Fundamentally altering cellular function at a genetic level is a major area of interest in the biologic sciences and the medical community. By engineering transfectable constructs that can be inserted to dysfunctional cellular systems, scientists can mitigate aberrant genetic behavior to produce proper molecular function. While viral vectors have been a mainstay in the past, there are many limitations, particularly related to safety, that have changed the focus of genome editing to incorporate alternative methods for gene delivery. Lance Array Nanoinjection (LAN), a second-generation microfabricated transfection biotechnology, is one of these alternative technologies. LAN works by utilizing both simultaneous electrostatic interaction with molecular loads and physical lancing of hundreds of thousands of target cell membranes. The purpose of this work is to demonstrate LAN in the context of in vitro transfection of immortalized culture cells and primary cells. As part of that exploration, three distinct areas of investigation are considered, which include: characterizing environmental factors that impact LAN transfection, demonstrating LAN genetic modification of immortalized HeLa 229 culture cells using an indicator marker, and lastly, investigating the effects of LAN on human primary, neonatal fibroblasts.

Abstract not available

<p>Heparan sulfate (HS) is a linear polysaccharide, located on the surface and in the extracellular matrix of most cells, that regulates functions of numerous proteins. HS-protein interaction is mainly mediated by sulfate groups found in N-sulfated (NS) regions of the HS, but may also involve rare HS substituents such as N-unsubstituted glucosamine (GlcNH₂) residues. The location of GlcNH₂ in an HS-epitope recognized by the monoclonal antibody 10E4, that specifically stains the prion lesions in scrapie-infected murine brain, suggests an involvement of GlcNH₂ in prion disease and other amyloid-related disorders. HS in general is strongly associated with amyloidosis, including Alzheimer’s disease (AD). Therefore, the aims of this thesis were to structurally characterize GlcNH₂-containing HS sequences found in native tissues, to further study HS epitopes recognized by 10E4, and to investigate the possible role(s) of GlcNH₂ and other HS structures in binding to amyloid β peptide (Aβ) (core material in AD plaque lesions, also stained by 10E4).</p><p>The GlcNH₂ content (0.7-4% of total disaccharide units) varied between HS from different tissues. Most GlcNH₂ units were found in poorly modified N-acetylated (NA-) or NA/NS-domains, located toward the polysaccharide-protein linkage region.</p><p>Binding of human cerebral cortex HS to Aβ(1–40) monomers requires N-, 2- and 6-O-sulfation of HS, while binding to Aβ fibrils requires N- and 2-O-sulfation only. GlcNH₂ units do not appreciably contribute to interaction with Aβ. Aβ fibril-binding HS domains also bind to fibroblast growth factor 2 (FGF-2), indicating that Aβ (neurotoxic) and FGF-2 (neuroprotective) may compete for common binding sites in HS. However, Aβ had no effect on FGF-2-induced MAPK signaling in NIH 3T3 fibroblasts.</p><p>Continued studies on 10E4-antigenic HS epitope(s) showed that binding of 10E4 to the previously identified antigenic tetrasaccharide, ∆UA-GlcNH₂-GlcA-GlcNAc, requires the nonreducing hexuronic acid (∆UA) to be 4,5 unsaturated (induced by lyase cleavage), and thus is artificial. Further studies are needed to clarify the potential involvement of GlcNH₂ in 10E4-recognition of the native HS epitope(s).</p>

<p>Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods.</p><p>Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. </p><p>In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF. </p>

Heparan sulfate (HS) is a linear polysaccharide, located on the surface and in the extracellular matrix of most cells, that regulates functions of numerous proteins. HS-protein interaction is mainly mediated by sulfate groups found in N-sulfated (NS) regions of the HS, but may also involve rare HS substituents such as N-unsubstituted glucosamine (GlcNH2) residues. The location of GlcNH2 in an HS-epitope recognized by the monoclonal antibody 10E4, that specifically stains the prion lesions in scrapie-infected murine brain, suggests an involvement of GlcNH2 in prion disease and other amyloid-related disorders. HS in general is strongly associated with amyloidosis, including Alzheimer’s disease (AD). Therefore, the aims of this thesis were to structurally characterize GlcNH2-containing HS sequences found in native tissues, to further study HS epitopes recognized by 10E4, and to investigate the possible role(s) of GlcNH2 and other HS structures in binding to amyloid β peptide (Aβ) (core material in AD plaque lesions, also stained by 10E4). The GlcNH2 content (0.7-4% of total disaccharide units) varied between HS from different tissues. Most GlcNH2 units were found in poorly modified N-acetylated (NA-) or NA/NS-domains, located toward the polysaccharide-protein linkage region. Binding of human cerebral cortex HS to Aβ(1–40) monomers requires N-, 2- and 6-O-sulfation of HS, while binding to Aβ fibrils requires N- and 2-O-sulfation only. GlcNH2 units do not appreciably contribute to interaction with Aβ. Aβ fibril-binding HS domains also bind to fibroblast growth factor 2 (FGF-2), indicating that Aβ (neurotoxic) and FGF-2 (neuroprotective) may compete for common binding sites in HS. However, Aβ had no effect on FGF-2-induced MAPK signaling in NIH 3T3 fibroblasts. Continued studies on 10E4-antigenic HS epitope(s) showed that binding of 10E4 to the previously identified antigenic tetrasaccharide, ∆UA-GlcNH2-GlcA-GlcNAc, requires the nonreducing hexuronic acid (∆UA) to be 4,5 unsaturated (induced by lyase cleavage), and thus is artificial. Further studies are needed to clarify the potential involvement of GlcNH2 in 10E4-recognition of the native HS epitope(s).

Collagenous colitis (CC) is an inflammatory bowel disease of unknown etiology. It is characterized by watery diarrhoea without blood, normal endoscopic findings but microscopically colonic mucosal inflammation and increased thickness of the subepithelial collagen band, the latter being a pathognomonic sign. The inflammatory infiltrate in the mucosa of CC contains lymphocytes, plasma cells, eosinophils, mast cells but few neutrophils. The pathophysiological roles of the thickened collagen band and the inflammatory infiltrate in CC are not fully understood. The aims of the present study were to develop a colonoscope based segmental perfusions technique and to analyze local intestinal secretion of inflammatory mediators: Eosinophilic Cationic Protein (ECP), Myeloperoxidase (MPO), Basic Fibroblast Growth Factor (bFGF), Vascular Endothelial Growth Factor (VEGF) and permeability marker albumin in CC patients without medication and also during steroid treatment. Furthermore, the colonic mucosal distribution of bFGF and VEGF were studied by immunohistochemical methods. Colonoscope-based segmental perfusions were performed in totally 22 patients and the success rate was 76% in both rectal and descending colon segments. The analysis showed high intraluminal concentrations of ECP, bFGF, VEGF and albumin in ten CC patients compared to 10 control patients. Further, albumin had correlations with ECP and VEGF. However, elevated concentrations of MPO, an important feature of ulcerative colitis, were only observed in a few CC patients. Immunohistochemistry visualized bFGF and VEGF in the colonic epithelium but also deeper in the lamina propria. The steroid treatment study (including 12 patients) showed that the perfusate concentrations of ECP, bFGF and VEGF declined significantly in parallel with decreased frequency of diarrhoea. In conclusion, a safe colonoscope-based, segmental perfusion technique was developed and perfusions of the rectum and descending colon were performed. CC patients had elevated perfusate concentrations of ECP, VEGF and bFGF. There was a marked reduction of these mediators during steroid treatment supporting the hypothesis that these inflammatory mediators separately or synergistically participate in the inflammatory reaction and tissue remodelling in CC patients. The finding of correlations between albumin and ECP or VEGF implies that permeability is increased in CC and may be triggered by ECP and VEGF.

Traumatic spinal cord injury (SCI) is a devastating condition for which there is no effective clinical treatment. Neuroprotective molecules that minimize tissue loss have shown promising results; however systemic delivery may limit in vivo benefits due to short systemic half-life and minimal passage across the blood-spinal cord barrier. To overcome these limitations, an injectable intrathecal delivery vehicle comprised of hyaluronan and methylcellulose (HAMC) was developed, and previously demonstrated to be safe and biocompatible intrathecally. Here, HAMC was determined to persist in the intrathecal space for between 4-7 d in vivo, indicating it as an optimal delivery system for neuroprotective agents to reduce tissue degeneration after SCI. HAMC was then investigated as an in vivo delivery system for two neuroprotective proteins: erythropoietin (EPO) and fibroblast growth factor 2 (FGF2). Both proteins demonstrated a diffusive release profile in vitro and maintained significant bioactivity during release. When EPO was delivered intrathecally with HAMC to the injured spinal cord, reduced cavitation in the tissue and significantly improved neuron counts were observed relative to the conventional delivery strategies of intraperitoneal and intrathecal bolus. When FGF2 was delivered intrathecally from HAMC, therapeutic concentrations penetrated into the injured spinal cord tissue for up to 6 h. Poly(ethylene glycol) modification of FGF2 significantly increased the amount of protein that diffused into the tissue when delivered similarly. Because FGF2 is a known angiogenic agent, dynamic computed tomography was developed for small animal serial assessment of spinal cord hemodynamics. Following SCI and treatment with FGF2 from HAMC, moderate improvement of spinal cord blood flow and a reduction in permeability were observed up to 7 d post-injury, suggesting that early delivery of neuroprotective agents can have lasting effects on tissue recovery. Importantly, the entirety of this work demonstrates that HAMC is an effective short-term delivery system for neuroprotective agents by improving tissue outcomes following traumatic SCI.

Traumatic spinal cord injury (SCI) is a devastating condition for which there is no effective clinical treatment. Neuroprotective molecules that minimize tissue loss have shown promising results; however systemic delivery may limit in vivo benefits due to short systemic half-life and minimal passage across the blood-spinal cord barrier. To overcome these limitations, an injectable intrathecal delivery vehicle comprised of hyaluronan and methylcellulose (HAMC) was developed, and previously demonstrated to be safe and biocompatible intrathecally. Here, HAMC was determined to persist in the intrathecal space for between 4-7 d in vivo, indicating it as an optimal delivery system for neuroprotective agents to reduce tissue degeneration after SCI. HAMC was then investigated as an in vivo delivery system for two neuroprotective proteins: erythropoietin (EPO) and fibroblast growth factor 2 (FGF2). Both proteins demonstrated a diffusive release profile in vitro and maintained significant bioactivity during release. When EPO was delivered intrathecally with HAMC to the injured spinal cord, reduced cavitation in the tissue and significantly improved neuron counts were observed relative to the conventional delivery strategies of intraperitoneal and intrathecal bolus. When FGF2 was delivered intrathecally from HAMC, therapeutic concentrations penetrated into the injured spinal cord tissue for up to 6 h. Poly(ethylene glycol) modification of FGF2 significantly increased the amount of protein that diffused into the tissue when delivered similarly. Because FGF2 is a known angiogenic agent, dynamic computed tomography was developed for small animal serial assessment of spinal cord hemodynamics. Following SCI and treatment with FGF2 from HAMC, moderate improvement of spinal cord blood flow and a reduction in permeability were observed up to 7 d post-injury, suggesting that early delivery of neuroprotective agents can have lasting effects on tissue recovery. Importantly, the entirety of this work demonstrates that HAMC is an effective short-term delivery system for neuroprotective agents by improving tissue outcomes following traumatic SCI.

Las membranas celulares contienen un canal de Cl- (Maxi-Cl-) que es modulado por estrógenos y antiestrógenos. El trabajo experimental de mi tesis doctoral ha consistido en el estudio de la modulación de este canal y su identidad molecular. El resultado de mi trabajo ha demostrado que la base molecular del canal Maxi-Cl- es una isoforma de la proteína mitocondrial VDAC y que la activación del canal por los antiestrógenos y su inhibición por los estrógenos implica procesos de defosforilación y fosforilación, respectivamente. / Cellular membranes contain a Maxi-Cl- channel that is modulated by oestrogen and antioestrogens. The experimental work of my PhD Thesis has focussed in the study of the molecular identity of the Maxi-Cl- channel and its regulation. My results demonstrated that an isoform of the mitochondrial protein VDAC is the molecular correlate of the Maxi-Cl- channel and that the activation of the channel by antioestrogens and its inhibition by oestrogen requires a dephosphorylation and phosphorylation process, respectively.

Genesis of life begins with the fusion of female and male haploid gametes through a process of fertilization leading to the formation of a diploid cell, the zygote. This undergoes successive cleavage divisions forming 2-, 4- and 8- cell embryos and their individual cells (blastomeres) are totipotent. As development proceeds, there is a gradual restriction in their totipotency, resulting in the generation of two distinct cell lineages i.e., the differentiated trophectoderm (TE) cells and the undifferentiated, inner cell mass (ICM) during blastocyst morphogenesis (Rossant and Tam 2009). During the course of development, the ICM cells can give rise to all cell types of an organism and can also provide embryonic stem (ES)-cells when cultured in vitro (Evan and Kaufman 1981). ES-cells are pluripotent cells, having the ability to self-renew indefinitely and differentiate into all the three primary germ layers (ectoderm, mesoderm and endoderm) derived-cell types. ES-cells are an excellent developmental model system to understand basic mechanisms of self-renewal, cell differentiation and function of various genes in vitro and in vivo (Capecchi 2001). Importantly, their cell derivatives could potentially be used for experimental cell-based therapy for a number of diseases. Although, human ES-cell lines have been successfully derived and differentiated to various cell types (Thomson et al., 1998; Odorico et al., 2001), their cell-therapeutic potential is far from being tested, in view of the lack of our understanding of lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked ES-cells and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse, under the control of ubiquitous chicken -actin promoter (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. We have been attempting to derive ES-cell line from this transgenic mouse. Because the derivation of ES-cell line is genetic strain-dependent, with some strains being relatively permissible for ES-cell derivation while others are quite resistant (non permissive), it has been extremely difficult to derive ES-cell line from the FVB/N mouse strain. There is a need to evolve experimental strategies to derive ES-cell line from FVB/N mouse, a strain extensively used for transgenesis. Thus, the aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked ES-cell line from a non-permissive FVB/N mouse strain; (2) characterize the established ES-cell line; (3) achieve differentiation of various cell types from EGFP-expressing ES-cell line and (4) understand role of FGF signaling in cardiac differentiation from the established ES-cell line. In order to have an appropriate and relevant literature background, the 1st chapter in this thesis describes a comprehensive up-to-date review of literature, pertaining to the early mammalian development and differentiation of blastocyst, followed by origin and properties of ES-cells. Various ES-cell derivation strategies from genetically permissive and non-permissive mouse strains are described and also the ES-cell differentiation potential to various progenitors and differentiated cell types. Subsequently, details on molecular basis of cardiac differentiation and the therapeutic potential of ES-cell derived differentiated cell types to treat disease(s) are described. This chapter is followed by three data chapters (II-IV). Chapter-II describes the issues related to non-permissiveness of FVB/N strain for ES-cell derivation and strategies to overcome this hurdle. This is followed by detailed results pertaining to generation of homozygous EGFP-expressing transgenic mice and development of a two-pronged ES-cell derivation approach to successfully establish a permanent ES-cell line (named ‘GS-2’ ES-cell line) from the EGFP-transgenic ‘green’ mouse. This chapter also provides results pertaining to detailed characterization of the ‘GS-2’ ES-cell line which includes colony morphology, expansion efficiency, alkaline phosphatase staining, expression analysis of pluripotent markers by RT-PCR and immunostaining approaches and karyotyping. Following this, the outcome of results and significance in the context of reported information are discussed in detail. Having successfully derived the ‘GS-2’ ES-cell line, it is necessary to thoroughly assess the differentiation competence of the ‘GS-2’ ES-cell line. Therefore, the Chapter-III describes detailed assessment of the in vitro and in vivo differentiation potential of the ‘GS-2’ ES-cell line. For in vitro differentiation, results pertaining to ES-cell derived embryoid body (EB) formation and their differentiation to ectodermal, mesodermal and endodermal cell types, expressing nestin, BMP-4 and α-fetoprotein, respectively, are described. Besides, the robustness of adaptability of ‘GS-2’ ES-cells to various culture conditions for their maintenance and differentiation are described. Also shown in the chapter is the relatively greater propensity of this cell line to cardiac differentiation. For in vivo differentiation, the ‘GS-2’ ES-cell derived teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives are described. Last part of the data described in this chapter, pertains to generation of chimeric blastocysts by aggregation method. Because the ‘GS-2’ ES-cell line exhibited a robust differentiation potential, including an efficient cardiomyocyte differentiation, it is of interest to enhance the efficiency of cardiomyocyte differentiation by exogenous addition of one of the key growth factors i.e., FGF8b since this has been implicated to be critical for cardiogenesis in non-mammalian verterbrate species. Therefore, Chapter-IV is focused on assessing the ability of ‘GS-2’ ES-cell line for its cardiomyocyte differentiation property with particular emphasis on the FGF-induced cardiac differentiation. Results pertaining to the expressions of various FGF ligands and their receptors during differentiation of ES-cells are described. Besides, increases in the cardiac efficiency, following FGF8b treatment and the associated up-regulation of cardiac-specific markers such as GATA-4, ISL-1 and α-MHC are shown. At the end of data chapters, separate sections are devoted for ‘Summary and Conclusion’ and for ‘Bibliography’.