Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Introduction: Mammalian circadian rhythms form an integral physiological system allowing for the synchronisation of all metabolic processes to daily light/dark cycles, thereby optimising their efficacy. Circadian disruptions have been implicated in the onset and progression of different types of cancers, including those arising in the breast. Several links between the circadian protein Per2 and DNA damage responses exist. Aberrant Per2 expression results in potent downstream effects to both cell cycle and apoptotic targets, suggestive of a tumour suppressive role for Per2. Due to the severe dose limiting side effects associated with current chemotherapeutic strategies, including the use of doxorubicin, a need for more effective adjuvant therapies to increase cancer cell susceptibility has arisen. We therefore hypothesize, that the manipulation of the circadian Per2 protein in conjunction with doxorubicin may provide a more effective chemotherapeutic strategy for the treatment of breast cancer. The aims of this project were thus to: (i) Characterize the role of Per2 in normal breast epithelial cells as well as in ER+ and ER- breast cancer cells; (ii) to determine the role of Per2 in doxorubicin-induced cell death, (iii) to determine the role of Per2 in autophagy and finally (iv) to assess whether the pharmacological inhibition of Per2 with metformin, can sensitize chemo-resistant MDA-MB-231 breast cancer cells to doxorubicin-induced cell death. Methods: An in vitro model of breast cancer was employed using the normal MCF-12A breast epithelial, estrogen receptor positive (ER+) MCF-7 and estrogen receptor negative (ER-) MDA-MB-231 breast adenocarcinoma cell lines. Circadian rhythmicity of Per2 protein expression was determined using western blotting, and Per2 cellular localization was assessed using fluorescent confocal microscopy. Per2 was then silenced by means of an endoribonuclease-prepared siRNA, and silencing efficiency was determined with the use of western blotting. The roles of Per2 in doxorubicin-induced cell death and autophagy were assessed by treating MDA-MB-231 breast cancer cells under the following conditions (1) Control, (2) 2.5 μM doxorubicin or 10 nM bafilomycin A1 (3) 30 nM esiPer2 and (4) 30 nM esiPer2 in combination with 2.5 μM doxorubicin or 10 nM bafilomycin A1. Following treatments cell viability was assessed using the MTT assay, western blotting for markers of apoptosis including p-MDM2 (Ser166), p-p53 (Ser15), cleaved caspase-3 and –PARP as well as markers of autophagy (AMPKα, mTOR and LC3). Furthermore, cell cycle analysis, G2/M transition and cell death (Hoechst 33342 and propidium iodide staining) were assessed by means of flow cytometry. The pharmacological inhibition of Per2 was achieved by treating MDA-MB-231 cells with 40 mM metformin as well as in combination with 2.5 μM doxorubicin. MTT cell viability assays, cell cycle analysis (flow cytometry) and western blotting for apoptosis (Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) were assessed. Results and discussion: A circadian pattern of Per2 protein expression was observed in the normal MCF-12A and MDA-MB-231 cancer cells with protein levels peaking at ±700% and ±500% of baseline was observed. However, no rhythmic expression was observed in the MCF-7 cancer cells. Immunostaining for Per2 showed localization OF Per2 in the cytoplasm as well as in the nucleus of both the MCF-12A and MDA-MB-231 cells. Concentration curves showed a significant reduction in cell viability following 2.5 μM doxorubicin treatment for 24 hours. Per2 protein expression was significantly reduced with both esiPer2 and metformin treatment. Silencing of Per2 in combination with doxorubicin treatment resulted in cell cycle arrest with a significant increase in apoptosis, indicating that Per2 silencing effectively sensitized the MDA-MB-231 cancer cells to the anti-carcinogenic properties of doxorubicin. Modulation of Per2 protein expression was effectively achieved with the use metformin although this decrease occurred independently of AMPKα phosphorylation. A significant increase in apoptosis was observed following treatment with metformin in combination with doxorubicin treatment. However, no changes in cell cycle regulation were observed. Per2 appears to be involved in the regulation of autophagy as a significant increase in autophagy flux was observed when Per2 was silenced. Additionally, this increase in autophagic flux resulted in a significant increase in MDA-MB-231 cancer cell death which was enhanced further when autophagy was inhibited with bafilomycin A1 subsequent to Per2 silencing. Conclusions: Per2 protein expression was shown to display a 24 hour circadian rhythm in the MCF-12A cells, and to a lesser extent in the MDA-MB-231 cells. However, the MCF-7 cells failed to show rhythmic changes in Per2 protein expression. Per2 was shown to be located predominantly in the cytoplasm, with nuclear localization observed when cytoplasmic fluorescent intensity was lower. Per2 silencing effectively sensitized the chemo-resistant MDA-MB-231 breast cancer cells to both doxorubicin-induced cell death and autophagic inhibition. / AFRIKKANSE OPSOMMING: Inleiding: Sirkadiese ritmes vorm ‘n integrale fisiologiese sisteem wat die sinkronisasie van alle metaboliese prosesse asook lig/donker siklusse se effektiwiteit optimaliseer. Onderbreking van hierdie sirkadiese ritmes word geïmpliseer in die ontstaan en bevordering van verskillende kankertipes, insluitend borskanker. Verskeie raakpunte bestaan tussen die sirkadiese proteïen, Per2, en die DNA skade-respons. Abnormale Per2 uitdrukking veroorsaak afstroom effekte op beide die selsiklus en apoptotiese teikens wat moontlik aanduidend van ‘n tumor-onderdrukkende rol vir Per2 kan wees. Daar bestaan ‘n groot nood vir meer effektiewe adjuvante terapieë om kankersel vatbaarheid vir chemoterapie te verhoog as gevolg van dosis-beperkende newe-effekte wat geassosieer word met huidige chemoterapeutiese strategieë, insluitende dié van doxorubicin. Ons hipotese is dus dat die manipulering van die sirkadiese Per2 proteïen tesame met doxorubicin ‘n meer effektiewe chemoterapeutiese strategie vir die behandeling van borskanker sal wees. Die doelwitte van hierdie projek was dus om: (i) Die rol van Per2 in normale borsepiteelselle sowel as in ER+ en ER- borsepiteel kankerselle te karakteriseer; (ii) die rol van Per2 in doxorubicin-geïnduseerde seldood te bepaal; (iii) te bepaal of Per2 ‘n rol in autofagie speel en laastens (iv) te bepaal of die farmakologiese inhibisie van Per2 met metformin chemo-weerstandbiedende MDA-MB-231 kankerselle kan sensitiseer vir doxorubicin-geïnduseerde seldood. Metodes: ‘n In vitro model vir borskanker is gebruik wat normale MCF-12A borsepiteelselle, estrogeen reseptor positiewe (ER+) MCF-7 en estrogeen reseptor negatiewe (ER-) MDA-MB-231 bors adenokarsenoomselle insluit. Sirkadiese ritmisiteit van Per2 proteïen uitdrukking is deur middel van die westelike kladtegniek bepaal en die sellulêre ligging van Per2 deur middel van fluoresensie mikroskopie. siPer2 is voorberei deur middel van endoribonuklease-siRNA en die effektiwiteit daarvan is deur middel van westelike kladtegniek getoon. Die rol van Per2 in doxorubicin-geinduseerde seldood en autofagie is bepaal deur MDA-MB-231 borskankerselle onder die volgende omstandighede te toets: (1) Kontrole, (2) 2.5 μM doxorubicin of 10 nM bafilomycin A1 (3) 30 nM esiPer2 en (4) 30 nM esiPer2 in kombinasie met 2.5 μM doxorubicin of 10 nM bafilomycin A1. Na die behandeling, is sellewensvatbaarheid bepaal deur gebruik te maak van ‘n MTT toets; westelike kladtegniek is gebruik om vir merkers van apoptose soos p-MDM2 (Ser166), p-p53 (Ser15), gekliefde caspase-3 en -PARP asook vir merkers van autofagie (AMPKα, mTOR en LC3) te toets. Die selsiklus, G2/M oorgang en seldood (Hoechst 33342 en propidium iodide kleuring) is deur middel van vloeisitometrie bepaal. Per2 is ook farmakologies geïnhibeer deur MDA-MB-231 selle met 40 mM metformin asook in kombinasie met 2.5 μM doxorubicin te behandel. Daarna is sellewensvatbaarheid (MTT) sowel as die selsiklus (vloeisitometrie) en apoptose (westelike kladtegniek vir Per2, p-AMPKα (Thr172), p53, caspase-3 and PARP) gemeet. Resultate en bespreking: ‘n Sirkadiese patroon vir Per2 proteïen uitdrukking is in die normale MCF-12A selle asook in die MDA-MB-231 kankerselle waargeneem met proteïenvlakke wat hul piek by ±700% and ±500% onderskeidelik in vergelyking met basislyn bereik het. Geen ritmiese patroon van Per2 proteïen uitdrukking is egter in die MCF-7 kankerselle waargeneem nie. Immunokleuring om Per2 ligging te bepaal het getoon dat Per2 in the sitoplasma sowel as in die nukleus in beide MCF-12A en MDA-MB-231 selle voorgekom het. Konsentrasie kurwes het aangetoon dat daar ‘n insiggewende vermindering in sellewensvatbaarheid voorgekom het na die behandeling van die selle met 2.5 μM doxorubicin vir 24 uur. Per2 proteïen uitdrukking is insiggewend verlaag met beide esiPer2 en metformin behandeling van die selle. esiPer2 aleen of in kombinasie met doxorubicin behandeling het selsiklus staking tot gevolg gehad asook ‘n beduidende toename in apoptose veroorsaak wat dus aangedui het dat esiPer2 effektief was om MDA-MB-231 kankerselle te sensitiseer vir die anti-karsinogeniese doxorubicin behandeling. Modulering van Per2 proteïen uitdrukking was effektief met metformin behandeling, alhoewel die afname onafhanklik van AMPKα fosforilasie plaasgevind het. ‘n Insiggewende toename in apoptose is waargeneem na metformin behandeling in kombinasie met doxorubicin. Geen veranderinge in die selsiklus is egter onder hierdie omstandighede waargeneem nie. Per2 blyk betrokke te wees in die regulering van autofagie aangesien ‘n insiggewende verhoging in autofagie omsetting waargeneem is na esiPer2 behandeling. Die toename in autofagie omsetting is geassosieer met ‘n insiggewende toename in seldood in MDA-MB-231 kankerselle wat verder verhoog is wanneer autofagie met bafilomycin A1 (autofagie inhibitor) in kombinasie met esiPer2 behandel is. Gevolgtrekkings: Per2 proteïen uitdrukking het ‘n 24 uur sirkadiese ritme in die MCF-12A normale selle, en tot ‘n mindere mate in die MDA-MB-231 selle getoon. Die MCF-7 selle het egter geen ritmiese patroon van Per2 proteïen uitdrukking getoon nie. Per2 kom hoofsaaklik in die sitoplasma voor en het slegs in die nukleus voorgekom wanneer die sitoplasmiese fluoresensie intensiteit laer was. esiPer2 was dus effektief om die chemo-weerstandbiedende MDA-MB-231 borskankerselle te sensitiseer vir doxorubicin-geïnduseerde seldood. / National Research Foundation

Thesis (MBA)--Stellenbosch University, 2008. / ENGLISH ABSTRACT: The cost of medical care, in particular the cost of cancer care, has seen significant increases globally in the last few years. These cost increases in part are a result of tremendous advancements in new health technologies to diagnose, treat and care for cancer sufferers. The development of these highly specialised treatment modalities is not expected to slow down in the next few years, as potentially new treatments are already in the pipeline. On the other hand, cancer is becoming more prevalent. affecting more people worldwide. The condition remains life threatening, causing patients to become dependent and desperately hopeful of their requested treatments. Managed care, which includes the processes of rationing, has been implemented by medical aid schemes in the private health care industry in an effort to curtail the escalating costs of health care. Currently medical aids in the country are under immense pressure to comply with financially demanding legislation as well as to increase their membership risk by keeping contributions low and subsequently improve access to private health care in the country. Notwithstanding the fact that rationing might be justified from an economic perspective, the implications of transposing free market principles into an almost sacred health care environment challenges current morals and ethics in this arena. The price consciousness in cancer care is almost creating a scenario where clinical reasons are becoming subservient to fiscal reasons or, put differently, it is placing a price tag on human lives. In its true glory, the rationale of rationing is to challenge the individual patient needs against that of the bigger medical aid society. The distributive justice principles of rationing are creating immense conflict between the virtue-based, principle-based and contemporary ethics, which are currently governing medical practice in the country. As a result rationing creates serious vexing funding decisions with long-ranging effects. Its against this background that the study further consider the implications of managed care and rationing as it creates serious questions about the fairness, decision-making power and authority of managed care organizations. The implication of this is that the treating physician seems to have lost all autonomy and control in trying to treat and care for his cancer patient. Hence the perception that managed care does not act in the best interest of the vulnerable and desperate cancer suffering patient. As a result of th is view of managed care it becomes important to ensure the fairness and or legitimacy of managed care and rationing decisions. Therefore, the final section of the study considers the fair and just rationing of medical care as well as setting limits that are morally and ethically acceptable, in a cancer related setting. The studies of Daniels and Sabin are utilized extensively in particular the suggested criteria required by managed care organisations to ensure their rationing decisions are fair and legitimate. The implications of this and the assurances to cancer sufferers in a medical scheme is that the decisions to fund new health technologies are based on a process that is transparent and collaborative and that cost consideration of treatment has merit if it is made within the confines of this process. / AFRIKAANSE OPSOMMING: Die koste van mediese sorg, en spesifiek die koste van kankersorg, het in die afgelope paar jaar wereldwyd aansienlik toegeneem. Hierdie toename in koste is gedeeltelik die resultaat van geweldige vooruitgang in nuwe gesondheidstegnologiee om kankerlyers te diagnoseer, te behandel en vir hulle te sorgo Daar word nie verwag dat die ontwikkeling van hierdie hoogs gespesialiseerde behandelingsmodaliteite oor die volgende paar jaar sal afneem nie, aangesien nuwe behandelings steeds geregistreer word. Aan die ander kant is die voorkomssyfer van kanker besig om toe te neem, en be"invloed dit mense oor die hele wereld. Die toestand is steeds lewensbedreigend, en veroorsaak dat pasiente afhanklik van en desperaat vol hoop is vir die nodige behandeling. Bestuurde sorg, wat die proses van rantsoenering insluit, is deur mediesefondsskemas in die privaat gesondheidsorgbedryf ge"lmplementeer in 'n poging om die stygende koste van mediese sorg te verminder. Mediese fondse in die land is tans onder geweldige druk om aan finansieel veeleisende wetgewing te voldoen en om hulle lidmaatskaprisiko te verhoog deur bydraes laag te hou en gevolglik toegang tot privaat gesondheidsorg in die land te verbeter. Ondanks die feit dat rantsoenering moontlik vanuit 'n ekonomiese perspektief geregverdig kan word, daag die implikasies van die omsetting van vryemarkbeginsels in 'n amper heilige gesondheidsorgomgewing huidige morele waardes en etiek in hierdie veld uit. Die prysbewustheid in kankersorg skep amper 'n scenario waar kliniese redes ondergeskik aan fiskale redes gestel word of, om dit anders te stel, dit plaas 'n prys op mense se lewens. In sy volle glorie is die rasionaal van rantsoenering om die individuele pasient se behoeftes teenoor die van die groter mediesefondssamelewing te stel. Die beginsels van verdelende regverdigheid van rantsoenering skep enorme konflik tussen die deug..gebaseerde, beginselgebaseerde en kontemporere etiek wat tans die mediese praktyk in die land beheer. Gevolglik skep rantsoenering ernstige, moeilike befondsingsbesluite met effekte oor die lang termyn. Oit is teen hierdie agtergrond dat die studie die verdere implikasies van bestuurde sorg en rantsoenering moet oorweeg, aangesien dit ernstige vrae rondom die billikheid , besluitneming en outoriteit van bestuurde sorg maatskappye lig. By implikasie beteken dit dat die geneesheer wat die pasient behandel, feitlik aile beheer verloor het om die pasient vir aile praktiese doeleindes optimaal te behandel. Oaarom die persepsie dat bestuurde sorg nie in die beste belang van die kwesbare en desperaat kanker pasiente is nie. As gevolg van die persepsie van bestuurde sorg, raak dit meer belangrik om die bilikheid en regverdigheid van gesondheid sorg besluite te verseker. Met dit in ag genome, oorweeg die finale deel van die studie die bilikheid en regverdigheid van mediese rantsoenering so-ook die set van perke wat eties en moreel aanvaarbaar is, in 'n kanker verwante agtergrond. Die werke van Daniels en Sabin word in aansienlike detail hersien in besonder hul voorgestelde kriteria wat vereis word deur bestuurde sorg organisasies om te verseker hul besluite ten opsigte van rantsoenering is redelik en regverdig. Die implikasies hiervan en die versekering tot kanker Iyers in 'n mediese skema is dat die besluite om nuwe gesondheidstegnologiee te befonds, is gebasseer op In deursigtige en samehorende proses en dat aile koste oorwegings vir behandeling meriete het, indien dit is gemaak is binne die raamwerk van hierdie proses.

Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Introduction – Due to spatial separation from the native vascular bed, solid tumours develop regions with limited access to nutrients essential for growth and survival. The promotion of a process known as macroautophagy may facilitate in the maintenance of intracellular amino acid levels, through breakdown of cytoplasmic proteins, so that they remain available for macromolecular biosynthesis and ATP production. Several studies point to the potential ability of some cancers to temporarily increase autophagy and thereby prolong cell survival during metabolic stress. The validity of these claims is assessed when a commonly used breast cancer cell line and an epithelial breast cell line are starved of amino acids in this study. Furthermore, we go on to hypothesize that acute amino acid deprivation during treatment will result in an elevated sensitivity of MDAMB231 cells to doxorubicin toxicity but limit its cytotoxic side-effects in MCF12A cells. Methods and study design- Human breast cancer cells (MDAMB231) and breast epithelial cells (MCF12A) cultured in complete growth medium were compared to those incubated in medium containing no amino acids. Steady state autophagy levels were monitored using classical protein markers of autophagy (LC3-II and beclin-1) and the acidic compartmentalization in cells (Lysotracker™ red dye) in conjunction with autophagy inhibition (bafilomycin A1 and ATG5 siRNA). Cell viability was monitored using several techniques, including caspase 3/7 activity. ATP levels were assessed using a bioluminescent assay, while mass spectrometry based proteomics was used to quantify cellular amino acid levels. Similar techniques were used to monitor autophagy during doxorubicin treatment, while cellular doxorubicin localization was monitored using immunofluorescence microscopy. Finally, a completely novel GFP-LC3 mouse tumour model was designed to assess autophagy and caspase activity within tumours in vivo, during protein limitation and doxorubicin treatment. Results - Amino acid deprivation resulted in a transient increase in autophagy at approximately 6 hours of amino acid starvation in MDAMB231 cells. The amino acid content was preserved within these cells in an autophagy-dependent manner, a phenomenon that correlated with the maintenance of ATP levels. Inhibition of autophagy during these conditions resulted in decreased amino acid and ATP levels and increased signs of cell death. MCF12A cells displayed a greater tolerance to amino acid starvation during 24 hours of amino acid starvation. Evidence indicated that autophagy was important for the maintenance of amino acid and ATP levels in these cells and helped prevent starvation-induced cell death. Furthermore, data showed that concomitant amino acid withdrawal resulted in decreased cellular acidity in MDAMB231 cells, and increased acidity in MCF12A cells, during doxorubicin treatment. These changes correlated with evidence of increased cell death in MDAMB231 cells, but a relative protection in MCF12A cells. A novel model was used to apply these techniques in vivo, and although mice fed on a low protein diet during high dose doxorubicin treatment had increased mean survival and smaller tumour sizes, evidence suggested that autophagy is protecting a population of cells within these tumours. Conclusions - This novel approach to tumour sensitization could have several implications in the context of cancer therapy, and given the delicate relationship that autophagy has with the cancer microenvironment, efforts to determine the mechanisms involved in autophagy and sensitization could lead to new and innovative treatment opportunities for cancer management. / AFRIKAANSE OPSOMMING: Inleiding – As gevolg van hul skeiding van die oorpronklike vaskulêre netwerk, ontwikkel soliede gewasse areas met beperkte toegang tot noodsaaklike voedingstowwe. Die bevordering van 'n proses wat as makro-autofagie bekend staan, kan die handhawing van intrasellulêre aminosuur vlakke fasiliteer. Voorafgenoemde proses word waarskynlik deur die afbreek van sitoplasmiese proteïene teweegebring om sodoende vir makro-molekulêre biosintese en ATP produksie beskikbaar te kan wees. Verskeie studies dui daarop dat sommige kankersoorte die vermoë het om autofagie tydelik te verhoog, en daarby sel oorlewing gedurende metaboliese stress te verleng. Die geldigheid van hierdie eise word evalueer wanneer 'n algemeen beskikbare borskanker sellyn, en 'n borsepiteelsellyn in hierdie studie van aminosure verhonger word. Verder, veronderstel ons dat akute aminosuur ontneming gedurende behandeling 'n verhoogde sensitiwiteit van MDAMB231 selle tot doxorubicin toksisiteit tot gevolg sal hê, maar terselfdetyd die middel se sitotoksiese newe-effekte in MCF12A selle sal beperk. Metodes en studie ontwerp – Menslike borskanker- (MDAMB231) en bors epiteel selle (MCF12A) wat in volledige groeimedium gekweek is, is vergelyk met selle wat in aminosuur vrye medium gekweek is. Basislyn autofagie-vlakke is gemonitor deur die gebruik van klassieke autofagie proteïen merkers (LC3-II en beclin-1) en die asidiese kompartementalisering in selle (Lysotracker™ rooi kleurstof) saam met autofagie inhibisie (bafilomycin A1 and ATG5 siRNA). Sellewensvatbaarheid is deur die gebruik van verskeie tegnieke, insluitend caspase 3/7 aktiwiteit, gemonitor. ATP-vlakke is deur die gebruik van 'n bioluminiserende tegniek gemeet, terwyl massa-spektrometrie-gebaseerde “proteomics” gebruik is om sel aminosuur vlakke te kwantifiseer. Soortgelyke tegnieke is gebruik om autofagie gedurende doxorubicin behandeling waar te neem, terwyl sellulêre doxorubicin lokalisasie deur die gebruik van immunofluoresensie mikroskopie gemonitor is. Ten slotte, is 'n unieke GFP-LC3 muismodel in hierdie studie ontwikkel. Hierdie model is gebruik om autofagie en caspase aktiwiteit in gewasse in vivo te bestudeer tydens proteïen beperking en doxorubicin behandeling. Resultate – Aminosuur ontneming het tot 'n tydelike verhoging in autofagie na ongeveer 6 ure van aminosuur verhongering in MDAMB231 selle gelei. Die aminosuur inhoud van hierdie selle het op 'n autofagie-afhanklike manier behoue gebly. Hierdie verskynsel het met die handhawing van ATP-vlakke gekorreleer. Autofagie inhibisie gedurende hierdie kondisies het 'n verlaging in aminosuur en ATP-vlakke teweeggebring, sowel as vermeerderde tekens van seldood tot gevolg gehad. MCF12A selle het 'n groter toleransie tot aminosuur verhongering tydens die 24 uur aminosuur verhongeringsperiode getoon. Getuienis het aangedui dat autofagie belangrik vir die handhawing van aminosuur en ATP-vlakke in hierdie selle was, en gehelp het om verhongerings-geïnduseerde seldood te voorkom. Verder het data gewys dat aminosuur ontrekking tot verminderde sellulêre asiditeit in MDAMB231 selle, en verhoogde asiditeit in MCF12A selle gedurende doxorubicin behandeling gelei het. Hierdie veranderinge stem ooreen met getuienis van toenemende seldood in MDAMB231 selle, maar 'n relatiewe beskerming in MCF12A selle. 'n Unieke model was gebruik om hierdie tegnieke in vivo toe te pas. Alhoewel verhoogde oorlewing en kleiner gewasse in muise op 'n lae proteïen dieet gedurende hoë dosis doxorubicin behandeling opgemerk is, het bewyse voorgestel dat autofagie 'n populasie selle binne die gewasse beskerm. Gevolgtrekkings – Hierdie unieke benadering tot tumor sensitisering kan verskeie implikasies in die konteks van kanker behandeling hê. Gegewe die delikate verhouding van autofagie met die kanker mikro-omgewing, kan pogings om die meganismes betrokke in autofagie en sensitisering te bepaal, tot nuwe en innoverende behandelings vir kanker lei.

Doctor of Philosophy / Department of Chemistry / Viktor Chikan / The research work in this thesis aims at investigating the basic physic-chemical properties of magnetic and metal nanoparticles (NPs) for biomedical applications such as magnetic hyperthermia and controlled drug release. Magneto-plasmonic properties of magnetic NPs are important to evaluate potential applications of these materials. Magnetic property can be used to control, monitor and deliver the particles using a magnetic field while plasmonic property allows the tracking of the position of the particles, but aggregation of NPs could pose a problem. Here, the aggregation of NPs is investigated via the Faraday rotation of gold coated Fe[subscript]2O[subscript]3 NPs in alternating magnetic fields. In addition, the Faraday rotation of the particles is measured in pulsed magnetic fields, which can generate stronger magnetic fields than traditional inductive heaters used in the previous experiments. In the second project, the formation of protein-NPs complexes is investigated for hyperthermia treatment. The interactions between gold and iron-platinum NPs with octameric mycobacterial porin A from Mycobacterium smegmatis (MspA) and MspA[superscript])cys protein molecules are examined to assemble a stable, geometrically suitable and amphiphilic proteins-NPs complex. Magnetic NPs show promising heating effects in magnetic hyperthermia to eliminate cancer cells selectively in the presence of alternating magnetic field. As a part of investigation, the heating capacity of a variety of magnetic NPs and the effects of solvent viscosity are investigated to obtain insight into the heating mechanism of these particles. Finally, the controlled drug release of magnetic NPs loaded liposomes by pulsed magnetic field is investigated. The preliminary data indicate about 5-10% release of drug after the application of 2 Tesla magnetic pulses. The preliminary experiments will serve as the initial stage of investigation for more effective magnetic hyperthermia treatment with the help of short magnetic pulses.

Le cancer pancréatique représente la cinquième cause de décès par cancer dans les pays occidentaux. Son mauvais pronostic (survie à 5 ans inférieure à 3,5 % des cas) est dû à l’absence de facteurs de risques spécifiques interdisant une prévention efficace, et à un diagnostic tardif qui révèle un cancer agressif chez environ 90% des patients. Actuellement, le seul traitement curatif de ce cancer est la chirurgie, mais celle-ci ne peut être envisagée que dans 10 à 15 % des cas. L’adressage de molécules thérapeutiques vers l’organe, le tissu ou la cellule malade constitue aujourd’hui un défi majeur pour le traitement des maladies humaines notamment infectieuses, cancéreuses ou d’origine génétique. C’est pour ces raisons que le développement de nanotechnologies, en tant que vecteurs de médicaments, a pris un essor considérable au cours des dernières années. Dans ce contexte, le concept de squalènisation repose sur le couplage chimique entre le squalène (SQ), un lipide naturel précurseur de la synthèse du cholestérol, et des principes actifs (notamment des molécules anticancéreuses). Les bioconjugués ainsi formés sont alors capables de s’auto-assembler en solution aqueuse pour former des nanoparticules stables de diamètres compris entre 100 et 300 nm. L’exemple de référence dans ce domaine est la nanoparticule de gemcitabine-squalène (SQdFdC) qui a donné lieu à des résultats spectaculaires in vitro sur des lignées de cellules cancéreuses humaines In vivo, les nanoparticules de gemcitabine-squalène se sont avérées beaucoup plus efficaces que la gemcitabine libre sur des tumeurs solides greffées par voie sous-cutanée ainsi que sur des modèles murins de leucémies agressives métastatiques.Au vu de ces résultats encourageants, le projet de thèse a été développé autour de deux axes de recherche. (I) Dans un premier temps, les nanoparticules de gemcitabine-squalène ont été fonctionnalisées par un peptide capable de reconnaître et de cibler spécifiquement les cellules cancéreuses pancréatiques. (II) Le deuxième axe de recherche a visé l’encapsulation d’un second principe actif au sein des nanoparticules de gemcitabine-squalène afin de développer le concept de nanoparticule « multi-thérapeutique ». / Pancreatic cancer is a lethal disease with the worst prognosis among all solid tumors. In the last decades, progresses in pancreatic cancer therapy had remained exceedingly slow and disappointing offering minimal benefits in median survival which remains of less than 6 months and the maximum of 5 years in the 6% of patients. One of the major requirements for a successful cancer therapy is its ability to selectively kill cancer cells with minimal damage to healthy tissues. In this context, a great deal of attention focused on advanced nanoscale systems (i.e., nanomedicines) with the aim to overcome the limits associated to the traditional drug delivery modalities. Nanomedicines can indeed enhance drug properties by (i) offering protection from degradation, (ii) enabling controlled release and distribution and increasing bioavailability while reducing undesired side effects.In the current work we aimed to propose novel nanoscale-based strategies to optimize pancreatic cancer treatment taking into account the specific physio-pathology of this tumor. The first approach relied on the design of a targeted nanomedicine able to specifically bind receptors mainly expressed onto pancreatic cancer cells in order to selectively increase drug accumulation in these cells saving healthy ones.In a second approach, by combining two therapeutic agents in the same nanoparticle we constructed a multi-therapeutic drug delivery system capable to increase the therapeutic index of the combined therapy. In particular, taking advantages from the “squalenoylation prodrug approach”, the research activity of this Ph.D. work lead to the to design of (i) a novel peptide-functionalized squalenoyl gemcitabine nanoparticle and (ii) a tyrosine kinase inhibitor-loaded squalenoyl gemcitabine nanoparticle. Obtained nanoparticles were investigated with respect to their physico-chemical properties and in vitro antitumor activity. The efficacy of peptide-functionalized nanoparticles in impairing tumor growth was assessed in vivo on an experimental model of pancreatic cancer.

Evaluation of dose optimization using the Pencil Beam (PB) and Monte Carlo (MC) algorithms may allow physicists to apply dosimetric offsets to account for inaccuracies of the PB algorithm for lung cancer treatment with Stereotactic Body Radiotherapy (SBRT). 20 cases of Non-Small Cell Lung Cancer (NSCLC) were selected. Treatment plans were created with Brainlab iPlanDose® 4.1.2. The D97 of the Planning Target Volume (PTV) was normalized to 50 Gy on the Average Intensity Projection (AIP) using the fast PB and compared with MC. This exact plan with the same beam Monitor Units (MUs) was recalculated over each respiratory phase. The results show that the PB algorithm has a 2.3-2.4% less overestimation at the maximum exhalation phase than the maximum inhalation phase when compared to MC. Significantly smaller dose difference between PB and MC is also shown in plans for peripheral lesions (7.7 ± 0.7%) versus central lesions (12.7±0.8%)(p< 0.01). / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

The purpose of this research is to accurately match the calculation environment, i.e. the treatment planning system (TPS) with the measurement environment (using a 2-D diode array) for lung Stereotactic Body Radiation Therapy (SBRT) patient-specific quality assurance (QA). Furthermore, a new phantom was studied in which the 2-D array and heterogeneities were incorporated into the patient-specific QA process for lung SBRT. Dual source dual energy computerized tomography (DSCT) and single energy computerized tomography (SECT) were used to model phantoms incorporating a 2-D diode array into the TPS. A water-equivalent and a heterogeneous phantom (simulating the thoracic region of a patient) were studied. Monte Carlo and pencil beam dose distributions were compared to the measured distributions. Composite and individual fields were analyzed for normally incident and planned gantry angle deliveries. The distributions were compared using γ-analysis for criteria 3% 3mm, 2% 2mm, and 1% 1mm. The Monte Carlo calculations for the DSCT modeled phantoms (incorporating the array) showed an increase in the passing percentage magnitude for 46.4 % of the fields at 3% 3mm, 85.7% at 2% 2mm, and 92.9% at 1% 1mm. The Monte Carlo calculations gave no agreement for the same γ-analysis criteria using the SECT. Pencil beam calculations resulted in lower passing percentages when the diode array was incorporated in the TPS. The DSCT modeled phantoms (incorporating the array) exhibited decrease in the passing percentage magnitude for 85.7% of the fields at 3% 3mm, 82.1% at 2% 2mm, and 71.4% at 1% 1mm. In SECT modeled phantoms (incorporating the array), a decrease in passing percentage magnitude were found for 92.9% of the fields at 3% 3mm, 89.3% at 2% 2mm, and 82.1% at 1% 1mm. In conclusion, this study demonstrates that including the diode array in the TPS results in increased passing percentages when using a DSCT system with a Monte Carlo algorithm for patient-specific lung SBRT QA. Furthermore, as recommended by task groups (e.g. TG 65, TG 101, TG 244) of the American Association of Physicists in Medicine (AAPM), pencil beam algorithms should be avoided in the presence of heterogeneous materials, including a diode array. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Bakgrund: Varje år insjuknar cirka 3000 kvinnor i gynekologisk cancer i Sverige, vilket innebär cancer i de kvinnliga könsorganen. Behandling mot gynekologisk cancer medför olika fysiska biverkningar. Sexuell hälsa är individuell och påverkas av faktorer som könsroller, njutning, könsidentitet och erotik. Sexuell lust, upphetsning och orgasm är delar av den sexuella hälsan. Sjuksköterskans roll innefattar personcentrerad vård med ett holistiskt perspektiv, där alla aspekter av patientens behov ingår, vilket innefattar den sexuella hälsan. Syfte: Syftet var att belysa upplevelsen av sexuell hälsa hos kvinnor som behandlats för gynekologisk cancer. Metod: Litteraturöversikten baserades på 11 kvalitativa artiklar som inhämtades från databaserna CINAHL Complete, Nursing and Allied health database och PubMed. Artiklarna som valdes att ingå i litteraturöversikten granskades i syfte att tematisera likheter och skillnader. Detta mynnade ut i litteraturöversiktens resultat. Resultat: Resultatet sammanställdes till fyra huvudteman som benämndes; Upplevelsen av ett förändrat sexuellt liv, En förändrad syn på sig själv, Förhållanden och En ny syn på sexuell hälsa. Under Upplevelsen av ett förändrat sexuellt liv identifierades två underteman; Den sexuella lusten och Den sexuella akten. Under En förändrad syn på sig själv identifierades Kvinnlighetens betydelse för kvinnan och Kroppsuppfattning som två underteman. Diskussion: En diskussion har förts kring metoden som användes i litteraturöversikten. Callista Roys adaptionsmodell, tidigare forskning och egna reflektioner har diskuterats i relation till litteraturöversiktens resultat för att stödja samt utöka förståelsen för resultatet. / Background: In Sweden approximately 3000 women fall ill in gynecological cancer each year. Gynecological cancer occurs in women’s genitals. Treatment for gynecological cancer can have different side effects. Sexual health is unique for each individual and is influenced by gender roles, identity, pleasure and erotism. Sexual desire, arousal and orgasm are segments of sexual health. The nurse’s role includes person centered care with a holistic perspective. All aspects of the patient’s needs should be included; therefore, sexual health should not be excluded.  Aim: The aim was to illustrate the experience of sexual health in women who have been treated for gynecological cancer. Method: The literature review was based on 11 qualitative studies collected from CINAHL Complete, Nursing and Allied health database and PubMed. The articles were reviewed in order to find similarities and differences. Results: The results were categorized into four main themes; The experience of a changed sexual life, An altered view on oneself, Relationship and A new view on sexual health. Within The experience of a changed sexual life two subthemes were identified; The sexual desire and The sexual act. Further on, two subthemes were identified in An altered view on oneself; The meaning of womanhood for women and Body image. Discussion: The chosen method for the literature review was discussed. To create understanding and support for the results Callista Roys adaptation model, previous research and our own thoughts were used to discuss the findings of the literature review.

Introdução: Além do catabolismo protéico acentuado, o paciente com câncer apresenta alterações no metabolismo lipídico. Objetivo: o objetivo do estudo foi avaliar as concentrações séricas de ácidos graxos (AG) antes, durante e após o tratamento oncológico de pacientes com neoplasia de estômago ou de esôfago. Casuística: O estudo prospectivo longitudinal foi conduzido com 14 pacientes com neoplasia de estômago ou de esôfago [62,1 anos (IC95% 55,6-68,6)], sob tratamento oncológico em unidade especializada. O estudo incluiu também 15 voluntários saudáveis [61,0 anos (IC95% 57,1-65,0)]. Métodos: Foram aplicados os questionários de ingestão alimentar (Recordatórios de 24h) e inquéritos relacionados com efeitos adversos e de toxicidade (CTCAE) que potencialmente interferem na ingestão alimentar e no estado nutricional. Foram feitas as medidas antropométricas, a impedância bioelétrica e coleta de sangue para os exames laboratoriais. Os AG foram determinados por cromatografia gasosa e expressos como porcentagem da área total. No Grupo Câncer, os procedimentos foram feitos antes do início, na metade e ao término do tratamento oncológico; o Grupo Controle foi submetido às mesmas avaliações em apenas uma ocasião. A análise estatística foi feita por meio do software Statistica 8.0, usando testes estatísticos não paramétricos. Resultados: As reações adversas relacionadas ao tratamento oncológico foram redução da ingestão de alimentos, saliva espessa com alteração no paladar e náuseas. Antes do início do tratamento, os pacientes com câncer já haviam perdido 17% do peso em relação ao usual; o peso corporal e o IMC reduziram entre a primeira e a terceira avaliação, mas não houve alteração na composição de massa corporal magra e gorda, na ingestão energética e da maioria dos macronutrientes no decorrer do estudo. Em relação ao Grupo Controle, o ácido nervônico foi maior enquanto que os ácidos gama-linolênico e alfalinolênico foram menores no Grupo Câncer. Na avaliação longitudinal, o ácido lignocérico reduziu durante o tratamento oncológico. Conclusão: os pacientes com câncer de esôfago e de estômago apresentam alteração discreta na concentração dos AG séricos em relação aos controles e o tratamento oncológico teve pouco impacto no perfil de AG circulantes / Introduction: In addition to enhanced protein catabolism, the cancer patient has alterations in lipid metabolism. Objective: The objective of the study was to evaluate serum concentrations of fatty acids (FA) before, during and after cancer treatment of patients with gastric or esophageal cancer. Subjects: The prospective longitudinal study was conducted with 14 patients with gastric or esophageal cancer [62.1 years (95% CI 55.6 to 68.6)], under cancer treatment in a specialized unit. The study also included 15 healthy volunteers [61.0 years (95% CI 57.1 to 65.0)]. Methods: The food intake questionnaires were applied (24-hour Dietary Recall) and inquiries related adverse effects and toxicity (CTCAE) that potentially interfere with food intake and nutritional status. Anthropometric measurements were made, the bioelectrical impedance and blood collection for laboratory tests. Gas chromatography determined the FA that was expressed as a percentage of the total area. In Cancer Group, the procedures were done before the start, the middle and at the end of cancer treatment; the control group underwent the same evaluations on only one occasion. Statistical analysis was performed using Statistica 8.0 software, using non-parametric statistical tests. Results: Adverse reactions related to cancer treatment have been reduced food intake, thick saliva with altered taste and nausea. Before the treatment, the patients with cancer had already lost 17% of weight with respect to the usual. Body weight and BMI reduced between the first and the third evaluation, but there was no change in the composition of lean and fat mass, energy intake and macronutrient most during the study. Compared to the control group, the nervonic acid was higher while the gamma-linolenic and alpha-linolenic acids were lower in the cancer group. In the longitudinal evaluation, the lignoceric acid reduced during cancer treatment. Conclusion: Patients with esophageal and stomach cancer have a mild change in the concentration of serum FA compared to controls and cancer treatment had little impact on the current FA profile

The purpose of this research is to validate various forms of mathematical modeling of glioblastoma multiforme (GBM) expressed as differential equations, numerically. The first work was involved in the numerical solution of the reaction-convection model, efficacy of which is expressed in terms of survival time. It was calculated using simple numerical scheme for the standard-of-care treatment in clinics which includes surgery followed by the radiation and chemotherapy. Survival time using all treatment options increased significantly to 57 weeks compared to that of surgery close to 14 weeks. It was also observed that survival time increased significantly to 90 weeks if tumor is totally resected. In reaction-diffusion model using simple numerical scheme, tumor cell density patterns due to variation in patient specific tumor parameters such as net proliferation rate and diffusion coefficient were computed. Significant differences were observed in the patterns while using dominant diffusion and proliferation rate separately. Numerical solution of the tumor growth model under the anti-angiogenic therapy revealed some impacts in optimum tumor growth control however it was not significant. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection