Experimental Studies of Electron Transfer between Wild Type and Mutagenic Cyanobacterial Cytochrome c\u3csub\u3e553\u3c/sub\u3e and Photosystem I

A mutant form of the protein complex cytochrome c553 (cyt c553) has been constructed by site-directed mutagenesis in Thermosynechococcus elongatus (T. elongates) to elucidate the binding and electron transfer properties between cyt c553 and photosystem I (PSI). The electron-transfer between wild type T. elongatus cyt c553 and a mutant form of cyt c553 and T. elongatus and Chlamydomonas reinhardtii (C. reinhardtii) PSI, has been studied as a function of cyt c553 concentration, ionic strength, pH and the detergents used to stabilize the protein. The effects of each of these variables were measured by an oxygen uptake assay. The mutated T. elongatus cyt c553 shows a higher electron transfer rate to C. reinhardtii PSI indicating that the insertion of acidic residues to the protein has facilitated the electrostatic interactions between cyt c553 and PSI. The effects of ionic strength and pH on the reaction indicate a strong influence of complementary charges on complex formation and stabilization

Reconstitution of CMV-specific T-cells following adoptive T-cell immunotherapy and haematopoietic stem cell transplantation

This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8$^+$ T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8$^+$ T cells and also reconstitution of CMV-specific CD4$^+$ and non-infused CMV-specific CD8$^+$ T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4$^+$ T-cells was studied using HLA-class II tetramers. CMV-specific CD4$^+$ T-cell count of >0.7x10$^3$/ml was found to protect from recurrent CMV reactivation. One third of specific CD4$^+$ T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4$^+$ T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells

BCR-ABL fusion genes and laboratory findings in patients with chronic myeloid leukemia in northeast Iran

Background: A specific chromosomal abnormality, the Philadelphia chromosome (BCR-ABL fusion), is present in all patients with chronic myeloid leukemia (CML). The b2a2 and b3a2 fusion mRNAs encode p210 fusion protein p210 and e1a2 encode p190. The aim of this study was to evaluate the frequency of BCR-ABL fusion transcript variants in Northeast of Iranian CML patients and to compare the laboratory results of our patients. Methods: This study was conducted in 85 peripheral blood and bone marrow samples of CML patients. Ribonucleic acid (RNA) was extracted by a commercial kit, RT- PCR for identifying BCR-ABL fusions was carried out by using designed primers and the PCR products were electrophoresed in agarose gels. Finally, statistical analysis was performed for variant frequency identification and their comparison was performed. Results: All patients examined were positive for BCR/ABL rearrangement. Fusion of b3a2 was detected in 53 (62.35%) patients, b2a2 in 25 (29.41), e1a2 in 1 (1.17%) and coexpression of b3a2 and e1a2 in 6 (7.05%) patients. There were significant differences between the mean age in patients with b3a2 positive ( 44.07 years) and in b3a2 negative group (50.35 years) however, no significant differences were seen between sex and b2a2 (P=0.61), b3a2 (P=0.79) and e1a2 (P=0.20). Conclusions: This study showed higher frequency b3a2 than b2a2 and e1a2 transcripts in CML patients in Northeast Iran and there was no association between e1a2 transcripts frequencies and monocytosis in peripheral blood

Exhaustion of bacteria-specific CD4 T cells and microbial translocation in common variable immunodeficiency disorders.

In the present study, we have investigated the functional profile of CD4 T cells from patients with common variable immunodeficiency (CVID), including production of cytokines and proliferation in response to bacteria and virus-derived antigens. We show that the functional impairment of CD4 T cells, including the reduced capacity to proliferate and to produce IFN-γ and IL-2, was restricted to bacteria-specific and not virus-specific CD4 T cells. High levels of endotoxins were found in the plasma of patients with CVID, suggesting that CD4 T cell dysfunction might be caused by bacterial translocation. Of note, endotoxemia was associated with significantly higher expression of programmed death 1 (PD-1) on CD4 T cells. The blockade of the PD-1-PD-L1/2 axis in vitro restored CD4 T cell proliferation capacity, thus indicating that PD-1 signaling negatively regulates CD4 T cell functions. Finally, we showed that intravenous immunoglobulin G (IVIG) treatment significantly reduced endotoxemia and the percentage of PD-1(+) CD4 T cells, and restored bacteria-specific CD4 T cell cytokine production and proliferation. In conclusion, the present study demonstrates that the CD4 T cell exhaustion and functional impairment observed in CVID patients is associated with bacterial translocation and that IVIG treatment resolves bacterial translocation and restores CD4 T cell functions

Patterning and cross-linking of functionalized polynorbornene polymers

A challenging application space exists for high-aspect-ratio, high-fidelity dielectrics in micro-electro-mechanical system (MEMS), microelectronic, and photonic applications. Photosensitive polymers are widely used in these fields because they are relatively easy to process and pattern, and have good mechanical properties. Photosensitive polynorbornene (PNB)-based dielectrics have been shown to have high sensitivity, excellent photodefinition properties, and high mechanical strength making them suitable for MEMS, microelectronic packaging, and photonic applications. PNB-based dielectrics can be functionalized with epoxide, carboxylic acid, or fluorinated alcohol groups. Epoxy or carboxylic acid groups can be used to provide cross-linkable sites, resulting in improved chemical and thermal properties while fluorinated alcohol groups can provide solubility in aqueous base. The focus of this study has been on the epoxy-based cross-linking of ultraviolet and electron beam (e-beam) sensitive negative-tone PNB-based dielectrics. The impact of multifunctional epoxy-based additives on the cross-linking, photolithographic properties, and adhesion properties of the photosensitive PNB dielectric was investigated. High aspect ratio features of 13:1 (height:width) were produced in 40 µm thick films (a single coat) with straight side-wall profiles and high fidelity. Contrast values as high as 33.4 were obtained at doses below 15 mJ/cm2. To evaluate the polymer's suitability to MEMS and microelectronics applications, epoxy cross-linking reactions were studied as a function of processing condition through Fourier transform infrared spectroscopy (FTIR), nanoindentation, swelling and dielectric measurements. The fully cross-linked films had an elastic modulus of 2.9 GPa and hardness of 0.18 GPa which can improve the mechanical compliance of the packaging device. To explore the feasibility of the PNB dielectric as a highly sensitive e-beam resist for nano scale fabrication, the e-beam initiated reaction between PNB cross-linking sites and the multifunctional epoxy cross-linkers was investigated. In this study, the interaction of an e-beam with the PNB mixture and its compounds was investigated. The contrast, photodefinability, and e-beam activation of the components in the PNB formulations were studied. The PNB polymer had very high e-beam sensitivity and contrast. It was shown that the addition of a photoacid generator (PAG) to the polymer-epoxy mixture enhanced the contrast and sensitivity. Formulations with the additional cross-linker showed improved contrast, sensitivity, and substrate adhesion. 100 nm structures with 13.5 nm line edge roughness (LER) were fabricated. The influence of the developing time, the developer concentration, PEB, and film thickness on the contrast and sensitivity were studied. Structures with contrast values as high as approximately 8 were fabricated at doses as low as 0.38 µC/cm2. The acid-catalyzed epoxy ring opening reaction of the PNB dielectric was studied using FTIR spectroscopy. The photo and thermal acid generation initiated epoxy ring opening reactions and subsequent cross-linking of polymer. Additionally, polymer properties were characterized as a function of processing conditions for this polymer system. It was shown that thermal cure conditions have a substantial impact on the mechanical and electrical properties of the polymer. The rate and ultimate conversion of the epoxy ring opening reaction increased with increasing cure temperature, resulting in a higher degree of cross-linking at cure temperatures above 140°C. Degradation reactions occurred at temperatures above 160°C, indicating loss of epoxide cross-linking groups and linkages. These hypotheses were supported by electrical and mechanical property studies. It was shown that curing the PNB polymer at 160°C for 1 h after develop resulted in full epoxy ring opening and highest cross-link density. This sample showed lower dielectric constant (3.9), residual stress (20 MPa), and solvent swelling (3.1%). Variable frequency microwave (VFM) processing of the PNB dielectric was studied to investigate the rapid curing of the polymer at lower temperatures. The FTIR results showed that the microwave reaction rates were higher at each isothermal cure temperature compared to convective heating, indicating that the rapid VFM curing of PNB at low temperatures is feasible. The PNB film was fully cross-linked after 15 min VFM cure at the low temperature of 150˚C. The shortest time to fully cure the polymer was found to be 5 min at 160°C. Also, the feasibility of rapid VFM curing of PNB in air was studied. All samples VFM-cured (140˚C-180˚C) in air showed no signs of oxidation. The electrical and mechanical properties of VFM-cured films were characterized and compared with thermally cured films to determine the effectiveness of the VFM processing. VFM-cured samples showed higher degree of cross-linking than thermally-cured samples, which was congruent with the FTIR results. Improved or equivalent properties were obtained for VFM-cured samples at shorter cure cycles and lower cure temperatures compared to thermally-cured films. The PNB dielectric was also used as an overcoat material to make micro and nano fluidic channels. In this work, incorporation of advanced micro/nano fluidics with high-sensitivity photonic sensors was demonstrated. 500 nm to 50 µm channels were fabricated by thermal decomposition of epoxy-based PNB polymers. Microdisks with quality factors of over 106 were presented in complementary metal-xide-semiconductor (CMOS) compatible SiN on oxide technology. These ultra-high quality factor SiN resonators were demonstrated in the visible range for the first time. The fluidic structures were interfaced with photonics for index and florescence sensing. This study was a collaboration with Dr. Ehsan Shahhosseini from the Photonics Group at Georgia Tech.PhDCommittee Chair: Paul Kohl; Committee Member: Dennis Hess; Committee Member: Michael Filler; Committee Member: Peter Ludovice; Committee Member: Sue Ann Bidstrup Allen; Committee Member: Yonathan Thi