There has been an influx of funding in science, technology, engineering and mathematics (STEM) allocated to adapting educational systems that engage, motivate and train learners with new and innovative techniques. This exploratory research project investigated the student outcomes associated with undergraduate biology learner' engagements in the ER Project. Thirty-one students interacted in small groups within an inquiry-learning environment supported by an innovative technology that introduced a database of images of green florescent endoplasmic reticulum and golgi apparatus. The aim of the ER Project was to increase learners' conceptual understanding of cell structure and movement and engage in scientific processes in an authentic inquiry setting. To identify relationships between and among independent and dependent variables in a causal model hypothesizing relationships among Prior Knowledge, Learning Preference, Attitudes toward Computers, Inquiry Task Performance and Conceptual Understanding were tested using path analysis. The study found that while prior knowledge was a strong predictor for conceptual understanding, it was not as effective for observing the inquiry task performance. But, the Motivation towards Computers and their Inquiry Task Performance indicated that learners understood the scientific processes and were able to communicate their results.

TSG101 was identified as a tumor susceptibility gene by Stanley Cohen. In a variety of human cancers, no genomic deletion in TSG101 gene has been reported but many aberrant TSG101 transcripts has been found. Some studies have revealed that TSG101 participates in MDM2/p53 regulatory circuitry¡Bmembrance trafficking and receptor recycling. Other reports also showed that TSG101 might be a transcription regulatory factor. However, mechanism of these TSG101 function awaits further characterization. To further scrutinize the function of TSG101 and its subcellular localization, a varieties of GFP-based recombinant plasmids which contain various length of TSG101 cDNA have been constructed and transfected into cells. Western blot analysis had shown that these constructs could express GFP-TSG101 fusion protein of expected size. The fluorescence and confocal microscopy have shown that wild type TSG101 localized in ER, Golgi and endosome compartments, also amino acid residues 136-233 and 316-390 of TSG101 are two important regions for its subcellular localization. Previous reports had shown that TSG101 interact with OP18 which is an important regulator for spindle formation in M phase. To elucidate the localization of TSG101 and OP18 in M phase cell, we have cloned OP18 and generate GST-OP18 fusion protein for anti-OP18 antiserum production.Then, pDsRed-OP18 fusion protein expressed in OP18/pDsRed recombinant plasmid transfected cell was detected by western blotting analysis using this anti-OP18 antiserum. The subcellular localization of DsRed-OP18 and GFP-TSG(1-390) fluorescence were recorded in double transfected cells which were arrested in M phase by nocodzole treatment. We observed the evenly distribution of pDsRed-OP18 red fluorescence and punctate vesicular localization of GFP-TSG(1-390) green fluorescence. Whether these two protein interact functionally awaits further investigation.

During co-translational integration, the transmembrane (TM) sequence of a nascent membrane protein moves laterally into the ER lipid bilayer upon reaching the translocon. Our lab has previously shown that this movement is a multistep process, but it was not clear whether the observed photocrosslinking of the TM segment to translocon proteins resulted from specific interactions or simply from TM-translocon proximity. If the latter, the TM α-helix will be oriented randomly with respect to translocon proteins, whereas, if the former, a specific TM helix surface would face TRAM and/or Sec61α. Integration intermediates were prepared by in vitro translation of truncated mRNAs in the presence of a Lys-tRNA analog with a photoreactive moiety attached to the lysine side-chain. When photoadduct formation was monitored as a function of probe location within the TM α-helix, we found that the extent of photocrosslinking to TRAM and Sec61α was non-random. Thus, the TM sequence occupies a distinct location within the translocon, a result that can only be achieved through protein-protein interactions that mediate the lateral movement, positioning, and integration of the TM sequence. In the case of multi-spanning membrane proteins, it was unknown how multiple hydrophobic regions integrated into the ER membrane. By placing photoprobes within each of several TM domains of a multi-spanning membrane protein, we were able to determine at what stage of integration each TM segment was no longer adjacent to translocon proteins. Using this approach we were able to establish a mechanism of integration for multi-spanning membrane proteins co-translationally inserted into the ER membrane.

In MCF7 and T47D cells cotreated with 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) plus 0.1-10 μM 3’,4’-dimethoxy flavone (DMF), there was a concentration-dependent decrease in the TCDD-induced ethoxyresorufin O-deethylase (EROD) activity. Gel mobility shift assays showed that 3’,4’-DMF inhibited TCDD-induced aryl hydrocarbon receptor (AhR) transformation in rat liver cytosol and blocked TCDD-induced formation of the nuclear AhR complex in MCF7 and T47D cells. The antiestrogenic activity of TCDD in estrogen-induced transactivation assays in MCF7 cells was reversed by 3’,4’-DMF, confirming the AhR antagonist activity of this compound in breast cancer cells. Cotreatment of T47D and MCF7 cells with TCDD and 10 μM resveratrol inhibited induction of CYP1A1 mRNA and EROD activity. Resveratrol did not inhibit TCDD-induced AhR transformation and reporter gene activity. Actinomycin D chase experiments in T47D cells showed that the mechanism of inhibition of CYP1A1 mRNA and EROD activity is due to an increased rate of CYP1A1 mRNA degradation, suggesting that resveratrol inhibits CYP1A1 via an AhR-independent post-transcriptional pathway. Vitamin D receptor-interacting protein 150 (DRIP150) coactivated estrogen receptor α (ER α)-mediated transactivation and the response was AF2-dependent in ZR75 breast cancer cells. C-and N-terminal NR-boxes (amino acids 1186-1182 and 73-69, respectively) were not necessary for coactivation of ERα. Analysis of DRIP150 deletion mutants identified a 23 amino acid sequence (811-789) required for coactivation. The 23 amino acid contained two regions at amino acids 789-794 and 795-804 which resembled α-helical motifs identified in Lanuguinosa lipase/histamine N-methyl transferase and hepatocyte nuclear factor 1, respectively. A squelching assay using specific point mutations within each α-helix showed that the NIFSEVRVYN (795-804) region was the critical sequence required for the coactivator activity of DRIP150.

Activation of the unfolded protein response follows induction of endoplasmic reticulum (ER) stress, resulting in widespread inhibition of protein expression. FLIP protein is particularly sensitive to stresses that perturb protein translation; as such, a reduction in FLIP is likely an early outcome of ER stress. Due to the anti-apoptotic role of FLIP, it is anticipated that potential decreases in FLIP would bring about an increase in sensitivity to death receptor stimuli and anoikis, a form of anchorage-dependent cell death. It was hypothesized that induction of ER stress results in downregulation of FLIP expression, resulting in sensitization of resistant tumour cells to death receptor stimuli and anoikis. From this hypothesis, it was determined that induction of ER stress through treatment of cells with brefeldin sensitized tumour cells to Fas-mediated cell death and anoikis. Moreover, over-expression of FLIP appeared to protect against ER stress-induced sensitization to cell death.

Neuronal excitability is controlled by multiple ion channels at the plasma membrane of neurons. Recently, the Na+ leak channel, NCA in C. elegans and NALCN in M. Musculus, has been identified as the molecular entity responsible for the background Na+ leak at rest in neurons. In this thesis, I show that NLF-1 (NCA Localization Factor) and mouse NLF-1, a group of newly defined, uncharacterized proteins, are endoplasmic reticular proteins required for the trafficking of NCA-1 and NCA-2 to their target axonal membrane. In primary mouse cortical neurons, knockdown of mNLF-1 partially abolishes the background Na+ leak current. Furthermore, NLF-1 and mNLF-1 directly interact with domain II S5/P-loop/S6 of NALCN through a membrane yeast-two-hybrid assay. In C. elegans, this region is required in vivo in NCA-1 for it’s trafficking. Finally, I identify novel NLF-1 interacting partners through a MYTH assay.

Neuronal excitability is controlled by multiple ion channels at the plasma membrane of neurons. Recently, the Na+ leak channel, NCA in C. elegans and NALCN in M. Musculus, has been identified as the molecular entity responsible for the background Na+ leak at rest in neurons. In this thesis, I show that NLF-1 (NCA Localization Factor) and mouse NLF-1, a group of newly defined, uncharacterized proteins, are endoplasmic reticular proteins required for the trafficking of NCA-1 and NCA-2 to their target axonal membrane. In primary mouse cortical neurons, knockdown of mNLF-1 partially abolishes the background Na+ leak current. Furthermore, NLF-1 and mNLF-1 directly interact with domain II S5/P-loop/S6 of NALCN through a membrane yeast-two-hybrid assay. In C. elegans, this region is required in vivo in NCA-1 for it’s trafficking. Finally, I identify novel NLF-1 interacting partners through a MYTH assay.

The combination of the arsenic trisulfide (As2S3) waveguide and titanium diffused lithium niobate (Ti:LiNbO3) waveguide provide us compact and versatile means for transmitting and processing optical signals, which benefits from the high index contrast between these two materials and the electro-optical properties of Ti: LiNbO3. Furthermore, waveguide gain is introduced through selective surface erbium (Er) doping which yields high quality loss-compensated or even amplifying waveguides without disturbing the excellent electrooptical, acoustooptical and nonlinear properties of the waveguide substrate LiNbO3. The integration of these waveguides allows the development of a whole class of new waveguide devices of higher functionality and complexity. As one kind of the hybrid waveguide devices, a new configuration consisting of an As2S3 channel waveguide on top of an Er doped titanium diffused x-cut lithium niobate waveguide has been investigated by simultaneous analytical expressions, numerical simulations, and experimentation. Both simulation and experimental results have shown that this structure can enhance the optical gain, as predicted by the analytical expressions. An As2S3 channel waveguide has been fabricated on top of a conventional Er:Ti:LiNbO3 waveguide, where the higher refractive index As2S3 waveguide is used to pull the optical mode towards the substrate surface where the higher Er concentration yields an improved propagation gain. The relationship between the gain and As2S3 layer thickness has been evaluated and the optimal As2S3 thickness was found by simulation and experimentation. Side integration was applied to reduce the extra propagation loss caused by the titanium diffusion bump. The propagation gain (dB/cm) has been improved from 1.1 to 2 dB/cm. Another hybrid device which combines the As2S3 and LiNbO3 is to make an As2S3 racetrack ring resonator on top of an x-cut y-propagation Er:Ti:LiNbO3 waveguide which is the potential structure for integrated lossless all-path filter. The ring was side-coupled with the Ti:LiNbO3 waveguide and the optical gain was achieved when the 5mm long coupling region where has been diffused with Er in advance pumped by 144mW pump laser. The free spectral range (FSR) of the measured ring response for TM mode is 0.0587nm (7.33GHz) at 1550nm. The roundtrip loss are 4.4dB (2.60dB/cm) when pump on and 5.8dB (3.44dB/cm) when pump off. The optical gain in the Er diffused area is 0.72dB/cm.

The variations in ER structure and motility under different stages of cell development remain largely unexplored. Here, I observe ER structure and the changes that take place in this structure over time in growing and non-growing live epidermal cells of the pea tendril. The ER was labelled by green fluorescent protein, fused to the KDEL-ER retention signal and confocal scanning laser microscopy was used to localize the fluorescent signal. I found both the structure and motility of growing cells to be different to non-growing cells. The growing cells had a more open arrangement of the cortical ER, fewer lamellae and showed greater tubular dynamics, while the non-growing cells had a denser arrangement of the cortical ER network, with more lamellae and less tubular dynamics. Furthermore, these differences in the cortical ER structure and dynamics were due to growth as, the ER in non-growing cells showed characteristics similar to those seen in growing cells when these cells were induced to grow by the exogenous application of auxin. These changes in ER structure and dynamics were dependant on both the microtubules and actin cytoskeleton networks.

Perturbations that derail the proper folding and assembly of proteins in the endoplasmic retriculum (ER) cause misfolded protein accrual in the ER – a toxic condition known as ER stress. The Unfolded Protein Response (UPR) is a signaling system evolved to detect and rectify ER stress. The work I present herein pertains to the most ancient member of the ER stress transducers, IRE1. ER stress stimulates IRE1 to activate a UPR-dedicated transcription factor called XBP1 in metazoans (or HAC1 in yeast) to bolster the productive capacity of the ER and purge misfolded proteins from the ER. To activate XBP1/HAC1, IRE1 cleaves XBP1/HAC1 mRNA twice to eliminate an inhibitory intron using a dormant nuclease function in its cytoplasmic effector region (IRE1cyto). My focus was to understand the mechanism of XBP1/HAC1 activation by IRE1, the regulation of IRE1 function and the manipulation of IRE1 signaling output using chemical tools. To better understand IRE1 mechanism, I determined the crystal structure of IRE1cyto bound to ADP. Structural and mutational analyses uncovered a probable novel IRE1 nuclease active site, allowing a catalytic mechanism of RNA cleavage to be inferred. Further genetic and biophysical experiments revealed that the ordered sequence of events: autophosphorylation, nucleotide binding and dimerization; orchestrates the assembly of the IRE1 nuclease active site to potentiate nuclease function. The flavanol quercetin was identified in a chemical screen as a potent stimulator of IRE1 nuclease output. To understand the mechanism of action of quercetin, I determined the crystal structure of IRE1cyto in complex with quercetin and ADP. Quercetin docked to a novel ligand binding site, termed the Q-site, at the interface of IRE1 dimers. Biophysical and genetic analyses revealed that quercetin engagement of the Q-site promotes IRE1 dimerization, thereby enhancing IRE1 nuclease activity. To gain insight on how IRE1 recognizes RNA, I performed bioinformatic analysis to identify a conserved sequence element in XBP1/HAC1 mRNA (termed XBP1mini) that may compose a higher-order structure recognized by IRE1. I developed an RNA production scheme to generate XBP1mini RNA for structural and biophysical studies. Preliminary X-ray diffraction studies indicate that XBP1mini may indeed adopt an ordered crystallizable tertiary structure.