Το κακόηθες μελάνωμα του δέρματος είναι το αποτέλεσμα της κακοήθους εξαλλαγής των μελανοκυττάρων της επιδερμίδας και χαρακτηρίζεται απο συνεχώς αυξανόμενη επίπτωση και θνησιμότητα παγκοσμίως. Η αξιοσημείωτη δε ανθεκτικότητα που επιδεικνύει το προχωρημένο μεταστατικό μελάνωμα στα χημειοθεραπευτικά σχήματα και στην ακτινοθεραπεία κάνει επιτακτική την ανάγκη για νέους, πιο αποτελεσματικούς θεραπευτικούς παράγοντες. Ένας αυξανόμενος όγκος δεδομένων υποστηρίζει τη παρουσία και ενεργό συμμετοχή καρκινικών κυττάρων με ιδιότητες stem κυττάρων (cancer stem cells, CSCs) στην ανάπτυξη και μετάσταση του μελανώματος. Οι μεταγραφικοί παράγοντες EZH2, SOX2 και Oct4 αποτελούν μόρια – κλειδιά στον έλεγχο του ρυθμιστικού δικτύου του stemness των εμβρυϊκών stem κυττάρων (ESCs). Είναι πλέον γνωστό ότι η χρωματίνη στα ESCs περιλαμβάνει περιοχές με «αντιμαχόμενες» τροποποιήσεις ιστονών (bivalent domain), οι οποίες φυσιολογικά σχετίζονται είτε με ενεργή (Η3Κ4me3) ή με ανενεργή κατάσταση της χρωματίνης (H3K27me3), ενώ η απορρύθμιση των επιγενετικών μηχανισμών ελέγχου σε συγκεκριμένους γονιδιακούς τόπους έχει συσχετισθεί με τη καρκινογένεση στον άνθρωπο. Σημαντικό ρόλο στη ρύθμιση αυτών των bivalent domain φαίνεται να έχουν οι πρωτεΐνες της οικογένειας Polycomb, και ιδιαίτερα ο EZH2 που δρα σαν μεθυλοτρανσφεράση στην επιγενετική τροποποίηση H3K27. Πρόσφατα, μια σειρά από μελέτες έδειξαν ότι CScs στη διηθητική παρυφή του όγκου ενδέχεται να συμμετέχουν ενεργά στη καρκινογένεση. Επιπλέον, η ανακάλυψη ότι η βιολογική διαδικασία της επιθηλιο-μεσεγχυματικής μετάβασης (EMT) οδηγεί υποπληθυσμούς καρκινικών κυττάρων εντός του όγκου να αποκτήσουν ιδιότητες stem κυττάρων, φαίνεται να αποτελεί τον σύνδεσμο μεταξύ μετάστασης και κατάστασης πολυδυναμίας (stemness). Σύμφωνα λοιπόν με τη θεώρηση αυτή, είναι πιθανό τα CSCs να εντοπίζονται κυρίως στη διηθητική παρυφή ενός όγκου, ενώ επιπλέον οι ιδιότητες stem κυττάρων που έχουν αποκτήσει είναι το αποτέλεσμα κυρίως της ΕΜΤ. Σε αυτό το πλαίσιο, παρουσιάζει επομένως εξαιρετικό ενδιαφέρον η προσεκτική και στοχευμένη εκτίμηση της ανοσοϊστοχημικής έκφρασης παραγόντων που σχετίζονται με τα stem κύτταρα στην διηθητική παρυφή του μελανώματος, και αυτός ήταν ένας από τους στόχους της παρούσας διαδακτορικής διατριβής. Σκοπός. Ο σκοπός της παρούσας διδακτορικής διατριβής είναι η μελέτη της έκφρασης των μεταγραφικών παραγόντων EZH2, Oct4, SOX2 όπως επίσης και την παρουσία των επιγενετικών τροποποιήσεων H3K4me2 and H3K27me3 (bivalent domain) στο κακόηθες μελάνωμα του δέρματος. Παράλληλα, μελετήθηκε η πιθανότητα αναγνώρισης και στοχοποίησης καρκινικών κυττάρων με ιδιότητες stem κυττάρων, με ιδιαίτερη έμφαση στη διηθητική παρυφή του όγκου. Υλικό και μέθοδος. Το ποσοστό κυττάρων με ανοσοθετικότητα για τα αντισώματα έναντι των μεταγραφικών παραγόντων EZH2, SOX2 και Oct4 όπως επίσης και των επιγενετικών τροποποιήσεων H3K4me2 and H3K27me3 εκτιμήθηκε σε 89 δείγματα ιστών από 79 ασθενείς με κακόηθες μελάνωμα 250 του δέρματος, εφαρμόζοντας τη μέθοδο της ανοσοϊστοχημείας. Για την επιλογή των κατάλληλων δειγμάτων έγινε ανασκόπηση των αρχείων του εργαστηρίου Παθολογικής Ανατομικής του Πανεπιστημιακού Γενικού Νοσοκομείου Πατρών των ετών 2001 έως 2010. Από αυτό το σύνολο των 89 δειγμάτων τα 70 αφορούν πρωτοπαθές μελάνωμα δέρματος, ενώ τα υπόλοιπα 19 προέρχονται από υλικό που εξαιρέθηκε κατά τη χειρουργική εκτομή του μεταστατικού μελανώματος. Επιπλέον 14 δείγματα περιείχαν εκτός από καρκινικά κύτταρα μελανώματος και κύτταρα σπίλων. Στην παρούσα μελέτη χρησιμοποιήθηκε το σύστημα ανίχνευσης EnVision (Envision, Dako, USA) ή MACH4 Universal HRP-Polymer Detection (Biocare Medical, USA) και πρωτογενή αντισώματα έναντι των EZH2 (Novocastra Laboratories Ltd, UK), SOX2 (R&D Systems, Inc.), Oct4 (Santa Cruz Biotechnology, Inc), H3K4me2 (Cell Signaling Technology, USA) και H3K27me3 (Cell Signaling Technology, USA). Σε κάθε περιστατικό και για κάθε δείκτη εκτιμήθηκε το ποσοστό των καρκινικών κυττάρων που εμφάνιζαν θετική ανοσοχρώση (Labeling Index, LI). Η καταμέτρηση των θετικών κυττάρων πραγματοποιήθηκε σε μεγάλης μεγέθυνσης πεδίο (400X). Η στατιστική ανάλυση έγινε με τη χρήση του SPSS στατιστικού πακέτου (SPSS©, Release 19.0). Τιμές p<0.05 θεωρήθηκαν ως στατιστικά σημαντικές. Αποτελέσματα. Πυρηνική χρώση ανιχνεύθηκε για τα αντισώματα έναντι των EZH2, H3K4me2 και H3K27me3, ενώ αντίθετα βρέθηκε πυρηνική και κυτταροπλασματική έκφραση για τους παράγοντες SOX2 και Oct4. Παρατηρήθηκε ανομοιογενές προφίλ ανοσοθετικότητας στα κύτταρα μελανώματος με σημαντικά αυξημένο ποσοστό καρκινικών κυττάρων με θετική ανοσοχρώση H3K4me2 και H3K27me3 στη διηθητική παρυφή του όγκου. Αντίστοιχη τάση για αυξημένη έκφραση έδειξε και ο μεταγραφικός παράγοντας EZH2, χωρίς όμως η διαφορά να είναι στατιστικά σημαντικά, ενώ παρατηρήθηκε και σε ορισμένες μεμονωμένες περιπτώσεις με τον SOX2. Όσον αφορά τον ΕΖΗ2, βρέθηκε σημαντική αύξηση των επιπέδων του παράγοντα στα κύτταρα μελανώματος σε σχεση με τα σπιλοκύτταρα (p=0.02). H πυρηνική έκφραση SOX2 ήταν σημαντικά υψηλότερη στα κύτταρα μελανώματος σε σχέση με τα κερατινοκύτταρα της βασική στιβάδας (p=0.02), όπως επίσης και στα σπιλοκύτταρα συγκριτικά με τα κερατινοκύτταρα της βασικής (p=0.0016) και της υπερβασικής στιβάδας (p=0.027). Η συσχέτιση της πυρηνικής έκφρασης με διάφορες παραμέτρους των ασθενών έδεξε υψηλότερα επίπεδα SOX2 στα πρωτοπαθή σε σχέση με τα μεταστατικά μελανώματα (p=0.045), στα κύτταρα μελανώματος με πάχος όγκου κατά Breslow <1mm (p=0.023), χωρίς εξέλκωση (p=0.009) και με αριθμό μιτώσεων ≤6 μιτ/mm2 (p=0.016). Τα επίπεδα πυρηνικής έκφρασης Oct4 βρέθηκαν υψηλότερα στα σπιλοκύτταρα σε σχέση με τα κερατινοκύτταρα (p<0.001) αλλά και με τα κύτταρα μελανώματος (p=0.004). Η μελέτη ωστόσο της κυτταροπλασματικής ανοσοθετικότητας έδειξε σημαντική μείωση των επιπέδων Oct στα κύτταρα μελανώματος σε σχέση με τα κερατινοκύτταρα της υπερβασικής στιβάδας (p<0.001), όπως επίσης στα μεταστατικά σε σχέση με τα πρωτοπαθή μελανώματα (p=0.025). Αξιοσημείωτο είναι επίσης ότι το προφίλ έκφρασης του Oct4 βρέθηκε σε ορισμένες περιπτώσεις να αυξάνεται τοπικά στα ενδοθηλιακά κύτταρα αγγείων εντός των μελανωμάτων. Τα μελανώματα με υψηλό επίπεδο διήθησης κατά Clark (IV-V) (p=0.038) ή μεγάλο 251 πάχος όγκου κατά Breslow (>1mm) (p<0.001) εμφάνισαν χαμηλότερο ποσοστό καρκινικών κυττάρων με ανοσοθετικότητα για το αντίσωμα H3K4me2 σε σχέση με τους όγκους με μικρότερο βαθμό διήθησης. Επιπλέον, παρατηρήσαμε ότι οι μεταστατικοί όγκοι είχαν χαμηλότερα ποσοστά θετικής ανοσοχρώσης και για τις δύο επιγενετικές τροποποιήσεις, H3K4me2 και H3K27me3, συγκριτικά με τους πρωτοπαθείς όγκους (p=0.0065 και p=0.027 αντίστοιχα). Τέλος, η ανάλυση της παράλληλης ανοσοϊστοχημικής έκφρασης στα κύτταρα μελανώματος έδειξε θετική συσχέτιση των δύο επιγενετικών τροποποιήσεων (p<0.01), όπως επίσης και μεταξύ του EZH2 και της επιγενετικής τροποποίησης H3K27me3 (p=0.03). Ισχυρή συσχέτιση βρέθηκε παρομοίως μεταξύ των επιπέδων έκφρασης Oct4 και SOX2, τόσο για την πυρηνική όσο και την κυτταροπλασματική εντόπιση (p<0.001 και p<0.001 αντίστοιχα). Συμπεράσματα. Λαμβάνοντας υπόψη τη λειτουργική σημασία των τριών υπό μελέτη μεταγραφικών παραγόντων και του ρόλου των επιγενετικών μηχανισμών στην καρκινογένεση, τα ευρήματα μας εισηγούνται ότι οι ΕΖΗ2, SOX2, Oct4 όπως επίσης και οι επιγενετικές τροποποιήσεις Η3Κ4me3 και H3K27me3 αποτελούν εν δυνάμει δείκτες καρκινικών stem κυττάρων στο κακόηθες μελάνωμα, και ιδιαίτερα στη διηθητική παρυφή του όγκου. Η υπόθεση αυτή πρέπει να διεριευνηθεί περαιτέρω, καθώς θα μπορούσε να αποτελέσει, σε συνδυασμό και με άλλες μελέτες, ένα μικρό βήμα προς τη κατεύθυνση της στοχευμένης αντικαρκινικής θεραπείας του μελανώματος στα πλαίσια της Ιατρικής του μέλλοντος. / Cutaneous malignant melanoma originates from melanocytes and is characterized by constantly growing incidence and mortality rates world-wide. The substantial unresponsiveness of advanced metastatic melanomas to most forms of chemotherapy and radiation indicates an urgent need for more effective agents to overcome chemoresistance. Accumulating evidence strongly suggests the presence and involvement of cancer cells with properties of stem cells (CSCs) in the initiation, progression and metastasis of malignant melanoma. EZH2, SOX2 and Oct4 represent crucial components of the reciprocal regulatory circuit that controls stemness. Genome-wide analyses of chromatin states of embryonic stem and progenitor cells suggest a ‘bivalent’ colocalization of the activating H3K4 methylation and the repressive H3K27me3 in development-associated genes, while the misregulation of histone modifications on specific residues actively contributes to human cancer. PcG proteins and mainly EZH2 are responsible for maintaining the balance of the bivalent chromatin domain through the methylation of H3K27. Recently a number of studies have shown that cancer cells with properties of stem cells at the tumor invasion front might be involved in the development of metastasis. The discovery that the epithelial to mesenchymal transition (EMT) generates cells with properties of stem cells and a more invasive and metastatic phenotype, brings a connection between metastasis and stem-cell state. According to this model, cells with stem cell properties are located predominantly at the invasion front of the tumor and can derive through the acquisition of transient EMT phenotype. In this context, a comparative analysis of the expression profile of putative CSC markers between the invasion front and the inner tumor mass could test this hypothesis in the case of cutaneous melanoma as well. Purpose. Taking these data into account, we performed the current study in order to evaluate the immunohistochemical expression of EZH2, SOX2 and Oct4 as well as H3K4me2 and H3K27me3, which constitute stem cell-like "bivalent"domains, in cutaneous malignant melanoma, investigating besides the potential identification of cancer cells with stem cells properties at the invasion front of the tumor. Materials and methods. Expression of EZH2, SOX2, Oct4, H3K4me2 and H3K27me3 was evaluated in 89 malignant melanoma (MM) lesions, deriving from 79 patients, using immunohistochemistry, on formalin-fixed paraffin-embedded tissue sections. The analyzed cases were accessioned over the time interval 2001-2010 and retrieved from an electronic database maintained by the Department of Pathology of the University General Hospital of Patras (Rion, Greece). The sample consists of 70 primary and 19 metastatic specimens. 14 specimens contained both melanoma cells and nevus cells. Analysis and comparative studies were carried out on the expression of the proteins tested in nevus cells (where existed), melanoma cells, melanoma cells at the invasion front, basal and suprabasal 253 keratinocytes as well. Polymer based technique (Envision, Dako, USA) or MACH4 Universal HRPPolymer Detection (Biocare Medical, USA) and primary antibodies against EZH2 (Novocastra Laboratories Ltd, UK), SOX2 (R&D Systems, Inc.), Oct4 (Santa Cruz Biotechnology, Inc), H3K4me2 (Cell Signaling Technology, USA) and H3K27me3 (Cell Signaling Technology, USA) were used. In each case, the percentage of cells exhibiting positive staining was determined. Cell counts were performed at a 400X magnification. Data were analyzed using the SPSS statistical package (SPSS©, Release 19.0). The level of significance was set at p-value <0.05. Results. The three markers studied, EZH2, H3K4me2 and H3K27me3, were identified in the cell nuclei of melanoma cells, nevus cells and normal epidermal keratinocytes, while SOX2 και Oct4 showed nuclear as well as cytoplastik expression. A specific distribution pattern of H3K4me2 and H3K27me3 was found, as stronger levels were localized at the invasion front of the tumor (p=0.034 and p<0.01 respectively). A similar trend was also observed for EZH2, whithout achieving however statistical significance (p=0.08), and similarly for SOX2 in a few sporadic cases. Significantly increased EZH2 immunohistochemical expression was observed in melanoma cells with respect to nevus cells (p=0.02). Nuclear SOX2 levels were also higher in melanoma cells than basal keratinocytes (p=0.02) and in nevus cells than basal keratinocytes (p=0.0016) and suprabasal keratinocytes (p=0.027). Furthermore LIs in melanoma cells presented significantly higher values in primary with respect to metastatic malignant melanoma lesions (p=0.045) as well as in melanoma cells with low Breslow’s depth (≤1mm) (p=0.023), under the absence of ulceration (p=0.009) and with low (≤6/mm2) mitotic rate (p=0.016). As well as nuclear expression of Oct4 is concerned, it was found increased in nevus cells with compared to keratinocytes (p<0.001) and melanoma cells (p=0.004). Cytoplastic expression of Oct4 followed an opposite trend, with decreasing levels in melanoma cells with respekt to suprabasal keratinocytes (p<0.001) and in metastic compared to to primary melanoma cases (p=0.025). Remarkably occasionally increased Oct4 expression in endothelial cells of the tumor microvasculature in melanoma tissues was observed. Furthermore, H3K4me2 and H3K27me3 levels were lower in metastatic with respect to primary melanoma cases (p=0.0065 and p=0.027 respectively). Advanced melanoma demonstrated significantly lower H3K4 immunohistochemical expression than cases of lowest Clark’s level (I) (p=0.038) or low Breslow’s depth (≤1 mm) (p<0.001). Moreover, EZH2 expression in melanoma cells was higher compared to nevus cells (p=0.02). Finally statistical analysis further revealed a positive correlation in melanoma cells betwenn EZH2 and H3K27me3 (p=0.03), H3K4me2 and H3K27me3 (p<0.01), as well as between Oct4 and SOX2 for both nuclear and cytoplastik expression (p<0.001 and p<0.001 respektively). Conclusions. Our results suggest the possibility that combined immunohistochemical expression of EZH2, SOX2, Oct4, H3K4me2 and H3K27me3 might identify cancer cells with potential stem cell properties, particularly at the invasion front of this malignancy. This hypothesis should be further investigated, as many of the epigenetic changes are reversible via pharmacologic manipulations and new CSC-directed therapies, overpassing the resistance of advanced melanoma, may be developed.

Salinity and oxygen availability have long been recognised as important factors influencing animal physiology and therefore species distribution. The maintenance of appropriate cellular ion levels is critical for many essential physiological processes, but at the same time is energetically expensive. Since hypoxia is likely to impose aerobic limitations for ATP generation, the maintenance of salt and water homeostasis could be at risk during hypoxia. The amphidromous inanga (Galaxias maculatus) is well known for its salinity tolerance and its life cycle that involves several salinity related migrations. During these migrations inanga also frequently encounters hypoxic waters, and therefore must maintain energy homeostasis when aerobic metabolism may be compromised. The present study has investigated behavioural, physiological, biochemical and molecular mechanisms by which inanga tolerate changes in salinity and hypoxia. After 14 days of acclimation to salinities ranging from freshwater to 43‰, inanga showed physiological acclimation. This was evident by no changes in metabolic rates or energy expenditures through this salinity range. Energy balance seemed to be tightly and efficiently controlled by changes in the proportion of protein and lipids used as energy substrate. No mortalities and only minor changes in plasma osmolality also indicated salinity acclimation. The remarkable osmoregulatory capacity of inanga was also evidenced after a seawater challenge. The osmotic balance of inanga was only disrupted during the first 24 hours after the challenge, evidenced by an increase in plasma osmolality and plasma Na+, and a decrease in muscle water content. These physiological changes were correlated with changes at the molecular level. Different isoforms of the catalytic subunit of the Na+,K+-ATPase (NKA) were isolated, partially sequenced and identified in inanga. Phylogenetic analysis grouped inanga isoforms (α-1a, α-1b, α-1c) with their respective homologues from salmonids. Patterns of mRNA expression were also similar to salmonids, with α-1a being downregulated and α-1b being up-regulated following seawater challenge. Previous to this study, NKA isoform switching was reported to occur only in salmonids and cichlids. The presence of NKA subunits that change with environmnetal salinity in inanga indicates that this isoform switching phenomenon is much more widespread among teleost lineages than previously thought. Aiming to elucidate the hypoxia tolerance of inanga, oxygen consumption rate as a function of decreasing external PO2 was evaluated. At no point did inanga regulate oxygen consumption, suggesting that this species is an oxyconformer. This is the first robust demonstration of the existence of oxyconforming in fish. Evaluation of the scaling relationship between oxygen consumption and fish size in normoxia, showed that the exponent of this relationship fell within the range previously reported for fish. However, in hypoxic conditions the scaling relationship was less clear suggesting different size-related mechanisms for tolerating hypoxia. Analysis of the aerobic and anaerobic metabolism of small and large fish, showed that smaller inanga were able to sustain aerobic metabolism for longer than larger inanga, which instead relied on anaerobic metabolism for extending their survival. This knowledge is likely to be of value for the conservation of this iconic fish species, by incorporating these size related differences in hypoxia tolerance in streams management. In light of the unusual oxyconforming response of inanga, a study examining the behavioural responses of this species to declining dissolved oxygen was performed. Inanga did not display a behaviour that might reduce energy expenditure during oxygen limitation; instead swimming activity and speed were elevated relative to normoxia. As hypoxia deepened inanga leaped out of the water, emersing themselves on a floating platform. Once emersed, fish exhibited an enhanced oxygen consumption rate compared to fish that remained in hypoxic water. Although this emersion behaviour was hypothesised to be of physiological advantage, both aquatic hypoxia and emersion resulted in similar physiological and biochemical consequences in inanga. While in hypoxic water oxygen availability seemed to be the limiting factor, in air failure of the circulatory system was hypothesised to be the cause of a similar metabolic signature to that found in aquatic hypoxia. Overall, inanga seemed to be not particularly well adapted to tolerate aquatic hypoxia. In light of the increasing likelihood of anthropogenic-induced hypoxia in inanga habitats, this is likely to have negative consequences for the future of inanga populations in the wild. Although this study provides the mechanisms behind the exceptional salinity tolerance of inanga, its susceptibility to hypoxia is likely to impose further constraints for the osmoregulatory processes that guarantee inanga survival during life cycle migrations. The results of the present study are relevant for understanding and managing the fishery of this economically- and culturally important fish species.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Michigan, 1983.

Thesis (M. Sc.)--Memorial University of Newfoundland, 1997. / Bibliography: leaves 95-108.

La proprioception permet de fournir des informations spatio-temporelles pour calibrer un espace moteur, ajuster la trajectoire des mouvements, l'équilibre, la direction des mouvements, et la coordination intermembres et multiarticulaires. Nous prenons à partir des récepteurs sensoriels une information liée à la vitesse du geste, à sa direction, à son amplitude et à son orientation. Bien que les muscles soient définis comme étant les récepteurs sensoriels contribuant principalement à fournir une information proprioceptive, les récepteurs cutanés avec l'étirement de la peau au cours du mouvement montrent des capacités à fournir une information redondante aux récepteurs musculaires. Cependant au regard de la contribution des récepteurs musculaires, le poids attribué aux informations cutanées est relativement faible hormis dans des situations précises où la contribution des récepteurs musculaires est altérée comme en situation de fatigue musculaire ou minimisée comme lors de l'évaluation du sens de la position articulaire. Le champ de la médecine sportive suggère que les différents supports de prévention des traumatismes améliorent l'acuité proprioceptive par une stimulation des récepteurs cutanés. Il reste néanmoins quelques points à éclaircir, en particulier comment pouvons nous stimuler la peau ? Au cours de trois expérimentations, nous avons testé deux moyens, la pression et les forces de cisaillement pour améliorer l'acuité proprioceptive. Dans l'ensemble nos résultats montrent une amélioration de la pertinence des informations cutanées par une diminution de l'incertitude dans le choix de la position articulaire ce traduisant par une amélioration de la précision de nos mouvements. Cette amélioration est indépendante du niveau de pression et serait en lien avec la création de force de cisaillement là où la peau s'étire le plus avec le mouvement. / Proprioception can provide spatiotemporal information for calibrating a motor space, adjust the trajectory of movement, balance, direction of movement, and coordination and Inter multiarticulaires. We take from the sensory receptors of information related to the speed of movement, its leadership, its amplitude and its direction. Although the muscles are defined as sensory receptors contributing primarily to provide information proprioceptive receptors in the skin of the tethering of the skin during movement show a capacity to provide redundant information to muscle receptors. However, in view of the contribution of muscle receptors, the weight given to information skin is relatively low except in specific situations where the contribution of muscle receptors is altered as experiencing muscle fatigue or minimized as in the evaluation of the meaning of joint position. The field of sports medicine suggests that different media Injury Prevention im prove proprioceptive acuity by stimulation of cutaneous receptors. It remains to clarify some points, especially how can we stimulate the skin? In three experiments, we tested two methods, pressure and shear forces to improve proprioceptive acuity. Overall our results show an improvement in the adequacy of the information skin by reducing the uncertainty in the choice of this joint position resulting in improved accuracy of our movements. This improvement is independent of pressure level and be linked with the creation of shear force where the skin stretches over with the movement.