Introdução: A exposição à poluição atmosférica durante a gestação provoca alterações nas características da placenta e pode resultar em restrição de crescimento intrauterino. Sabe-se que o transforming growth factor beta 1 (TGFbeta1), o sistema renina-angiotensina uteroplacentário (SRA) e os fatores angiogênicos, tais como vascular endothelial growth factor A (VEGF-A) participam do processo de placentação e regulação do fluxo sanguíneo uteroplacentário. Assim, o objetivo do presente estudo foi investigar o efeito da exposição à poluição do ar sobre a morfologia, função e SRA placentários. Métodos: Ratas Wistar fêmeas foram expostas ao ar filtrado (F) ou ao material particulado 2.5um (P) durante 15 dias. Depois o cruzamento, as ratas foram divididas em 4 grupos e novamente expostas a F ou P (FF, FP, PF, PP). No 19º dia de gestação, as porções maternas e fetais das placentas foram coletadas. Estrutura da placenta, TGFbeta1, VEGF-A e seus receptores e os componentes do SRA foram avaliados. Resultados: A exposição ao material particulado diminuiu massa, tamanho e área de superfície placentária, um indicativo da interação materno-fetal. As concentrações placentárias de TGF beta1, VEGF-A e Flk-1 e os componentes do SRA foram alterados e isso pode indicar um prejuízo na invasão do trofoblasto, angiogênese placentária e troca de nutrientes e gases entre mãe e fetos. Discussão: Os resultados indicam que a exposição a partículas compromete a interação materno-fetal e pode refletir sobre a nutrição e crescimento fetal / Introduction: Exposure to air pollution during pregnancy causes alterations in placental characteristics and may result in intrauterine growth restriction. It was suggested that transforming growth factor beta 1 (TGFbeta1), the uteroplacental renin-angiotensin system (RAS) and angiogenic factors, such as vascular endothelial growth factor A (VEGF-A) participates of the placentation process and regulation of the uteroplacental blood flow. Thus, the aim of the present study was to investigate the effect of exposure to air pollution on the placental morphology, function and placental RAS. Methods: Female Wistar rats were exposed to filtrated air (F) or to concentrated particulate matter 2.5um (P) for 15 days. After mating, rats were divided in 4 groups and again exposed to F or P (FF, FP, PF, PP). At 19th day of pregnancy, maternal and fetal portions of placenta were collected. Placental structure, TGFbeta1, VEGF-A and its receptors and RAS components were evaluated. Results: Exposure to particulate matter decreased placental mass, size and surface area, an indicative of maternal-fetal interaction. Placental TGFbeta1, RAS components and VEGF-A and receptors Flk-1 concentrations were altered and this may indicate a prejudice in the trophoblast invasion, placental angiogenesis and maternal-fetal nutrients and gases exchange. Discussion: These findings indicate that exposure to particulate matter compromises the maternal-fetal interaction and may reflect on fetal nutrition and growth

Para identificar, quantificar e correlacionar os eosinófilos, mastócitos, miofibroblastos e células TGF-beta positivas nos pólipos nasais de pacientes com e sem asma foi realizado a imunoistoquímica. A quantidade de eosinófilos, miofibroblastos e células TGF-beta positivas esteve aumentada no pólipo nasal de indivíduos asmáticos. O número de mastócitos não mostrou diferença entre os grupos. O miofibroblasto foi o denominador comum na correlação entre eosinófilos, mastócitos, células TGF-beta positivas e presença de asma / Introduction: Nasal polyposis is a chronic inflammatory disease of the nasal mucosa or paranasal sinuses characterized by the formation of benign polyps. The pathogenesis is not known, although nasal polyps are associated with several systemic diseases, with asthma being the most frequent. The aim of the present study was to identify, quantify, compare and correlate eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells in nasal polyps of patients with and without asthma. Material and Methods: Seventy-eight subjects with nasal polyps undergoing endoscopic sinus surgery were selected. Control specimens were obtained from eight subjects with a normal sinus mucosa. One group consisted of polyps from 56 patients with asthma and the other of polyps from 22 patients without asthma. Immunohistochemistry was performed using monoclonal antibodies against eosinophil cationic protein to stain eosinophils, against tryptase to stain mast cells, against alpha-smooth muscle actin to stain myofibroblasts, and against TGF-ß to stain TGF-ß-positive cells. Results: The number of eosinophils, myofibroblasts and TGF-ß-positive cells was significantly higher in the asthma group than in the nonasthma group, whereas no significant difference in the number of mast cells was observed between the two groups. The number of eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells was significantly higher in nasal polyps than in the control group. Myofibroblasts showed a significant correlation with eosinophils, mast cells, TGF-ß-positive cells, and asthma. Conclusion: Eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells were identified in all nasal polyps, although the number of eosinophils, myofibroblasts and TGF-ß-positive cells was higher in the asthma group. The number of mast cells was similar regardless of the presence or absence of asthma. Myofibroblasts were a common denominator in the correlation between eosinophils, mast cells, TGF-ß-positive cells, and asthma

Para identificar, quantificar e correlacionar os eosinófilos, mastócitos, miofibroblastos e células TGF-beta positivas nos pólipos nasais de pacientes com e sem asma foi realizado a imunoistoquímica. A quantidade de eosinófilos, miofibroblastos e células TGF-beta positivas esteve aumentada no pólipo nasal de indivíduos asmáticos. O número de mastócitos não mostrou diferença entre os grupos. O miofibroblasto foi o denominador comum na correlação entre eosinófilos, mastócitos, células TGF-beta positivas e presença de asma / Introduction: Nasal polyposis is a chronic inflammatory disease of the nasal mucosa or paranasal sinuses characterized by the formation of benign polyps. The pathogenesis is not known, although nasal polyps are associated with several systemic diseases, with asthma being the most frequent. The aim of the present study was to identify, quantify, compare and correlate eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells in nasal polyps of patients with and without asthma. Material and Methods: Seventy-eight subjects with nasal polyps undergoing endoscopic sinus surgery were selected. Control specimens were obtained from eight subjects with a normal sinus mucosa. One group consisted of polyps from 56 patients with asthma and the other of polyps from 22 patients without asthma. Immunohistochemistry was performed using monoclonal antibodies against eosinophil cationic protein to stain eosinophils, against tryptase to stain mast cells, against alpha-smooth muscle actin to stain myofibroblasts, and against TGF-ß to stain TGF-ß-positive cells. Results: The number of eosinophils, myofibroblasts and TGF-ß-positive cells was significantly higher in the asthma group than in the nonasthma group, whereas no significant difference in the number of mast cells was observed between the two groups. The number of eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells was significantly higher in nasal polyps than in the control group. Myofibroblasts showed a significant correlation with eosinophils, mast cells, TGF-ß-positive cells, and asthma. Conclusion: Eosinophils, mast cells, myofibroblasts and TGF-ß-positive cells were identified in all nasal polyps, although the number of eosinophils, myofibroblasts and TGF-ß-positive cells was higher in the asthma group. The number of mast cells was similar regardless of the presence or absence of asthma. Myofibroblasts were a common denominator in the correlation between eosinophils, mast cells, TGF-ß-positive cells, and asthma

La fibrose pulmonaire idiopathique (FPI) et le syndrome de détresse respiratoire aiguë (SDRA) de l’adulte constituent des affections sévères du poumon distal, avec un pronostic sombre pour les patients. A ce jour, aucun traitement n’est réellement efficace. De manière intéressante, une hypoxie alvéolaire est retrouvée dans ces pathologies.La thérapie cellulaire utilisant des cellules souches mésenchymateuses humaines (CSMh) pourrait représenter un intérêt thérapeutique chez l’Homme. Cependant, leurs mécanismes d’action sont multiples et encore mal définis. Aussi, nous avons testé in vitro l’hypothèse selon laquelle les CSMh pourraient exercer un effet cytoprotecteur paracrine sur les cellules épithéliales alvéolaires (CEA) soumises à l’hypoxie.Dans une première étude, nous avons montré qu’une exposition prolongée à l’hypoxie telle que celle rencontrée au cours de la FPI induisait des modifications phénotypiques des CEA primaires de rat, évocatrices d’une transition épithélio-mésenchymateuse (TEM). On observe une perte progressive d’expression des marqueurs épithéliaux (TTF1, AQP5, ZO-1 et E-Cadhérine), couplée à l’apparition tardive de marqueurs mésenchymateux (α-SMA et Vimentine). Ces modifications phénotypiques s’accompagnent de l’expression dès les premières heures d’hypoxie de facteurs de transcription impliqués dans la TEM (SNAI1, TWIST1 et ZEB1) ou induits par l’hypoxie (HIF-1α et HIF-2α), et de protéines induisant la TEM (TGF-β1 et CTGF). La co-culture des CEA avec des CSMh en fond de puits prévient les modifications phénotypiques induites par l’hypoxie ainsi que l’expression des facteurs pro-TEM TWIST1, ZEB1, TGF-β1 et CTGF. Cet effet bénéfique des CSM est en partie expliqué par la sécrétion d’un facteur de croissance épithélial, le KGF.Dans une deuxième étude, nous avons confirmé que les CEA entraient en apoptose en condition hypoxique, via l’induction de deux voies de signalisations hypoxiques pro-apoptotiques. D’une part, les facteurs de transcription induits par l’hypoxie HIF sont stabilisés, et une cible pro-apoptotique, Bnip3, est induite. D’autre part, l’hypoxie induit une accumulation d’espèces réactives à l’oxygène délétère pour la cellule, perturbant l’équilibre redox de la cellule, endommageant l’ADN, et conduisant à l’apoptose. Cette accumulation pourrait résulter notamment d’une diminution de l’activité des enzymes anti-oxydantes SOD, en hypoxie. Le manque d’oxygène entraine également l’expression de CHOP, facteur de transcription pro-apoptotique impliqué dans le stress du réticulum endoplasmique, qui va13inhiber l’expression de la protéine anti-apoptotique Bcl-2. Nous avons montré que la culture des CEA en présence de milieu conditionné de CSMh (mc-CSMh) permet de prévenir partiellement l’apoptose des CEA en hypoxie, en modulant la voie de signalisation HIF, et en prévenant l’accumulation et les effets délétères des ROS. L’effet protecteur des CSM impliquerait le KGF comme observé lors de la première étude, mais également le HGF.Ces deux études indiquent que les CSMh sont susceptibles d’exercer des effets cytoprotecteurs paracrines vis-à-vis des CEA soumises à l’hypoxie aiguë ou prolongée, en limitant d’une part les modifications phénotypiques évocatrices de TEM, et d’autre part l’apoptose des CEA via la modulation des voies de signalisations hypoxiques. La sécrétion par les CSMh de KGF et de HGF, facteurs de croissance épithéliaux connus pour leurs effets bénéfiques sur les CEA, explique en partie les effets protecteurs paracrines des CSMh. Nos résultats suggèrent que les effets cytoprotecteurs des CSMh vis-à-vis des CEA pourraient contribuer aux effets bénéfiques des CSMh observés in vivo dans différents modèles animaux de fibrose induite, ou lors d’agressions alvéolaires aiguës. / Acute or chronic alveolar injuries provoke massive apoptosis of alveolar epithelial cells (AEC) that compromises an efficient repair of the alveolar epithelium and leads to lung diseases such as ARDS or IPF. These disorders are commonly associated with local alveolar hypoxia aggravating their progression through the stimulation of AEC apoptosis. Administration of allogenic mesenchymal stem cells (MSCs) has been shown to limit lung inflammation and fibrosis in murine models of alveolar injury, through a still poorly understood paracrine mechanism. In a first study, we showed that long term exposure of AEC in hypoxia leads to phenotypic alterations which looks like epithelio-mesenchymal transition (EMT). Co-culture with MSCs prevent hypoxia-induced EMT.In a second work, we studied whether MSC could protect AEC from hypoxia-induced apoptosis and the mechanisms involved. hMSC-conditioned media (hMSC-CM) significantly reduced hypoxia-induced apoptosis of AEC. Such a anti-apoptotic effect was also obtained with ROS scavenger N-acetylcystein or HIF1a inhibitor YC-1. hMSC-CM decreased the protein expression of HIF1α and HIF2α and of their pro-apoptotic target Bnip3 in hypoxic AEC. hMSC-CM also reduced ROS accumulation in hypoxic AEC by enhancing the activity of anti-oxidant enzymes and prevented the induction of CHOP, a pro-apoptotic factor induced by ROS signaling. The paracrine effect of hMSC was partly dependent on KGF and HGF secretion. hMSC prevent via a paracrine effect hypoxia-induced apoptosis of AEC by modulating hypoxic and ROS signaling.These two studies show that MSCs exert cytoprotective effects in vitro against hypoxia-induced apoptosis and EMT in AEC

Einen Aspekt dieser Arbeit stellt die Analyse der Stimulation von Ratten-Calvaria-Osteoblasten (RCA) mit den beiden Wachstumsfaktoren TGF-b1 und BMP-4 während der Proliferations- sowie Differenzierungs- und Mineralisierungsphase dar. Hierfür soll die Phosphorylierung und Aktivierung von Erk1 und Erk2, sowie von Smad1 und Smad2 mit Hilfe eines Kinase-Aktivitätsassays sowie der Westernblot-Analyse untersucht werden. Im Rahmen dieser Arbeit soll weiterhin untersucht werden, welche Bedeutung die Wachstumsfaktoren TGF-b1 und BMP-4 auf die Aktivität der alkalischen Phosphatase (ALP), einem wichtigen Differenzierungsmarker in Osteoblasten, ausüben. Enzymatische Aktivitätsbestimmungen und zytochemische Färbung aktiver ALP sollen darüber Aufschluss geben. Weiterhin soll der Gehalt an ALP-mRNA durch PCR bestimmt werden. Ein weiteres wichtiges Ziel dieser Arbeit ist die Analyse der Bedeutung von Erk1, Erk2, Smad1 und Smad2 auf die Aktivität der ALP. Dafür sollen Inhibitoren eingesetzt werden. Die enzymatische Aktivitätsbestimmung soll darüber aufklären. Außerdem soll mit Hilfe von kurzen, doppelsträngigen RNA-Molekülen (siRNA) ein knock down der Kinasen herbeigeführt werden und dessen Auswirkung auf die Aktivität der ALP enzymatisch bestimmt werden. Dafür muss zunächst die Wirksamkeit der siRNA auf RNA-Ebene mittels PCR und auf Proteinebene mittels Westernblot-Analysen überprüft werden. Zusätzlich soll die Bedeutung der Wachstumsfaktoren und der Kinasen Erk1 und Erk2 auf die Mineralisierung der RCA analysiert werden. Dafür wird die Menge des zellassoziierten Kalziums und Phosphats experimentell bestimmt, wodurch der Mineralisationsgrad der Zellen wiedergegeben werden kann.

Η Ιδιοπαθής Μεμβρανώδης Σπειραματονεφρίτιδα (ΙΜΣ) ή νεφροπάθεια, η πιο συχνή αιτία νεφρωσικού συνδρόμου στους ενήλικες, συνήθως αντιμετωπίζεται με τη χορήγηση κορτικοειδών και κυτταροτοξικών φαρμάκων ή κυκλοσπορίνης (cyclosporine-A, CsA). Σκοπός της μελέτης ήταν η εκτίμηση της αποτελεσματικότητας της μακροχρόνιας χορήγησης CsA στην πρόκληση ύφεσης του νεφρωσικού συνδρόμου και των ιστολογικών αλλοιώσεων σε επαναληπτικές βιοψίες νεφρού μετά τη χορήγηση του δυνητικά νεφροτοξικού αυτού φαρμάκου. Επιπλέον, εκτιμήθηκε η αποτελεσματικότητα του Mycophenolate Mofetil (MMF) ως σχήματος θεραπείας της ΙΜΣ σε μικρό αριθμό ασθενών και η προγνωστική αξία των επιπέδων του αυξητικού παράγοντα TGF-β1 στα ούρα και στο πλάσμα ως δεικτών εξέλιξης της νόσου. Μελετήθηκαν 32 ασθενείς με ΙΜΣ οι οποίοι εμφάνιζαν νεφρωσικό σύνδρομο και είχαν ικανοποιητική νεφρική λειτουργία κατά τη διάγνωση της νόσου και στους οποίους χορηγήθηκε συνδυασμός πρεδνιζολόνης και CsA. Παρατηρήθηκε πλήρης ύφεση του νεφρωσικού συνδρόμου σε 18 (56%) και μερική ύφεση σε 10 ασθενείς (31%) μετά από 12 μήνες θεραπείας (συνολικά στο 87% των ασθενών). Επεισόδια υποτροπών παρατηρήθηκαν στο 39% και 60% των ασθενών με πλήρη ή μερική ύφεση αντίστοιχα, και πολλαπλές υποτροπές στο 25% των ασθενών, οι οποίοι παρουσίασαν βαθμιαία μείωση της απαντητικότητας στη CsA και επιδείνωση της νεφρικής λειτουργίας. Επαναληπτική βιοψία νεφρού έγινε σε 18 ασθενείς με ύφεση του νεφρωσικού συνδρόμου μετά από 24 μήνες θεραπείας για να εκτιμηθεί η δραστηριότητα της νόσου και οι πιθανές ιστολογικές αλλοιώσεις σε πλαίσια τοξικότητας από κυκλοσπορίνη. Στις επαναληπτικές βιοψίες παρατηρήθηκαν: εξέλιξη του σταδίου της νόσου, επιδείνωση της σπειραματοσκλήρυνσης και της διαμεσοσωληναριακής βλάβης στο 60% των ασθενών. Δεν παρατηρήθηκαν χαρακτηριστικές αλλοιώσεις νεφροτοξικότητας από την κυκλοσπορίνη. Η βαρύτητα των ιστολογικών αλλαγών συσχετίστηκε με το χρονικό διάστημα που είχε παρέλθει από την πρώτη βιοψία νεφρού (r = 0.452, p < 0.05) και θεωρήθηκε ως φυσική εξέλιξη της νόσου. Ικανοποιητικά αποτελέσματα διαπιστώθηκαν από τη χορήγηση Mycophenolate Mofetil σε 6 ασθενείς με ΙΜΣ, στους οποίους το MMF χρησιμοποιήθηκε σε συνδυασμό με μικρή δόση πρεδνιζολόνης, είτε λόγω ανθεκτικότητας του νεφρωσικού συνδρόμου στην CsA, είτε ως αρχική θεραπεία σε περιπτώσεις αντένδειξης στην χορήγηση CsA. Ύφεση του νεφρωσικού συνδρόμου παρατηρήθηκε σε 4 από τους 6 ασθενείς. Τα επίπεδα του TGF-β1 στα ούρα ασθενών με ΙΜΣ και λευκωματουρία ήταν σημαντικά υψηλότερα συγκριτικά με αυτά υγιών εθελοντών και ασθενών με άλλες σπειραματοπάθειες που δεν παρουσίαζαν λευκωματουρία και μειώθηκαν σημαντικά μετά από χορήγηση κορτικοειδών και κυκλοσπορίνης. Η συγκέντρωση του TGF-β1 στο πλάσμα δε διέφερε σημαντικά μεταξύ υγιών εθελοντών και ασθενών με ΙΜΣ και νεφρωσικό σύνδρομο, καθώς και μεταξύ ασθενών με ή χωρίς ύφεση της λευκωματουρίας μετά από τη θεραπευτική αγωγή. / Idiopathic membranous nephropathy (IMN), the most common cause of nephrotic syndrome in adults, is usually treated with a combination of corticosteroids with cytotoxic drugs or cyclosporin A (CsA). The aim of this study was the estimation of the effectiveness of long-term use of CsA in the remission and relapse rate of nephrotic syndrome along with histological changes in repeat renal biopsies after treatment with this potentially nephrotoxic drug, and the evaluation of Mycophenolate Mofetil (MMF) as a treatment regimen for IMN. In addition, urinary and plasma TGF-β1 levels were evaluated as markers of progression of kidney disease. Thirty-two nephrotic patients with well-preserved renal function treated by prednisolone and CsA were studied. Complete remission of nephrotic syndrome was observed in 18 (56%) and partial remission in 10 patients (31%) after 12 months of treatment (total 87%). Relapses were observed in 39% and 60% of patients with complete and partial remission, respectively, and multiple relapses in 25% of patients, who showed gradual unresponsiveness to CsA and decline of renal function. A repeat biopsy was performed in 18 patients with remission of nephrotic syndrome, after 24 months of treatment, to estimate the activity of the disease and features of CsA toxicity. Progression of the stage of the disease, more severe glomerulosclerosis and tubulointerstitial injury were recognized in 60% of patients in repeat renal biopsies. Features of CsA nephrotoxicity were not observed. The severity of histological changes was related to the time elapsed from the first biopsy (r = 0.452, P < 0.05). MMF was proved effective in a small number of nephrotic patients with IMN and well-preserved renal function. MMF in combination with small dose of prednisolone was given in 6 patients with either persistent nephrotic syndrome to CsA or as initial therapy because of contraindication to CsA administration. Remission of nephrotic syndrome was observed in 4 out of 6 MMF treated patients. Urinary and plasma TGF-β1 levels were examined as markers of progression of the disease. TGF-β1 levels in the urine of patients with proteinuria were significantly higher compared with those of healthy individuals and patients with other types of nephropathy without proteinuria. Furthermore, urinary TGF-β1 of nephrotic patients with membranous nephropathy significantly reduced after treatment with CsA and corticosteroids. Plasma TGF-β1 levels showed no difference between patients and healthy subjects as well as between patients with and without remission of proteinuria after treatment.

Scar-free wound healing and regeneration are uncommon phenomena permitting the near complete restoration of damaged tissues, organs and structures. Although rare in mammals, many lizards are able to undergo scarless healing and regeneration following loss of the tail. This study investigated the spontaneous and intrinsic capacity of the leopard gecko (Eublepharis macularius) tail to undergo scar-free wound healing and regeneration following two different forms of tail loss: autotomy, a voluntary and evolved mechanism of tail shedding at fracture planes; and surgical amputation, involuntary loss of the tail outside the fracture planes. Furthermore, I investigated the ability of the regenerate tail to regenerate by amputating a regenerate tail (previously lost by autotomy). To investigate these phenomena I imaged wound healing and regenereating tails daily (following autotomy and amputation) to document gross morphological changes. I used histochemistry to document tissue structure and immunohistochemistry to determine the tissue/cellular location of my five proteins of interest (PCNA, MMP-9, WE6, α-sma, TGF-β3). Each of these proteins of interest has been previously documented during wound healing and/or regeneration in other wound healing/regeneration model organisms (e.g. mice, urodeles, lizards, zebrafish). Scar-free wound healing and regeneration occurred following autotomy, amputation of the original tail and amputation of the regenerate tail, indicating that the leopard gecko tail has an instrinsic scar-free wound healing and regenerative capacity that is independent of the mode of tail loss (autotomy or amputation). Furthermore immunohistochemistry revealed a conserved sequence and location of the expression of the five proteins of interest following both forms of tail loss. These results provide the basis for further studies investigating scar-free wound healing and regeneration in a novel amniote model, the leopard gecko. / NSERC

Les protéines GILZ (Glucocorticoid-Induced Leucine Zipper) et TSC-22 (Transforming growth factor-beta Stimulated Clone-22) appartiennent à la famille de protéines TSC-22D (TSC-22 Domain). GILZ a été décrit précédemment comme étant induit au cours de la déprivation en interleukine-2 (IL-2) des lymphocytes de la lignée cellulaire CTLL-2, permettant ainsi de retarder leur apoptose. Le but de notre travail était de déterminer les rôles respectifs de GILZ et TSC-22 au cours de l'apoptose des cellules CTLL-2.Nos résultats ont permis de montrer que TSC-22 augmentait l'apoptose induite par la déprivation en IL-2 des cellules CTLL-2. Nous avons mis en évidence une augmentation de l'activation des caspases ainsi qu'une régulation positive de l'expression de BIM. Nous avons en outre montré que l'expression de GILZ, protéine anti-apoptotique, induite lors de la déprivation en IL-2, était régulée négativement en présence de TSC-22. Enfin, nous avons montré que l'expression de l'ARNm de gilz était régulée négativement par TSC-22, mais que la stabilité de son ARNm n'était pas modifiée.Notre travail a donc permis de montrer que TSC-22 accélère l'entrée en apoptose des lymphocytes T en régulant négativement l'expression de la protéine anti-apoptotique GILZ.

The TGFβ family of cytokines are potent signalling molecules that regulate tissue development, inflammation and immunity. Previous studies in mice with a null mutation in the Tgfb1 gene (TGFβ1-/- mice) implicate a key role for TGFβ1 in male reproductive function. These mice show profound infertility due to an inability to copulate successfully, associated with reduced testosterone and sperm production. The focus of this project was to 1) further characterize mechanisms underpinning reproductive deficiency in male TGFβ1-/- mice, 2) identify a reliable physiological marker of TGFβ1 availability in vivo, and 3) to determine whether exogenous TGFβ1 administration influences TGFβ1 availability and restores fertility. To investigate the causes of unsuccessful copulation by TGFβ1-/- mice, penis morphometry was examined. Penile organ structure, as assessed by scanning electron microscopy, was comparable between genotypes however a superfluous epidermal covering that impeded penile spine protrusion was evident in TGFβ1-/- mice. The epidermal covering was not due to increased epithelial cell proliferation, as measured by Brdu labelling and immunohistology. Behavioural observations of erectile activity showed that TGFβ1-/- mice achieved spontaneous erections albeit at reduced frequency compared to TGFβ1+/+ mice. The efficacy of exogenous TGFβ1 replacement was evaluated by first identifying measures of in vivo TGFβ1 availability and/or function and selecting an effective route of administration. Serum TGFβ1 and testosterone levels were reliable discriminators of TGFβ1 genotype. Gene expression and phagocytic function of peritoneal macrophages revealed no differences between genotypes. Exogenous sources of TGFβ1 for replacement studies included colostrum, naturally occurring in breast milk and recombinant human latent TGFβ1 (rhLTGFβ1). Colostrum did not increase circulating levels and rhTGFβ1 injection caused only transient elevation of serum levels. Thus mini-osmotic pumps were used to deliver a constant supply of cytokine to TGFβ1-/- mice. The fertility status of TGFβ1-/- mice receiving exogenous TGFβ1 was investigated. Reproductive behaviour in response to normal receptive female mice was assessed twice during treatment, on day 7 and day 14. Blood, liver and reproductive tissues were collected at sacrifice. Circulating TGFβ1 was increased in TGFβ1 treated TGFβ1-/- mice above TGFβ1-/- control levels, although this did not affect circulating testosterone. Erectile activity and sperm production were unchanged. Videotaping behaviour with estrous females revealed that the TGFβ1+/+ mice successfully mounted and intromitted, unlike the TGFβ1-/- controls. The TGFβ1-/- mice receiving exogenous TGFβ1 displayed moderately enhanced mounting and intromission behaviour although this remained less frequent than in the TGFβ1+/+ controls. Ejaculation behaviour was not observed in any TGFβ1-/- mice regardless of TGFβ1 replacement, compared to TGFβ1+/+ controls where >90% mice displayed ejaculated. Modest improvement in the copulation activity of the TGFβ1-/- mice receiving exogenous TGFβ1 suggests that systemic TGFβ1 availability can influence reproductive performance in male TGFβ1-/- mice. However since fertility was not restored, locally produced TGFβ1 in the reproductive tract and/or hypothalamic pituitary axis are also implicated in regulating fertility. These findings advance our knowledge of the role of the TGFβ1 cytokine in male reproductive physiology and may have relevance for devising new treatments for infertility and erectile dysfunction in men. / Thesis (Ph.D.) - University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 117-147). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;