The p53 tumour suppressor protein has a highly complex pattern of regulation at transcriptional and posttranslationallevels. The discovery of p53 isoforms has added another layer of complexity to the mechanisms thatregulate p53 functions. Indeed, p53 is expressed as 12 isoforms that differ in their N- and C-terminus due toalternative splicing, promoter or codon initiation usage. So far, there is limited understanding of the patterns ofexpression and of the functions of each of these isoforms.In this Thesis, we have focused on the two major p53 N-terminal isoforms, Δ40p53 and Δ133p53. We haveanalysed their patterns of interactions with the full-length p53 and we have investigated whether their expressioncould be deregulated in melanoma, a cancer type in which TP53 mutations are rare. Our results show that Δ40p53 can modulate p53 function with a bi-phasic effect, acting as a repressor or activator of p53 to control itslevels and activity. Moreover, we demonstrate that the internal P2 promoter produces Δ133p53 and is regulatedby p53 in response to genotoxic stress, identifying a novel auto-regulatory loop by which p53 may control theexpression of an isoform acting as an inhibitor of p53 activities. Finally, we show that mRNAs encoding Nterminalisoforms are often over-expressed in highly metastatic melanoma when compared to non-invasiveforms, suggesting that N-terminal isoforms contribute to functionally inactivate p53. Thus, we propose that Δ40p53 and Δ133p53 modulate p53 functions within dynamic fluctuations of aprotein network. Hence, p53 isoforms may have a major role in basal p53 activities as well as in the functionalinactivation of p53 in cancer cells.

Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Pigmentation disorders occur in multiple conditions (Hakozaki et al., 2006:105). Although many modalities of treatments are available, none are completely satisfactory (Briganti et al., 2003:101). Two cytokines normally present in the skin, transforming growth factor–beta1 (TGF–81) and tumour necrosis factor–alpha (TNF–9), have been shown to inhibit melanin synthesis (Martinez–Esparza, 2001:972). The stratum corneum has been commonly accepted as the main barrier to percutaneous absorption. Many techniques have been applied to overcome this barrier properties and to enhance penetration with varying success (Pellet et al., 1997:92). The objective of this study was to investigate the topical delivery of the above mentioned peptide drugs with aid of the Pheroid drug delivery system. Pheroid technology is a delivery system that promotes the absorption and increases the efficacy of dermatological, biological and oral medicines in various pharmacological groups (Grobler et al., 2008:4). Pheroid entraps drugs with high efficiency and delivers them with remarkable speed to target sites (Grobler, 2004:4). In order to avoid degradation of these peptides, bestatin hydrochloride (an aminopeptidase inhibitor), was used (Lkhagvaa et al., 2008:386). Topical drug delivery was achieved by means of vertical Franz cell diffusion studies performed over a 6 and 12 h period. ELISA (enzyme linked immunosorbent assay) detection was used to detect cytokine concentrations. Entrapped cytokine solutions were monitored by confocal laser scanning microscopy (CLSM). Upon removal of donor and receptor compartments, skin discs were subjected to tape stripping in order to establish the amount of active present within the stratum corneum and epidermis as well as the remaining dermis (Pellet et al., 1997:92). When comparing the two studies with each other, it is evident that the diffused concentration values obtained with PBS (phosphate buffer solution, pH 7.4) was lower than that obtained with the Pheroid drug delivery system. Both cytokine concentrations were successfully delivered topically as a minimum of concentrations for both actives were detected. This positive result was confirmed as well by the amount of active detected in stratum corneum–epidermis and epidermis–dermis solutions. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2011.

Introduction Tumour-infiltrating lymphocytes (TIL) are an important prognostic indicator in high-grade serous ovarian carcinoma (HGSC). Certain types of TIL (in particular CD8+ effector T cells) predict better outcomes, whereas others (most notably CD4+CD25+FoxP3+ regulatory T cells; Tregs) predict worse outcomes. An unconventional subset of CD8+FoxP3+ T cells has been reported to be involved in autoimmunity and in immune response to several cancers. While the functional significance of CD8+FoxP3+ TIL remains poorly understood, they were associated with effective anti-tumour responses in a murine tumour model. Hypothesis CD8+FoxP3+ TIL are present in a subset of cases of HGSC and correlate with a strong immune response and increased patient survival. Experimental Design Multi-colour immunohistochemistry (IHC) was performed on a cohort of 44 primary HGSC specimens to enumerate and locate CD8+FoxP3+ TIL in comparison to CD8+FoxP3- and CD8-FoxP3+ TIL. Triple-colour IHC methodology was developed to further assess the phenotype of CD8+FoxP3+ TIL, including the measurement of additional markers CD4 and CD25 (classical markers of Tregs), Ki-67 (a marker of proliferation), and TIA-1 (a marker of cytotoxic potential). Intraepithelial versus stromal location was determined by staining adjacent sections for the epithelial marker pan-cytokeratin. Survival analysis was performed using a cohort of 188 cases of HGSC. Multi-colour staining was resolved using the Nuance™ multispectral imaging system in conjunction with Metamorph™ software. Survival analysis was performed using Kaplan-Meier and log rank tests. Results CD8+FoxP3+ cells were found in 60% of 44 cases of HGSC, in variable proportions ranging from 0.2 - 7.9% of CD8+ TIL and 0.5 – 12.7% of FoxP3+ TIL. CD8+FoxP3+ TIL were found to be either CD4+ (38.8%) or CD4- (61.2%). The majority of CD8+FoxP3+ TIL were also found to be CD25-TIA-1+Ki-67-, more closely resembling their CD8+FoxP3- counterparts. CD8+FoxP3+ TIL were found mainly in intraepithelial regions and were positively associated with patient survival (progression free survival; P = 0.0396). Conclusions CD8+FoxP3+ TIL are a component of the host immune response to HGSC. They appear to have a non-proliferative effector phenotype, consistent with an active role in the anti-tumour response. CD8+FoxP3+ TIL are associated with increased patient survival. An improved understanding of this new TIL subset may inform future immunotherapeutic strategies for this challenging malignancy. / Graduate / 0982 / sarakost@hotmail.com

This work investigates the mechanisms of binding of T cell receptors (TCRs) to Class I MHC-peptide complexes (pMHC). The structure of a TCR specific for the Melan-A tumour antigen bound to its cognate pMHC was solved to a resolution of 2.5 Å which gives insight into how this TCR could be mutated to optimize binding and subsequently used as a cancer vaccine. Detailed sequence and geometric analyses of all currently available structures of Class I TCR-pMHC complexes revealed that TCRs can bind to pMHC with a range of orientations, yet always focus on the central portion of the peptide and use a specific subset of six residues on the MHC helices for binding. The most striking finding was the use of aromatic residues in the TCR CDR loops to bind to residue Q155 on the MHC α2 helix. Attempts were also made to express and purify Toll-like receptors (TLRs) with the aim of solving one or more of these structures. However, despite testing of over 50 different constructs from 12 different TLRs or associated proteins, insufficient soluble protein expression was obtained for crystallization trials. Finally, a protein disorder prediction tool was developed to aid construct design for structural biology studies and improve the chances of obtaining protein crystals. This tool is based on a novel type of neural network and blind tests comparing it to 8 other disorder prediction tools showed it is one of the best in the field. It is freely available at www.strubi.ox.ac.uk/RONN. Analysis of large datasets revealed that the position of order/disorder transitions is quite precisely defined in amino-acid sequences and that transition regions have an amino acid composition distinct from that of bulk ordered and disordered sequences. There is a steady decrease in order-promoting residues on the ordered side of boundaries as well as a weak sequence signal, both of which signify the approaching disorder and may prove useful for improving existing disorder prediction tools.

Galvos smegenų pilnavertiškam funkcionavimui turi būti palaikoma optimali galvos smegenų kraujotaka ir įsotinimas deguonimi. Darbo tikslas - nustatyti ir palyginti galvos smegenų kraujotakos pokyčius neurochirurginių operacijų metu naudojant sevofluraną ir propofolį. Mokslinio darbo tikslui pasiekti nustatyti uždaviniai: nustatyti galvos smegenų kraujotakos pokyčius transkranijiniu ultragarsiniu doplerografu matuojant vidurinės smegenų arterijos kraujo tėkmės greitį viršpadangtinių galvos smegenų navikų ir galvos smegenų arterinių aneurizmų operacijų metu bei operuojant ligonius, neturinčius galvos smegenų susirgimų (kontrolinė grupė); palyginti galvos smegenų kraujotakos pokyčius viršpadangtinių galvos smegenų navikų šalinimo operacijų metu naudojant sevofluraną ir propofolį; palyginti galvos smegenų kraujotakos pokyčius sevoflurano anestezijoje atliekant galvos smegenų navikų šalinimo, arterinių aneurizmų išjungimo operacijas ir operuojant ligonius, neturinčius galvos smegenų susirgimų (kontrolinė grupė); nustatyti ir palyginti sisteminės kraujotakos pokyčius bei įvertinti ir palyginti ligonių būklę po neurochirurginių operacijų naudojant sevofluraną ir propofolį. Klinikinio tyrimo metu ištirta 260 ligonnių. Nustatyta, kad bendrosios anestezijos palaikymui, atliekant neurochirurgines operacijas, sevofluranas gali būti naudojamas taip pat saugiai, kaip ir propofolis. Sevofluranas, naudojamas dozėmis iki 2,0 proc. tūrio iškvėpiamose dujose chirurginiam anestezijos gyliui... [toliau žr. visą tekstą] / Optimal cerebrovascular circulation and oxygen saturation must be maintained for full cerebral functioning. The aim of the study is to determine the changes of the cerebral circulation during supratentorial tumour resection or cerebral arterial aneurysm clipping surgery under sevoflurane and propofol anaesthesia. The following objectives were established: to determine and evaluate changes of the cerebral circulation by measurements of cerebral blood flow velocity in the middle cerebral artery using transcranial Doppler ultrasonography during supratentorial brain tumour resection or cerebral arterial aneurysm clipping surgery or operations for patients without cerebral pathology (control group); to compare the changes of the cerebral circulation during supratentorial brain tumour resection surgery under sevoflurane and propofol anaesthesia; to compare the changes of the cerebral circulation during supratentorial brain tumour resection, cerebral arterial aneurysm clipping or operations for patients without cerebral pathology (control group) under sevoflurane anaesthesia; to determine and compare the changes of the systemic hemodynamic during neurosurgery under sevoflurane and propofol anaesthesia; to determine and compare recovery period after sevoflurane and propofol anaesthesia.After giving written informed consent 260 eligible patients were enrolled in the study. The obtained results showed, that sevoflurane can be used as safely as propofol for maintaining general... [to full text]

The statin family of hydroxymethylglutaryl coenzyme A reductase (HMGCR) inhibitors, used to control hypercholesterolemia, triggers apoptosis of various human tumour cells. HMGCR is the rate-limiting enzyme of the mevalonate (MVA) pathway, a fundamental metabolic pathway required for the generation of a number of biochemical end-products including cholesterol and isoprenoids, but the contribution of the MVA pathway to human cancer remains largely unexplored. Furthermore, as only a subset of tumour cells has been shown to be highly responsive to statins, the identification of appropriate subsets of patients will be required to successfully advance these agents as anticancer therapeutics. To this end, there were two major aims to this work: 1) Elucidate a molecular rationale for the observed therapeutic index of statin-induced apoptosis in normal and tumour cells; 2) Identify molecular determinants of sensitivity for statin-induced apoptosis in human tumour cells. To address the first aim we demonstrated that dysregulation of the MVA pathway, achieved by ectopic expression of either full length HMGCR (HMGCR-FL) or its novel splice variant lacking exon 13 (HMGCR-D13), increases transformation. Ectopic HMGCR promotes growth of transformed and non-transformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice. We also show that high mRNA levels of HMGCR and four out of five other MVA pathway genes correlate with poor prognosis in primary breast cancer, suggesting the MVA pathway may play a role in the etiology of human cancers. To address the second aim, we show that dysregulation of the MVA pathway is a key determinant of sensitivity to statin-induced apoptosis in multiple myeloma. In a panel of 17 distinct myeloma cell lines, half were sensitive to statin-induced apoptosis and the remainder were insensitive. Interestingly, in sensitive cells, the classic feedback response to statin exposure is lost, a feature we demonstrated could distinguish a subset of statin-sensitive primary myeloma cells. We further illustrated that statins are highly effective and well tolerated in an orthotopic model of myeloma using cells harboring a dysregulated MVA pathway. Taken together, this work provides a molecular rationale and determinants of sensitivity for statin-induced apoptosis of human tumour cells.

Malgré un intérêt grandissant pour la question du devenir des survivants de tumeur cérébrale pédiatrique, l’évaluation de leur équilibre est souvent négligée. Les objectifs de nos travaux étaient de 1) examiner les écrits portant sur l’équilibre chez les survivants de tumeur cérébrale pédiatrique; 2) comparer l'équilibre debout et la qualité de vie entre les survivants et un groupe d’enfants témoins; et 3) examiner l'association entre l'équilibre debout et qualité de vie chez les survivants. Notre recension des écrits démontra que les survivants de tumeur cérébrale présentent des troubles de l’équilibre, mais les limites méthodologiques des études nous empêchent de conclure de manière définitive. Ensuite, nous avons recruté un groupe d’enfants survivants d’une tumeur cérébrale de la fosse postérieure et un groupe d’enfants sains. Leur équilibre était évalué à l’aide du Bruininks-Oseretsky Test of Motor Proficiency-2nd edition (BOT-2) et du Pediatric Balance Scale (PBS). Certains participants ont aussi été évalués avec une plate-forme de force où les limites de stabilité étaient documentées. Finalement, tous les enfants et leurs parents remplissaient le Pediatric Quality of Life Inventory (PedsQL4.0). Nos résultats démontrent que les survivants présentent une diminution de l’équilibre mise en évidence par le BOT-2, mais que leur qualité de vie est similaire aux enfants sains. La performance au BOT-2 est associée à la dimension physique du PedsQL4.0, suggérant une relation entre l’équilibre et la qualité de vie. Nos résultats suggèrent qu’une évaluation de l’équilibre pourrait être bénéfique chez cette clientèle afin de mieux cerner ses besoins de réadaptation. / There is growing interest in studying outcomes in survivors of pediatric brain tumours. Physical outcomes, especially balance abilities, are less investigated. Objectives of this thesis are to: 1) examine the literature for balance outcomes in survivors of pediatric brain tumours, 2) compare standing balance and health-related quality of life (HRQOL) between survivors of pediatric posterior fossa brain tumours (PFBT) and typically-developing controls and 3) explore the association between balance and HRQOL. A comprehensive review demonstrated that although the literature suggests that survivors of pediatric brain tumours display ongoing balance deficits, studies have limitations, emphasizing the need for further research. Survivors of pediatric PFBT and typically-developing children were recruited and their balance assessed with the Bruininks-Oseretsky Test of Motor Proficiency-2nd edition (BOT-2) and Pediatric Balance Scale (PBS). Dynamic balance was also evaluated for some participants using a force platform. The Pediatric Quality of Life Inventory measured HRQOL. Statistical analyses included Mann-Whitney U tests to compare results between groups and Spearman's rank correlation coefficient to determine the association between balance and HRQOL. Balance abilities were significantly worse in survivors as measured by the BOT-2. The PBS displayed a ceiling effect. Certain laboratory outcome measures suggested balance difficulties. All participants’ HRQOL scores were within normative values. In survivors, an association was found between BOT-2 scores and the physical dimension of HRQOL. Survivors of PFBT demonstrate persistent balance difficulties, best assessed by the BOT-2; however, they report normal HRQOL. Future research should be collaborative and focus upon the best ways to manage balance deficits.

Für die Therapie maligner intranasaler Neoplasien beim Hund existieren nur mäßig be-friedigende Behandlungsstrategien. Als Therapiemodalität der Wahl wird die Radiothe-rapie angesehen, die gegenwärtig v.a. in Form einer perkutanen Bestrahlung (Telethe-rapie) mit aufwendigen, bis zu 20 Fraktionen umfassenden Protokollen kurativer Intenti-on angewendet wird. Die erreichbaren Überlebenszeiten sind meist limitiert durch das Auftreten eines Rezidivs des Nasentumors innerhalb des Bestrahlungsfeldes, sodass eine Erhöhung der applizierten Gesamtdosis nötig erscheint. Dies ist jedoch im Rahmen einer Teletherapie aufgrund nicht vertretbarer akuter Nebenwirkungen nicht möglich. Alternativ steht die Brachytherapie zur Verfügung, die aufgrund ihrer physikalischen Charakteristika zur besseren Schonung des umliegenden Normalgewebes beiträgt. Ge-genwärtig existieren keine anderen Untersuchungen zur Anwendung der fraktionierten High-Dose-Rate-Brachytherapie bei Nasentumoren des Hundes. Ziel dieser Studie war es daher, die Durchführbarkeit dieser Therapiemodalität beim Hund erstmals zu unter-suchen und die akuten und chronischen Nebenwirkungen sowie die erzielbare progres-sionsfreie Zeit und die Überlebenszeit zu dokumentieren. Im Zeitraum von 2001 bis 2007 gingen 18 Hunde in die Studie ein. Das diagnostische Vorgehen beinhaltete neben einer klinischen Untersuchung und der Röntgenuntersu-chung von Nase und Thorax auch die kernspintomographische Beurteilung der Nasen-höhlen und eine nachfolgende Rhinoskopie inklusive Biopsie. Die Therapie bestand aus zwei wöchentlichen Fraktionen, bei denen in Vollnarkose über einen in der Nasenhöhle applizierten Katheter mithilfe des Radioisotops 192Iridium jeweils 5 Gy appliziert wurden. Die damit über vier Wochen erreichte Gesamtdosis lag bei 40 Gy, und entsprach damit der biologischen Effizienz einer perkutan applizierten konventionell fraktionierten Ge-samtdosis von circa 60 Gy. Im Anschluss an die Therapie wurden die Hunde monatlich klinisch untersucht und die auftretenden Nebenwirkungen anhand des Radiation Morbi-dity Scores der VRTOG beschrieben. Es wurden außerdem weiterführende Untersu-chungen in Form von MRT, Rhinoskopie und Biopsie durchgeführt. Die aufgetretenen Nebenwirkungen waren mit denen in der Literatur nach Teletherapie beschriebenen vergleichbar, beziehungsweise fielen im Bereich von Augen und Maulschleimhaut ge-ringer aus. Nebenwirkungen im Bereich der Haut traten in Form von Alopezie, Hyper-pigmentation oder Leukotrichie auf. Im Bereich der Nasenschleimhaut zeigten fast alle Hunde eine leichte chronische Rhinitis. Als problematische Nebenwirkungen traten bei drei Patienten Osteoradionekrosen auf, die einer aufwendigeren chirurgischen Versor-gung bedurften. Die mediane progressionsfreie Zeit lag bei 13 Monaten, die mediane Überlebenszeit bei 17 Monaten. Die Adenokarzinome wiesen die längste Überlebens-zeit auf, dies war jedoch aufgrund der insgesamt kleinen Patientenzahl nicht signifikant. Ein Zusammenhang zwischen dem Tumorstadium und der progessionsfreien Zeit oder Überlebenszeit bestand nicht. Bei dem beschriebenen Protokoll handelt es sich um eine unter klinischen Bedingungen praktikable Therapieform, die mit ihren insgesamt acht Fraktionen für Besitzer und Tier wesentlich weniger belastend ist als teletherapeutische kurative Protokolle mit 12-20 Fraktionen. Gleichzeitig gelingt es, eine Gesamtdosis von verhältnismäßig hoher biolo-gischer Effizienz zu applizieren, ohne jedoch stärkere Nebenwirkungen in Kauf nehmen zu müssen. Im Bereich von Auge und Maulschleimhaut sind die Nebenwirkungen sogar geringer. Bei einem kleinen Teil der Patienten treten jedoch auch hier, ebenso wie nach teletherapeutischen Protokollen, problematische chronische Nebenwirkungen auf, die die Lebensqualität der betroffenen Tiere beeinträchtigen und die einer aufwendigeren Therapie zwingend bedürfen. Die mit diesem Protokoll erreichten Remissions- und Ü-berlebenszeiten sind mit denen aus der Literatur vergleichbar bis tendenziell besser. Aufgrund der oben genannten Vorteile erscheint die vorgestellte Therapie daher als Al-ternative zu Teletherapie bei der Behandlung kaniner Nasentumoren durchaus geeig-net. Weitere Studien mit größeren Patientenzahlen unter Einbeziehung einer anders therapierten Kontrollgruppe sind jedoch notwendig

Neuroendocrine tumors (NETs), originally described as carcinoids, represent a rare and heterogeneous group of neoplasms associated with intensive secretion of hormones, bioactive peptides and amines. Most of the patients are diagnosed at a late stage of disease, often with liver metastases. Surgery remains the main treatment to control metastatic disease, but is not curative. Oncolytic virotherapy represents a promising approach to treat cancer and different strategies have been exploited to restrict viral replication to tumor cells. We developed an oncolytic adenovirus based on serotype 5, Ad5[CgA-E1A], where the chromogranin A (CgA) promoter controls expression of the E1A gene and thereby virus replication. We found that Ad5[CgA-E1A], selectively replicates in NET cells and it is able to suppress fast-growing human BON carcinoid tumors in nude mice. The activity of Ad5[CgA-E1A] was not completely blocked in liver cells. We further repressed virus replication in hepatocytes by targeting E1A with miR122, an miRNA specifically expressed in the liver. miRNAs bind to mRNA and induce its cleavage or translational blockage. By insertion of tandem repeats of miR122 target sequences in 3’UTR of E1A gene, we observed reduced E1A protein expression and replication arrest in miR122 expressing liver cells. The oncolytic potency of the miR122-targeted virus was not affected in NET cells. Since some NET and neuroblastoma cells express high levels of somatostatin receptors (SSTRs), we introduced in the virus fiber knob cyclic peptides, which contain four amino acids (FWKT) and mimic the binding site of somatostatin for SSTRs. The FWKT-modified Ad5 transduces midgut carcinoid cells from liver metastases about 3-4 times better than non-modified Ad5. Moreover, FWKT-modified Ad5 overcomes neutralization in an ex vivo human blood loop model to a greater extent than Ad5, indicating that the fiber knob modification may prolong the systemic circulation time. NETs represent a huge therapeutic challenge and novel diagnostic markers are needed for early detection and effective treatment of NETs. We have profiled primary tumors and liver metastases of ileocaceal NETs, using Affymetrix microarrays and advanced bioinformatics. We have identified six novel marker genes and show high similarity between primary lesions and liver metastases transcriptome by hierarchical clustering analysis.