Vor Einführung der direkt antiviralen Kombinationstherapien war die Kombination aus pegyliertem Interferon plus Ribavirin die Standardbehandlung für Patienten mit chronischer Hepatitis-C-Infektion. Bei 30% der Patienten zeigten sich neurokognitive sowie depressive Nebenwirkungen, die das dauerhafte Therapieansprechen negativ beeinflussen können. Vor diesem Hintergrund untersuchten wir in unserer Arbeit bei 93 Patienten mit chronischer Hepatitis-C-Infektion den Zusammenhang zwischen drei Single Nucleotide Polymorphismen im Bereich des IL28B-Gens und der Verträglichkeit sowie dem Therapieerfolg einer interferonbasierten Behandlung. Der Vergleich zwischen den Ergebnissen im HADS-(Hospital Anxiety and Depression Scale) sowie TAPS- (Testbatterie zur Aufmerksamkeitsprüfung) Testverfahren mit den Genotypen der drei SNPs zeigte im Studienkollektiv keinen signifikanten Zusammenhang. Hinsichtlich des Therapieerfolges konnten wir bei einem der drei SNPs das C-Allel als positiven Prognosefaktor für das dauerhafte Therapieansprechen nachweisen. / Prior to the introduction of direct antiviral combination therapies, the combination of pegylated interferon plus ribavirin was the standard treatment for patients with chronic hepatitis C infection. In 30% of the patients, neurocognitive and depressive side effects were observed, which could negatively influence the sustained virological response. Against this background, the central question that motivates our work was to investigate the association between three single nucleotide polymorphisms in the IL28B gene and the tolerability and therapeutic outcome of interferon-based treatment in 93 patients with chronic hepatitis C infection. The comparison between two test procedures, the HADS (hospital anxiety and depression scale) and TAPS (test battery for attention testing), with the genotypes of the three SNPs showed no significant association in the study population. In terms of therapeutic success, we were able to demonstrate the C allele in one of the three SNPs as a positive prognostic factor for the sustained virological response.

The phenomenon of the increase in alpha EEG activity associated with eye closure has been shown to be successful for implementing environmental control for disabled persons. Studies in this thesis investigate strategies which improve the reliability, robustness, and ease of use of alpha EEG control systems. Primarily, research covers the effectiveness of alpha EEG detection algorithms (with regard to detection time and susceptibility to artifact) and the construction and use of EEG sensing electrodes. Many new techniques for the detection of the increase of alpha EEG associated with eye closure are researched, developed, implemented and evaluated. All detection techniques are compared to a conventional method using a novel performance parameterisation criterion. In conjunction with the application of the same EEG data sets to all techniques, the use of the performance criteria enables a fair and quantitative comparison to be made between alpha detection methodologies. Detection techniques employed include enhanced versions of conventional methods, localisation of apparent alpha sources in the brain, and preprocessing methods (such as spatial filtering, adaptive filtering and independent component analysis). The best performance of alpha EEG detection was given by the source power alpha localisation technique, which showed statistically significant and practically important improvements in performance over conventional techniques. Additionally, this localisation technique is convenient and fast to implement. In situations in which electrodes are intended for unsupervised use with environmental control systems, the evaluation of alternative electrode types to the conventional wet electrodes is required, as the use of wet electrodes has several drawbacks. The performance of wet, dry and insulating electrodes is compared in this research. One aspect of the quantitative comparison of electrodes types is the measurement of contact impedance. To enable the fast and accurate measurement of impedance spectra, a new impedance spectroscopy system was developed as part of this thesis. In addition to comparison of impedance criteria, electrodes were evaluated in the presence movement-based, and electric field induced, artifacts. The electrode comparisons were carried out in a direct and quantitative manner in a controlled test environment for the first time. Results indicate that, in contrast to earlier reports, both dry and insulating electrode perform well with respect to artifact and offer a viable alternative to wet electrodes for long-term monitoring of biosignals from the surface of the skin. More improvements are required before such electrodes are suitable for EEG usage.

This study investigates the Monte Carlo calculations of cell survival in metastatic subcutaneous melanoma cancer tumours. To achieve this goal, a Monte Carlo program called SLAB.FOR was developed by Prof. David Charlton. The program randomly places alphas from 213Bi in the medium, which is a cancer cell sized micro dosimeter with a SiO2 converter on the top and Si as the sensitive volume. Then the Monte Carlo program calculates the energy deposited by alphas and their chord length and also the dose deposited in the sensitive volume. To be able to use this program, some information was taken from phase one of a clinical trial conducted by the Centre of Experimental Radiation Oncology (CERO) in 2001. During the course of this study the administered activities on tumours with different diameters are determined. Using this information the number of alpha particles going through each m3 of the tumour was found. Based on this number, the program SLAB.FOR was run for different administered activities in the tumours. The output of the program yielded the energy deposited and the number of hits by the alpha particles as they go through the tumours. The output data was also used to calculate the cell survival values, energy and hit distribution probabilities. The cell survival values were then used to plot the cell survival curves. They were plotted against dose, number of hits and injected activity per volume of the tumours. These data were also used to plot the energy and hit distribution probability curves. Our results show that survival is dependent on the diameter of the cell and decreases when the dose deposited in the tumour increases. The survival also has a relationship with the number of hits that a cell receives and it also depends on the injected activity to the volume. The survival decreases as the number of hits and injected activity increases. Our results confirmed what was stated in the clinical trial conducted by the Centre of Experimental Radiation Oncology (CERO) in 2001.

The phenomenon of the increase in alpha EEG activity associated with eye closure has been shown to be successful for implementing environmental control for disabled persons. Studies in this thesis investigate strategies which improve the reliability, robustness, and ease of use of alpha EEG control systems. Primarily, research covers the effectiveness of alpha EEG detection algorithms (with regard to detection time and susceptibility to artifact) and the construction and use of EEG sensing electrodes. Many new techniques for the detection of the increase of alpha EEG associated with eye closure are researched, developed, implemented and evaluated. All detection techniques are compared to a conventional method using a novel performance parameterisation criterion. In conjunction with the application of the same EEG data sets to all techniques, the use of the performance criteria enables a fair and quantitative comparison to be made between alpha detection methodologies. Detection techniques employed include enhanced versions of conventional methods, localisation of apparent alpha sources in the brain, and preprocessing methods (such as spatial filtering, adaptive filtering and independent component analysis). The best performance of alpha EEG detection was given by the source power alpha localisation technique, which showed statistically significant and practically important improvements in performance over conventional techniques. Additionally, this localisation technique is convenient and fast to implement. In situations in which electrodes are intended for unsupervised use with environmental control systems, the evaluation of alternative electrode types to the conventional wet electrodes is required, as the use of wet electrodes has several drawbacks. The performance of wet, dry and insulating electrodes is compared in this research. One aspect of the quantitative comparison of electrodes types is the measurement of contact impedance. To enable the fast and accurate measurement of impedance spectra, a new impedance spectroscopy system was developed as part of this thesis. In addition to comparison of impedance criteria, electrodes were evaluated in the presence movement-based, and electric field induced, artifacts. The electrode comparisons were carried out in a direct and quantitative manner in a controlled test environment for the first time. Results indicate that, in contrast to earlier reports, both dry and insulating electrode perform well with respect to artifact and offer a viable alternative to wet electrodes for long-term monitoring of biosignals from the surface of the skin. More improvements are required before such electrodes are suitable for EEG usage.

Thesis (Ph. D.)--Université de Paris, 1925. / At head of title: Series A. No. 1004 No d'ordre 1839. Thèses présentée a la faculté de sciences pour obtenir le grade le docteur ès sciences physiques. Printed paper cover.

The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Å (volume ~32,600 Å3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Ã (volume ~32,600 Ã 3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

Breast cancer is the second leading cause of mortality among women in the US. A contributing factor to such dire statistics is that conventional therapies are all too often compromised due to tumor relapse. Clearly there is an urgent need for agents that can circumvent resistance when combined with conventional therapies. RRR-α-tocopherol ether-linked acetic acid analog (α-TEA), a small bioactive lipid, exhibits in vitro and in vivo anticancer actions in a variety of cancers, including breast, prostate, and ovarian with little or no effect on normal cells and tissues, which potentially makes it an ideal chemotherapeutic agent. My studies investigated the anticancer actions of α-TEA alone and in combination with therapeutic agents using human breast cancer cell lines. Data show that: (i) Endoplasmic reticulum (ER) stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling, (ii) α-TEA plus tamoxifen act cooperatively to circumvent acquired and de novo tamoxifen resistance, resulting in cancer cell death by apoptosis. Mechanistically, the circumvention of tamoxifen resistance involved induction of DR5/caspase-8 pro-apoptotic mediators and suppression of anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling. (iii) α-TEA alone or with tamoxifen circumvents tamoxifen resistance via disruption of membrane cholesterol rich lipid raft microdomains. Cholesterol blocked the ability of α-TEA + tamoxifen to circumvent tamoxifen resistance. (iv) α-TEA in combination with PI3K, MEK or mTOR inhibitors acted cooperatively to induce apoptosis, by down-regulation of IRS-1/PI3K mediators via JNK. (v) α-TEA plus doxorubicin or cisplatin enhanced apoptosis in p53 mutant human breast cancer cells via targeting p53-inducible genes in a p73-dependent manner; namely, via up-regulation of death receptor-5 (DR5), CD95/APO-1 (Fas), Bax and Noxa, as well as down-regulation of anti-apoptotic mediator Bcl-2. Data showed that p73 responses were downstream of c-Abl, JNK and Yap. (vi) FASN inhibitor alone or with Tamoxifen or α-TEA circumvents tamoxifen resistance, thereby, providing novel strategies for restoring tamoxifen sensitivity to tamoxifen resistant cancers. In summary data show, α-TEA alone and in combination with multiple clinically-relevant anticancer agents is a promising anticancer agent. / text

Parkinson’s disease can be caused by A53T or A30P mutations in the α-synuclein (SNCA) gene, or by multiplication of the gene locus. Patients often experience depression and anxiety. We investigated affect, serotonin content and bioenergetic homeostasis of mice expressing human wild-type (WT), A53T, A30P or A53T+A30P (DM) SNCA transgenes. A30P-Tg mice displayed altered affect, increased serotonin turnover and reduced ATP and complex I+III activity. To determine whether murine α-synuclein (Snca) might mask effects SNCA transgenes we re-examined effects of SNCA transgenes in Snca-/- mice. SNCA transgenes rescued anxiety, serotonin levels and ATP content in Snca-/- mice. Only A53T SNCA abrogated behavioural despair associated with decreased norepinephrine in Snca-/- brains. The A53T residue is the natural sequence of murine Snca, and appears to be important for synuclein function in mice. The Snca-/- mouse provides a means to study the effects of SNCA mutants, and the physiologic roles of Snca in vivo.

Dictyostelium myosin heavy chain kinase A (MHCK A) and mammalian transient receptor potential melastatin-related 7 (TRPM7) are two divergent members of a family of atypical protein kinases called the alpha kinases. The crystal structures of the alpha-kinase domains of MHCK A (A-CAT, residues 552-841) and mouse TRPM7 (TRPM7-CAT, residues 1548-1862) are very similar. In both cases a C-terminal tail (C-tail) sequence (A-CAT, residues 806-841 and TRPM7-CAT, residues 1819-1862) is missing from the crystal structure. Here I show that the unstructured C-tail is required for the catalytic activity of A-CAT and TRPM7-CAT. Truncation of the C-tail of A-CAT to residue 823 decreased kinase activity by ~98% and ATPase activity by ~97%. Truncation of the C-tail of TRPM7-CAT to residue 1827 decreased kinase activity by ~97% and ATPase activity by ~58%. Ligation of the C-tail sequence of MHCK B (residues 326-354) to A-CAT-802 (residues 552-802), fully rescued kinase activity. Alignment of the C-tail sequences of MHCK A-D revealed a conserved Gly-Thr-hydrophobic motif. Previous work has shown that in A-CAT, the conserved threonine (T825) is a site of autophosphorylation. Mutation of the T825 to alanine reduced A-CAT kinase and ATPase activities by 97%, whereas mutation to serine decreased kinase and ATPase rates by 85% and 60%, respectively. This result is consistent with the finding that A-CAT strongly prefers to phosphorylate threonine residues. Surprisingly, mutation of T825 to glutamic acid reduced kinase activity by ~93% and ATPase activity by ~96%. This result suggests that glutamic acid does not properly mimic phosphothreonine in this situation, or that the free hydroxyl group of T825 is required for the catalytic activity of A-CAT. Mutation of T825 to alanine or glutamic acid in full-length MHCK A reduced kinase activity by ~90% and ATPase activity by ~40%. Further studies are required to determine if the C-tail of TRPM7-CAT also contains an essential threonine residue. / Thesis (Master, Biochemistry) -- Queen's University, 2010-02-11 11:23:04.195