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.

Baigiamajame darbe įvertintas skirtingų titano implantų paviršiaus modifikavimo strategijų poveikis pirminių dantenų fibroblastų savybėms – adhezijai, proliferacijai ir diferenciacijai. Tyrimuose naudoti titano mėginiai, kurių paviršiaus šiurkštumas pakeistas fizikiniais metodais – smėliasrove ir lazeriu. Iš gingivektomijos metu paimto ţmogaus dantenų subepitelinio audinio buvo išskirtos ir charakterizuotos dantenų fibroblastų ląstelės. Tiriant šių ląstelių adheziją ant modifikuotų titano mėginių paviršių nustatyta, kad ankstyvuoju laikotarpiu dantenų fibloblastų adhezija buvo panaši ant visų tirtų įvairiai modifikuotų titano mėginių paviršių, tačiau vėliau efektyviausias ląstelių prikibimui paviršius buvo lazeriu suformuotos grotelės. Siekiant pagerinti modifikuotų titano paviršių adhezines savybes, jie papildomai buvo padengti tarpląstelinio uţpildo baltymais – kolagenu ir lamininu, įvertintas tokio padengimo poveikis ląstelių prikibimui ir augimui. Analizuojant mechanizmus, reguliuojančius ląstelių adhezijos ant modifikuotų paviršių procesus, buvo tirta FAK ir Akt kinazių raiška ir aktyvinimas. Vertinant karkasų paviršiaus topografijos poveikį ląstelių diferenciacijai, buvo palygintas osteogeninės diferenciacijos laipsnis ląstelėse augintose ant įvairiai modifikuotų titano mėginių paviršių. Darbą sudaro 6 dalys: įvadas, literatūros apţvalga, medţiagos ir metodai, rezultatai ir jų aptarimas, išvados, literatūros sąrašas. Darbo apimtis – p. teksto be priedų, 24 pav., 2 lent... [toliau žr. visą tekstą] / The influence of different modification strategies of titanium implant surfaces on gingival fibroblasts properties - adhesion, proliferation, and differentiation was studies in this final master thesis. Different titanium surface roughness modifications using physical methods such as sand-blasting as well as laser irradiation were developed. Gingival fibroblasts derived from human gingival subepithelial tissues obtained during gingivectomy were isolated and characterized. The data suggested that the initial adhesion between tested cells and various modified titanium surfaces was similar, but the most efficient surface for subsequent cell attachment was laser-ablated holey arranged in grid-like structures. Additionally, in order to improve the modified titanium surface adhesion properties, these surfaces were coated by extracellular matrix proteins - collagen and laminin. The coating influence on the cell growth and adhesion was evaluated. To analyze the mechanisms regulating cell adhesion processes on the modified surfaces, FAK and Akt kinase expression as well as activation were studied. In order to evaluate the effect of surface topography on cell differentiation, the level of osteogenic differentiation was compared. Structure: introduction, literature review, materials and methods, results and discussion, conclusions, references.

<p>Massive loss of cardiac tissue as a result of myocardial infarction can create a poorly-conducting substrate with impaired contractility, ultimately leading to heart failure and lethal arrhythmias. Recent advances in pluripotent stem cell research have provided investigators with potent sources of cardiogenic cells that may be transplanted into failing hearts to provide electrical and mechanical support. Experiments in both small and large animal models have shown that standard cell delivery techniques suffer from poor retention and engraftment of cells. In contrast, the transplantation of engineered cardiac tissues may provide improved cell retention at the injury site, creating a more localized paracrine effect and yielding more efficient structural and functional repair. However, tissue engineering methodologies to assemble cardiomyocytes or cardiac progenitors into aligned, 3-dimensional (3D) myocardial tissues capable of physiologically relevant electrical conduction and force generation are lacking. The objective of this thesis was thus to develop a methodology to generate highly functional engineered cardiac tissues starting from pluripotent stem cells.</p><p>To accomplish this goal, we first derived purified populations of cardiac myocytes from mouse embryonic stem cells (mESC-CMs) by antibiotic selection driven by an &#945;-myosin heavy-chain promoter. Culture conditions that yielded robust mESC-CM electrical coupling and fast action potential propagation were optimized in confluent cell monolayers. We then developed a microfabrication-based tissue engineering approach to create engineered cardiac tissues ("patches") with uniform 3D cell alignment. We found that, unlike in monolayers, mESC-CMs required a population of supporting cardiac fibroblasts to enable the formation of 3D engineered tissues. Detailed structural, electrical and mechanical characterization demonstrated that engineered cardiac patches consisted of dense, uniformly aligned, highly differentiated and electromechanically coupled mESC-CMs and supported rapid action potential conduction velocities between 22 - 25cm/s and contractile force amplitudes of up to 2mN. </p><p>Next, we sought to circumvent the use of primary cardiac fibroblasts by utilizing a single pluripotent stem cell-derived source, multipotent cardiovascular progenitors (CVPs) capable of differentiating into vascular smooth muscle and endothelial cells in addition to cardiomyocytes. CVPs were derived from mouse embryonic stem cells and induced pluripotent stem (iPS) cells by antibiotic selection driven by an Nkx2-5 enhancer element. Similar to mESC-CMs, CVPs formed highly differentiated cell monolayers with electrophysiological properties that improved with time in culture to levels achieved with pure mESC-CMs. However, unlike mESC-CMs, CVPs formed highly functional 3D engineered cardiac tissues without the addition of cardiac fibroblasts, enabling engineered cardiac tissues to be formed from a single, entirely stem cell-derived source.</p><p>Finally, we explored mechanisms of synergistic cardiac fibroblast/myocyte signaling in 3D engineered tissues by using cardiac fibroblasts of different developmental stages in the settings of direct 3D co-culture as well as in conditioned media studies. When co-cultured with fetal cardiac fibroblasts, mESC-CMs were capable of two-fold faster action potential propagation and 1.5-fold higher maximum contractile force generation than when co-cultured with adult cardiac fibroblasts. These functional improvements were associated with enhanced mESC-CM spreading and upregulation of important ion channel, coupling, and contractile proteins. Conditioned medium studies revealed that compared to adult fibroblasts, fetal cardiac fibroblasts secreted distinct paracrine factors that promoted mESC-CM spreading and spontaneous contractility in 3D engineered tissues and acted via the MEK-ERK pathway. Quantitative gene expression analysis revealed paracrine factor candidates that may mediate this action.</p><p>In summary, this thesis presents methods and underlying mechanisms for generation of highly functional cardiac tissues from pluripotent stem cell sources. These techniques and findings provide foundation for future engineering of human ES and iPS cell-based cardiac tissues for therapeutic and drug screening applications.</p> / Dissertation

Cell-substrate interactions influence various cellular processes such as morphology, motility, proliferation and differentiation. Actin dynamics within cells have been shown to be influenced by substrate stiffness, as NIH 3T3 fibroblasts grown on stiffer substrates tend to exhibit more prominent actin stress fiber formation. Circular dorsal ruffles (CDRs) are transient actin-rich ring-like structures within cells, induced by various growth factors, such as the platelet-derived growth factor (PDGF). CDRs grow and shrink in size after cells are stimulated with PDGF, eventually disappearing ten of minutes after stimulation. As substrate stiffness affect actin structures and cell motility, and CDRs are actin structures which have been previously linked to cell motility and macropinocytosis, the role of substrate stiffness on the properties of CDRs in NIH 3T3 fibroblasts and how they proceed to affect cell behavior is investigated. Cells were seeded on Poly-dimethylsiloxane (PDMS) substrates of various stiffnesses and stimulated with PDGF to induce CDR formation. It was found that an increase in substrate stiffness increases the lifetime of CDRs, but did not affect their size. A mathematical model of the signaling pathways involved in CDR formation is developed to provide insight into this lifetime and size dependence, and is linked to substrate stiffness via Rac-Rho antagonism. CDR formation did not affect the motility of cells seeded on 10 kPa stiff substrates, but is shown to increase localized lamellipodia formation in the cell via the diffusion of actin from the CDRs to the lamellipodia. To further probe the influence of cell-substrate interactions on cell behavior and actin dynamics, a two dimensional system which introduces a dynamically changing, reversible and localized substrate stiffness environment is constructed. Cells are seeded on top of thin PDMS nano-membranes, and are capable of feeling through the thin layer, experiencing the stiffness of the polyacrylamide substrates below the nano-membrane. The membranes are carefully re-transplanted on top of other polyacrylamide substrates with differing stiffnesses. This reversible dynamic stiffness system is a novel approach which would help in the investigation of the influence of reversible dynamic stiffness environments on cell morphology, motility, proliferation and differentiation in various cells types.

This study revolves around a family in which 4 male members have metabolic profiles similar to that of atypical 3–methylcrotonyl–CoA carboxylase (MCC) deficiency, an inborn error of leucine catabolism. This profile consists of high urinary 3–hydroxyisovaleric acid (3–HIVA) and trace amounts of 3–methylcrotonylglycine. One of the individuals also had clinical symptoms of chronic fatigue and muscle weakness, symptoms also related to MCC–deficiency. Further investigation showed that these individuals were negative for MCC–deficiency. The inheritance pattern of the abnormal metabolic profile seemed to indicate a link to the X–chromosome. In this study the single nucleotide polymorphism (SNP) and copy number variation (CNV) profiles of the X–chromosomes of participating members of the family were investigated for a possible link to the abnormal metabolic profile, using SNP6 DNA microarrays. The data generated by the SNP6 arrays was of good quality. The small sample size available for this study necessitated an unorthodox method for analysing the SNP6 data. No clear link between the SNP6 data and the abnormal metabolic profile was found. Selected SNP calls made by the SNP6 arrays were verified by sequencing. The origin of the elevated 3–HIVA detected in the urine of the male family members was also investigated. This was done by culturing fibroblasts from case individuals in culture medium supplemented with deuterium labelled leucine. The culture medium was analysed using GC–MS after an organic acid extraction. The resulting data seems to indicate at least two sources of 3–HIVA formation by the cells, one originating from leucine and another from a source other than leucine. The mevalonate shunt is one possible source of 3–HIVA, which does not originate from leucine catabolism. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Plasmacytoid dendritic cells (pDC) are a specialized group of circulating dendritic cells that respond to viral nucleic acids with Type I IFN production as well as other cytokine and chemokines. These pDC responses lead to the production of antiviral molecules and recruitment of defense cells. During zoonotic B virus infection, a simplex virus of the subfamily Alphaherpesviridae, our lab has observed that infected individuals who succumb to infection have little-to-no-antibody or cell-mediated defenses. To identify whether this was partly due to failure of pDCs to produce antiviral interferon responses or produce chemokine and cytokines, we tested the hypothesis that B virus modulates the IFN response during zoonotic infection by blocking pDC activation and subsequent IFN signaling pathways to circumvent host defenses, while these pathways remain intact in the macaque hosts. We showed that human pDCs respond to B virus through the production of IFN-a, IL-1a, IL-6, TNF-a, MIP-1a/b and IP-10. Human pDCs co-cultured with B virus infected fibroblasts produced fewer cytokines and at lower levels. The macaque response to B virus was measured using PBMCs, as there are no specific reagents available to enrich macaque pDCs. Human and macaque PBMCs produced IFN-a when exposed directly to B virus infected lysates. Co-cultures of PBMCs with B virus infected fibroblasts from both hosts failed to produce any significant amounts of IFN-a. To quantify the antiviral effects of PBMC induced IFN-a, we measured B virus titers after exposure to supernatants from B virus exposed PBMCs, PBMC co-cultures with infected fibroblasts and exogenous recombinant Type I IFN. Our data further suggest that B virus resistance was not due to virus specific blockade of the Type I IFN signaling pathway because STAT-1 was activated in infected fibroblasts when treated with Type I IFNs. These data demonstrate for the first time that B virus replication is unimpeded in the presence of any source of IFN-a in either host cell type. In conclusion, this dissertation shows that the IFN-a production by both hosts in response to B virus is similar and that IFN-a treatment of B virus infected fibroblasts did not reduce B virus replication.

This study revolves around a family in which 4 male members have metabolic profiles similar to that of atypical 3–methylcrotonyl–CoA carboxylase (MCC) deficiency, an inborn error of leucine catabolism. This profile consists of high urinary 3–hydroxyisovaleric acid (3–HIVA) and trace amounts of 3–methylcrotonylglycine. One of the individuals also had clinical symptoms of chronic fatigue and muscle weakness, symptoms also related to MCC–deficiency. Further investigation showed that these individuals were negative for MCC–deficiency. The inheritance pattern of the abnormal metabolic profile seemed to indicate a link to the X–chromosome. In this study the single nucleotide polymorphism (SNP) and copy number variation (CNV) profiles of the X–chromosomes of participating members of the family were investigated for a possible link to the abnormal metabolic profile, using SNP6 DNA microarrays. The data generated by the SNP6 arrays was of good quality. The small sample size available for this study necessitated an unorthodox method for analysing the SNP6 data. No clear link between the SNP6 data and the abnormal metabolic profile was found. Selected SNP calls made by the SNP6 arrays were verified by sequencing. The origin of the elevated 3–HIVA detected in the urine of the male family members was also investigated. This was done by culturing fibroblasts from case individuals in culture medium supplemented with deuterium labelled leucine. The culture medium was analysed using GC–MS after an organic acid extraction. The resulting data seems to indicate at least two sources of 3–HIVA formation by the cells, one originating from leucine and another from a source other than leucine. The mevalonate shunt is one possible source of 3–HIVA, which does not originate from leucine catabolism. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Telomeres are nucleoprotein complexes located at the end of chromosomes. They have an essential role in protecting chromosome ends. Telomerase or ALT (alternative lengthening of telomeres) mechanisms maintain telomeres by compensating natural telomeric loss. We have set up a flow-FISH method and using mouse lymphoma cell lines we identified unexpectedly the presence of subpopulations of cells with different telomere lengths. Subpopulations of cells with different telomere lengths were also observed in a human ALT and non-ALT cell line. Differences in telomere length between subpopulations of cells were significant and we term this phenomenon TELEFLUCS (TElomere LEngth FLUctuations in Cell Subpopulations). By applying flow-FISH we could successfully measure telomere lengths during replicative senescence in human primary fibroblasts with different genetic defects that confer sensitivity to ionising radiation (IR). The results from this study, based on flow-FISH and Southern hybridisation measurements, revealed an accelerated rate of telomere shortening in radiosensitive fibroblasts. We also observed accelerated telomere shortening in murine BRCA1 deficient cells, another defect conferring radiosensitivity, in comparison with a BRCA1 proficient cell line. We transiently depleted BRCA1 by siRNAs in two human mammary epithelial cell lines but could not find changes in telomere length in comparison with control cells. Cytological evidence of telomere dysfunction was observed in all radiosensitive cell lines. These results suggest that mechanisms that confer sensitivity to IR may be linked with mechanisms that cause telomere dysfunction. Furthermore, we have been able to show that human ALT positive cell lines show dysfunctional telomeres as detected by either the presence of DSBs at their telomeres or cytogenetic analysis and usually cells with dysfunctional telomeres are sensitive to IR. Finally, we assessed hTERT mRNA splicing variants and telomerase activity in brain tumours, which exhibit considerable chromosome instability suggesting that DNA repair mechanisms may be impaired. We demonstrated that high levels of hTERT mRNAs and telomerase activity correlate with proliferation rate. The presence of hTERT splice variants did not strictly correlate with absence of telomerase activity but hTERT spliced transcripts were observed in some telomerase negative brain tumours suggesting that hTERT splicing may contribute to activation of ALT mechanisms.

Background: Airway fibroblasts have become a critical addition to all facets of structural lung tissue changes such as in human asthma and chronic obstructive pulmonary disease, but little is known about their role in the equine recurrent airway obstruction, a disease that resembles to the human asthma. Since the equine bronchial fibroblasts (EBF) have not been isolated and characterized yet, the use of defined medium was investigated. Results: Primary EBF were cultured on non-collagen coated flasks without serum or in the presence of feta bovine serum (FBS) or horse serum (HS) or in serum depleted medium. EBF cultured in serum-free basal media and those serum deprived were not able to proliferate and even exhibited considerable cell death. In media containing FBS or HS, proliferation of the cells was reproducible between different primary cultures and cells demonstrated expression of vimentin. Large variations were found in the ability of FBS and HS to support growth and differentiation of EBF in monolayer culture. Indications of growth-promoting actions, increasing passage number as well as maintaining fibroblast morphology were found rather in FBS than in HS. EBF culturing in HS needed longer doubling and confluence time. The protein content of the cell pellets was higher in EBF cultured in medium containing HS than FBS. Alpha-smooth muscle actin seemed to be less expressed in EBF cultured in medium containing FBS than those in HS. Conclusions: In sum, serum addition to basal EBF medium enhanced EBF differentiation into myofibroblasts, and these findings are useful to develop in vitro fibroblast culture models that mimic in vivo physiological processes and to study airway disease mechanisms and remodeling.

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.