Toxoplasma gondii is an obligate intracellular parasite that can cause lifethreatening disease in immunocompromised individuals. Host cell invasion is therefore central to the pathology of the disease and parasite survival. Unlike many intracellular pathogens, T. gondii does not enter cells by manipulating the host’s phagocytic machinery; instead, the parasite enters the cell by a process of active penetration. Gliding motility and active penetration are driven by a complex of proteins termed the glideosome. The glideosome consists of four major proteins: TgMyoA, an unconventional myosin XIV, myosin light chain (TgMLC1) and glideosome-associated proteins 45 and 50 (TgGAP45, TgGAP50). TgMyoA has been shown to be essential for parasite motility, but the role of TgMLC1 in regulating myosin function remains unknown. Our lab has identified an inhibitor of T. gondii motility and invasion that results in a post-translational modification (PTM) to TgMLC1. Using molecular genetic and mass spectrometry methods we have shown cysteine 53 and cysteine 58 of TgMLC1 are essential for the modification to occur. To determine if the TgMLC1 PTM alters TgMyoA activity, glideosomes were isolated from DMSO- and 115556-treated parasites. Using an in vitro motility assay we have shown that the TgMyoA actin filament displacement velocities are decreased after 115556 treatment. This is the first evidence that TgMLC1 plays a role in regulating TgMyoA activity. The TgMLC1 PTM is responsible, at least in part, for the invasion and motility defects seen in the parasite after compound treatment. During the course of our investigations we have shown that TgMLC1 is dimethylated on lysine 95. This is an unusual modification for cytosolic proteins and has not been previously described for MLCs. Experiments using parasites expressing a non-methylatable form of TgMLC1 (TgMLC1-K95A) show that dimethylation is not necessary for TgMLC1 peripheral localization, TgMLC1 protein-protein interactions and is not required for TgMyoA activity in vitro. However, TgMLC1-K95A does not appear to be phosphoryalted indicating that TgMLC1 dimethylation is necessary for efficient phosphorylation of TgMLC1. These experiments will provide new insight into the ways in which TgMLC1 regulates this unconventional myosin motor complex.

No description available.

It is not often that a single experiment opens up a new direction of research. In this report, the optical properties of a series of organic molecules, of the form 2,4-bis((N-methyl-N-alkylamino)phenyl)squarine, which are symmetrical about their centre point, are investigated. By conventional thinking, these molecules should not show any even order non-linear optical effects; the generation of second, fourth or sixth harmonics of the incident light. In fact, significant second harmonics are generated by the materials, x(2)=25-pm v-1. This fact in itself is noteworthy, but the additional fact that the efficiency of the conversion is over two times greater than any other previously reported material is exceptional. The door is now open to an entirely new class of non-linear optical materials. In this report a thorough characterisation of the organic molecules is made by a variety of techniques; polarised second harmonic generation, quartz crystal micro balance and grazing angle X-ray diffraction. A mechanism for this new phenomenon, based on intermolecular charge transfer is developed. In a world of ever tightening pollution control legislation the need for sensors which can accurately measure the concentrations of various pollutant gases is becoming more important. In this report the pollutant sensing properties of a series of organic molecules, 2,4-bis((N-methyl-N-alkylamino)phenyl)squarine, are investigated. The series of compounds was found to be sensitive to nitrogen dioxide in concentrations of as little as 1 part per million in air, but the changes produced in the molecules were non-reversible. It is very rare to accidentally stumble across a molecule with ideal properties. A molecule must usually be modified to achieve optimum properties. In this report the steps taken in the molecular engineering of a new selective and reversible gas sending molecule are also reported. 1-(4-hydroxyphenyl)-2-(1-octadecyl-4-quinolinium iodide) ethene shows sensitivity to ammonia gas in concentrations as low as 25 parts per million in air. The response is fully reversible and can be detected using a surface plasmon resonance based sensor. In the search for new technical solutions to old problems simple solutions are often overlooked. The final section off this report describes a simple gas sensing technique, which had been available in many research institutions, but overlooked in favour of more complex solutions.

University of Macau / Institute of Chinese Medical Sciences

The first part of this thesis reports the synthesis of five calixarene molecules and their incorporation into ISEs as thallium(I) selective ionophores for use in clinical and environmental analysis. Four calix[4]arene molecules were successfully synthesized. Two were isolated in the cone conformation with iso-propyl groups attached to the lower rim. The other two were in the 1,3-alternate conformation with allyl groups attached to the phenolic oxygen. The final calixarene synthesized was a calix[6]arene that was partially substituted with iso-propyl groups on the lower rim. The parallel orientation of the aromatic rings was confirmed using single crystal X-ray structure determination. Nuclear Magnetic Resonance (NMR) complexation studies were conducted on the four calix[4]arene derivatives and showed that thallium binds within the aromatic cavity, and that the complexation/decomplexation kinetics and molecule flexibility are affected by the incorporated ion and the attached groups on the calixarene. Once the binding characteristics of the synthesized calixarenes had been examined they were then incorporated into plasticized PVC together with an ion-exchanger to make ISEs capable of determining thallium activities in solutions. Three calixarenes were successfully incorporated into the membranes and produced Nernstian responses over the concentration range 10-2-10-6 M TlNO3. The lower detection limit of the above electrodes lies in the micro-molar range, which is a common characteristic of the experimental setup used. Since the electrodes behaved ideally with respect to thallium(I), it was then decided to test the complex formation constants and selectivities of the three ionophores. / The complex formation constant (Log βILn) of two of the calixarene derivatives with thallium(I) were determined to be 6.44 and 5.85 respectively, through the use of the sandwich membrane technique. The selectivities were measured with a new protocol, whereby the electrode had not previously come into contact with the primary ion. This helps to remove ion fluxes of the primary ion and subsequent biased selectivity coefficient of highly discriminated ions. The three ionophores showed excellent selectivity against Zn2+, Ca2+, Ba2+, Cu2+, Cd2+ and Al3+, and moderate selectivity against Pb2+, Li+, Na+, H+, K+, NH4+ and Cs+. Silver was the only common high interferent in all three ionophores tested. As the detection limits of current thallium(I) ISEs in the literature would be insufficient in practical samples, attempts were made to lower the detection limits of the above ISEs with the application of relatively new experimental techniques. The lower detection limit of the three ISEs was successfully lowered by an order of magnitude from the original values through the use of an EDTA-buffered inner filling solution. The lowest achieved detection limit was obtained with the iso-propyl functionalized calix[4]arene, which reached a value of 8.32 nM (IUPAC definition). / The second part of the thesis investigated the incorporation of one of the calixarenes into a solid-contact ISE (SC-ISEs), which are seen as the future in this field due to their potential for miniaturisation and use in lab-on-a-chip applications. Four different solid-contact designs were tested to evaluate which was the best to pursue for future testing. The chosen calixarene was successfully incorporated into all four designs with Nernstian responses recorded in each case. The best response was recorded for an electrode which had a solid gold substrate, poly(3-octylthiophene) (POT) intermediate layer and a methyl methacrylate/decyl methacrylate (MMA-DMA) co-polymer membrane. This electrode exhibited a slope of 58.4 mV decade-1 and a lower detection limit of 30.2 nM. The other three solid-contact electrodes, which consisted of a graphite contact, a plasticised PVC membrane on a gold substrate, and a plasticized PVC membrane on a gold substrate with a polypyrrole intermediate layer, exhibited detection limits that were inferior to the MMA-DMA/POT SC-ISE. Further tests were used to assess one of the main problems associated with SC-ISEs, being the presence of water layers of droplets between the membrane and the solid substrate. Potential tests, electrochemical impedance spectroscopy, small angle neutron scattering and the electrode’s reactivity to changes in the concentration of dissolved oxygen were used to study water uptake and the concomitant formation of water layers in solid-contact ISEs. Water was confirmed at the surface of the membrane that consisted only of the membrane and gold substrate, but was not confirmed for the other three electrode designs.

Thesis (Ph. D.)--Washington State University, December 2007. / Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, Dept. of Biochemistry and Molecular Biology, June 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (M. Sc.)--Cambridge. / Typescript.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.