and activator of transcription 6 (STAT6) (Paper II). Constitutional expression of IL-6, LIF and another member of the IL-6 family of cytokines, IL-11, was demonstrated in human gingival fibroblasts. IL-6 type cytokine expression levels were found to be enhanced by IL-1β and tumour necrosis factor-α (TNF-α) (Paper III), whereas IL-4 and IL-13 inhibited IL 11 and LIF release from gingival fibroblasts (Paper IV). In conclusion, IL 6 type cytokines were found to be stimulators and IL-4 and IL-13 inhibitors of bone resorption in vitro via mechanisms involving RANK/RANKL/OPG interactions. Additionally, gingival fibroblasts were able to secrete several cytokines in the IL-6 family. Secretion was further enhanced by pro-inflammatory mediators and inhibited by IL-4 and IL- 13. These findings support the view that resident cells may influence the pathogenesis of periodontal disease through osteotropic cytokine production.

Tissue engineering applies principles of biology and engineering to the development of functional substitutes for damaged or lost tissues. Tools for the neo-generation of tissue in tissue engineering research include cells, biomaterials and soluble factors. One main obstacle in tissue engineering is the limited availability of autologous tissue specific progenitor cells. This has led to interest into using autologous cells with stem cell plasticity. Bone marrow derived stem cells were the first adult stem cells shown to have multilineage potential. Since, several reports have been published indicating that cells from other tissues; fat, muscle, connective tissue e.g., possess potential to differentiate into lineages distinct from their tissue of origin. The optimal cell type for use in tissue engineering applications should be easy to obtain, cultivate and store. The human dermal fibroblast is an easily accessible cell source, which after routine cell expansion gives a substantial cell yield from a small skin biopsy. Hence, the dermal fibroblast could be a suitable cell source for tissue engineering applications.The main aim of this thesis was to investigate the differentiation capacity of human dermal fibroblasts, and their possible applications in bone and cartilage tissue engineering applications. Human dermal fibroblasts were shown to differentiate towards adipogenic, chondrogenic, and osteogenic phenotypes upon subjection to specific induction media. Differentiation was seen both in unrefined primary cultures and in clonal populations (paper I). Fibroblasts could be used to create three-dimensional cartilage- and bone like tissue when grown in vitro on gelatin microcarriers in combination with platelet rich plasma (paper II). 4 weeks after in vivo implantation of osteogenic induced fibroblasts into a fracture model in athymic rats, dense cell clusters and viable human cells were found in the gaps, but no visible healing of defects as determined by CT-scanning (paper III). After the induction towards adipogenic, chondrogenic, endotheliogenic and osteogenic lineages, gene expression analysis by microarray and quantitative real-time-PCR found several master regulatory genes important for lineage commitment, as well as phenotypically relevant genes regulated as compared to reference cultures (paper IV). In conclusion, results obtained in this thesis suggest an inherent ability for controllable phenotype alteration of human dermal fibroblasts in vitro. We conclude that dermal fibroblasts could be induced towards adipogenic, chondrogenic, endotheliogenic or osteogenic novel phenotypes which suggest a genetic readiness of differentiated fibroblasts for lineage-specific biological functionality, indicating that human dermal fibroblasts might be a suitable cell source in tissue engineering applications.

Fibroblasts continuously strive to organize and compact the surrounding extracellular matrix (ECM). Recent data suggest that this cellular contractility controls interstitial fluid homeostasis in loose connective tissues (CT). The aim of this thesis was to study the effects of prostaglandins on fibroblast contractility and to investigate whether fibroblasts in the interstitial CT surrounding the human endolymphatic duct (ED) can modulate inner ear fluid pressure and endolymph resorption. Paper I shows that prostaglandin E1 (PGE1) and prostacyclin inhibit fibroblast-mediated collagen matrix compaction in vitro and lower the interstitial fluid pressure in vivo in rat dermis. Paper II demonstrates that the inhibition of collagen matrix compaction by PGE1 is protein kinase A-dependent. Furthermore, PGE1 induces a complete but reversible actin depolymerization in human dermal fibroblasts by affecting the phosphorylation state of regulatory actin-binding proteins. Paper III describes that the cells of the interstitial CT encompassing the human ED are organized in a network based on intercellular- and cell-ECM contacts. Paper IV shows that two distinct cell phenotypes populate this interstitial CT: one expressing the lymph endothelial marker podoplanin and the other a fibroblast marker. Furthermore, CT cells isolated from human ED tissues exhibited the same tissue compacting properties in vitro as dermal fibroblasts. In conclusion, PGE1 inhibits fibroblast contractility by interfering with the stability and dynamics of the actin cytoskeleton, which leads to a loss of integrin-mediated adhesion to the ECM. These mechanisms are supposedly involved in edema formation in skin during inflammation and might be involved in the formation of endolymphatic hydrops in the inner ear of patients with Ménière’s disease.

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes can induce osteoblast proliferation in vitro, but do so only when direct cell-to-cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of megakaryocytes with another cell-type of mesenchymal origin - the fibroblast. Our findings implicate the involvement of fibronectin/RGD-binding integrins including á3â1 (VLA-3) and á5â1 (VLA-5) as well as glycoprotein IIb (CD41), all of which are known to be expressed on megakaryocyte membranes. Furthermore, we demonstrate that IL-3 can enhance megakaryocyte-induced osteoblast activation in vitro, as demonstrated in the megakaryocyte-fibroblast model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic-mesenchymal cell activation are mechanistically analogous.

The human gammaherpesviruses EBV and KSHV realize their oncogenic potential during latent infection. The species specificity of the human gammaherpesviruses has hindered the study of latency in animal models. Murine gammaherpesvirus MHV-68 (MHV-68) may be used as a representative gammaherpesvirus for the study of latency. The goal was to establish an in vitro model of MHV-68 latency using replication defective MHV-68. ORF50 has been identified as the major viral trans-activator essential for entry into the lytic replication cycle and necessary and sufficient for reactivation of MHV-68 virus from latency. ORF50 null mutants (A50) can theoretically be used to infect cells in vitro to facilitate an analysis of virus gene expression and episome maintenance during latency. In this project A50 mutants containing the luciferase or green fluorescence protein (GFP) under OW50 promoter control were used to infect a variety of cell types. 3T3 fibroblasts are a permissive cell line and were used for an initial characterization of the ability of A50 MHV-68 to establish latency. B lymphocytes and macrophages are the major reservoirs of persistence in vivo thus the ability of A50 mutants to establish latency in NSO B and RAW macrophage cell lines was explored. Latency was readily established and maintained in 3T3 and RAW cells. The low infectability of NSO B- cells restricted the utility of this cell line in studies of latency. Examination of patterns of lytic and latent transcription in 3T3 and RAW cells coordinately infected with A50 MHV-68 revealed reactivation efficiencies of 40-60%. Following long-term passage A50 exhibited stable transcription of two latency related genes M2 and ORF73, with episomal maintenance of the viral genome, in the absence of contaminating lytic infection. The results demonstrate the utility of A50 mutants for studies of gammaherpesvirus latency in vivo.

Thesis--University of Oklahoma. / Includes bibliographical references (leaves 51-55).

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Medical Sciences

Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra of the brain. The pathogenesis and etiology of PD are unclear. Mitochondrial dysfunction occurs in PD, causing a decrease in complex I activity in postmortem brain, and exacerbating reactive oxygen species production and ATP deficiency contributing to neuronal cell death. Mutation of leucine-rich-repeat kinase 2 (LRRK2) gene is the most common genetic factor identified in both familial and sporadic PD cases. Several mutations in LRRK2 have been linked to PD, in which R1441G is the second commonest mutation after G2019S. LRRK2 protein is ubiquitously expressed in human body, in which a portion is localized to the mitochondria. Mutations of LRRK2 directly or indirectly cause mitochondria dysfunction. Dysfunction of mitochondrial respiratory complexes has been described in skin fibroblasts and skeletal muscle of PD patients. Therefore, these clinically accessible tissues are good for monitoring disease progression. The objectives of this study were to investigate how LRRK2 R1441G mutation affects normal mitochondrial function, and whether this specific LRRK2 mutation potentiates age-dependent deterioration of mitochondrial function. To achieve these aims, colonies of skin fibroblast carrying LRRK2 R1441G mutation or wild-type LRRK2 were derived from a novel LRRK2 R1441G knock-in (KI) mouse model and its wild-type (WT) littermates. Skeletal muscles were dissected from the hind legs of WT and KI mice. The effects of aging and LRRK2 R1441G mutation on mitochondrial function were investigated in vitro using these derived skin fibroblast cultures, and ex vivo using skeletal muscle obtained from young (3-month-old) and aged (18-month-old) WT and KI mice. Reduction-oxidation activities of mitochondrial complex I and complex II in skin fibroblasts and skeletal muscle were measured spectrophotometrically. Intracellular ATP levels in skin fibroblasts were determined by bioluminescent assay. Phase-contrast microscopy showed that aging and LRRK2 R1441G mutation did not affect cell morphology of the derived skin fibroblast cultures. Complex I activity determined in skin fibroblasts and skeletal muscle derived from KI and their WT littermates revealed that, aging caused a significant increase in complex I activity in WT but not KI skin fibroblasts. Conversely, a significant decrease in complex I activity was observed in both WT and KI skeletal muscle, demonstrating an aging effect ex vivo. LRRK2 R1441G mutation did not affect complex I activity in WT and KI skin fibroblasts and skeletal muscle. Moreover, complex II activity in these two tissues was neither affected by aging nor R1441G LRRK2 mutation. Intracellular ATP levels in the skin fibroblast cultures were also unaltered by aging and LRRK2 R1441G mutation. In conclusion, my current findings indicated a significant aging effect on mitochondrial complex I activity ex vivo, supporting the role of age-dependent deterioration of complex I activity in mitochondrial dysfunction of PD. LRRK2 R1441G mutation did not affect complex I and II activities in both skin fibroblasts and skeletal muscle. Also, this mutation did not potentiate the age-dependent deterioration of complex I activities as observed in skin fibroblasts and skeletal muscle of the LRRK2 R1441G knock-in mice. / published_or_final_version / Medicine / Master / Master of Philosophy

Tendons, essential tissues that connect muscles to bones, are susceptible to rupture/degeneration due to their continuous use for enabling movement. Often surgical intervention is required to repair the tendon; relieving the pain and fixing the limited mobility that occurs from the damage. Unfortunately, post-surgery immobilization techniques required to restore tendon properties frequently lead to scar formation and reduced tendon range of motion. Our ultimate goal is to create an optimal tendon prosthetic that can stabilize the damaged muscle-bone connection and then be remodeled by resident cells from the surrounding tissues over time to ensure long-term function. To achieve this, we must first understand how cells respond to and interact with candidate replacement materials. The most abundant extracellular matrix (ECM) protein found in the body, collagen, is chosen as the replacement material because it makes up the majority of tendon dry mass and it can be remodeled by cell-based homeostatic processes. Previous studies found that Di-catechol nordihydroguaiaretic acid (NDGA) cross-linked fibers have greater mechanical strength than native tendons; and for this reason this biomaterial could be used for tendon replacement. This work focuses on investigating the behavior of fibroblasts on NDGA cross-linked and uncross-linked collagen samples to determine if cross-linking disrupts the cell binding sites affecting cell spreading, attachment, and migration. The in-vitro platform was designed by plasma treating 25 mm diameter cover slips that were exposed to 3-aminopropyl-trimetoxysilane/toluene and glutaraldehyde/ethanol solutions. The collagen solution was then dispensed onto the glutaraldehyde-coated cover slip and incubated for fibrillar collagen matrix formation. The collagen matrices were submerged in NDGA cross-linking solution for 24 hours to ensure the surface was completely cross-linked. Collagen films were made by allowing the uncross-linked gels to dry overnight before and after NDGA treatment, resulting in a more compacted structure. A spinning disk device was employed to quantify the ability of cells to remain attached to the collagen samples when exposed to hydrodynamic forces. To avoid any cell-cell interaction and focus on cell-surface interactions, 50-100 cells/mm2 were seeded carefully on each sample. Temporal studies demonstrated that cell adhesion strength and spreading area reached steady-state by 4 hr. Adhesion and spreading studies along with migration experiments demonstrated that NDGA treatment affects cellular behavior on films, partially reducing adhesion strength, migration, and spreading area. However, on the cross-linked gels which are less dense, the only change in cell behavior observed was in migration speed. We hypothesize that these differences are due to the collapsing of the collagen films. This compaction suggests a less open organization and could be allowing the collagen fibers to form more inter-chain bonds as well as bonds with the small NDGA cross-linker; while NDGA treatment of the fully hydrated gels may rely more on NDGA polymerization to span the greater distance between collagen fibrils. From these results, we can determine that the chemical/physical masking of the adhesion sites by NDGA on collagen films affects cellular behavior more than the masking that occurs in the cross-linked gels. Although this study shows an effect in cell behavior on the cross-linked films, it also demonstrates that cells can adhere and migrate to this NDGA biomaterial supporting the idea that this biomaterial can be utilized for tendon replacement.