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Skin related injuries are some of the most dangerous forms of wounds. In addition to treating the wound itself, health care providers must be cautious of microbial infections. In this study, we evaluate a novel all-natural antimicrobial gel compound (AMG) designed to kill planktonic bacteria, penetrate bacterial biofilms, and accelerate wound healing. In -vitro experiments demonstrate that AMG is effective in inhibiting planktonic growth and biofilm development of eight common pathogens. LIVE/DEAD staining and confocal microscopy reveal that planktonic growth and three-dimensional structure of biofilms were significantly reduced. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to investigate a small panel of genes (PrsA, Sprx) and showed potential targets for future study. A physiologically relevant wound model was created for treating S. aureus infections by using AMG alone or in combination with a common topical antibiotic, Mupirocin. AMG is a safe and effective treatment option for skin related infection.

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The potential for private sector healthcare business in Africa has been forecasted to reach $35 billion by 2016, with South Africa being regarded as the most industrially advanced country on the continent. South Africa’s entry to modern biotechnology is fairly recent, though, with companies in the private sector still in a developmental phase, and most having limited bioproduct ranges. While considerable research has been conducted in the past to attempt to define the biotechnology environment of South Africa, as yet, a concise overview is lacking. In particular, a synopsis of the biomedical or commercial health technology environment has not been forthcoming for entrepreneurs to refer to as a ‘roadmap’. The purpose of this study was to perform a comprehensive study on the attributes that should be met for a successful, sustainable health technology venture (HTV) to be started in South Africa; while identifying the opportunities and threats that have existed in the South African market; thereby, affecting their success and sustainability to date. In this study, two phases of research were conducted. The first was a small-sampled mixed-methods (both qualitative and quantitative) study involving 21 medical devices, biogenerics, diagnostics, and contract services companies. The second was a quantitative study, involving 107 vaccines, biogenerics, therapeutics, nutraceuticals, reagents, diagnostics, medical devices, biotools, contract services and public services companies. Inferential statistical tests were conducted on the data, including Pearson’s Chi-Square, ANOVA, bivariate correlation, linear regression, logistic regression and multinomial logistic regression. From the study, the overall proportion of business sustainability for HTVs was found to be 66.7%, and at least 30% were unsustainable (or not yet at a level of sustainability). Variations were observed in the overall rate of sustainability for companies, based on their core functional classification, location, production type, size and start-up or R&D spending. By converting the observed frequencies of activity level, as an indication of sustainability, into a probability, it was possible to observe the company type that was most, and least likely to succeed in South Africa. Based on the statistical observations in this study, the HTV type most likely to succeed in South Africa, with a 63.7% probability of reaching sustainability, is a ‘vaccines’, ‘biotools’ or ‘public services’ company from Johannesburg with at least 20 employees; that has developed its goods or services internally, but manufactured externally and spent between R20 million–and–R30 million on its R&D or start-up. Conversely, least likely to succeed (3.2% probability) is a nutraceutical company from Cape Town with between six and 20 employees, that has developed and produced internally, and which has spent between R1 million–and–R10million on its start-up. / Life and Consumer Sciences / M.Sc (Life Sciences)

Οι καρκίνοι του γαστρεντερικού συστήματος είναι από τους πιο συνηθισμένους τύπους καρκίνου στον αναπτυγμένο κόσμο. Ο καρκίνος του παχέος εντέρου είναι εκείνος με τη μεγαλύτερη συχνότητα εμφάνισης, αλλά αντιμετωπίζεται με αρκετά καλή πρόγνωση. Αντίθετα, ο καρκίνος του παγκρέατος είναι εκείνος με τη χειρότερη πρόγνωση και πολλές φορές δεν αντιμετωπίζεται. Τα τελευταία χρόνια αναπτύσσεται εκτεταμένη έρευνα στα εξωκυττάρια μακρομόρια των καρκινικών ιστών και στο ρόλο τους στην ανάπτυξη και εξέλιξη του καρκίνου, όπως επίσης και στις δυνατότητες επηρεασμού των παραγόντων αυτών φαρμακευτικά. Η παρούσα Διατριβή έχει δύο στόχους, ο πρώτος σχετίζεται με τη μελέτη των εξωκυττάριων πρωτεογλυκανών στον καρκίνο του παγκρέατος και ο δεύτερος με τη μελέτη του μεταβολισμού του υαλουρονικού οξέος στον καρκίνο του παχέος εντέρου μέσω της μελέτης των βιοσυνθετικών και καταβολικών του ενζύμων. Ο πρώτος στόχος διερευνήθηκε με συνδυασμό βιοχημικών και ανοσοϊστοχημικών τεχνικών, από τα αποτελέσματα των οποίων διαπιστώθηκε ότι μόνο δύο πρωτεογλυκανικά μόρια ανευρίσκονται στο παγκρεατικό καρκίνωμα, η versican και η decorin. Και οι δύο πρωτεογλυκάνες εντοπίστηκαν στο στρώμα και απουσίαζαν πλήρως από τα καρκινικά κύτταρα, γεγονός που υποστηρίζει ότι παράγονται από τις ινοβλάστες του στρώματος. Ήταν σημαντική η αύξηση των ποσοτήτων των δύο πρωτεογλυκανών στο παγκρεατικό καρκίνωμα, σε σχέση με το φυσιολογικό πάγκρεας και μεγάλη η διαφορά που εμφάνιζαν μεταξύ τους. Η versican αυξήθηκε 27 φορές και η decorin 7 φορές, σε σχέση με το φυσιολογικό πάγκρεας. Η μεγαλύτερη αύξηση της versican σχετίζεται με τις ιδιότητες της πρωτεογλυκάνης, τόσο δομικές, ενυδατικές, χωροπληρωτικές, όσο και λειτουργικές, εφ’ όσον πρόκειται για πολυλειτουργικό μόριο. Επί πλέον, η versican συμβάλλει στην κατακόρυφη αύξηση του κυτταρικού πολλαπλασιασμού, ενώ παράλληλα συμβάλλει και στις αντι-προσκολλητικές ιδιότητες των κυττάρων, παρέχοντας τη δυνατότητα για υπέρμετρη, και πολλές φορές ανεξέλεκτη, κυτταρική ανάπτυξη σε τοπικό επίπεδο. Το γεγονός της χαμηλότερης αύξησης της συγκέντρωσης της decorin, σε σχέση με εκείνη της versican, θα μπορούσε να αποτελεί ένα μέτρο της επιθετικότητας του καρκίνου ή ακόμα, ένα μέτρο της κακοήθειας. Η αύξηση των πρωτεογλυκανών συνοδευόταν από σημαντικότατες αλλαγές στη βιοχημική δομή τους σε επίπεδο υδροδυναμικού μεγέθους, βαθμού και προτύπου θείωσης, όπως επίσης και επιμερίωσης του γλυκουρονικού σε ιδουρονικό. Οι αλλαγές αυτές θα μπορούσε να οφείλονται σε πολλαπλές αλλοιώσεις των βιοσυνθετικών μονοπατιών τους. Ο δεύτερος στόχος διερευνήθηκε με συνδυασμό ενζυμολογικών, ανοσοενζυμικών και μοριακών τεχνικών, από τα αποτελέσματα των οποίων διαπιστώθηκε σε όλα τα δείγματα η παρουσία των ισομορφών υαλουρονιδάσης Hyal1 και ΡΗ20, και σε πολλαπλές μορφές, αλλά και των Hyal2 και Hyal3, όμως μόνο σε προχωρημένο στάδιο, ως επίσης και η παρουσία των τριών συνθασών του υαλουρονικού. Παρατηρήθηκε σημαντική μεταβολή της έκφρασης με το καρκινικό στάδιο. Η Hyal1 εκφραζόταν σε πολύ χαμηλά επίπεδα σε μακροσκοπικώς φυσιολογικά δείγματα παχέος εντέρου με καρκίνο σταδίου Α, όμως η έκφρασή της ήταν πάνω από δέκα φορές μεγαλύτερη στα αντίστοιχα καρκινικά, υποστηρίζοντας ότι η Hyal1 παράγεται από τα καρκινικά κύτταρα. Το γεγονός ότι η έκφραση της Hyal1 στα καρκινικά δείγματα εμφάνιζε σταδιο-εξαρτώμενη μείωση, σε συνδυασμό με το δεδομένο ότι η δράση της οδηγεί στην παραγωγή αγγειογενετικών θραυσμάτων υαλουρονικού, έρχεται σε συμφωνία με το δεδομένο ότι η διαδικασία της αγγειογένεσης απαιτείται κυρίως στα αρχικά στάδια της καρκινικής εξαλλαγής. Από την άλλη πλευρά, η ΡΗ20 εμφάνιζε υψηλότερη έκφραση, σε σχέση με την Hyal1, στα μακροσκοπικώς φυσιολογικά δείγματα σταδίου Α, η οποία όμως αυξανόταν τέσσερις φορές στα αντίστοιχα καρκινικά, υποδεικνύοντας και τη δική της συμμετοχή στη 10 διαδικασία της αγγειογένεσης. Στα επόμενα στάδια υπήρχε μεν αύξηση στην έκφραση της ΡΗ20, αυτή όμως ήταν μικρή, και πιθανόν να λειτουργεί με σκοπό την ακόμα μεγαλύτερη αποικοδόμηση του υαλουρονικού ώστε να χαλαρώσει η δομή του εξωκυττάριου χώρου και να δοθεί η δυνατότητα ανάπτυξης του καρκίνου ή μετάστασης των καρκινικών κυττάρων. Παράλληλα, διαπιστώθηκε μικρή αύξηση της έκφρασης της ΡΗ20 στα μακροσκοπικώς φυσιολογικά δείγματα σταδίου Β και πολύ μεγαλύτερη σε εκείνα σταδίου C. Από τον έλεγχο της έκφρασης των συνθασών του υαλουρονικού διαπιστώθηκε ότι υπάρχει σημαντική σταδιο-εξαρτώμενη αύξηση της HAS1, η οποία οδηγεί στη βιοσύνθεση υαλουρονικού μεγάλου μοριακού μεγέθους, υποστηρίζοντας ότι ο καρκίνος συντονίζει την παραγωγή υαλουρονικού με μέγεθος τέτοιο που, σύμφωνα με τις χωροπληρωτικές και ενυδατικές του ιδιότητες, μπορεί να βοηθήσει την ενυδάτωση του εξωκυττάριου χώρου και την κυτταρική ανάπτυξη. Από την άλλη πλευρά, η HAS2 εμφάνιζε μικρή σταδιο-εξαρτώμενη αύξηση, μόνο στα καρκινικά δείγματα, η οποία μπορεί να οδηγήσει στη βιοσύνθεση μεγαλομοριακού υαλουρονικού, που απαιτείται για τη σωστή και οργανωμένη ανάπτυξη του καρκινικού όγκου. Τέλος, η HAS3 εμφάνιζε μικρή σταδιο-εξαρτώμενη μείωση, υποδηλώνοντας ότι η παρουσία της είναι απαραίτητη σε σχεδόν σταθερό βαθμό για την ανάπτυξη και εξέλιξη του καρκίνου και τούτο γιατί το προϊόν της είναι μικρότερου μοριακού μεγέθους σε σχέση με τις υπόλοιπες συνθάσες. Αυτό εξ άλλου υποστηρίζεται και από το γεγονός ότι η HAS3 εμφανίζει τη μεγαλύτερη έκφραση σε σχέση με τις άλλες συνθάσες σε δείγματα σταδίου Α, στάδιο που είναι κρίσιμο για την αγγειογένεση και την υποβοήθηση της διατροφής των καρκινικών κυττάρων. Συμπερασματικά, γίνεται φανερό ότι τα καρκινικά κύτταρα συνδυάζουν πολλούς μηχανισμούς για την ανάπτυξη, τη διήθηση και την επέκταση του καρκίνου και τα εξωκυττάρια μακρομόρια μπορεί να έχουν κομβικό ρόλο σ’ αυτούς. Οι μηχανισμοί αυτοί, όπως μελετήθηκαν στην παρούσα διατριβή, είναι: ελεγχόμενη αύξηση της συγκέντρωσης των πρωτεογλυκανών versican και decorin, εξειδικευμένες τροποποιήσεις στη βιοχημική δομή των γλυκοζαμινογλυκανών, ελεγχόμενη έκφραση των βιοσυνθετικών και καταβολικών ενζύμων του υαλουρονικού. Όλες αυτές οι μεταβολές συντονίζονται κατά τέτοιο τρόπο ώστε να εξυπηρετήσουν τις απαιτήσεις του καρκίνου είτε στο επίπεδο της αγγειογένεσης είτε στο επίπεδο της οργάνωσης του εξωκυττάριου χώρου. Οι μελλοντικές μελέτες θα πρέπει να προσανατολιστούν ακόμα περισσότερο στη συσχέτιση χημικής δομής και λειτουργικότητας των αλυσίδων θειικής χονδροϊτίνης/δερματάνης, ώστε να γίνουν πλήρως αντιληπτοί οι ρόλοι αυτών των μακρομορίων, όπως επίσης στους ρυθμιστικούς μηχανισμούς που ελέγχουν την έκφραση των ενζύμων του μεταβολισμού του υαλουρονικού, με απώτερο στόχο την ειδικότερη και πληρέστερη φαρμακευτική αντιμετώπιση του καρκίνου. / Cancers of gastrointestinal tract are the most common type of cancers in developed world. Colorectal cancer has the highest incidence, however it is treated with rather good prognosis. On the other hand, pancreatic cancer has very bad prognosis and in most cases it cannot be treated. The last years extended research focused to the extracellular macromolecules of cancer, their role in cancer progression and their possible use as drug targets. The present Thesis aimed to study the extracellular proteoglycans structure in pancreatic cancer and the hyaluronan metabolism in colorectal cancer. The first was investigated by using a combination of biochemical and immunohistochemical techniques, and from their results it was found that two extracellular proteoglycans were present in pancreatic carcinoma, versican and decorin. Both proteoglycans were detected in the stroma and were absent from cancer cells, suggesting their biosynthesis from normal fibroblasts. In addition, their amounts were highly increased in pancreatic carcinoma, as compared with normal pancreas. Their increase in cancer was disproportional. Versican was increased 27 times and decorin 7 times, as compared with normal pancreas. Versican increase is related to its structural, hydration and space-filling properties, as well as to its function, since it is a multifunctional macromolecule. Versican enhances cell proliferation, and together with its anti-adhesive properties, allows mainly uncontrolled cellular growth locally. The lower increase of decorin, as compared with that of versican, could explain aggressiveness and malignancy of pancreatic cancer. Proteoglycans increase was followed by extensive alterations in their biochemical structure, namely, hydrodynamic size, sulphation pattern, and epimerization of glucuronate to iduronate. These should be attributed to multiple alterations of their biosynthetic pathways. The second was investigated by using a combination of enzymatic, immunoenzymatic and molecular techniques, and from their results it was found that multiple forms of Hyal1 and PH20 were present in all samples, as well as all hyaluronan synthases (HASs). Hyal2 and Hyal3 were present in some samples of advanced stage of cancer. Changes in expression with cancer stage were observed. Hyal1 expressed in low levels in apparently macroscopically normal parts of stage A samples, and its expression was 10 times greater in the respective cancerous. This finding suggested that Hyal1 is produced from cancer cells. Hyal1 expresssion showed a stage-related decrease and since its activity results to hyaluronan fragments of angiogenetic size, it could be concluded that Hyal1 is required at early stage of cancer. PH20 expressed in a higher rate than Hyal1 in apparently macroscopically normal parts of stage A samples, and its expression was 4 times greater in the respective cancerous, suggesting its participation in angiogenesis. In advanced stages, PH20 expression was slightly increased, suggesting its participation in extracellular matrix disorganization to permit cancer growth and progression, as well as metastasis. This was also observed in apparently macroscopically normal parts of samples of advanced stages. From the examination of the expression of the various HASs, a great and stage-related increase of HAS1 was found. This enzyme is responsible for the biosynthesis of hyaluronan of very high molecular size and thus it could be suggested that cancer regulates hyaluronan biosynthesis in such a way to fulfill cancer requirements in matrix hydration. HAS2 expression was also increased in a stage-related order only in cancerous samples, but the increase was lower than that of HAS1. This enzyme is responsible for the biosynthesis of hyaluronan of high molecular size which might be required for well-organized cancer growth. HAS3 expression was slightly decreased in a stage-related order, suggesting that its presence is stably required for the correct cancer growth and progression, since its biosynthetic product is of lower hydrodynamic size, as compared with that of HAS1 and 12 HAS2. Moreover, HAS3 expression was higher than that of both other HASs in samples of stage A, a stage very critical for angiogenesis. From the results obtained, it could be concluded that cancer cells combine a variety of multiple mechanisms for cancer growth, progression and invasion, and that extracellular macromolecules might play a very critical role. The mechanisms, as studied in the present Thesis, are: selective and highly regulated increase of the proteoglycans versican and decorin, selective modifications of glycosaminoglycans biochemical structure, selective and highly regulated expression of biosynthetic and catabolic enzymes related to hyaluronan. All these changes coordinate to fulfill cancer requirements for either angiogenesis or extracellular matrix organization, depending on its stage. Future studies will be oriented to chondroitin/dermatan sulphate structure/function relationship for better understanding of the role of these macromolecules in cancer, and of the regulatory mechanisms implicated in the expression of the enzymes involved in hyaluronan metabolism, aiming at the cancer treatment.

INTRODUÇÃO: Poucos estudos têm focado as modificações fisiológicas que ocorrem no trato genital de mulheres menopausadas infectadas pelo HIV e sua associação com a excreção genital do vírus. Nesse estudo de corte transversal, comparou-se a excreção genital do HIV em mulheres menopausadas e em idade fértil em acompanhamento em um centro especializado em São Paulo, Brasil. Investigou-se também a associação entre a excreção genital de RNA de HIV e a viremia em ambos os grupos. Fatores associados com a intensidade da excreção genital de HIV também foram pesquisados, incluindo achados ginecológicos e marcadores de progressão da infecção por HIV. MÉTODOS: 146 mulheres infectadas pelo HIV [73 menopausadas (M)/73 em idade fértil (F)] foram selecionadas em Serviço de Extensão ao Atendimento de Pacientes com HIV/Aids Casa da Aids do Hospital das Clínicas da FMUSP, São Paulo, Brasil. As mulheres menopausadas referiram tempo médio de 8,17 anos (DP=6 anos) de menopausa. A contagem de linfócitos T CD4+ foi obtida por citometria de fluxo e a quantificação do RNA do HIV no plasma e no lavado cervicovaginal (LCV) foi realizada por RT-PCR quantitativo, utilizando-se o kit Cobas Amplicor HIV-1 Monitor Test®, no método ultrasensível. Cloreto de lítio foi introduzido no tampão para obtenção do LCV e quantificado antes e depois da coleta do lavado, a fim de determinar o fator de diluição de cada amostra. A deteção do gene SRY por PCR também foi realizada a fim de eliminar amostras com eventual contaminação espermática. A prevalência de excreção genital foi estimada para ambos os grupos e os fatores associados à intensidade da excreção viral foram investigados, utilizando-se modelo de regressão linear múltipla. As variáveis com p<0,2 na análise bivariada foram incluídas na análise multivariada, assim como o grupo em estudo (M ou F). O modelo final incluiu fatores que se mostraram independentemente associados com a intensidade da excreção genital de HIV. RESULTADOS: A prevalência de excreção genital de HIV-RNA foi similar em ambos os grupos (M: 17,8%, IC 95% 9,8 28,5; F: 22%, IC 95% 13,1 33,1, p=0,678). Similarmente, a intensidade de excreção genital do HIV também não se mostrou diferente entre os grupos (mediana - M: 1,4log/mL; F: 1,4log/mL, p=0,587). A carga viral plasmática foi detectável em 34,2% das pacientes menopausadas (IC 95% 23,5 46,3) e em 42,5% entre as pacientes em idade fértil (IC 95% 31 54,6, p=0,395). Três pacientes (2 M/1 F) exibiram excreção genital de HIV-RNA na ausência de viremia detectável. Existe evidência de correlação entre a carga viral plasmática e a genital em ambos os grupos (rM: 0,658; rF: 0,684, p<0,01). Adicionalmente, o número de células CD4+ periféricas mostrou-se negativamente correlacionada à excreção genital do HIV em ambos os grupos (rM: -0,250; rF: -0,248, p<0,05). À análise multivariada, a carga viral plasmática mostrou-se independentemente associada à ocorrência de excreção genital do HIV em ambos os grupos (OR 4,03, IC 95% 2,52 6,45, p<0,001). Já a intensidade de excreção genital mostrou-se independentemente associada ao pH vaginal (p<0,001), concentração de TNF- no LCV (p=0,01), e à carga viral plasmática (p=0,001), todos com correlação positiva. CONCLUSÕES: Apesar das modificações significativas que ocorrem na mucosa vaginal da mulher menopausada, a excreção cervicovaginal do HIV parece não ser significativamente influenciada por esse estado. A carga viral plasmática e o número de células CD4+ periféricas estão correlacionadas com a excreção genital do vírus. A frequência de excreção genital mostrouse independentemente associada à intensidade de viremia. Além disso, o aumento do pH vaginal e evidência de inflamação genital, associada à concentração de TNF- no LCV, independentemente aumentam a intensidade de excreção genital nas mulheres estudadas. / BACKGROUND: Few studies have focused on physiological modifications that occur in the genital tract of HIV-infected postmenopausal women and their association with HIV cervicovaginal shedding. In this cross-sectional study we evaluated and compared HIV genital shedding among postmenopausal and fertile-aged women under care at a specialized center in Sao Paulo, Brazil, investigating the association between HIV-RNA shedding and HIV plasma viral loads in both groups. Factors associated with higher HIV shedding were also investigated, including gynaecological features and HIV disease progression markers. METHODS: 146 women living with HIV [73 postmenopausal (PM)/73 in fertile-aged (F)] were enrolled at the HIV Clinic, University of São Paulo Medical School, Brazil. Postmenopausal women referred a mean duration of 8.17y (SD=6y) since menopause. CD4+ cell counts were obtained by flow cytometry and HIV-RNA was quantified in plasma and in cervicovaginal lavages (CVL) by RT-PCR, using Cobas Amplicor HIV-1 Monitor Ultrasensitive Test. Lithium chloride was introduced into the CVL buffer and measured before and after CVL collection in order to determine the dilution factor for each specimen. SRY gene detection by PCR was also performed in all samples in order to rule out sperm contamination. Prevalence of HIV genital shedding was estimated for both groups and factors associated with the intensity of viral shedding were investigated, using a multiple linear regression model. Variables with p<0.2 in bivariate analysis were included in multivariate analysis, as well as the study group (PM and F). The final model included factors shown to be independently associated with intensity of HIV genital shedding. RESULTS: The prevalence of HIV-RNA genital shedding was similar in both groups. (PM: 17.8%, 95%CI 9.8 28.5; F: 22%, 95%CI 13.1 33.1, p=0.678). Likewise, the intensity of HIV shedding was shown not to differ between PM and F women (means - PM: 1.4log/mL; F: 1.4log/mL, p=0.587). Plasma viral loads were detectable in 34.2% of PM patients (95%CI 23.5 46.3), as compared to 42.5% among F women (95%CI 31 54.6) (p=0.395). Three patients (2 PM/1 F) exhibited HIV-RNA genital shedding in the absence of detectable viremia. We found evidence of correlation between HIV plasma viral load and HIV cervicovaginal shedding in both groups (rPM: 0.658; rF: 0.684, p<0.01). In addition, CD4+ cell counts were shown negatively correlated to HIV shedding in both groups (rPM: -0.250; rF: -0.248, p<0.05). In multivariate analysis, HIV plasma viral load was shown independently associated with occurrence of HIV genital shedding in both groups (OR 4.03, 95%CI 2.52 6.45, p<0.001). In addition, the intensity of HIV shedding was shown independently associated with vaginal pH (p<0.001), TNF- concentrations in CVL (p=0.01), and with HIV plasma viral loads (p=0.001), all of them with positive correlation. CONCLUSION: Despite the significant changes that occur in the vaginal mucosa of postmenopausal women, HIV cervicovaginal shedding does not seem to be significantly influenced by this state. Plasma viral loads and CD4+ cell counts are correlated to HIV genital shedding. The frequency of HIV genital shedding was shown independently associated with viremia intensity. Moreover, increased vaginal pH and evidence of genital inflammation associated with TNF- concentration independently enhanced the intensity of HIV shedding in postmenopausal and fertile-aged women.