Rozprawy doktorskie na temat „Medical applications potential”

Fracture non-unions and bone defects represent a recalcitrant problem in the field of orthopaedic surgery. Although the current gold-standard treatment, autologous bone grafting, has a relatively high success rate, the technique is not without serious problems. The emerging field of regenerative medicine may have the potential to provide an alternative treatment. One promising strategy involves the delivery of both cells and multiple growth factors with different release profiles. A range of scaffolds was developed from Poly( -caprolactone) (PCL), Poly(lactideco- glycolide) (PLGA), and two blends of PCL (Mn 42,500) and PLGA. The scaffolds were manufactured utilising a novel modified fused deposition modelling system, using polymer/dichloromethane solutions. The scaffolds were found to have pore sizes suitable for bone regenerative medical applications (373±9.5 μm in the Ydirection and 460±13 μm in the X-direction). However, the scaffolds were found to be only 52±3 μm in height. This means that the two-layer scaffolds were relatively flat. This was undesirable, as direct control of the complete 3D geometry was the favoured strategy, though it may not be a necessary requirement. Five scaffold coatings were also developed from alginate, chitosan (crosslinked using sodium hydroxide or tripolyphosphate), Type-I collagen and Type-A gelatin. The scaffold coatings were screened in vitro for their cell-compatibility with human marrow stromal cells (hMSCs), human osteoblasts and MG63 cells. This was assessed using an assay for cell death, and assessing total cell counts. From these studies, Type-I collagen was found to be the optimum coating. For hMSCs, their death rates were found to be 19.1±6.3% for alginate, 5.3±3.6% and 2.9±1.4% for chitosan crosslinked with tripolyphosphate and sodium hydroxide respectively, compared to 0.11±0.07% for Type-I collagen, and 0.15±0.13% and 0.16±0.12% for 0.1% and 0.2% gelatin respectively. Type-I collagen was found to be the most cellcompatible coating, as it was consistently associated with higher cell counts than Type-A gelatin. Similarly, PCL scaffolds vacuum dried for 1 hr were found to be cell-compatible. No detectable clinically significant difference was found in either total cell counts, or the proportion of cell death in; hMSCs exposed to PCL scaffolds processed with dichloromethane, hMSCs either exposed to scaffolds known to be biocompatible, or hMSCs cultured in the absence of scaffolds. When cell morphology was compared, scaffolds vacuum dried for 1 hr or more were found to have a similar morphology to the cells cultured in the absence of scaffolds. It was therefore concluded that a vacuum drying time of 1 hr was sufficient for cell-compatibility. The scaffold materials were screened both for their encapsulation efficiencies and release characteristics using the model drug, methylene blue. The encapsulation efficiency was found to be both relatively high and consistent for both Mn 42,500 and 80,000 PCL as well as PCL:PLGA 66:33, at 71±6%, 71±5%, and 78±10% respectively, relative to the low efficiencies recorded for both PCL:PLGA 66:33 and PLGA: 57±5% and 38±10% respectively. The release rate of methylene blue from PCL (Mn 42,500), was found to be relatively slow, controlled, and consistent between batches (between 21±2% and 20±3% released in the first 24 hr). Despite the release rate being consistent for PCL (Mn 80,000), the release rate was thought to be too high, since between 29±3% and 39±5% of the test compound was released in the first 24 hr period. The release rate of methylene blue from the PCL/PLGA blends (between 17±2% – 30±7% and 18±4% – 31±6% in the first 24 hr) and PLGA (between 7.1±3.4% – 9.3±2.9% in the first 24 hr) were found to be inconsistent, and low in the case of PLGA, even taking the different loading efficiencies into account. Therefore, PCL (Mn 42,500) was selected as the favoured candidate scaffold material. The loading content and release profiles from methylene blue loaded collagen scaffold coatings were also evaluated. The drug loading capacity was found to be suitable for use as a drug delivery system (65±5 μg/g of methylene blue per unit scaffold mass). The release of methylene blue was observed to be rapid (between 54±10% – 70±17% in the first 24 hr), which was thought to be desirable for the coating delivery system. Recombinant human bone morphogenetic protein-7 (rhBMP-7) was used as a representative growth factor of interest for bone regenerative medical applications. It was loaded in collagen scaffold coatings (CoatBMP 1.25) and encapsulated within PCL (Mn 42,500) scaffolds (ScaffBMP 1.25). Control coatings and scaffolds were designated CoatPBS and ScaffPBS respectively. Both delivery systems were found to release detectable quantities of rhBMP-7 (releasing 2.8±0.2 μg/g and 87±7 ng/g respectively in the first 24 hr), even after 14 days. The release rate of the growth factor from the scaffold coating was higher than that from the encapsulating scaffolds. However, the cumulative release profiles were found to deviate from the desired ideal release profiles, and burst release was observed from both delivery systems. Although differences were observed for the two delivery systems, this difference may not be of clinical significance. Nevertheless, scaffolds with less than ideal delivery properties may still be of potential clinical use. The bioactivity of the rhBMP-7 released from the test scaffolds was therefore assessed by quantifying the area of normalised ALP staining of hMSCs. The release of rhBMP-7 from the collagen coating of the PCL (Mn 42,500) scaffolds (CoatBMP 1.25ScaffPBS) was capable of statistically significantly increasing hMSC normalised ALP expression, although the actual differences were often relatively small. Therefore, at least a proportion of the growth factor released is likely to have been bioactive. The release from scaffolds encapsulating rhBMP-7 (CoatPBSScaffBMP 1.25) did not have this effect on the hMSCs, indicating that either the concentration released was too low, or the growth factor released was no longer bioactive. However, when the cells were seeded directly onto the scaffolds, the activity of ALP, normalised by a DNA assay, was statistically significantly increased for the CoatPBSScaffBMP 1.25 scaffolds, in hMSCs from all three test patient donors (by 35±10% on the control). ALP activity was also significantly increased in hMSCs from two of the three patients seeded onto CoatBMP 1.25ScaffBMP 1.25 scaffolds (by 39±10% on the control). ALP activity was only statistically significantly increased for one of the hMSC patients when seeded onto CoatBMP 1.25ScaffPBS scaffolds (by 35±14% on the control). The functional osteoinductive capacity of Type-I collagen coated PCL (Mn 42,500) scaffolds loaded with rhBMP-7 was assessed using C2C12 cells seeded onto the scaffolds, and quantified using qRT-PCR. The genes of interest were; Type-I collagen (Col1), osteopontin (OP), ALP, osteocalcin (OC) and runt related transcription factor 2 (Runx2). The CoatBMP 1.25ScaffPBS scaffolds had an early osteoinductive effect on the C2C12 cells, as ALP, OC and Runx2 were elevated during the first 2 days only, compared to the control (e.g. by 44±12%, 128±42%, 60±25% and 46±25% respectively at the 24 hr mark). The CoatPBSScaffBMP 1.25 scaffolds also had an osteoinductive effect on the cells, which was more sustained than that observed for the CoatBMP 1.25ScaffPBS group. While OP, ALP and Runx2 were up-regulated in the first 24 hr compared to the control (by 38±10%, 208±82% and 72±31% respectively), statistically significant up-regulation of the late marker OC was delayed until the 48 hr mark (by 73±49%). The effect was found to be sustained until day 7, when OC and Runx2 were both statistically significantly up-regulated compared to the control (by 151±91% and 93±27% respectively). The CoatBMP 1.25ScaffBMP 1.25 scaffolds were found to combine the early effect of the CoatBMP 1.25ScaffPBS scaffolds, with the more sustained effect of the CoatPBSScaffBMP 1.25 scaffolds. ALP, OC and Runx2 were all up-regulated at the 24 hr mark (by 312±56%, 329±39% and 96±25% respectively). This osteoinductive effect was sustained until day 7 when Col1, ALP and Runx2 were still up-regulated compared to the control (by 174±78%, 72±24% and 178±78% respectively). These data suggest that the scaffolds containing rhBMP-7 have a weak osteoinductive effect on the cells seeded onto them. The different delivery systems were found to affect the cells differently. The clinical significance of this was not assessed in these studies. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was used as a model drug to assess the feasibility of releasing lipid-soluble active factors from the scaffolds. This was assessed by quantifying the area of normalised ALP staining of hMSCs. The release of 1,25(OH)2D3 from the loaded collagen scaffold coatings and the encapsulating scaffolds significantly increased ALP expression compared to the control scaffold groups (by 115±28% and 69±25% respectively). Furthermore, ALP expression was significantly increased when the two delivery systems were used together, when compared to either delivery system on its own. These data suggest that the delivery of lipid-soluble active factors is feasible from collagen coated PCL scaffolds, and that the coating and encapsulating delivery systems are mutually compatible.

This thesis examines the toxicity of metallic and organometallic nanoparticles which have applications in the medical field. Commercially available Silver particles} used to create bactericidal surfaces} are tested against two human in vitro cell models to investigate the sensitivity of the models and the toxicity of the particles. No toxicity to a human blood brain barrier in vitro cell model arises from concentrations of particles likely to be encountered. Significant toxicity however is demonstrated in a human placental in vitro cell model} raising concerns for maternal exposure to Silver particles and selecting the placental cell model for further study. A novel polymer nanoparticle drug delivery system capable of encapsulating a wide variety of lipophilic drugs is described. Extensive characterisation demonstrates successful encapsulation of fluorescent} water insoluble} Tris-(8-hydroxyquinolinato) Aluminium (III) (AlQ3) a molecule with antibiotic potential. The AlQ3 nanoparticles are tested against a human placental in vitro cell model} at physiologically relevant doses} finding no significant toxicity to the cell membrane} metabolism} nucleus or viability. Confocal experiments with concomitant organelle staining confirm cellular internalisation and examine the AlQ3 intracellular localisation. The antibacterial properties of AlQ3 nanoparticles are then demonstrated by the treatment of human keratinocytes infected with Methicillin Resistant Staphylococcus Aureus (MRSA). The polymer nanoparticle delivery system is then further examined by synthesising nanoparticles of the chemotherapeutic analogue} Tris-(8-hydroxyquinolinato) Gallium (lit) (Ga~). Ga~ having undergone phase I human clinical trials} provides an ideal example of an insoluble drug molecule whilst allowing for direct comparison to the AlQ3 nanoparticles. Characterisation confirms the formation of nanoparticles suitable for passive disease targeting with the potential for extended blood circulation. Toxicity testing against a human placental in vitro cell model shows significant toxicity to the cell membrane} metabolism} nucleus and viability. Confocal experiments confirm the GaQ3 nanoparticles follow the same intracellular pathway as the AlQ3 nanoparticles elucidating further the mechanism of toxicity.

This thesis explores the potential of a compact low energy (<10MeV) proton accelerator for medical applications such as the production of neutrons for cancer neutron therapy and the production of SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography) radioisotopes. During the course of this study the simulation code GEANT4 was used to study yields of these neutrons and isotopes from the typically low threshold high cross-­‐section (p,n) reactions. Due to the limits of the current models within GEANT4 some development of a new data-­‐driven model for low energy proton interactions was undertaken and has been tested here. This model was found to be suitably reliable for continued study into the low energy production of positron emitting, PET, isotopes of copper and gallium as replacements for the main SPECT isotope technetium-­‐99m. While 99mTc is currently the most popular radioisotope being used in over 90% of the worlds nuclear medicine diagnostic procedures supply is under threat by the impending shut down of the current reactor based sources. Simulations of both thin and thick targets were carried out to study the potential of low energy production of these isotopes. The final activity of the radioisotopes after irradiation of these targets produced by the simulations has been shown here to be sufficient for multiple doses. The useable activity is dependent on the efficiency of the extraction process and the time between irradiation and administration.

Methacrylate-terminated poly(dimethylsiloxane) (PDMS) macromonomers have been obtained through a flexible synthetic method which has been tailored into an efficient 1 pot, 2 stage reaction. Linear and star-shaped analogues have been synthesised and 3 molecular weights of each architecture were produced for use in subsequent curing investigations. The PDMS macromonomers were photo cured into films by exposure to UV irra- diation in the presence of a free-radical initiator. A curing time study was carried out and the rheological properties, swelling ratio, Young's modulus, surface tack and macroscopic extension of the cured films were investigated. At short irradiation times, samples were sensitive to irradiation time and an increase in exposure produced a signif- icantly more solid-like film. However, after longer irradiation times, the film properties reached a plateau and films did not become significantly more solid-like despite further irradiation. Ideal network models are proposed which support the observation that the cured film properties were strongly affected by the macromonomer molecular weight. Low molecular weight macromonomers produced densely cross-linked films which were hard, elastomeric solids. Films formed from medium molecular weight macromonomers were softer materials with tacky surfaces, and films formed from the highest molecular weight were softer still and some samples exhibited viscous flow. Introducing branching into the macromonomers decreased the irradiation time re- quired to form a cohesive film. Increasing the degree of macromonomer branching increased the solid-like nature of the film in comparison to those formed from linear species, although this did not outweigh the effect of macromonomer molecular weight. The materials investigated in this study may have potential for use as non-degradable, curable materials in medical applications and have scope for in vivo curing.

Since early 1960s, ultrasound has become one of the most widely used medical imaging device as a diagnostic tool or an image guider for surgical intervention because of its high portability, non-ionization, non-invasiveness and low cost. Although continuous improvements in commercial equipments have been underway for many years, almost all systems are developed with pulse-echo geometry. In this research, a newly invented ultrasound sensor array was incorporated into the developments of a projection imaging system. Three C-scan prototypes, which included prototypes #1, #2 and an ultrasound mammography system, were constructed. Systematic and Evaluative studies included ultrasound CT, 3-D ultrasound, and multi-modality investigations were also performed. Furthermore, a new analytical method to model ultrasound forward scattering distribution (FSD) was developed by employing a specific annular apparatus. After applying this method, the scattering-corrected C-scan images revealed more detail structures as compared to unprocessed images. This new analytical modelling approach is believed to be effective for most imaging systems operating in projection geometry. In summary, while awaiting additional clinical validation, the C-scan ultrasound prototypes with the state-of-the-art PE-CMOS sensor arrays can provide veritable value and holds real and imminent promise in medical diagnostic imaging. Potential future uses of C-scan ultrasound include but not limit to computerized tomography, biopsy guidance, therapeutic device placing, foreign object detection, pediatric imaging, breast imaging, prostate imaging, human extremities imaging and live animal imaging. With continuous research and development, we believe that C-scan ultrasound has the potential to make a significant impact in the field of medical ultrasound imaging.
Ph. D.

Includes abstract.
Includes bibliographical references.
In South Africa, resource-poor farmers mainly depend on livestock farming for their livelihoods, with cattle production being the most important livestock sector. As a consequence of natural selection in stressful conditions, Nguni cattle have been reported to be metabolically superior to other cattle breeds under unfavourable conditions. Using proteomics, with mass spectrometry at the core of the analysis, the objective of this study was to establish a reliable set of methods for the protein profiling of Nguni cattle livers. To achieve this several alternative technologies were employed and their outcomes compared namely, two-dimensional electrophoresis, fractionation by solution phase iso-electric focusing-reversed phase chromatography (IEF-RP), offline strong cation exchange- low pH reversed phase chromatography (SCX-RP) and offline high pH reverse phase-low pH reverse phase chromatography (RP-RP). All solution based methods were coupled to a tandem mass spectrometer. Protein identification was performed using the ParagonTMAlgorithm of Protein Pilot v4.0 as well as PEAKS v6. The IEF-RP and RP-RP methods achieved similar results in terms of number of proteins identified. In addition, proteins that play a role in the urea cycle (which is believed to contribute to the Nguni cattle’s enhanced metabolic ability) were all identified with both techniques. The RP-RP method was selected as the most appropriate method for future research linked to this work and will be used in the next phase of this project, on the basis that it is easier to automate compared to the IEF-RP method. It will be used beyond the scope of this work to compare levels of expression and modification of the liver proteins and their isoforms in Nguni and Hereford cattle grown under adverse environmental conditions, in order to identify those that may contribute to enhanced liver metabolism in Nguni cattle. This will be complemented by the identification and characterisation of potential polymorphisms with in such proteins that can be used to select for this trait during breeding.

The ability of the electronic portal imaging device (EPID) to acquire a large two-dimensional array of digitized x-ray data in real time is extremely attractive for dosimetric measurements. To evaluate the potential use of an EPID for portal dose measurement in Wellington Blood and Cancer Centre, some dosimetric characteristics of the Varian's PortalVisionTM aS500 were investigated. PortalVisionTM incorporates an amorphous silicon detector (aSi). Some potential applications of EPID in linac QA were also explored. The EPID's performance for linearity with MU and dose rate was verified and it was found to be proportional over the entire measured range. Short term repeatability was found to be excellent. An investigation of calibration method to improve dosimetric accuracy demonstrated two methods of avoiding detector saturation. Firstly, acquiring flood field with the use of additional buildup and secondly, increasing the source to detector distance for calibration. A study of EPIDs behaviour under conditions of varying dose rate which commonly arise in EDW treatment techniques was carried out. The EPID exhibited a field size dependence in addition to a 8% discrepancy on the `hot edge' of EDW profiles. Further investigation into the field size dependence and the discrepancy at hot edge is required. EPIDs ability to acquire asymmetric field profile was also investigated. The profiles acquired using EPID deviated in shape and magnitude by upto 16% from the ion chamber profiles. Some potential applications of EPID to perform QA of linac beam properties, its ability to perform optical and mechanical linac QA have been explored. The EPID's capability to give constant output, flatness, symmetry, wedge angle and wedge factors with high level of accuracy and reproducibility was demonstrated. EPID was also found to be objective, efficient and feasible for performing optical linac QA. The use of EPID for linac QA could be simplified by improving the available software analysis tools thus making it more efficient.

Proton Transfer Reaction Mass Spectrometry (PTR-MS) was applied to determine which volatile organic compounds in breath are associated with cirrhosis and hence diagnostically useful. A two-stage biomarker procedure was used. In the first-stage, alveolar breath samples of 31 cirrhotic patients and 30 controls were analysed and compared. In the second-stage, 12 of the patients had their breath analysed after liver transplant. The first-stage study showed that seven volatiles were elevated in patients’ breath compared to controls. Of these, limonene, methanol, 2-pentanone showed a statistically significant decrease post-transplant and hence can unequivocally be used as biomarkers for chronic liver disease. Limonene which is not produced in the body showed washout characteristics and the best diagnostic capability. These findings suggest that limonene, methanol and 2-pentanone are potential biomarkers for early-stage liver disease. Limonene was detected in higher levels in patients with symptoms of hepatic encephalopathy (HE) in comparison to those with no symptoms. Limonene discriminates patients suffering from HE, but not methanol or 2-pentanone. The elimination characteristics of post-operative isoflurane levels in breath of 5 patients were investigated. High concentrations of isoflurane remained in their breath for several weeks. This study raises the question about the effect of isoflurane in the neurocognitive function of patients after major surgery.

Cationic steroid antimicrobials (CSAs or ceragenins) are a novel class of synthetic, cholic acid-based mimics of endogenous antimicrobial peptides. These small molecule compounds display broad bactericidal activity against gram-negative and gram-positive bacteria, potent ability against fungal pathogens, and cidal effects against drug resistant and multidrug resistant microbes. Implantable medical devices provide an abiotic surface upon which bacteria and fungi can accumulate--thereby leading to localized or systemic infection. We proposed that CSA antibiotics can be incorporated into medical device surface coatings which can be optimized for the active release or elution of the CSA compounds over time to prevent device-associated infections. This report will discuss the progress of developing and testing coating systems for 3 such devices: cardiac implantable electronic devices (CIED), silicone nasal splints, and breast tissue expanders. In the case of CIEDs, an envelope material containing CSA was created using bioresorbable polymers. We found that this envelope elutes CSA antibiotics and kills all surrounding bacteria or fungi in both planktonic and biofilm forms within 1 hour of exposure. We also developed a nasal splint coating which is directly adhered to the surface of the silicone splint. This coating system demonstrated more than 8 days of protective ability (full microbicidal activity to the detection limit) against Candida albicans, and reduced microbial growth of P. aeruginosa, Candida auris, and MRSA for approximately 6 days. Lastly, in the case of tissue expanders, we developed a layered coating which displays fully-reductive antimicrobial activity against MRSA for 8 days with reintroduction of bacteria every 24 hours. Additionally, this work will discuss our investigations into the secondary properties of ceragenin compounds. On the basis of studies which have demonstrated the pro-osteogenic properties of CSA, we probed the mechanism of this effect. We studied the potential effects of ceragenins on the proliferation, differentiation, and migration of bone-derived mesenchymal stem cells (MSCs). We have determined the absence of any positive proliferative effects of ceragenins on these cells; however, we have demonstrated the significant migration-promoting chemoattractant properties of CSA. In the case of CSA-13, we have observed up to a 400% increase in migration compared to the control. Also, we demonstrated that the P2X7 receptor is strongly implicated in the cellular mechanism of this effect. Our studies of the differentiation-promoting properties of CSA on MSCs have been largely inconclusive, but further investigations are proposed in this report. Lastly, this work includes a report on our investigations into the potential synergistic interactions between CSA-131/CSA-44 with amphotericin B or caspofungin, two commonly used antifungal agents.

Doctor of Philosophy
Department of Biochemistry and Molecular Biophysics
John M. Tomich
Branched Amphiphilic Peptide Capsules (BAPCs) are peptide nano-spheres comprised of equimolar proportions of two branched peptide sequences bis(FLIVI)-K-KKKK and bis(FLIVIGSII)-K-KKKK that self-assemble in water to form bilayer delimited poly-cationic capsules capable of trapping solutes. We examined the lipid-like properties of this system including assembly, fusion, solute encapsulation, and resizing by membrane extrusion as well as their capability to be maintained at a specific size by storage at 4˚C. These studies along with earlier work from the lab (Gudlur et al. (2012) PLOS ONE 7(9): e45374) demonstrated that the capsules, while sharing many properties with lipid vesicles, were much more robust. We next investigated the stability, size limitations of encapsulation, cellular localization, retention and, bio-distribution of the BAPCs. We demonstrated that the BAPCs are readily taken up by epithelial cells in culture, escape or evade the endocytotic pathway, and accumulate in the peri-nuclear region where they persist without any apparent degradation. The stability and persistence of the capsules suggested they might be useful in delivering radionuclides. The BAPCs encapsulated alpha particle emitting radionuclides without any apparent leakage, were taken up by cells and were retained for extended periods of time. Their potential in this clinical application is being currently pursued. Lastly we studied the temperature dependence of capsule formation by examining the biophysical characteristics of temperature induced conformational changes in BAPCs and examined the structural parameters within the sequences that contribute to their remarkable stability. A region in the nine-residue sequence was identified as the critical element in this process. The ability to prepare stable uniform nano-scale capsules of desired sizes makes BAPCs potentially attractive as delivery vehicles for various solutes/drugs.

Pancreatic cancer is especially a deadly form of cancer with a survival rate less than 2%. Pancreatic cancers respond poorly to existing chemotherapeutic agents and radiation, and progress for the treatment of pancreatic cancer remains elusive. To address this unmet medical need, a better understanding of critical pathways and molecular mechanisms involved in pancreatic tumor development, progression, and resistance to traditional therapy is therefore critical. Reduction¿oxidation (redox) signaling systems are emerging as important targets in pancreatic cancer. AP endonuclease1/Redox effector factor 1 (APE1/Ref-1) is upregulated in human pancreatic cancer cells and modulation of its redox activity blocks the proliferation and migration of pancreatic cancer cells. Modulation of APE1/Ref-1 using the siRNA increases the HIF target, HMOX1 and NQO1,two genes that play key roles in tumor adaptation to a variety of stresses, including anticancer drugs.Ape inhibition activates the NRF2 regulator of redox stress response, which is known to regulate HMOX1 and NQO1. As all these genes are of high relevance for the acquired resistance to various antineoplastic drugs.

Antimicrobial resistance (AMR) is concerning because it limits antimicrobial drug treatment options. AMR occurs by the overuse and misuse of antimicrobial drugs. In environmental settings, AMR can disseminate from places of high use, which leads to increased exposure to humans and animals. A previous study from our laboratory group showed extended-spectrum cephalosporinase-producing Escherichia coli/Klebsiella pneumoniae were isolated from fecal samples obtained in rural Cambodian community settings. Based on these isolates, this study has two aims. The first aim was characterization of selected Cambodian isolates with random amplification polymorphic DNA (RAPD) and antibiotic susceptibility test. From RAPD, the selected six isolates are diverse, except for C61 and C66 bacteria isolates with potential clonality. Additionally, the selected isolates are multidrug resistant (MDR) with reduced susceptibility to beta-lactams and fluoroquinolones. The second aim was to assess two developed methodologies, multiplex automated genome engineering (MAGE) and One-Step Curing Plasmid, by validation in bacteria laboratory strain and development for six Cambodian isolates. To modify AMR genetic elements, MAGE uses pMA7-SacB for homologous recombination with oligos for chromosomal gene disruption. Meanwhile, One-Step Curing Plasmid uses pFREE with the CRISPR/Cas9 system for plasmid and self-curing. Validation showed that MAGE can modify 8% of E. coli MG1655 with lacZ control screening oligos and almost 90% are cured from pFREE. Selected Cambodian isolates have antibiotic-resistance plasmids of IncR or IncFII replicon. For usage in Cambodian isolates, pFREE was modified to be pCAM-FREE by cloning IncR and IncFII plasmid as gRNA1 and gRNA5, respectively. Sequencing results showed pCAM-FREE have gRNA5. In conclusion, our study managed to characterize selected Cambodian isolates as MDR and diverse. In a laboratory strain, MAGE and One-Step Curing Plasmid are functional methods. Furthermore, pCAM-FREE was constructed to target IncFII and in the future, MAGE and pCAM-FREE could be tested in Cambodian isolates.

This thesis reports on two graphene-based structures that have been proposed and fabricated as possible prototypes for high-spatial-resolution Hall sensors with potential application in research on high-density magnetic recording technology such as bit patterned magnetic recording (BPMR) and other areas where the measurement of highly inhomogeneous fields is required. There is a direct graphene-metal contact in the first structure, which is named as TYPE I in this thesis, so that the anomalous Hall effect (AHE) in the ferromagnetic islands deposited on the graphene could be detected. Meanwhile, the graphene and the metal are isolated by an h-BN layer in the second structure which is named as TYPE II, so that only the stray field from the islands can be detected using the ordinary Hall effect (OHE).The transport measurements performed on TYPE I devices revealed there is no AHE or stray field signal detectable, and their Hall resistance relations are non-linear and do not pass through the origin point. A finite element simulation comparing the resistance of the empty graphene cross and the island-occupied cross indicates that the current in the graphene may not redistribute through the metallic islands due to interface current blocking, resulting in the non-appearance of the expected AHE signal. Moreover, an analysis on the data of the longitudinal magnetoresistance (MR) reveals that a two-fluid model and effective medium theory (EMT) model might be the major graphene MR mechanisms in the regime away from and near to the charge neutrality point (CNP) respectively. As a combined result of the above findings, a joint MR-Hall effect model under the condition of the presence of a pre-existing transverse offset current, is proposed to explain the unusual behaviour of the Hall measurement data of the TYPE I devices. The model gives qualitatively correct fitting for all longitudinal and transverse transport data of TYPE I devices. In addition, the nature of the graphene/metal contact is considered as the reason responsible for the non-appearance of the expected AHE and stray field signal, although further experimental work is needed, and suggested in the thesis, to clarify this issue. On the other hand, the TYPE II devices have shown their potential to be developed as a Hall sensor being able to detect a sub-micron magnetic island in the future, but there is still a large space for the performance of the devices to be improved. At the end of the thesis, future experimental work, which could lead to the eventual development of a high-sensitivity high-spatial-resolution Hall sensor on the basis of TYPE I and TYPE II structures, are suggested and described.

Le processus metastatique reste une des causes majeures de l'echec des traitements anticancereux. Les differentes etapes impliquees dans la formation de metastases font intervenir la migration cellulaire. De nombreuses etudes s'interessent a la correlation pouvant exister entre l'invasion tumorale et la migration cellulaire. Nous avons utilise une methode de quantification basee sur une approche de flot optique qui permet d'estimer le mouvement a partir de sequences d'images en contraste de phase sans segmentation prealable. Nous avons valide la pertinence de cette approche sur des images de synthese puis dans differents contextes biologiques : au niveau individuel ou nous avons observe l'existence d'une periodicite dans la deformation spontanee de cellules et au niveau de la population ou nous avons pu quantifier la dynamique de l'ensemble de la couche cellulaire lors du processus de recolonisation apres blessure in vitro, en presence ou non d'un marqueur de l'adn le hchst 3342. Cette approche a ete appliquee a un systeme experimental constitue de cellules issues d'un carcinome colorectal et de cellules issues de metastase peritoneale, provenant d'un meme patient. Nous avons etudie la migration de ces cellules en relation avec l'expression de la proteine cd9/mrp1. Il s'agit d'une proteine transmembranaire qui a ete decrite comme etant associee au potentiel metastatique de certaines tumeurs. Dans notre cas, cette proteine est plus fortement exprimee dans les cellules issues de la tumeur primaire et a une contribution positive dans la migration cellulaire.

Pancreatic cancer is one of the deadliest diseases in the world. The best hope for reducing mortality by cancer lies in its early diagnosis and treatment, namely in the detection of lesions that can evolve into cancer stages. On the other hand, traditionally applied therapeutics to fight pancreatic cancer have several drawbacks, namely related with limited efficacy, toxicity to patients, and acquisition of drug resistance. As to counteract this trend, a greater effort has been devoted to the discovery of new anti-pancreatic cancer drugs from biological natural resources. In this context, organisms from marine environments (e.g., microbial symbionts, algae, invertebrates) possess a wide genetic potential for the biosynthesis of natural compounds. However, marine ecosystems are yet severely overlooked for that purpose. Marine bacterial symbionts, in particular, can produce several secondary metabolites with high biotechnological interest, especially for biomedical applications like those directed to anticancer treatment. Therefore, the major goals of this work were to: (1) develop a brief review on pancreatic cancer and current therapeutics, as well as on the pool of natural products and associated biosynthetic pathways in marine microbes; (2) unravel the potential of bacterial symbionts isolated from marine cnidarians to synthesize bioactive compounds against pancreatic cancer. Some bacteria have the potential to synthesize new products through the activity of the modular enzymes polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), or a combination of both. These multifunctional enzymes are involved in the production of polyketides (PKs), nonribosomal peptides (NRPs) or PKs-NRPs hybrid compounds that can have various chemical structures and bioactivities (e.g., antimicrobial, antioxidant, antiinflammatory, anticancer). The molecular screening of NRPS gene fragments in marine bacterial symbionts of cnidarians showed their presence in 22.3% of the isolates, which belong to Proteobacteria and Actinobacteria phyla. Most aminoacid sequences obtained for a selected group of these potential NRPsbacterial producers presented a relatively high homology with deposited aminoacid sequences of NRPS clusters. Furthermore, crude extracts prepared from the cell and cell-free fractions of cultures from the selected bacteria could generally significantly inhibit the activity of a human pancreatic ductal adenocarcinoma cell line, according to the MTT assay. Thereby, the anticancer potential of cnidarian bacterial symbionts was promising, hence reinforcing the need for deepening its study in the future.
O cancro do pâncreas é uma das doenças mais letais do mundo. A melhor esperança para reduzir a mortalidade provocada por este cancro reside no diagnóstico e tratamento precoces, mormente na deteção de lesões que podem conduzir ao seu desenvolvimento. Por outro lado, as terapias tradicionalmente aplicadas para combater o cancro do pâncreas têm vários inconvenientes, relacionados nomeadamente com uma eficácia limitada, toxicidade para os pacientes e aquisição de resistência aos medicamentos. Para contrariar esta tendência, tem sido dedicado um maior esforço na descoberta de novos fármacos anti-cancro pancreático, a partir de recursos naturais biológicos. Neste âmbito, organismos provenientes de ambientes marinhos (e.g., microorganismos simbiontes, algas, invertebrados) possuem um amplo potencial genético para a biossíntese de compostos naturais. No entanto, os ecossistemas marinhos estão ainda consideravelmente subexplorados para este propósito. Os simbiontes bacterianos marinhos, em particular, podem produzir vários metabolitos secundários com alto interesse biotecnológico, especialmente para aplicações biomédicas como as direcionadas para o tratamento anticancerígeno. Portanto, os principais objetivos deste trabalho foram: (1) desenvolver uma breve revisão sobre o cancro do pâncreas e suas atuais terapias, bem como sobre os produtos naturais bioativos e vias biossintéticas existentes em microorganismos marinhos; (2) desvendar o potencial de simbiontes bacterianos isolados de cnidários marinhos para sintetizar compostos bioativos contra o cancro do pâncreas. Algumas bactérias têm o potencial de sintetizar novos produtos através da atividade das enzimas policetídeo sintases (PKS), sintetases peptídicas não ribossomais (NRPS), ou uma combinação de ambas. Estas enzimas multifuncionais produtoras de policetídeos (PKs), péptidos não ribossomais (NRPs) ou híbridos PKs-NRPs podem ter várias estruturas químicas e bioatividades (e.g., antimicrobiana, antioxidante, anti-inflamatória, anticancerígena). O rastreio molecular de fragmentos do gene NRPS em simbiontes bacterianos marinhos de cnidários evidenciou a sua presença em 22,3% dos isolados, os quais pertencem aos filos Proteobacteria e Actinobacteria. A maioria das sequências de aminoácidos obtidas para o grupo de bactérias selecionadas por serem potenciais produtores de NRPs, apresentou uma homologia relativamente elevada em relação a sequências de aminoácidos depositadas para NRPS. Além disso, os extratos preparados a partir das frações celulares e sobrenadante de culturas das bactérias selecionadas inibiram significativamente a atividade de uma linha celular humana de adenocarcinoma do ducto pancreático, mediante o ensaio MTT. Assim, o potencial anticancerígeno dos simbiontes bacterianos de cnidários demonstrou ser promissor, pelo que o seu estudo deve ser mais aprofundado no futuro.
Mestrado em Biologia Molecular e Celular

This work describes a methodology of employment of solid-state NMR spectroscopy as tool for the atomic detailed characterization of bioconjugates with potential pharmaceutical interest (such as PEGylated, nanoparticle-conjugated and glycosylated proteins) and bioinspired materials used as medical devices or enzyme-entrapped based targets for drug screening and discovery.

病原體感染是包括植物的高等生物的主要健康危害之一。為抵禦入侵者，大多數植物會製造防禦蛋白，包括凝集素、蛋白酶抑製劑、抗真菌蛋白、核糖核酸酶和核糖體失活蛋白，並分佈在不同的器官，如葉、根、種子和塊莖。一些植物防禦蛋白被發現能表現出多種生物活性，如抗腫瘤活性、抗細菌活性和抗病毒活性，能抵抗多種植物病原菌和人類病原體。因此，一些植物防禦蛋白可能有潛力用於治療人類疾病，或保護農作物免受感染。
我們在研究中從不同的植物來源成功純化出各種防禦蛋白，包括：小芋頭塊莖中的血凝素、日本長芋中的凝集素、東北紅豆中的血凝素和抗真菌多肽、棕色芸豆中的凝集素、抗真菌多肽和胰蛋白酶抑製劑，玉豆一號中的凝集素以及小斑豆中的胰蛋白酶抑製劑。小芋頭血凝素被發現能誘導脾細胞的有絲分裂反應。日本長芋凝集素和東北紅豆血凝素被發現能對一些腫瘤細胞株（如乳腺癌MCF7細胞及鼻咽癌CNE2細胞）發揮抗增殖的作用。棕色芸豆凝集素能誘導脾臟細胞的有絲分裂反應以及抑制腫瘤細胞株（如乳腺癌MCF7細胞、肝癌HepG2及鼻咽癌CNE1和 CNE2細胞）的生長，而棕色芸豆抗真菌蛋白能抑制數種病原真菌物種的生長。研究這些防禦蛋白的生物活性有助找出其潛在應用價值，如藥用前景。
Infection from pathogens is one of the major health hazards in higher organisms including plants. To defend against harmful invaders, most plants produce a variety of defense proteins including lectins, protease inhibitors, antifungal proteins, ribonucleases and ribosome-inactivating proteins. They may be present in different organs of the plants, such as leaves, roots, seeds and tubers. Some of the plant defense proteins were found to exhibit a variety of biological activities such as anti-tumor activity, anti-bacterial activity and anti-viral activity that act against various plant pathogens and also some human pathogens. Therefore, some plant defense proteins may have potential for therapeutic applications in human diseases, or protecting the crops from infections.
This study involved purification of defense proteins from different plant sources. The proteins that were successfully isolated included a hemagglutinin from small taro tubers, a lectin from Japanese yam tubers, a lectin and an antifungal peptide from northeast red beans, a lectin, an antifungal peptide and a trypsin inhibitor from brown kidney beans, a lectin from French bean cultivar no. 1 and a trypsin inhibitor from mini pinto beans. The small taro hemagglutinin was found to induce mitogenic response in splenocytes. The Japanese yam lectin and northeast red bean hemagglutinin were found to exert anti-proliferative activity toward some tumor cell lines including MCF7 and CNE2 cells. The brown kidney bean lectin induced a mitogenic response from murine splenocytes as well as inhibited the growth of tumor cell lines including MCF7, HepG2, CNE1 and CNE2 cells, while the brown kidney bean antifungal protein inhibited the growth of several pathogenic fungal species including M. arachidicola, S. turcica and B. maydis. Studying the biological activities of these defense proteins helps to find out their potential applications like therapeutic uses.
Detailed summary in vernacular field only.
Detailed summary in vernacular field only.
Chan, Yau Sang.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves i-xvii).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstract --- p.i-ii
論文摘要 --- p.iii
Acknowledgements --- p.iv
List of Publications --- p.v
Table of Contents --- p.vi-vii
List of Figures --- p.viii-ix
List of Tables --- p.x
List of Abbreviations --- p.xi
Chapter Chapter 1 --- Introduction on plant defense proteins
Chapter 1.1 --- General introduction to plant defense proteins --- p.1-2
Chapter 1.2 --- An overview on lectins --- p.3-18
Chapter 1.2.1 --- History of lectins --- p.3-6
Chapter 1.2.2 --- Classification of lectins --- p.7-11
Chapter 1.2.3 --- Biological activities of lectins --- p.12-16
Chapter 1.2.4 --- Applications of plant lectins --- p.16-18
Chapter 1.3 --- An overview on defensins --- p.18-25
Chapter 1.3.1 --- Types of defensins --- p.18-21
Chapter 1.3.2 --- Mechanism of anti-microbial activity of defensins --- p.22-23
Chapter 1.3.3 --- Application of defensins --- p.23-25
Chapter 1.4 --- An overview on trypsin inhibitors --- p.25-38
Chapter 1.4.1 --- Serpins --- p.26-28
Chapter 1.4.2 --- Kunitz-type protease inhibitors --- p.29-31
Chapter 1.4.3 --- Bowman-Birk protease inhibitors --- p.32-34
Chapter 1.4.4 --- Physiological functions of protease inhibitors --- p.35-38
Chapter 1.5 --- Aim of study --- p.38-41
Chapter Chapter 2 --- Isolation and characterization of a hemagglutinin from small taros and a lectin from yam tubers
Chapter 2.1 --- Introduction --- p.42-45
Chapter 2.2 --- Materials and Methods --- p.46-55
Chapter 2.3 --- Results --- p.56-78
Chapter 2.4 --- Discussion --- p.79-84
Chapter Chapter 3 --- Isolation and characterization of two defense proteins from seeds of Phaseolus vulgaris cv. “northeast red bean“
Chapter 3.1 --- Introduction --- p.85-86
Chapter 3.2 --- Materials and Methods --- p.87-93
Chapter 3.3 --- Results --- p.93-119
Chapter 3.4 --- Discussion --- p.120-129
Chapter Chapter 4 --- Isolation and characterization of three defense proteins from seeds of Phaseolus vulgaris cv. “brown kidney bean“
Chapter 4.1 --- Introduction --- p.130-131
Chapter 4.2 --- Materials and Methods --- p.131-136
Chapter 4.3 --- Results --- p.136-175
Chapter 4.4 --- Discussion --- p.176-189
Chapter Chapter 5 --- Isolation and characterization of a lectin from French bean cultivar no. 1 beans and a trypsin inhibitor from mini pinto beans
Chapter 5.1 --- Introduction --- p.190-191
Chapter 5.2 --- Materials and Methods --- p.191-194
Chapter 5.3 --- Results --- p.195-212
Chapter 5.4 --- Discussion --- p.213-221
Chapter Chapter 6 --- General discussion
Chapter 6.1 --- Summary on purification protocols of the defense proteins in the study --- p.222-228
Chapter 6.2 --- Chemical properties of the defense proteins in the study --- p.228-232
Chapter 6.3 --- Biological activities of the defense proteins in the study --- p.232-238
Chapter 6.4 --- Potential application of these defense proteins and future perspectives --- p.238-242
References --- p.i-xvi