Exploring and understanding how the smallest scale features of a cell affect biochemical reactions has always been a challenge. Nanoscale fabrication advancements have allowed scientists to create small volume reaction containers that resemble the physical scale of cell membranes. Engineers seek to use biological design principles to manipulate information and import new functionality to such synthetic devices, which in turn, play a crucial role in allowing them to explore the effects of physical transport and extreme conditions of temperature and pH on reaction systems. Engineered reaction containers can be physically and chemically defined to control the flux of molecules of different sizes and charge. The design and testing of such a container is described here. It has a volume of 19 pL and has defined slits of 10-200 nm. The device successfully contains DNA and protein molecules and has been used to conduct and analyze enzyme reactions under different substrate concentrations and a continuous cell-free protein synthesis. The effect of DNA concentration and slit size on protein yield is also discussed. Glucose oxidase and horseradish peroxidase were loaded in the small volume container and fed with a solution containing glucose and Amplex Red™ to produce Resorufin. Fluorescent microscopy was used to monitor the reaction, which was carried out under microfluidic control. Enzyme kinetics were characterized and compared with conventional scale results. Continuous cell free protein synthesis in arrays of nanoporous, picoliter volume containers has also been achieved. A multiscale fabrication process allows for the monolithic integration of the containers and an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the nanoporous containers continues beyond 24 hours and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. These picoliter, nanoporous containers provide new ways for quick determination of enzyme kinetics and continuous protein synthesis in microfluidic systems. They can be used in a wide variety of applications such as drug discovery, clinical diagnostics and high-throughput screening.

This thesis describes the design, synthesis and structure-activity relationships analysis of potential inhibitors targeting the hepatitis C virus (HCV) NS3 protease. Also discussed is the disease caused by HCV infection and the class of enzymes known as proteases. Furthermore are explained why such enzymes can be considered to be suitable targets for developing drugs to combat diseases in general and in particular HCV, focusing on the NS3 protease. Moreover, some strategies used to design protease inhibitors and the desired properties of potential drug candidates are briefly examined. Synthesis of linear and macrocyclic NS3 protease inhibitors comprising a designed trisubstituted cyclopentane moiety as an N-acyl-(4R)-hydroxyproline bioisostere is also addressed, and several very potent and promising compounds are evaluated. / Report code: LIU-TEK-LIC-2006:46.

[FeFe]-Hydrogenases (H2ases) are metalloenzymes that can catalyze the reversible reduction of protons to molecular hydrogen as part of the metabolism of certain cyanobacteria and green algae. Due to the low availability of the enzyme, synthetic complexes that mimic the natural active site in structure, function and activity are highly sought after. In this thesis, a number of [FeFe]-H2ases active site model complexes were synthesized to answer open questions of the active site and to develop unprecedented bio-inspired proton reduction catalysts. The first part describes the synthesis and the protonation properties of a [Fe2(μ-adt)(CO)4(PMe3)2] (adt = azadithiolate) complex which contains two basic sites that are similar to those found in the enzyme active site. Unusual kinetic factors give rise to four discrete protonation states. The twofold protonated state is the first model complex that simultaneously carries a proton at the azadithiolate nitrogen and a bridging hydride at the Fe-Fe bond. In the second part, a model complex with an unprecedented amine ligand was synthesized and studied. In analogy to the enzyme active site, the labile amine ligand is expelled after electrochemical reduction. The third part describes a series of model complexes with electronically different aromatic dithiolate ligands. It is demonstrated in one case that the tuning of the ligand by electron-withdrawing substituents results in proton reduction catalysis at an overpotential that is lower than that required by the non-substituted parent compound. The design and the synthetic work towards a new ruthenium-diiron dyad for light-driven hydrogen production are presented in the fourth part. In the final part, differently isotope-labelled mixed valent Fe(I)-Fe(II) model complexes were synthesized, in particular the unprecedented 15N labelled analogue, with the aim to provide EPR-spectroscopic references that will allow the elucidation of the nature of the central atom in the dithiolate bridge of the [FeFe] hydrogenase active site.

This thesis is concerned with the synthesis and evaluation of regenerable chalcogen containing antioxidants. Variously substituted 2,3-dihydrobenzo[b]selenophene-5-ol antioxidants were evaluated in order to gain information about structure/reactivity-relationships. Within the series explored, the most regenerable unsubstituted compound inhibited lipid peroxidation for more than 320 minutes when assayed in a two-phase lipid peroxidation model in the presence of N-acetylcysteine (NAC). α-Tocopherol which could inhibit lipid peroxidation for 90 minutes under similar conditions was therefore easily outperformed. The antioxidant activity of the parent was also documented in an aqueous environment. The best catalyst quenched/inhibited ROS production by neutrophils and PMA-stimulated macrophages more efficiently than Trolox. In addition, over a period of seven days, no disruption in proliferation for the cell lines used was observed when exposed to our synthetic compound or Trolox at a concentration of 60 µM. 3-Pyridinols substituted with alkyltelluro groups in the ortho-position were more regenerable in the two-phase model than their corresponding para-substituted analogues in the presence of NAC and also inhibited autoxidation of styrene in a catalytic fashion in homogenous phase in the presence of N-tert-butoxycarbonyl cysteine methyl ester (LipCys), a lipid-soluble analogue of NAC. The best inhibitors quenched peroxyl radicals more efficiently than α-tocopherol. They could also catalyze reduction of organic hydroperoxides in the presence of thiols and therefore mimic the action of the glutathione peroxidase enzymes. Mechanisms for the catalysis are proposed. Octylthio, octylseleno and octyltelluro analogues of butylated hydroxyanisole (BHA) were synthesized and evaluated. Among these, the tellurium compound was superior to α-tocopherol in the presence of NAC both when it comes to quenching capacity and regenerability.  Organochalcogen substituent effects in phenolic compounds were studied by using EPR, IR and computational methods.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

The development of reversible and irreversible inhibitors of steroid sulfatase (STS) and protein tyrosine phosphatase 1B (PTP1B) is reported herein. STS belongs to to the aryl sulfatase family of enzymes that have roles in diverse processes such as hormone regulation, cellular degradation, bone and cartilage development, intracellular communication, and signalling pathways. STS catalyzes the desulfation of sulfated steroids which are the storage forms of many steroids such as the female hormone estrone. Its crucial role in the regulation of estrogen levels has made it a therapeutic target for the treatment of estrogen-dependent cancers. Estrone sulfate derivatives bearing 2- and 4-mono- and difluoromethyl substitutions were examined as quinone methide-generating suicide inhibitors of STS with the goal of developing these small molecules as activity-based probes for proteomic profiling of sulfatases. Kinetic studies suggest that inhibition by the monofluoro derivatives is a result of a quinone methide intermediate that reacts with active-site nucleophiles. However, the main inhibition pathway of the 4-difluoromethyl derivative involved an unexpected process in which initially formed quinone methide diffuses from the active site and decomposes to an aldehyde in solution which then acts as a potent, almost irreversible STS inhibitor. This is the first example where this class of inactivator functions by in situ generation of an aldehyde. 6- and 8-mono- and difluoromethyl coumarin derivatives were also examined as quinone methide-generating suicide inhibitors of STS. The 6-monofluoromethyl derivative acted as a classic suicide inhibitor. The partition ratio of this compound was found to be very large indicating that this class of compounds is not likely suitable as an activity-based probe for proteomic profiling of sulfatases. Boronic acids derived from steroid and coumarin platforms were also examined as STS inhibitors with the goal of improving our understanding of substrate binding specificity of STS. Inhibition constants in the high nanomolar to low micromolar range were observed for the steroidal derivatives. The coumarin derivatives were poor inhibitors. These results suggest that the boronic acid moiety must be attached to a platform very closely resembling a natural substrate in order for it to impart a beneficial effect on binding affinity compared to its phenolic analog. The mode of inhibition observed was reversible and kinetic properties corresponding to the mechanism for slow-binding inhibitors were not observed. PTP1B catalyzes the dephosphorylation of phosphotyrosine residues in the insulin receptor kinase and is a key enzyme in the down regulation of insulin signaling. Inhibitors of PTP1B are considered to have potential as therapeutics for treating type II diabetes mellitus. The difluoromethylenesulfonic (DFMS) acid group, one of the best monoanionic phosphotyrosine mimics reported in the literature, was examined as a phosphotyrosine (pTyr) mimic in a non-peptidyl platform for PTP1B inhibition. The DFMS-bearing inhibitor was found to be an approximately 1000-fold poorer inhibitor than its phosphorus analogue. It was also found that the fluorines in the DFMS inhibitor contributed little to inhibitory potency. In addition, [sulfonamido(difluoromethyl)]-phenylalanine (F2Smp) was examined as a neutral pTyr mimic in commonly used hexapeptide and tripeptide platforms. F2Smp was found to be a poor pTyr mimic. These inhibition studies also revealed that the tripeptide platform is not suitable for assessing pTyr mimics for PTP1B inhibition. Taken together, the kinetic data on the inhibition of STS and PTP1B provide valuable information relevant for future design of inhibitors of these two therapeutic targets.

With the advances in computer technology and the changing needs of people&rsquo / s daily lives, robots start to offer alternative solutions. As one of these solutions, the branch of humanoid robots emerged as advanced robots that can interact with people. Robot faces are one of the most effective means of interacting with people / since they can express their emotions and reactions through facial mimics. However, the development of realistic robot faces necessitates the knowledge of the trajectories and displacements of actual face mimics. In this study, a setup (both hardware and software), that can be used for tracking critical points on human face while exhibiting mimics, is developed. From the outputs of this setup, the mimic trajectories are going to be extracted. The setup is designed and manufactured to be durable to external effects so that with a single camera calibration procedure the 3D reconstruction can be carried out several times. The setup consists of two webcams that are specially oriented for mimic tracking. The images taken from the cameras are corrected / their features are extracted using image processing algorithms / the centroids of the features are found / correspondence is carried out and the reconstruction is made. This system can also be used for any special point tracking or volumetric measurement purposes.

Technological initiatives should be contextualized in the social, cultural and economic sectors. As information and communications technologies (ICT) have evolved with the process of globalization, ICT has played a significant role in supporting the development for both the marginal group and developing countries. In spite of the increasing numbers of ICT development projects, there is a lack of the robust research and evaluation regarding to ICT project outcomes. This study attempts to examine the ICT projects carried out in the Aboriginals community in Taiwan. Drawing the attention to the issues of power, identity, culture, and society, the postcolonial perspective is adopted. As the nature of the problem and the lack of previous understanding of the phenomenon, the interpretive ethnographic method is deployed in order to explore the cultural and social issues involved. During the two-year research, three ICT projects are examined through participate observation, interviews and related data retrieve. From the reviews of these ICT projects, the findings present in three themes: ¡§Technological Utopianism¡¨; ¡§Power Struggle¡¨ and ¡§Polarization¡¨. Although project Principal-initiator, teachers-project members and Parents- the community agree on the potential of ICT, they approach and expect ICT differently as their disparities in their position in the ICT projects, ICT literacy, and limitation from their social/economic reality. Each stakeholder has faced different context and carried different perception and vision toward ICT and ICT projects. After the ICT projects are carried out, the unanticipated and hidden gap of perception results in dissatisfied stakeholders. Although the media cover the project outcomes with positive tone, and both TY School and Principal obtains the reputation in ICT education, teachers are reluctant to execute ICT project and parents are disappointed and think the projects don¡¦t really help them. In order to accomplish these ICT projects under the disharmony, Principal takes advantage of his power from administration of school, knowledge legitimacy, and the control over resources. The power-laden process occurs and the communication gap is unable to close. Moreover, the relationships between stakeholders are eventually polarized. As failing to taking account of the dynamic context difference and lacking of communication, ICT projects in this study fail to deliver its promise to the people they intended to serve.

Optical coherence tomography (OCT) is a technology that enables 2D cross-sectional images of tissue microstructure. This interferometric technique provides resolutions of approximately 10-20 um with a penetration depth of 1-2 mm in highly scattering tissues. With the use of fiber optics, OCT systems have been developed for intravascular imaging with a demonstrated improvement in both resolution and dynamic range compared to commercial intravascular ultrasound systems. OCT studies of normal, atherosclerotic, and stented arteries indicate the ability of OCT to visualize arterial structures. These results suggest OCT may be a valuable tool for studying luminal structures in tissue engineered constructs.In the present study, new endoscopic OCT systems and analysis techniques were developed to visualize the growth and response of the cellular lining within a tissue engineered blood vessel mimic (BVM). The BVM consists of two primary components. A biocompatible polymeric scaffold is used to form the tubular structure. Human microvessel cells from adipose tissue are sodded on to the inner surface of the scaffold. These constructs are then developed and imaged within a sterile bioreactor.Three specific aims were defined for the present study. First, an OCT longitudinal scanning endoscope was developed. With this endoscope, a study of 16 BVMs was performed comparing images from OCT and corresponding histological sections. The study demonstrated that endoscopic imaging did not visually damage the mimic cellular lining. OCT images showed excellent correlation with corresponding histologicalsections. Second, a concentric three element endoscope was developed to provide radial cross-sections of the BVM. OCT images using this endoscope monitored lining development on three types of polymeric scaffolds. In the third specific aim, automated algorithms were developed to assess the percent cellular coverage of a stent using volumetric OCT images.The results of the present study suggest that OCT endoscopic systems may be a valuable tool for assessing and optimizing the development of tissue engineered constructs. Conversely, the BVMs modeled the arterial response to deployed stents allowing the development of automated OCT analysis software. These results suggest that blood vessel mimics may be used to advance OCT technology and techniques.

abstract: Measurement invariance exists when a scale functions equivalently across people and is therefore essential for making meaningful group comparisons. Often, measurement invariance is examined with independent and identically distributed data; however, there are times when the participants are clustered within units, creating dependency in the data. Researchers have taken different approaches to address this dependency when studying measurement invariance (e.g., Kim, Kwok, & Yoon, 2012; Ryu, 2014; Kim, Yoon, Wen, Luo, & Kwok, 2015), but there are no comparisons of the various approaches. The purpose of this master's thesis was to investigate measurement invariance in multilevel data when the grouping variable was a level-1 variable using five different approaches. Publicly available data from the Early Childhood Longitudinal Study-Kindergarten Cohort (ECLS-K) was used as an illustrative example. The construct of early behavior, which was made up of four teacher-rated behavior scales, was evaluated for measurement invariance in relation to gender. In the specific case of this illustrative example, the statistical conclusions of the five approaches were in agreement (i.e., the loading of the externalizing item and the intercept of the approaches to learning item were not invariant). Simulation work should be done to investigate in which situations the conclusions of these approaches diverge. / Dissertation/Thesis / Masters Thesis Psychology 2016