<p>Monoclonal antibodies (mAbs) as therapeutic proteins have shown great potential in treatment of various human diseases because of their highly specific nature. This has attracted worldwide attention leading to increased demand for such mAb products. To meet this demand large scale manufacturing is carried out using recombinant mammalian cell culture techniques for high yields and faster production. mAb products are worth the investment if produced in their native state. The quantity of mAb present in such cell cultures is very less and therefore special care is needed while handling them. Purifying antibody molecules from heterogeneous cell culture impurities and maintaining their native functional state is a critical task mainly because these antibodies are labile in nature. Care also need to be exercised during processing because mAbs have inherent tendancy to aggregate which is undesirable since such aggregates in antibody formulation produces immunogenic reaction when injected in humans. The other important factor in mAb purification is the processing cost involved since majority of the total production cost is utilized for purification of mAb. Protein-A chromatography is the first choice for purifying antibodies and is widely adopted. However failure in distinguishing between monomer and aggregate antibody molecules along with harsh acidic processing conditions necessitates the use of further purification steps.</p> <p>In this work various techniques for mAb processing are discussed and are outlined below:</p> <p>Removal of impurities from mAbs is a major challenge and this thesis discusses various processing options available to purify these mAbs. Impurities in mAb products are usually the aggregate byproducts formed due to unfolded monomer antibody molecules. These molecules are naturally hydrophobic in nature and display great differences in hydrophobicity on aggregation. Hydrophobic interaction membrane chromatography (HIMC) makes use of this hydrophobicity difference and helps in removal of aggregate impurities from monomer antibody.</p> <p>Heavy chain mAbs (hcmAbs) are promising new developments in the area of biopharmaceuticals because of their unique structural composition. Similar to conventional mAbs these hcmAbs are also rapidly finding their way into therapeutic markets. Purifying hcmAbs will be an important step in their development and for this purpose we use HIMC technique for removing impurities and obtain pure product.</p> <p>Antibody molecules are almost always lost as aggregates which leads to great economic losses and the ability to disaggregate these mAb oligomers would be of significant practical and scientific interest. In this work a novel thermalcycling technique is discussed to disaggregate such mAb oligomers and potentially recover functional monomer mAb molecules.</p> / Master of Applied Science (MASc)

<p>Lectin-functionalized, Poly [oligo(ethylene glycol) methyl ether methacrylate] (<em>POEGMA</em>) loaded with 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide (PAOPA) and poly(ethylene glycol)–block-poly(D,L-lactic-co-glycolic acid) (PEG-PLGA) nanoparticles loaded with haloperidol were prepared with narrow size distributions and sizes < 135 nm. The microgels and nanoparticles exhibited high <em>Solanum tuberosum </em>lectin (STL) conjugation efficiencies, encapsulation efficiencies, and drug loading capacities. The <em>in vitro</em> release of PAOPA and haloperidol was slow in physiological conditions over 96 hours, demonstrating minimal drug leakage and the potential for efficient drug transport to the targeted brain tissue. POAPA, POEGMA and the STL-functionalized POEGMA microgels were found to be non-toxic in both cell lines, indicating that they would not be toxic when administered intranasally or when they reach the brain. The nasal epithelial cell uptake of rhodamine-labelled microgels was higher in cells when the STL-functionalization was present. All haloperidol-loaded nanoparticle formulations were found to be highly effective at inducing catalepsy, while intranasal administration of STL-functionalized nanoparticles using the intranasal spray device increased the brain tissue haloperidol concentrations by 2-3.5 fold compared to STL-functionalized particles administered intranasally with a pipette. For the first time, brain tissue concentrations of rhodamine-labelled microgels confirmed that microgels are capable of passing the blood-brain barrier and that this uptake is size dependent. These formulations demonstrate promise in the reduction of the drug dose necessary to produce a therapeutic effect with antipsychotic drugs for the treatment of schizophrenia using a non-invasive route of administration.</p> / Master of Science (MSc)

The growing prevalence of antibiotic-resistant bacteria is a global health crisis that threatens the effectiveness of antibiotics in medical treatment. Increases in the number of antibiotic-resistant bacteria and a drop in the pharmaceutical development of novel antibiotics have combined to form a situation that is rapidly increasing the likelihood of a post-antibiotic era. The development of antibiotics with novel enzymatic targets is critical to stall this growing crisis. In silico methods of molecular modeling and drug design were utilized in the development of novel tryptamine analogs as potential antibiotics through their inhibition of the bacterial enzyme tryptophan synthase. Following the creation of novel tryptamine analogs, the molecules were analyzed in silico to determine their binding affinity to human MAOB and the E. coli α-subunit, E. coli β2-dimer and the M. tuberculosis β2-dimer of tryptophan synthase. Ten tryptamine analogs displayed significant increases in tryptophan synthase binding affinity and show promise as potential antibiotics and antibiotic adjuvants. Further in silico modeling determined that the binding sites of the tryptamine analogs were similar to wild-type tryptamine in the E. coli β2-dimer, the M. tuberculosis β2-dimer and human MAOB, while the analogs’ binding sites to the E. coli α-subunit differed. Although no tryptamine analogs increased tryptophan synthase binding affinity while decreasing human MAOB binding affinity, related increases in MAOB binding affinity warrants further research into the analogs’ potentials as MAO inhibitors. Given the increases in tryptophan synthase binding affinity and similar β2-dimer binding sites, a provisional patent was filed on the ten identified tryptamine analogs. Moving forward, we recommend the synthesis of the ten identified tryptamine analogs. Following synthesis, further research should be conducted to determine the in vitro and in vivo antibiotic properties of the ten tryptamine analogs.

The expression of C-X-C Chemokine Receptor 4 (CXCR4) has been correlated with increased metastatic potential of cancer cells. CXCR4 increases tumor malignancy by encouraging tumors cells to migrate to distal organs expressing its cognate ligand, CXCL12, facilitating metastasis. Thus, targeting the CXCR4/CXCL12 signaling axis provides a good strategy to inhibit the metastatic spread of tumor cells and slow cancer progression. Various studies suggest that cannabis may have anti-proliferative as well as anti-metastatic properties, though a biochemical mechanism describing how this occurs has yet to be discovered. Our lab has confirmed that agonist-bound CXCR4 and agonist-bound Cannabinoid Receptor 2 (CB2) can form heterodimers that play a role in decreasing cancer cell migration. Simultaneous treatment of the breast cancer cell line, MDA-MB-231 and the prostate cancer cell line PC-3, with CXCL12 and AM1241, a synthetic ligand for CB2, desensitizes the intrinsic cellular response to migrate toward areas of high CXCL12 concentration. Furthermore, through co-immunoprecipitation and proximity ligation assays (PLA), we have determined that there is increased interaction between the two receptors with co-stimulation of respective agonists, providing evidence for the therapeutic notion that treating tumors that endogenously secrete CXCL12 with exogenous ligands for the cannabinoid can induce dimerization. Moreover, when CXCR4 and CB2 were activated simultaneously with various agonists, decreases in migration were observed, confirming that the regulatory activity was receptor-based, not agonist-based. Finally, to determine whether simultaneously–treated, dimerized receptors inhibited activity of respective receptors, calcium mobilization assays to determine G-protein coupled receptor activation were employed. Results showed that transiently activated calcium levels were significantly lower in response to simultaneous treated cells when compared to cells treated with their individual ligands. Phosphorylation of ERK and AKT were abrogated in response to simultaneous stimulation indicating loss in downstream signaling. Therefore, we believe that the interaction of CB2 with CXCR4 may play a role in inhibiting the cells response to CXCL12, leading to a loss in metastatic potential of cells expressing these receptors.

Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it's interaction with the γ TuRC.

In response to the growing rates of antibiotic resistance in human bacterial pathogens, this study explores the natural products involved in the defensive symbiosis between actinobacteria and fungus-growing ants to uncover new potential antibiotics. This study also seeks to understand the function of natural antibiotics in their ecological contexts, especially those involved in defensive symbioses. Defensive symbiosis can be a beneficial platform for discovering useful antibiotics, because antibiotics in these relationships must be able to selectively inhibit enemies without harming hosts, and are therefore likely more specific and less toxic. Pseudonocardia sp. associated with Trachymyrmex septentrionalis ants demonstrated antibiotic activity against several gram-positive bacteria. Therefore, the natural products from this strain were extracted and purified through activity-guided fractionation. Using mass spectrometry, the structure of the active compound was elucidated as GE37468A, an antibiotic that has been previously identified from Streptomyces sp. ATCC 55365 from Italy. This compound had never before been characterized in a defensive symbiosis, which demonstrates the use of the molecule in a new context. Antibiotic GE37468A is a thiopeptide, which is a group of antibiotics that has previously demonstrated strong activity against many gram-positive bacteria, including bacterial human pathogens. Due to its potency against dangerous bacteria and its likely low toxicity, this antibiotic could therefore hold potential pharmacological uses.

Voltage-gated K+ channels associate with multiple regulatory proteins to form complexes with diverse gating properties and pharmacological sensitivities. Small molecules which activate or inhibit channel function are valuable tools for dissecting the assembly and function of these macromolecular complexes. My thesis focuses on the discovery and use of small molecules to probe the structure and function of the KCNQ family of voltage-gated K+ channels. One protein that obligatorily assembles with KCNQ channels to mediate proper assembly, trafficking, and gating is the calcium sensor, calmodulin. Although resolution of the crystal structures of calmodulin associated with isolated peptide fragments from other ion channels has provided some insight into how calmodulin interacts with and modulates KCNQ channels, structural information for calmodulin bound to a fully folded ion channel in the membrane is unknown. In Chapter II, I developed an intracellular tethered blocker approach to determine the location of calmodulin binding with respect to the KCNQ ion-conducting pathway. Using distance restraints from a panel of these intracellular tethered blockers we then generated models of the KCNQ-calmodulin complex. Our model places calmodulin close to the gate of KCNQ channels, providing structural insight into how CaM is able to communicate changes in intracellular calcium levels to KCNQ channel complexes. In addition to pore blockers, chemical modification of ion channels has been used to probe ion channel function. During my initial attempt to chemically activate KCNQ channels, I discovered that some boronates modulate KCNQ complexes. In Chapter III, the activating derivative, phenylboronic acid, is characterized. Characterization of activation by phenylboronic acid showed that it targeted the ion conduction pathway of KCNQ channels with some specificity over other voltage-gated K+ channels. The commercial availability of thousands of boronic acid derivatives provides a large class of compounds with which to systematically dissect the mechanisms of KCNQ gating and may lead to the discovery of a potent activator of KCNQ complexes for the treatment of channelopathies. All of the electrophysiological studies presented in this thesis were conducted in Xenopus oocytes. Unexpectedly, during the studies described above, the quality of our Xenopus oocytes declined. The afflicted oocytes developed black foci on their membranes, had negligible electric resting potentials, and poor viability. Culturing the compromised oocytes determined that they were infected with multi-drug resistant Stenotrophomonas maltophilia, Pseudomonas fluorescens and Pseudomonas putida. Antibiotic testing showed that all three species of bacteria were susceptible to amikacin and ciprofloxacin, which when included in the oocyte storage media prevented the appearance of black foci and resulted in oocytes that were usable for electrophysiological recordings. This study provides a solution to a common issue that plagues many electrophysiologists who use Xenopus oocytes. Taken together, these findings provide new insights into activation of KCNQ channel complexes and provide new tools to study the structure-function relationship of voltage-gated K+ channels.

Dynactin is a multi-subunit complex which was initially identified in 1991 as an activator of cytoplasmic dynein-driven microtubule-based organelle motility in vitro. Although genetic studies also supported the involvement of both complexes in the same functional pathways in yeast, filamentous fungi, and Drosophila, none of these findings yielded significant insights into dynactin's mechanism of action. The full range of cytoplasmic dynein functions in vertebrate cells has also remained poorly understood, due, in large part, to the lack of a specific method of inhibition. The present thesis work was designed to investigate these issues through a study of the 50 kDa subunit of dynactin. As a first step (Chapter 1), I cloned mammalian p50 and characterized its expression at the tissue and subcellular levels. Rat and human cDNA clones revealed p50 to be a novel α-helix-rich protein containing several highly-conserved structural features including one predicted coiled-coil domain. Immunofluorescence staining of p50, as well as other dynactin and cytoplasmic dynein components in cultured vertebrate cells showed that both complexes are recruited to kinetochores during prometaphase and concentrate near spindle poles thereafter. These findings represented the first evidence for dynactin and cytoplasmic dynein co-localization within cells, and for the presence of dynactin at kinetochores. The second major phase of the thesis (Chapter 2) was focused on investigating dynactin and cytoplasmic dynein function in cultured cells in vivo using a dominant negative inhibition approach based on transient transfections of p50 constructs. Overexpression of wild type human p50 in cultured cells resulted in a dramatic fragmentation and dispersal of the Golgi apparatus. Time-lapse fluorescence microscopy analysis of p50-overexpressing cells revealed that microtubule-based vesicle transport from the endoplasmic reticulum to the Golgi was inhibited. Also, the interphase microtubule organizing center was found to be less well-focused in some but not all transfected cells. Overexpression of p50 also disrupted mitosis, causing cells to accumulate in a prometaphase-like state. Chromosomes were condensed but unaligned, and spindles, while still generally bipolar, were dramatically distorted. Sedimentation analysis revealed the dynactin complex to be dissociated in the transfected cultures. Furthermore, both dynactin and cytoplasmic dynein staining at prometaphase kinetochores was markedly diminished in cells expressing high levels of p50. These findings provided the first in vivoevidence for the role of dynactin in cytoplasmic dynein function, i.e. mediating the motor's binding to at least one "cargo" organelle, the kinetochore, and probably also to others such as vesicles destined for the Golgi complex. These data also strongly implicated both dynactin and dynein in Golgi organization during interphase, and chromosome alignment and spindle organization during mitosis. Based on the remarkable disruptive phenotypic effects associated with overexpressing of p50, the name of dynamitin was proposed for this polypeptide. In the third and last phase of the thesis (Chapter 3), two issues were addressed: first, the dynamitin-induced mitotic arrest phenotype was studied in greater detail to better understand the exact sites of dynactin and cytoplasmic dynein activity throughout mitosis. Second, a domain analysis of dynamitin was performed to gain insight into its function within the dynactin complex. A time-lapse fluorescence microscopy study of mitosis in living dynamitin-overexpressing COS-7 cells strongly suggested specific defects in interactions of astral microtubules with the cell cortex, and in both spindle pole assembly and maintenance. Analysis of the mitotic arrest phenotype in a second cell line revealed a second arrest point at metaphase, and a clear effect of dynamitin overexpression on spindle axis orientation, again consistent with defects in interactions between microtubules and the cell cortex. Refined analyses of kinetochore and spindle pole components also confirmed specific defects in kinetochore function and spindle pole organization. Taken together, these findings support three main sites of dynactin and cytoplasmic dynein activity during vertebrate mitosis: prometaphase kinetochores, spindle poles, and the cell cortex. Finally, the domain analysis revealed dynamitin to be capable of self-association through at least two separate interaction domains, consistent with models of the mechanism underlying dynamitin-induced dynactin dissociation, and therefore, yielding important new insights into dynactin assembly. This study also indicated that a third region within dynamitin, residues 105 to 154, is essential for dynamitin and dynactin function. An independent study confirmed this finding, implicating this region in binding to ZW10, an upstream kinetochore protein. Dynamitin has therefore been revealed to be the kinetochore-targeting subunit of dynactin, and indirectly, cytoplasmic dynein. Through the body of this thesis work, dynamitin has also emerged as a powerful new tool for studying vertebrate dynactin and cytoplasmic dynein function in vivo and in vitro.

Hindbrain patterning requires many factors involved in early segmentation and later segment identity of the specific domains of the hindbrain. Hox proteins and their cofactors are of great importance during segmentation of the hindbrain, because segmentation and/or segment identity are lost when any of them are lost. Previously, we have reported that Meis proteins synergize with Pbx, another Hox cofactor, and Hox proteins expressed in the hindbrain. To further investigate Meis function during hindbrain development, we utilized a Meis dominant-negative molecule, ΔCPbx4, and expressed it in zebrafish embryos. We find that ΔCPbx4 affects gene expression and neuronal differentiation especially in r3 through r5. Further, we combined ΔCPbx4 with another Meis dominant-negative molecule (ΔHDCMeis) to disrupt Meis function more extensively. Under these conditions, we find that the entire hindbrain loses gene expression as well as its complement of neuronal differentiation. This phenotype is strikingly similar to that of loss of Pbx function, suggesting that Meis proteins act in the same pathway as Pbx. Therefore, Meis family proteins are indispensable for the entire hindbrain segmentation. In addition to the milder effect on hindbrain patterning, we also found upon expressing ΔCPbx4 that the caudal hindbrain transforms to r4-like fates, supported by expression of r4-specific marker gene (hoxbla) and specification of r4-specifc Mauthner neurons in the domain. This phenotype is not reported upon loss of Pbx function, suggesting that Meis proteins may play a more modulatory role, while Pbx is absolutely required during hindbrain development. Through several in vivo assays, we find that this r4 transformation is induced by Hox PG1 proteins and that vhnf1 represses r4 fates in the caudal hindbrain to further specify caudal fates in this region. Based on these results, we propose a model by which hindbrain patterning is achieved. Initially, un-segmented hindbrain is segmented into two domains wherein the caudal domain displays an r4 fate. This caudal r4 fate is then repressed by vhnf1 function which restricts the r4 fate to the presumptive r4 domain and specifies r5 and r6 by inducing its downstream genes such as valentino and hox PG3. Taken together, we conclude that Meis family proteins are essentially involved in function of Hox complexes to specify distinct rhombomeres during segmentation of the zebrafish hindbrain.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli O157:H7 (EHEC) form characteristic lesions on infected mammalian cells called actin pedestals. Each of these two pathogens injects its own translocated intimin receptor (Tir) molecule into the plasma membranes of host cells. Interaction of translocated Tir with the bacterial outer membrane protein intimin is required to trigger the assembly of actin into focused pedestals beneath bound bacteria. Despite similarities between the Tir molecules and the host components that associate with pedestals, recent work indicates that EPEC and EHEC Tir are not functionally interchangeable. For EPEC, Tir-mediated binding of Nck, a host adaptor protein implicated in actin signaling, is both necessary and sufficient to initiate actin assembly. In contrast, for EHEC, pedestals are formed independently of Nck, and require translocation of bacterial factors in addition to Tir to trigger actin signaling.