Yes / Non-healing wounds are a major area of unmet clinical need remaining problematic to treat. Improved understanding of pro-healing mechanisms is invaluable. The enzyme arginase1 is involved in pro-healing responses with its role in macrophages best characterized. Arginase1 is also expressed by keratinocytes; however, arginase1 function in these critical wound repair cells is not understood. We characterized arginase1 expression in keratinocytes during normal cutaneous repair and reveal de novo temporal and spatial expression at the epidermal wound edge. Interestingly, epidermal arginase1 expression was decreased in both human and murine delayed healing wounds. We therefore generated a keratinocyte specific arginase1-null mouse model (K14-cre;Arg1fl/fl) to explore arginase function. Wound repair, linked to changes in keratinocyte proliferation, migration and differentiation, was significantly delayed in K14-cre;Arg1fl/fl mice. Similarly, using the arginase inhibitor nor-NOHA, human in vitro and ex vivo models further confirmed this finding, revealing the importance of the downstream polyamine pathway in repair. Indeed, restoring the balance in arginase1 activity via addition of putrescine, proved beneficial in wound closure. In summary, we demonstrate that epidermal arginase1 plays a, to our knowledge, previously unreported intrinsic role in cutaneous healing, highlighting epidermal arginase1 and downstream mediators as potential targets for the therapeutic modulation of wound repair.

Submitted by Suelen Reis (suziy.ellen@gmail.com) on 2013-04-23T13:47:41Z No. of bitstreams: 1 Tese - Ana Paula Cavalcanti.pdf: 9447670 bytes, checksum: 44f05935b4d040bdf7dacb56067b1fe3 (MD5) / Approved for entry into archive by Rodrigo Meirelles(rodrigomei@ufba.br) on 2013-05-08T11:47:47Z (GMT) No. of bitstreams: 1 Tese - Ana Paula Cavalcanti.pdf: 9447670 bytes, checksum: 44f05935b4d040bdf7dacb56067b1fe3 (MD5) / Made available in DSpace on 2013-05-08T11:47:47Z (GMT). No. of bitstreams: 1 Tese - Ana Paula Cavalcanti.pdf: 9447670 bytes, checksum: 44f05935b4d040bdf7dacb56067b1fe3 (MD5) Previous issue date: 2008 / O objetivo do presente estudo foi avaliar histologicamente o reparo de feridas cutâneas em dorso de ratos submetidos ao tratamento com Laser e/ou LED, utilizando laser de diodo GaAlAs nos comprimentos de onda de λ660nm e λ790nm e LEDs em três comprimentos de onda. Foram utilizados 44 ratos da linhagem Wistar adultos jovens, machos, pesando entre 200 e 250 gramas. Após a realização de uma ferida excisional no dorso, os animais foram distribuídos aleatoriamente em onze grupos de estudo, com quatro animais cada: G0- Controle; G1- Laser λ660nm, 10J/cm2; G2- Laser λ790nm, 10J/cm2; G3- LED ~λ700nm, 15mW, 10J/cm2; G4- Laser λ660nm + LED ~λ700nm, 10J/cm2; G5- LED ~λ700nm, 16mW, 10J/cm2; G6- Laser λ790nm + LED ~λ700nm, 10J/cm2; G7- LED ~λ530nm, 10J/cm2; G7- Laser λ790nm + LED ~λ530nm, 10J/cm2; G8 - LED ~λ460nm, 10J/cm2; G10- Laser λ790nm + LED ~λ460nm, 10J/cm2. As irradiações foram realizadas durante sete dias com intervalos de 48 horas, e a morte animal ocorreu no oitavo dia. As peças foram removidas, processadas histologicamente e coradas com HE e Picrosírius, e analisadas por microscopia óptica. Os resultados indicam que as radiações laser λ790nm, LED vermelha (~λ700nm)(15mW) e as associações LED vermelho com laser λ790nm e LED verde com laser λ790nm apresentaram biomodulação positiva sobre a quantidade de fibroblastos e de fibras colágenas. A associação LED verde com laser λ790nm, dentre todos os tratamentos, resultou na deposição de colágeno mais significante e as radiações LED vermelha (15 e 16mW) e laser λ660nm exerceram o efeito de biomodulação positiva mais expressivo na proliferação de fibroblastos e na angiogênese. As radiações laser, LED e suas associações, dentro dos parâmetros especificados, exercem, de maneira geral, biomodulação positiva sobre a proliferação de fibroblastos, a formação de tecido de granulação, a deposição de fibras colágenas e a angiogênese. / Salvador

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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

The ovarian surface epithelium (OSE) is a monolayer of cells surrounding the ovary that is ruptured during ovulation. After ovulation the wound is repaired, however this process, and the mechanisms to maintain OSE homeostasis after the wound is repaired are poorly understood. We have shown the mouse OSE (mOSE) contains a stem cell population that is expanded by Transforming Growth Factor Beta 1 (TGFB1), a factor present in follicular fluid. These data suggest that components in the follicular fluid such as TGFB1 may promote wound repair and OSE homeostasis through maintenance of the OSE stem cell population. Additionally, TGFB1 may promote wound repair through induction of an epithelial-to-mesenchymal transition (EMT) and activation of pro-survival pathways, as seen in other tissues. To elucidate the mechanism for TGFB1-mediated ovulatory wound repair, mOSE cells were treated with TGFB1, which induced an EMT seen with increased Snai1 expression and cell migration. Snai1 overexpression also increased cell migration and sphere formation (a stem cell characteristic). RNA sequencing results suggest this is at least in part through elevated collagen deposition in SNAI1 overexpressing cells. A TGFB signalling targets array identified Cox2 induction following TGFB1 treatment. Constitutive Cox2 expression did not promote an EMT, but enhanced sphere formation and cell survival. Finally, TGFB1 treatment decreased Brca1 expression, which when deleted from mOSE cells also increased sphere formation. RNA sequencing results suggest that Brca1 deletion promotes stemness through activation of the stem cell genes Ly6a and Lgr5. RNA sequencing was also used to compare mOSE cells cultured as monolayers and as spheroids, with and without TGFB1. These results validate our findings that TGFB1 promotes an EMT partially through Snail induction and the upregulation of Cox2. mOSE cells cultured as spheroids acquire a mesenchymal transcriptional profile that is further enhanced with TGFB1 treatment. These data suggest that TGFB1 may promote ovulatory wound repair and maintain OSE homeostasis through the induction of an EMT, maintenance of the stem cell population and activation of a pro-survival pathway. Interestingly, mOSE spheroids also decrease Brca1 expression and upregulate cancer associated genes such as Pax8 and Greb1. The induction of survival pathways, while simultaneously increasing stemness and repressing Brca1 could render cells more susceptible to transformation. This work provides novel insights as to why ovulation is the primary non-hereditary risk factor for ovarian cancer.

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Yes / Cytokines are extremely potent biomolecules that regulate cellular functions and play multiple roles in initiation and inhibition of disease. These highly specialised macromolecules are actively involved in control of cellular proliferation, apoptosis, cell migration and adhesion. This work, investigates the effect of transforming growth factor-beta2 (TGF-β2) on the biological regulation of chondrocyte and the repair of a created model wound on a multilayer culture system. Also the effect of this cytokine on cell length, proliferation, and cell adhesion has been investigated. Chondrocytes isolated from knee joint of rats and cultured at 4 layers. Each layer consisted of 2 × 105 cells/ml with and without TGF-β2. The expression of mRNA and protein levels of TGF-β receptors and Smad1, 3, 4, and 7 have been analysed by RT-PCR and western blot analysis. The effect of different supplementations in chondrocyte cell proliferation, cell length, adhesion, and wound repair was statistically analysed by One-way ANOVA test. Our results showed that the TGFβ2 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. Also the TGF-β2 caused an increase in chondrocyte cell length, but decreased its proliferation rate and the wound healing process. TGF-β2 also decreased cell adhesion ability to the surface of the culture flask. Since, TGF-β2 increased the cell size, but showed negative effect on cell proliferation and adhesion of CHC, the effect of manipulated TGF-β2 with other growth factors and/or proteins needs to be investigated to finalize the utilization of this growth factor and design of scaffolding in treatment of different types of arthritis.

No / Wound infection is a major clinical problem, yet understanding of bacterial host interactions in the skin remains limited. Microbe-derived molecules, known as pathogen-associated molecular patterns, are recognised in barrier tissues by pattern-recognition receptors. In particular, the pathogen-associated molecular pattern, lipopolysaccharide (LPS), a component of microbial cell walls and a specific ligand for Toll-like receptor 4, has been widely used to mimic systemic and local infection across a range of tissues. Here we administered LPS derived from Klebsiella pneumoniae, a species of bacteria that is emerging as a wound-associated pathogen, to full-thickness cutaneous wounds in C57/BL6 mice. Early in healing, LPS-treated wounds displayed increased local apoptosis and reduced proliferation. Subsequent healing progression was delayed with reduced re-epithelialisation, increased proliferation, a heightened inflammatory response and perturbed wound matrix deposition. Our group and others have previously demonstrated the beneficial effects of 17β-estradiol treatment across a range of preclinical wound models. Here we asked whether oestrogen would effectively promote healing in our LPS bacterial infection model. Intriguingly, co-treatment with 17β-estradiol was able to promote re-epithelialisation, dampen inflammation and induce collagen deposition in our LPS-delayed healing model. Collectively, these studies validate K. pneumoniae-derived LPS treatment as a simple yet effective model of bacterial wound infection, while providing the first indication that oestrogen could promote cutaneous healing in the presence of infection, further strengthening the case for its therapeutic use.