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Viruses are pathogenic agents which affect all varieties of organisms, including plants, animals and humans. These microscopic particles are genetically simple organisms which encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope coat that surrounds them. Lipid enveloped viruses comprise a diverse range of pathogenic viruses, known to cause disease in both animals and human which often leads to high fatality rates, many of which lack effective and approved therapeutics. This report focuses on learning how a multifunctional protein within lipid enveloped viruses, the matrix protein, interacts with the plasma membrane of cells to enter and exit cells. Specifically, four viruses are investigated, Measles virus and Nipah virus (within the <i>Paramyxoviridae</i> family) and Ebola virus and Marburg virus (within the <i>Filoviridae</i> family). Through numerous <i>in vitro </i>experiments, functional cellular assays, a myriad of microscopy techniques, and experiments in high containment bio-safety level 4 settings, this report identifies specific lipids at play during the viral assembly process for each virus. Moreover, mechanistic insight is presented as to how each matrix protein interacts with the plasma membrane to facilitate: membrane association, viral matrix protein oligomerization and assembly, the rearrangement of lipids within the plasma membrane, and viral production. Lastly, numerous small molecule inhibitors targeting specific lipids, (e.g. phosphatidylserine and phosphatidylinositol 4,5 bisphosphate) within the cell were investigated for their efficacy in inhibiting matrix protein-dependent viral like particle production and viral spread in cells. As a whole, these projects lend credence to the significant role that lipids and the plasma membrane play throughout lipid enveloped viral life cycles, and provide compelling evidence for the merit of future drug-development research geared at targeting the matrix protein-plasma membrane interaction.

The extracellular matrix is a multi-functional environment that cells inhabit to form living tissue. To maintain the tissue, cells require constant telemetry with the matrix and respond to a variety of cues by remodeling matrix architecture. In this study the physical and biochemical manipulation of the matrix by resident cells is explored to better understand how these are used to remodel tissue. Cell-populated collagen hydrogels are used as a controllable in vitro tissue model. To directly measure mechanical forces involved with gel contraction, a culture force monitor was designed and built. Measuring dimensional changes together with contractile forces presents a method of separating mechanisms that influence tissue remodeling. Together, these techniques revealed a correlation between contractile force and gel deformation, suggesting a novel method for examining the material properties of the matrix. Limiting matrix metalloproteinase (MMP) activity altered the correlation as predicted, indicating a stiffer matrix. Contractile force was found to be regulated independent of MMP activity. In contrast, contractile force was found to be dependent on α2β1 integrin function. Collagen gel contraction correlated with both α2β1 function and MMP activity, and was significantly enhanced when combined. The results of this study indicate cells have the capacity to use multiple mechanisms for remodeling the extracellular matrix and may alternately use them together or independently to vary the rate of matrix contraction.

No / This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and β1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Αν και η Αυχενική Σπονδυλωτική Μυελοπάθεια (ΑΣΜ) αποτελεί την πιο κοινή αιτία δυσλειτουργίας νωτιαίου μυελού στους ενήλικες άνω των 55 ετών, οι μοριακοί μηχανισμοί παραμένουν άγνωστοι. Μέχρι σήμερα, πολλές προσπάθειες έχουν διενεργηθεί για την ανάπτυξη ενός αξιόπιστου πειραματικού μοντέλου AΣΜ. Ωστόσο, αρκετά μειονεκτήματα εμφανίζονται σε αυτές τις μελέτες. Στη παρούσα μελέτη έχουμε σκοπό τη δημιουργία ενός νέου, πρωτότυπου πειραματικού μοντέλου ΑΣΜ, το οποίο εξομοιώνει τα ιστολογικά και κλινικά χαρακτηριστικά της ανθρωπίνης νόσου. Mεθοδολογία: Μετά από αφαίερεση του πετάλου του έβδομου αυχενικού σπονδύλου, ένα λεπτό τεμάχιο αρωματικού πολυαιθέρα τοποθετήθηκε κάτω από το πέταλο του έκτου αυχενκού σπονδύλου σε κόνικλους Νέας Ζηλανδίας (Ομάδα ΧΠΠ). Σε μία άλλη ομάδα πειραματόζωων ο αρωματικός πολυαιθέρας αφαιρέθηκε 30 δευτερόλεπτα μετά την εμφύτευση (ομάδα ελέγχου). Νευρολογική εκτίμηση πραγματοποιήθηκε χρησιμοποιώντας τη κλίμακα του Tarlov μετά το πέρας της χειρουργικής διαδικασίας και ακολούθως εβδομαδιαίως. Ηλεκτροφυσιολογικές μελέτες πραγματοποιήθηκαν στις 20 εβδομάδες μετά το χειρουργείο και πριν από τη θυσία των πειραματόζωων. Ακολούθησαν ιστολογικές και ανοσοιστοχημικές μελέτες. Αποτελέσματα: Τα πειραματόζωα που άνηκαν στην ομάδα ελέγχου δεν εμφάνισαν νευρολογικά ελλείμματα κατά τη διάρκεια της μελέτης. Αντιθέτως τα πειραματόζωα που άνηκαν στη ΧΠΠ εμφάνισαν νευρολογικά ελλείματα. Στους νωτιαίους μυελούς προερχόμενους από την ΧΠΠ ομάδα ανεδείχθησαν οι χαρακτηριστικές ιστοπαθολογικές αλλοιώσεις της χρόνιας μυελοπάθειας. Ειδικότερα, ανεδείχθη σπογγώδης εκφύλιση της λευκής ουσίας, διάμεσο οίδημα και αποπλάτυνση των πρόσθιων κεράτων της φαιάς ουσίας. Επίσης ανεδείχθη κατακρήμνιση του μυελικού σάκου και διόγκωση του δακτυλίου της μυελίνης. Τέλος, η χρόνια πίεση του νωτιαίου οδήγησε σε ενεργοποίηση της απόπτωσης και διαταραχή της αρχιτεκτονικής του μικροαγγειακού συστήματος του νωτιαίου μυελού Συμπέρασμα: Το πρωτότυπο μοντέλο ΑΣΜ στους κονίκλους ποσομοιώνει το χωρικό και χρονικό προφίλ της ανθρώπινης νόσου στο σημείο της πίεσης του νωτιαίου μυελού. ΜΕΛΕΤΗ B Εισαγωγή: Η φλεγμονή, η δημιουργία ουλώδους ιστού και η διαταραχή του μικροαγγειακού συστήματος του νωτιαίου μυελού είναι ορισμένα από τα κύρια παθοφυσιολογικά φαινόμενα της ΑΣΜ. Ωστόσο οι μοριακοί μηχανισμοί που εμπλέκονται σε αυτά τα φαινόμενα κάτω από τη χρόνια και προοδευτική πίεση του νωτιαίου μυελού παραμένουν ανεξερεύνητα. Mεθοδολογία: Στη συγκεκριμένη μελέτη χρησιμοποιήθηκε το πειραματικό μοντέλο ΑΣΜ που περιγράφεται στη μελέτη Α με σκοπό να διερευνηθεί ο ρόλος του NF-κB και των πρωτεινών της εξωκυττάριας ουσίας στην ΑΣΜ. Εν συντομία, κόνικλοι Νέας Ζηλανδίας (διαφορετικά πειραματόζωα από εκείνα της μελέτης Α) χωρίστηκαν τυχαία σε δύο ομάδες: την ομάδα ΧΠΠ (n=15) και την ομάδα ελέγχου (n=15). Η έκφραση των πρωτεινών των υπομονάδων p50 και p65 του NF-kB, όπως επίσης και των ενζύμων διάσπασης της εξωκυττάριας ουσίας (MMP-2, MMP-9) και του ενεργοποιητή του πλασμινογόνου τύπου ουροκινάσης (urokinase-type plasminogen activator; u-PA) αξιολογήθηκαν σε τομές νωτιαίων μυελών προερχόμενων και από τις δύο ομάδες χρησιμοποιώντας ανοσοιστοχημική τεχνική. Στατιστική ανάλυση πραγματοποιήθηκε χρησιμοποιώντας SPSS για Windows, release 12.0 (SPSS Inc., Chicago, IL). Αποτελέσματα: Σε τομές νωτιαίων μυελών που προέρχονταν από πειραματόζωα που έπασχαν από ΑΣΜ αναδείχθηκε στατιστικά σημαντικά αυξημένη έκφραση των υπομονάδων του NF-κB (p50 & p65), όπως επίσης και των ενζύμων MMP-2, MMP-9, and u-PA σε σύγκριση με εκείνες που προέρχονταν από την ομάδα ελέγχου. Τέλος, σημαντικά θετική συσχέτιση παρατηρήθηκε μεταξύ των επιπέδων έκφρασης του NF-κB και εκείνων των MMP-9, MMP-2, and u-PA. Συμπέρασμα: Τα ευρήματα αυτά αποτελούν ισχυρές ενδείξεις πως η χρόνια και προοδευτική πίεση του αυχενικού νωτιαίου μυελού οδηγεί σε αυξημένη έκφραση των MMP-2, MMP-9 και u-PA πιθανόν μέσω της δράσης του μεταγραφικού παράγοντα NF-κB. Είναι βέβαιο ότι περισσότερες μελέτες απαιτούνται για την εξακρίβωση του ρόλου των πρωτεινών αυτών στην ΑΣΜ. / Although cervical spondylotic myelopathy (CSM) represents the most common cause of spinal cord impairment among individuals over 55 years old, the molecular mechanisms of the disease remain mainly unknown. To date, many experimental studies have been conducted to establish a reliable model of CSM, however most of them appear some limitations. In this study we aim to create a new animal model of CSM, which will reproduce the temporal course of the human disease and the local microenvironment at the site of spinal cord compression. Methods: Following C7 posterior laminectomy, a thin sheet of aromatic polyether was implanted underneath C5–C6 laminae of the New Zealand rabbits. A sham group in which the material was removed 30 sec after the implantation was also included. Motor function evaluation was performed after the material implantation and weekly thereafter using the Tarlov classification. At 20 weeks post-material implantation electrophysiological studies were also conducted. All the animals were sacrificed 20 weeks post-material implantation and histological and immunohistochemical studies were performed. Results: Clinical evaluation of animals after operation reveals no symptoms and signs of acute spinal cord injury. Moreover, no neurological deficits were noticed in the sham group during the course of the study. However, the animals which underwent implantation of compression material exhibited progressive neurological deficits throughout the study. Rabbits of the compression group experienced significant increased axonal swelling and demyelination, interstitial edema and myelin sheet fragmentation. Histological evaluation of C5 and C6 laminae (at the site of implantation) reveals osteophyte formation. Moreover, the chronic and progressive compression of the cervical spinal cord resulted in induction of apoptosis as well as in disruption of the basement membrane of vessels. Conclusion: The proposed rabbit CSM model reproduces the temporal evolution of the disease and creates a local microenvironment at the site of spinal cord compression, which shares similar features with that of human disease. STUDY B Introduction: Inflammation, glial scar formation and disruption of spinal cord microvasculature represent some of the principal neuropathological features of CSM. However, the molecular mechanisms which are implicated in these pathophysiological phenomena under the chronic and progressive compression of the cervical spinal cord remain interestingly unexplored. Methods: In this study (B) in order to evaluate the role of NF-κB and extracellular matrix proteins in cervical myelopathy we used the rabbit CSM model which was extensively characterized in study A. Briefly New Zealand rabbits (different cohort of animals than that of the study A) were randomly and blindly divided into the following two groups: CSM (n=15) and sham group (n=15). The expression pattern of p50 and p65 subunits of NF-kB, as well as that of MMP-2, MMP-9, and u-PA, was evaluated in spinal cord sections coming from both groups using immunohistochemistry technique. Statistical analysis was performed using SPSS for Windows, release 12.0 (SPSS Inc., Chicago, IL). Results: CSM animals exhibited statistically significant increased immuoreactivity in both NF-κB subunits, p50 and p65. Moreover, the levels MMP-2, MMP-9, and u-PA were found to be significantly increased in CSM animals compared to controls. Finally, strong positive correlation between NF-κB subunits immunoreactivity and that of MMP-9, MMP-2, and u-PA was demonstrated. Conclusion: The NF-κB pathway as well as the extracellular matrix proteins (MMP-2 and MMP-9) are involved in CSM. However, more studies are needed to clarify the functional role of these molecules in the pathobiology of CSM.

Aggregatibacter actinomycetemcomitans está associado à etiologia da periodontite agressiva localizada. A colonização de tecidos do hospedeiro é necessária para patogênese e a adesão é fundamental. A proteína autotransportada Aae faz a adesão da bactéria a células epiteliais gengivais. Proteínas autotransportadas de diferentes espécies apresentam múltiplas funções e podem ser antígenos vacinais na prevenção de infecções. Para entender o papel de Aae na ligação ao hospedeiro e efeito de anticorpos contra Aae, o polimorfismo de aae na região que codifica o domínio de ligação a células epiteliais foi determinado e relacionado à adesão a células epiteliais KB . Aae recombinante foi obtida, e a capacidade de ligação a proteínas da matriz extracelular e soro foi determinada em ensaios com a recombinante e com uma amostra deficiente na expressão de Aae obtida (ensaios comparativos com amostra selvagem). Títulos de IgG contra Aae em pacientes com periodontite agressiva e saudáveis foram determinados e relacionados à resposta humoral contra sorotipos de A. actinomycetemcomitans. Por fim, o efeito de anticorpos contra Aae e/ou seu domínio efetor, produzidos em modelo animal, foi determinado na inibição da adesão mediada por Aae e opsonização por fagócitos. / Aggregatibacter actinomycetemcomitans is related with etiology of localized aggressive periodontitis. Colonization of host tissues is necessary to pathogenesis and adhesion is essential. The autotransporter protein Aae mediates the adhesion of bacteria to gingival epithelial cells. Autotransporter proteins from different species have multiple functions and could be vaccine antigens to prevent infections. To understand the role of Aae in host interaction and the effect of antibodies against Aae, polymorphism of aae in codifying effector domain region of ligation to epithelial cells was determined and related with adhesion to these cells. Recombinant Aae was obtained and the ability of interaction with Extracellular matrix and serum proteins was determined through assays using the recombinant and an obtained defective sample in Aae expression (comparative assays with wild type). IgG titters against Aae were determined in patients with aggressive periodontitis and healthy and related to humoral response against A. actinomycetemcomitans serotypes. At last, the effect of antibodies against Aae and/or its effector domain, obtained in animal model, was determined in inhibition of adhesion to epithelial cells and macrophages oopsonization.

Leptospirose é uma zoonose causada pela espiroqueta pertencente ao gênero Leptospira. LipL32 é um antígeno de superfície altamente conservado somente entre as espécies de leptospiras patogênicas e é expresso em altos níveis tanto in vitro com in vivo. HlyX é relatada como sendo uma proteína que possui um provável peptídeo sinal e cinco tetratricopeptídeos repetidos (TPR) em sua sequência de aminoácidos. Neste trabalho, mostrou-se que HlyX é expressa somente em cepas patogênicas, não sendo detectada a sua expressão na cepa saprofítica. HlyX foi reconhecida somente por soros de pacientes da fase convalescente da doença. Em constraste, LipL32 foi reconhecida por soros de pacientes colhidos tanto na fase aguda quanto na fase convalescente da infecção. Nossos resultados de immunoblot indicam que os domínios imunodominantes da proteína são os fragmentos C-terminal e intermediário. Uma resposta IgM foi detectada exclusivamente contra o fragmento C-terminal de LipL32 em ambas as fases da infecção. Com relação à capacidade de LipL32 e HlyX de interagir com componentes de matriz extracelular (CME), foi observada uma interação específica e dose-dependente de LipL32 e HlyX com colágeno tipo IV e fibronectina plasmática. O fragmento C-terminal de LipL32 é responsável por esta interação. Tanto a heparina quanto a gelatina foram capazes de inibir a ligação de LipL32 à fibronectina plasmática de forma dose-dependente, indicando que os domínios de ligação à heparina (30 kDa) e gelatina (45 kDa) da fibronectina estão envolvidos nesta interação. Por outro lado, apenas o domínio de ligação à heparina participa da interação da fibronectina com a proteína HlyX. A capacidade protetora das duas proteínas estudadas foi avaliada através de ensaios de imunização e desafio realizados em modelo animal (hamsters). A proteína HlyX induziu altos títulos de anticorpos IgG (1:128.000), mas somente a co-administração HlyX e LipL32 e a proteína LipL32 pura conferiram proteção, 100% e 80% respectivamente. HlyX não foi capaz de conferir proteção quando administrada apenas com o adjuvante Al(OH)3. Em conclusão, os resultados indicam que o domínio C-terminal de LipL32 é reconhecido desde o início da infecção e este domínio é responsável por mediar a interação de LipL32 com CME. Os dados obtidos com HlyX demonstram um possível papel desta proteína na patogênese, pelo fato de ser expressa e conservada em cepas patogênicas, e também por interagir com CME. Porém, apesar de HlyX apresentar altos títulos de anticorpos IgG, não conferiu atividade protetora quando administrada individualmente. / Leptospirosis, a spirochaetal zoonotic disease caused by Leptospira, has been recognized as na important emerging infectious disease. LipL32 is a surface lipoprotein which is highly conserved among pathogenic Leptospira species and is also expressed at high levels either during cultivation and natural infection. Regarding HlyX, it has been annotated as a protein containing a signal peptide and five tetratricopeptide repeats (TPR). Immunoblot analyses concerning HlyX distribution on Leptospira spp. indicate that this protein is expressed exclusively by pathogenic species. Moreover, HlyX was only recognized by sera of patients in the second week of leptospirosis infection. In contrast, LipL32 was recognized by acute and convalescent sera from leptospirosis patients. Our immunoblot results indicate that both the C-terminal and the intermediate domains of LipL32 are recognized by sera of patients. An IgM response was detected exclusively against the LipL32 C-terminus in both the acute and convalescent phases of illness. Concerning the capacity of LipL32 and HlyX to interact with extracellular matrix (ECM) components, a dose-dependent specific binding of LipL32 and HlyX to collagen IV and plasma fibronectin was observed. The LipL32 binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin- and the 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. However, HlyX binding to fibronectin could only be inhibited by heparin in a concentration-dependent manner. We also evaluated whether HlyX and LipL32 could induce protective immunity against the challenge with a homologous serovar in hamsters. Although high anti-HlyX (IgG) titers (1:128,000) have been achieved upon immunization, no protection was observed. However, a combined HlyX and LipL32 immunization could induce a protective response (100%). The protection observed for LipL32 immunization was 80%. Altogether, the results provide evidence that the LipL32 C-terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins. HlyX protein may contribute to the pathogenesis of the disease by interacting with host proteins. However, HlyX is not a protective antigen when administered alone.

Zielsetzung: Verschiedene chirugische Techniken sind für die Deckung entblößter Wurzeloberflächen vorgeschlagen worden. Die vorliegende Studie sollte die Ergebnisse koronaler Verschiebelappen mit bzw. ohne Einsatz von Schmelzmatrixprotein (SMP) bei der Therapie fazialer Rezessionen vergleichen. Material und Methode: Die Studie war als intraindividueller longitudinaler Vergleich über 12 Monate in einem doppelt verblindeten plazebo-kontrollierten randomisierten Design gestaltet. 22 Patienten im Alter von 24-64 Jahren, die 2 paarige faziale Rezessionen von mindestens 3 mm aufwiesen, wurden untersucht. Beide Rezessionen wurden in derselben Sitzung nach der Technik des koronalen Verschiebelappens chirurgisch gedeckt. Eine Rezession wurde dabei zusätzlich mit einem kommerziell erhältlichen SMP (Emdogain) und die jeweils andere mit dem entsprechenden Trägergel (Vehikel: Propylen-glykol-alginat) behandelt. Die Zuweisung der Therapien erfolgte zufällig. Präoperativ sowie 1 und 3 Wochen, 3, 6 und 12 Monate postoperativ wurden durch einen verblindeten Untersucher klinische Parameter (Höhe und Breite der Rezession, Breite der keratinisierten Gingiva, Sondierungstiefe, Attachmentniveau, Knochenniveau) mittels manueller sowie elektronischer Parodontalsonde bzw. Schieblehre auf 0,5 mm genau erhoben. Ergebnisse: 12 Monate postoperativ zeigten beider Therapievarianten signifikante Rezessionsdeckungen und Attachmentgewinne. Die fazialen Rezessionen verringerten sich von 4,5 mm auf 1,5 mm in der SMP- und von 4,4 mm auf 1,5 mm in der Vehikel-Gruppe was einer Rezessionsdeckung von 71,7% bzw. 73,6% entspricht. Der Unterschied zwischen den zwei Gruppen war nicht signifikant. Alle anderen klinische Parameter zeigten keine Unterschiede zwischen den Gruppen. Schlußfolgerungen: Der Einsatz von SMP zusätzlich zum koronalen Verschiebelappen zur chirurgischen Rezessionsdeckung ergab keine wesentliche Unterschiede in den klinischen Resultaten im Vergleich zum koronalen Verschiebelappen in Kombination mit dem Trägergel. / Objectives: Various surgical techniques have been proposed for root coverage of denuded root surfaces. The aim of this study was to evaluate a comparison of coronally advanced flap procedure with or without the use of enamel matrix proteins in the treatment of recession defects. Material and methods: This study was an intra-individual longitudinal test of 12 months duration conducted as a blinded, split-mouth, placebo-controlled and randomised design. 22 patients, aged 24-64 years, with 2 paired buccal recession defects of at least 3 mm participated. Surgical recession coverage was performed as coronally advanced flap technique at both sites in the same session. One site was additionally treated with commercially available enamel matrix proteins (Emdogain) and the other site with placebo (propylene glycol alginate) in accordance with the randomisation list. A blinded examiner assessed pre- and post-surgical measurements. Clinical measurements and photographs were taken pre-surgically and after 1 week, 3 weeks, 3 months, 6 months and 12 months postoperatively. Measurements comprised height and width of the gingival recession, height of keratinized tissue, probing attachment level, probing pocket depth and alveolar bone level by periodontal probe, Florida Probe or caliper to the nearet 0.5 mm. Results: Twelve months after therapy, both treatment modalities showed significant root coverage and probing attachment gain. Gingival recession decreased from 4,5 mm to 1,5 mm for the Emdogain treated sites and from 4,4 mm to 1,5 mm for the control sites, corresponding to mean root coverages of 71,7% and 73,6%, respectively. This difference was not significant. All other clinical variables were not different in the between-group comparison. Conclusions: The use of Emdogain together with coronally advanced flap technique for recession coverage appeared to be equally effective in the overall clinical outcome, there is no clear benefit to combine Emdogain with this surgical technique.

The peroxisome is a subcellular organelle that carries out a diverse range of metabolic functions, including the b-oxidation of very long chain fatty acids, the breakdown of peroxide and the a-oxidation of fatty acids. Disruption of peroxisome metabolic functions leads to severe disease in humans. These diseases can be broadly grouped into two categories: those in which a single enzyme is defective, and those known as the peroxisome biogenesis disorders (PBDs), which result from a generalised failure to import peroxisomal matrix proteins (and consequently result in disruption of multiple metabolic pathways). The PBDs result from mutations in PEX genes, which encode protein products called peroxins, required for the normal biogenesis of the peroxisome. PEX1 encodes an AAA ATPase that is essential for peroxisome biogenesis, and mutations in PEX1 are the most common cause of PBDs worldwide. This study focused on the identification of mutations in PEX1 in an Australasian cohort of PBD patients, and the impact of these mutations on PEX1 function. As a result of the studies presented in this thesis, twelve mutations in PEX1 were identified in the Australasian cohort of patients. The identified mutations can be broadly grouped into three categories: missense mutations, mutations directly introducing a premature termination codon (PTC) and mutations that interrupt the reading frame of PEX1. The missense mutations that were identified were R798G, G843D, I989T and R998Q; all of these mutations affect amino acid residues located in the AAA domains of the PEX1 protein. Two mutations that directly introduce PTCs into the PEX1 transcript (R790X and R998X), and four frameshift mutations (A302fs, I370fs, I700fs and S797fs) were identified. There was also one mutation found in an intronic region (IVS22-19A>G) that is presumed to affect splicing of the PEX1 mRNA. Three of these mutations, G843D, I700fs and G973fs, were found at high frequency in this patient cohort. At the commencement of these studies, it was hypothesised that missense mutations would result in attenuation of PEX1 function, but mutations that introduced PTCs, either directly or indirectly, would have a deleterious effect on PEX1 function. Mutations introducing PTCs are thought to cause mRNA to be degraded by the nonsense-mediated decay of mRNA (NMD) pathway, and thus result in a decrease in PEX1 protein levels. The studies on the cellular impact of the identified PEX1 mutations were consistent with these hypotheses. Missense mutations were found to reduce peroxisomal protein import and PEX1 protein levels, but a residual level of function remained. PTC-generating mutations were found to have a major impact on PEX1 function, with PEX1 mRNA and protein levels being drastically reduced, and peroxisomal protein import capability abolished. Patients with two missense mutations showed the least impact on PEX1 function, patients with two PTC-generating mutations had a severe defect in PEX1 function, and patients carrying a combination of a missense mutation and a PTC-generating mutation showed levels of PEX1 function that were intermediate between these extremes. Thus, a correlation between PEX1 genotype and phenotype was defined for the Australasian cohort of patients investigated in these studies. For a number of patients, mutations in the coding sequence of one PEX1 allele could not be identified. Analysis of the 5' UTR of this gene was therefore pursued for potential novel mutations. The initial analyses demonstrated that the 5' end of PEX1 extended further than previously reported. Two co-segregating polymorphisms were also identified, termed –137 T>C and –53C>G. The -137T>C polymorphism resided in an upstream, in-frame ATG (termed ATG1), and the possibility that the additional sequence represented PEX1 coding sequence was examined. While both ATGs were found to be functional by virtue of in vitro and in vivo expression investigations, Western blot analysis of the PEX1 protein in patient and control cell extracts indicated that physiological translation of PEX1 was from the second ATG only. Using a luciferase reporter approach, the additional sequence was found to exhibit promoter activity. When examined alone the -137T>C polymorphism exerted a detrimental effect on PEX1 promoter activity, reducing activity to half that of wild-type levels, and the -53C>G polymorphism increased PEX1 promoter activity by 25%. When co-expressed (mimicking the physiological condition) these polymorphisms compensated for each other to bring PEX1 promoter activity to near wild-type levels. The PEX1 mutations identified in this study have been utilised by collaborators at the National Referral Laboratory for Lysosomal, Peroxisomal and Related Genetic Disorders (based at the Women's and Children's Hospital, Adelaide), in prenatal diagnosis of the PBDs. In addition, the identification of three common mutations in Australasian PBD patients has led to the implementation of screening for these mutations in newly referred patients, often enabling a precise diagnosis of a PBD to be made. Finally, the strong correlation between genotype and phenotype for the patient cohort investigated as part of these studies has generated a basis for the assessment of newly identified mutations in PEX1.

Decorin, is an extracellular matrix proteoglycan with important biological functions. Decorin deficiency affects collagen fibrillogenesis, airway mechanics, airway-parenchymal interdependence, and airway smooth muscle proliferation and apoptosis. We questioned whether decorin deficiency would alter allergen-induced asthma in a mouse model. Decorin-/- and decorin+/+ mice (C57Bl/6) were sensitized and challenged with ovalbumin. Control animals received saline. Responsiveness was assessed at baseline and after delivery of increasing concentrations of methacholine. Histological analyses were also performed. Decorin deficiency resulted in more modest hyperresponsiveness. Respiratory resistance and elastance along with tissue damping and tissue elastance, were increased in ovalbumin decorin +/+ and decorin-/-, but more so in decorin+/+ . Airway resistance was increased in ovalbumin decorin+/+ only. Inflammation and collagen staining within the airway wall, were increased in ovalbumin decorin+/+ mice only; whereas biglycan was significantly increased in ovalbumin decorin-/- mice only. These results reflect the role of decorin in the development of allergen-induced asthma.