Fibrosis of vital organs remains one of the leading causes of death in the developed world, where it occurs predominantly in soft tissues (liver, lung, kidney, heart) through fibroblast proliferation and deposition of extracellular matrix (ECM). In the process of fibrosis, remodeling and deposition of ECM results in stiffening of cellular microenvironment; cells also respond to these changes in the stiffness through engagement of their cytoskeleton and signaling via cell-ECM contacts. Thus, understanding to what extent the stiffness of the cellular microenvironment changes as a consequence of fibrotic progression, and how cells respond to this change, is critical. In this thesis, we quantitatively measured stiffness of the lung parenchyma and its changes during fibrosis. We find that the average stiffness increases by approximately 10-fold. We then investigated how changes in ECM rigidity affect the cytoskeletal phenotype of lung fibroblasts. We find a complex relation between expression of the glycoprotein Thy-1 (CD90) and ECM rigidity-dependent cytoskeletal phenotype (i.e. “mechanotransduction”). Finally, we investigate a mechanism for the regulation of rigidity sensing by Thy-1 and its involvement in intracellular signaling through cell-ECM contacts. Taken together, this work helps define in vivo parameters critical to the fibrogenesis program and to define unique cellular phenotypes that may respond or contribute to mechanical homeostasis in fibrotic diseases.

Background: Chronic wounds are a significant health problem in various populations. Psychosocial stress is a lifestyle factor that has been shown to directly influence wound healing. Current findings support roles for assessment and reduction of psychosocial stress in the comprehensive management of chronic wounds, however, a gap remains between current research and current clinical practice. Purpose: To develop a novel method by which to educate nurses about the effects of psychosocial stress on wound healing while incorporating state-of-the-art technology that is sensitive to the needs of individuals with various learning styles. Objectives: To review current literature documenting the relationship between chronic psychosocial distress and delayed wound healing to identify essential content to include in educational modules for nurses. To develop three educational modules for nurses in inpatient and outpatient settings that address the relationship between chronic psychosocial distress and delayed wound healing, and the effects of stress-reduction interventions in formats that meet the needs of different learning styles. To conduct a focus group discussion with nurse-participants regarding educational module content and delivery methods in order to evaluate and improve these educational modules. Methods: A series of literature reviews were performed between June, 2010 and October, 2013, using articles identified through searches using the databases PubMed and CINAHL. Essential content regarding psychosocial stress and its impact on wound healing was identified, and was used in the development of educational modules, designed to meet the basic needs of individuals with different learning styles. A purposive sample of nurses was recruited through the use of flyers, reviewed the educational modules online, and met for a focus group to discuss their experiences with these modules. Outcomes: A novel method was developed by which to deliver educational material to nurses about psychosocial stress and delayed wound healing. Participants were motivated to learn, had self-awareness of their preferred learning styles, and responded positively to this method of education delivery; they were able to articulate the basic concepts presented in the modules. These findings may be generalizable to a larger audience and may inform the development of future education-delivery approaches in this area.

Leader cells are a fundamental biological process that have only been investigated since the early 2000s. These cells have often been observed emerging at the edge of an artificial wound in 2D epithelial cell collective invasion, created with either a mechanical scrape from a pipette tip or from the removal of a plastic, physical blocker. During migration, the moving cells maintain cell-cell contacts, an important quality of collective migration; the leader cells originate from either the first or the second row, they increase in size compared to other cells, and they establish ruffled lamellipodia. Recent studies in 3D have also shown that cells emerging from an invading collective group that also exhibit leader-like properties. Exactly how leader cells influence and interact with follower cells as well as other cells types during collective migration, however, is another matter, and is a subject of intense investigation between many different labs and researchers. The majority of leader cell research to date has involved epithelial cells, but as collective migration is implicated in many different pathogenic diseases, such as cancer and wound healing, a better understanding of leader cells in many cell types and environments will allow significant improvement to therapies and treatments for a wide variety of disease processes. In fact, more recent studies on collective migration and invasion have broadened the field to include other cell types, including mesenchymal cancer cells and fibroblasts. However, the proper technology for picking out dynamic, single cells within a moving and changing cell population over time has severely limited previous investigation into leader cell formation and influence over other cells. In line with these previous studies, we not only bring new technology capable of dynamically monitoring leader cell formation, but we propose that leader cell behavior is more than just an epithelial process, and that it is a critical physiological process in multiple cell types and diseases.

The investigation of complex biological processes, such as wound healing, cell migration, cancer cell invasion, and gene regulatory networks can be benefited tremendously by novel biosensing techniques with high stability and spatiotemporal resolution. In particular, molecular probes with qualities including high stability, sensitivity, and specificity are highly sought-after for long-term monitoring of gene expression in individual cells. Among different single-cell analysis techniques oligonucleotide optical probes is a promising detection method to monitor the dynamics of cellular responses. Herein, the design and optimization of double-stranded LNA probes are first investigated. With alternating DNA/LNA monomers for optimizing the stability and specificity, we show that the probe is highly stable in living cells and is capable of detecting changes in gene expression induced by external stimuli. Using dsLNA probes we then demonstrate the novel approaches to monitor the spatiotemporal gene expression response during cell injury. Our results also suggest a potential autoregulatory role of Nrf2 in injury induced EMT. We also show that the signaling level of dsLNA probe can serve as a molecular signature for the leader cells near the wound which allows us to track the behaviors of leader cells during collective cell migration. Finally multimodal GNR-LNA approach is proposed to map spatiotemporal gene expression profile and reveal dynamic characteristics of heat shock response in photothermal operations.

The healing of tympanic membrane (TM) perforations is a complex wound healing process including inflammation, migration of keratinocytes and tissue remodelling. Most TM perforations in human heal spontaneously, however some perforations become chronic, and the reason to why is still largely unknown. In cutaneous wound healing plasminogen (plg) has been shown to play an important role. Plg is converted into the protease plasmin regulated by two plasminogen activators (PA), urokinase type PA (uPA) and tissue-type PA (tPA). The aim of the present thesis was to evaluate the role of plg in healing of TM perforations, both in vivo and in vitro. The main objectives were to determine the healing capacity of the TM, the involvement of keratinocytes, fibrin(ogen) and inflammatory cells in the healing process. The studies were performed in plg deficient and uPA deficient mice, with littermate wild type (wt) mice as controls It was shown that myringotomies of the TMs in plg deficient mice still remained open 143 days following a perforation. The wound area was characterized by an abundant recruitment and accumulation of inflammatory cells; mainly macrophages and neutrophils, an arrested keratinocyte migration and a fibrin deposition covering the surface of the TM. The TM perforations in the wt mice all healed within 11 days. Interestingly, the myringotomies of the plg deficient mice could be closed by reconstitution with systemic injections of plg, whereas injections of PBS had no affect on the healing. To characterize mechanisms involved in the development of persistent TM perforations in plg deficient mice after a myringotomy the early inflammatory response during the first 48 hours was studied. The recruitment and accumulation of inflammatory cells in the perforated TMs was found to be similar between the plg deficient and the wt mice. Myringotomized TMs in uPA deficient mice healed similar to perforations of wt controls. Neither did the keratinocyte migration nor the occurrence of inflammatory cells differ between these genotypes. In the in vitro experiments TMs from plg deficient and wt mice, were dissected out, perforated and cultured in absence or surplus of plg. A decrease in perforation size was seen in all groups regardless of genotype or amount of plg in the medium. In conclusion, the present studies show: • Plg is essential for the healing of TM perforations in mice. • The altered healing process after a myringotomy in plg deficient mice involves a disturbed keratinocyte migration, a massive deposition of fibrin and an abundant accumulation of inflammatory cells in the wound area. • Plasminogen deficiency does not alter the early inflammatory response, following a myringotomy. • Deficiency of uPA does not influence the healing of TM perforations. • During in vitro conditions healing of TM perforations is initiated irrespectively of genotype of the explant (plg deficient or wt) or supply of plg. The increased knowledge of the involvement of plg in the healing of TM perforations may open therapeutical possibilities in the treatment of chronic TM perforations in humans.

Šio darbo tikslas – įvertinti žmogaus rekombinantinio augimo hormono Biosoma (UAB „Biotechna“, Vilnius, Lietuva) įtaką eksperimentinių gyvūnų (žiurkių) odos paviršinės mechaninės, visos odos mechaninės ir visos odos nudegimo žaizdos gijimui. Tyrimo uždaviniai: mechaniškai sukėlus eksperimentiniams gyvūnams odos paviršiaus iki tinklinio sluoksnio defektą, įvertinti Biosoma poveikį žaizdos gijimui, atliekant žaizdos dugno kolageno tinklo histomorfometrinius matavimus; eksperimento su gyvūnais metu mechaniškai sukėlus visos odos storio defektą, nustatyti Biosoma poveikį žaizdos gijimo greičiui, pagal jos kontūro kitimą; nustatyti temperatūros ir ekspozicijos parametrus siekiant sukelti standartizuotą visos odos nudegimo žaizdos sąlyčio modelį; įvertinti Biosoma įtaką eksperimentinių gyvūnų visos odos nudegimo žaizdos gijimui bei palyginti jį su visos odos mechanines žaizdos gijimu; įvertinti Biosoma įtaką gyvūnų visos odos nudegimo gijimui atliekant žaizdos dugno kolageno tinklo histomorfometrinius matavimus. Mūsų tyrimo duomenimis, eksperimentinių gyvūn���� (žiurkių), gavusių sisteminį anabolinį preparatą – žmogaus rekombinantinį augimo hormoną Biosoma 5,6 TV (2,0 mg/kg per parą injekcijas) visų tipų žaizdos – odos paviršinė mechaninė, visos odos mechaninė ir visos odos nudegimo – gijo greičiau, jos dugno vienas pagrindinių struktūrinių komponentų – fibrilinio kolageno tinklas buvo sintetinamas ir „brendo“ greičiau. / The aim of this study is to estimate the influence of recombinant human growth hormone Biosoma (UAB „Biotechna“, Vilnius, Lietuva) on different types of rats skin wound healing. We examined part and full thickness skin mechanical lesions and full thickness skin burns. The objectives: to estimate recombinant human growth hormone Biosoma impact on mechanically inflicted animal part thickness cutaneous wound healing according to histomorphometrical evaluation of the wound bed collagen; to evaluate recombinant human growth hormone Biosoma influence on full thickness skin mechanical wound healing in accordance with the wound contour changing; to choose the thermal exposition parameters for inflicting standard full thickness contact skin burn in animal; to evaluate the influence of recombinant human growth hormone Biosoma on animal full thickness skin burn healing and to compare it with the full thickness skin mechanical wound healing; to estimate the influence of recombinant human growth hormone Biosoma on animal full thickness burn ECM synthesis according to histomorphometrical wound bed collagen measurements. According to the research data, all types skin wounds (part and full thickness skin mechanical and full thickness burn) healed faster, their wound bed collagen fibers synthesis and maturation were faster in the experimental animals (rats) treated with rhGH Biosoma 5.6 IU (2.0 mg/kg a day) injections.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成25年1月31日 匠原龍太郎氏の博士論文として提出された

The transforming growth factor beta (TGFβ)/activin signaling pathway has a number of documented roles during wound healing and is becoming increasingly appreciated as a vital component of multi-tissue regeneration. The leopard gecko (Eublepharis macularius) is able to spontaneously, and repeatedly, regenerate its tail following tail loss. We thus examined the expression and localization of several key components of the TGFβ/activin signaling pathway during tail regeneration of the leopard gecko. We observed a marked increase in phosphorylated-Smad2 expression among regenerating tissues corresponding to the location of the regenerate blastema. Interestingly, we observe that during early regeneration there appears to be an absence of TGFβ family member TGFβ1 and instead a strong upregulation of activin-βA. We also observe the expression of EMT transcription factors Snail1 and Snail2 in blastemal tissue. These observations combined with other data provide strong support for the importance of unique and non-overlapping expression patterns of different TGFβ ligands during multi-tissue regeneration

Macrophages are active participants in many important biological processes, including antimicrobial activity, tumour surveillance, apoptotic cell clearance, homeostasis and wound healing. The activity of all cells is under the direct influence of their genetic makeup and macrophages are no exception. Natural resistance-associated macrophage protein 1 (Nramp1, also known as SLC11A1) is a macrophage-restricted gene that confers resistance to intracellular pathogens in mice. Mitogen activated protein kinase activated protein kinase 2 (MAPKAPK-2 or MK2), a substrate of p38 MAPK, is known to influence the activation of macrophages in response to stressors, including the Toll-like receptor (TLR)-4 ligand LPS. Like NRAMP1, MK2 has also been shown to influence the efficiency of the antibacterial response. The present study evaluates the role of NRAMP1 and MK2 in TLR-mediated cytokine induction and their role in cutaneous wound healing. Mice lacking NRAMP1 are severely impaired in their rate of cutaneous wound healing. Nramp1 gene ablation has been associated with lower levels of SLPI, a protein previously demonstrated to influence the rate of wound healing in a non-redundant fashion. Macrophages derived from Nramp1-null mice are less efficient in activating p38 MAPK signaling, which results in lower levels of MK2 phosphorylation. The reduced level of p38 MAPK and MK2 activation in Nramp1-null macrophages also correlates with decreased cytokine induction in response to TLR7 ligand stimulation of these cells. Using p38 MAPK inhibitor and MK2-deficient macrophages, we demonstrate that TLR7- and TLR9-mediated cytokine induction is directly under the control of this signaling pathway. Furthermore, cytokine induction is regulated by MK2 at the post-transcriptional level. Macrophage-induced cytokines play an important role in cutaneous wound healing. Since MK2-deficient macrophages are severely impaired in their ability to induce cytokines following activation, we next evaluated the role of MK2 in cutaneous wound healing. Our results demonstrate that the rate of wound healing is significantly delayed in the absence of MK2. The level of cytokine expression in the wounds is impaired and macrophages are major players in cutaneous wound healing. Our data also show that intradermal transfer of macrophages with intact MK2 significantly improved wound healing kinetics. Overall, the studies presented in this dissertation demonstrate the importance of NRAMP1 and MK2 in the modulation of macrophage gene expression, and their important role in the control of cutaneous wound healing.

Transcutaneous oxygen pressure measurement (TCp02) using a miniaturised Clarke electrode and a heating thermistor was developed independently by Huch et al and Eberhardt et al in 1972. After its initial use to non invasively monitor arterial partial pressure (Pa02) in neonates it was proposed as a useful test of skin blood flow and possibly amputation wound healing level selection in patients with peripheral vascular disease. Unfortunately a wide range of predictive values emerged with some authors reporting amputations healing when the TCp02 value was 0 mmHg. The investigation, while still considered useful, has not gained widespread support. This study investigates the use of TCp02, establishes a value for the use of the TCp02 Index to predict amputation wound healing potential and examines the hypothesis that the use of the TcpO Index to select amputation level can reduce patient morbidity and mortality. The literature is reviewed and a series of studies evaluating TCp02 use, undertaken in the Durban Metropolitan Vascular Service Non-Invasive Laboratories, are presented. TCp02 measurements were performed in a standardised manner with the subject supine breathing room air. Measurements were taken at fixed sites, on the mid dorsum of the foot (Foot), 10 cm distal to the tibial tuberosity and 2 cm lateral to the anterior tibial margin (BKA), 10 cm proximal to the patella in the midline (AKA) and on the chest in the mid-clavicular line. A TCp02 Index, the limb to chest ratio was defined. TCp02 data derived from control subjects asymptomatic of peripheral vascular disease were shown to be similar to age matched pooled data derived from the literature. In patients with peripheral vascular disease, absolute TCp02 and the TCp02 Index were shown to fall from proximal to distal sites and again were no different to pooled data derived from the literature. Based on presenting symptoms, the fall in TCp02 and the TCp02 Index was significant from proximal to distal sites. The reduction in absolute TCp02 and the TCp02 was also related to the most distal pulse present. TCp02 values were found to be no different in patients with peripheral vascular disease with or without diabetes. When comparing TCp02 and the TCp02 Index with Doppler pressure measurements at the Popliteal artery and at the foot, and the Doppler ankle brachial index (ABI), Doppler derived data were significantly higher in diabetic patients than in non-diabetic patients. No differences were noted in TCp02 data. TCp02 was compared with the 133Xe radio-isotope skin washout test. The best correlation was (r = 0.46) was obtained with a logarithmic curve y = 10.862Ln(x) + 38.751. TCp02 was compared with antibiotic concentrations (Cefoxitin) in muscle obtained from the site of amputation and the Cefoxitin Index, the ratio of muscle antibiotic concentration to plasma concentration, as an indication of the relationship of skin TCp02 to muscle blood flow. A significant correlation was shown between the Cefoxitin Index and TCp02 (r = 0.67, p = 0.035) and the TCp02 Index (r = 0.64, P = 0.045), suggesting that skin oxygen delivery may reflect muscle antibiotic delivery and hence blood flow. TCp02 and the TCp02 Index were compared with heated and unheated laser Doppler fluxmetry (LDF) in 35 patients undergoing amputation wound healing assessment. Significant correlations were shown between heated LDF, heated LDF Index and the TCp02 Index (r = 0.63 and r = 0.69, P < 0.0001). TCp02 Index values of 0.5 and 0.55 showed an accuracy of 96.2 % in predicting amputation outcome while LDF values of 3, 4 and 5 arbitrary units gave an accuracy of 88.5 %. Using receiver operator curves, a TCp02 Index of 0.55 was shown to be the best test. Over the years 1987 and 1988, TCp02 data were gathered on 193 patients undergoing lower limb amputation for peripheral vascular disease. Information on the outcome of the amputation was available for 152 amputations. Circumstances which might result in a reduced pre-operative TCp02 reading were identified and criteria were set for the use of TCp02 to predict amputation wound healing potential. 122 amputations which met the defined entry criteria were available for evaluation. A TCp02 Index of 0.50 gave a definitive predictive value below which no amputation healed. Similarly no amputation with an absolute TCp02 of less than 27 mmHg healed. Receiver operator characteristic curves showed the TCp02 Index to be a better test than absolute TCp02. A TCp02 Index of 0.55 was shown to have the best sensitivity of96.7 %, with a specificity of79.8 % and an accuracy of 90.2 %. When introduced to clinical practice, correct use of the TCp02 Index of 0.55 resulted in a reduction in amputation revision rate from 40.3 % in 1987, to 8.2 % in 1990. Initially some surgeons felt that the TCp02 Index predicted amputation wound failure at distal sites at which healing could be expected on clinical criteria, and chose amputate at sites with a TCp02 Index value less than 0.55. These amputations failed to heal. As surgeons gained confidence in the test, they chose to follow the TCp02 data more often and the percentage of amputations performed at sites predicted by the TCp02 Index to fail , fell from 35.5 % in 1987 to 6.6 % in 1990. Over a 15 year period at King Edward VIII Hospital, the amputation revision rate has fallen from an average of 32.7 % in the first five years when Tcp02 data were not available to the surgeon, to 21.4 % and 22.9 % in the two subsequent 5 year periods when Tcp02 data were available. The mortality rates were unchanged. The decline in revision rates was less than expected and relates to the fact that approximately only 42 % of patients requiring amputation undergo the test. This is because it is time consuming and available only during weekday office hours. These studies have confirmed the usefulness of Tcp02 measurement in the non-invasive vascular laboratory. The index is shown to be superior to absolute Tcp02 as a predictive test of amputation wound healing. The introduction of several criteria to define when Tcp02 use is appropriate has refined the investigation and made it clinically useful in our setting. A Tcp02 Index of 0.55 in the appropriate patient is a useful test to predict amputation wound healing and its use has resulted in reduced patient morbidity and mortality, confirming the hypothesis tested. / Thesis (M.D.)-University of Natal, 2001.