Members of the Argonaute family of proteins, which interact with small RNAs, are the key players of RNAi and other related pathways. The C. elegans genome encodes 27 members of the Argonaute family. During this thesis research, we sought to understand the functions of the members of this gene family in C. elegans. Among the Argonaute family members, rde-1 and alg-1/2have previously been shown to be essential for RNAi and development, respectively. In this work, we wanted to assign functions to the remaining members of this large family of proteins. Here, we describe the phenotype of 31 deletion alleles representing all of the previously uncharacterized Argonaute members. In addition to rde-1, our analysis revealed that two other Argonaute members csr-1 and prg-1 are also essential for development. csr-1 is partially required for RNAi, and essential for proper chromosome segregation. prg-1, a member of PIWI subfamily of Argonaute genes, exhibits reduced brood size and temperature-sensitive sterile phenotype, implicating that it is required for germline maintenance. Additionally, we showed that RDE-1 interacts with trigger-derived sense and antisense siRNAs (primary siRNAs) to initiate RNAi, while several other Argonaute proteins, SAGO-1, SAGO-2, and perhaps others, functioning redundantly, interact with amplified siRNAs (secondary siRNAs) to mediate downstream silencing. Moreover, our analysis uncovered that another member of Argonaute gene family, ergo-1, is essential for the endogenous RNAi pathway. Furthermore, we built an eight-fold Argonaute mutant, MAGO8, and analyzed its developmental phenotype and sensitivity to RNAi. Our analysis revealed that the genes deleted in the MAGO8 mutant function redundantly with each other, and are required for RNAi and the maintenance of the stem cell totipotency.

Myosin 10 is a vertebrate specific actin-based motor protein that is expressed in a variety of cell types. Cell biological evidences suggest that myosin 10 plays a role in cargo transport and filopodia extension. In order to fully appreciate these physiological processes, it is crucial to understand the motor property of myosin 10. However, little is known about its mechanoenzymatic characteristics. In vitro biochemical characterization of myosin 10 has been hindered by the low expression level of the protein in most tissues. In this study, we succeeded in obtaining sufficient amount of recombinant mammalian myosin 10 using the baculovirus expression system. The movement directionality of the heterologously expressed myosin 10 was determined to be plus end-directed by the in vitro motility assay with polarity-marked actin filament we developed. The result is consistent with the proposed physiological function of myosin 10 as a plus end-directed transporter inside filopodia. The duty ratio of myosin 10 was determined to be 0.6~0.7 by the enzyme kinetic analysis, suggesting that myosin 10 is a processive motor. Unexpectedly, we were unable to confirm the processive movement of dimeric myosin 10 along actin filaments in a single molecule study. The result does not support the proposed function of myosin 10 as a transporter. One possible explanation for this discrepancy is that the apparent nonprocessive nature of myosin 10 is important for generating sufficient force required for the intrafilopodial transport by working in concert with numbers of other myosin 10 molecules while not interfering with each other. Altogether, the present study provided qualitative and quantitative biochemical evidences for the better understanding of the motor property of myosin 10 and of the biological processes in which it is involved. Finally, a general molecular mechanism of myosin motors behind the movement directionality and the processivity is discussed based on our results together with the currently available experimental evidences. The validity of the widely accepted ‘leverarm hypothesis’ is reexamined.

In the course of experiments designed to identify and characterize structural proteins of the nuclear matrix, one antibody was generated which recognized an extraction-resistant cytoplasmic protein. This antibody was used as the starting point in the cloning and molecular characterization of a novel protein of the inter-membrane space of the mitochondrion which has been named mitofilin. Mitofilin is expressed in all human cell types, and murine homologues also exist. Mitofilin associates only with mitochondria and not with other membrane-bounded organelles such as Golgi or endoplasmic reticulum. This observation has been confirmed both by biochemical fractionation and multi-label fluorescence microscopy. Recombinant mitofilin, purified to homogeneity by affinity chromatography and preparative electrophoresis, was used to raise second-generation antibodies. Results of Western blot and immunofluorescence microscopy experiments, identical to those obtained using the original monoclonal antibody, verify the cloning and biochemical characterization. The mitofilin polypeptide contains several regions which are predicted to interact by forming coiled coils; a mitochondrial targeting signal; and a hydrophobic, membrane-spanning domain. During the course of this work, a sequence match was found with a cDNA reported by Icho, et al (1994) for a mRNA preferentially expressed in heart muscle, which they have called HMP. Evidence is presented which contradicts those authors' contention that HMP is a kinesin-like motor protein. In the course of these investigations, methods were developed to detect and quantitate the expression of solubilization-resistant proteins of the nuclear matrix and the nuclear matrix-intermediate filament scaffold. This was accomplished by combining SDS-PAGE, high sensitivity chemiluminescent Western blots, and scanning densitometry. Sensitivity in the picogram range was obtained, and reproducibility was assessed. For semi-quantitative measurements of protein expression in tissue samples, cell number was normalized by measurement of lamin B, the major protein of the nuclear envelope. Results of screening several cell and tissue types for the expression of mitofilin and for the nuclear matrix proteins NuMA, the nucleoporin tpr, and lamin B are presented. These preliminary data suggest a potential connection of over-expression of NuMA, tpr, and mitofilin with ovarian carcinoma. In addition, quantitative analysis of mitofilin expression in a variety of human cell types was done using purified recombinant protein antigen as the standard. The presence of coiled coil domains in these and other proteins associated with cellular sub-structures gave rise to the third area of investigation described here. Experimental observations of the nuclear matrix-intermediate filament scaffold (NMIF), a tissue-wide structure greatly enriched in coiled coil proteins, led to the following hypothesis: that the differentiated cell and tissue architecture which characterizes Metazoa has evolved through the propagation and selective expression of genes encoding a wide variety of coiled coil proteins, and the integration of the gene products into a tissue-wide matrix based on coiled coil interactions. This hypothesis was explored by computer searches of sequence data files. The GenBank phylogenetic sequence files were examined with a heptad repeat analysis program to assess the occurrence of coiled coil proteins. how heptad repeat domains are organized within these proteins, and what structural/functional categories they comprised. Of 102,007 proteins analyzed, 5.95% (6074) contained coiled coil domains: 1.26% (1289) contained "extended" (> 75 amino acid) domains. While the frequency of proteins containing coiled coils was surprisingly constant among all biota, extended coiled coil proteins were 4-fold more frequent in the animal kingdom, and may reflect early events in the divergence of plants and animals. Structure/function categories of extended coils also revealed phylogenetic differences. In pathogens and parasites, many extended coiled coil proteins are external and bind host proteins. In animals, the majority of extended coiled coil proteins were identified as constituents of two categories: 1) myosins and motors, or 2) components of the NMIF. This scaffold, produced by sequential extraction of epithelial monolayers in situ, contains only 1-2% of the cell mass while accurately retaining morphological features of living epithelium. The NMIF incorporates many proteins with extensive, interrupted coiled coil forming domains. The increased occurrence of this type of protein in Metazoa compared to plants or protists supports the hypothesis that a tissue-wide matrix of coiled coil interactions underlies metazoan differentiated cell and tissue structure.

The minimal element of the nervous system, the synapse, is a plastic structure that has the ability to change in response to various internal and external factors. This property of the synapse underlies complex behaviors such as learning and memory. However, the exact molecular and cellular mechanisms involved in this process are not fully understood. To understand the mechanisms that regulate synapse development and plasticity I took advantage of a powerful model system, the Drosophila larval neuromuscular junction (NMJ). In this system, both anterograde and retrograde signaling pathways critical for coordinated synapse development and plasticity have been documented. An anterograde WNT/Wingless (Wg) signaling pathway plays a crucial role in both developmental and activity-dependent synaptic plasticity at the NMJ. Presynaptic motor neuron terminals secrete highly hydrophobic Wg, which travels to relatively distant postsynaptic sites where it activates a signal transduction pathway required for postsynaptic development. In the first half of my thesis I unraveled a previously unrecognized cellular mechanism by which Wg is shuttled to postsynaptic sites. In this mechanism Wg rides on secreted microvesicles or exosomes that contain a dedicated WNT secretion factor, the WNT-binding transmembrane protein, Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). To our knowledge, this was the first in vivo study demonstrating that neurons release exosomes, which are involved in trans-synaptic communication. Moreover, this was the first study showing that hydrophobic WNT signals are transported to the extracellular space on exosomes to reach WNT-receptor containing target cells. Retrograde signals are also critical during development and plasticity of synaptic connections. These signals function to adjust the activity of presynaptic cells according to postsynaptic cell outputs, to maintain synaptic function within a dynamic range. However, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are not clear. In the second half of my thesis, I provided evidence that a crucial component of retrograde signaling at the fly NMJ, Synaptotagmin-4 (Syt4), is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Drosophila Syt4 is known to reside on postsynaptic vesicles at the NMJ and function as a calcium sensor to release a retrograde signal upon synaptic activity. This event is required for coordinated maturation of the presynaptic terminal. We demonstrated that retrograde Syt4 function in postsynaptic muscle is required for activity-dependent presynaptic growth. However, surprisingly, Syt4 protein was not synthesized in postsynaptic muscles. Instead, Syt4 was produced in motorneurons and transferred to postsynaptic muscle cells via exosome secretion by presynaptic cells. The above study provided evidence for a presynaptic control of postsynaptic retrograde signaling through exosomal transfer of an essential retrograde signaling component. In summary, this body of work reveals a novel mechanism of trans-synaptic communication through exosomes. While intercellular communication through exosomes had been demonstrated during antigen presentation in the immune system, our studies were the first to substantiate this mode of communication in the nervous system. Thus, these studies provide a significantly deeper and novel understanding of the mechanisms underlying synapse development and plasticity.

The formation of the anterior-posterior axis requires a symmetry-breaking event that starts gastrulation. Ultimately, the morphogenetic movements of gastrulation reshape the embryo to its final tri-dimensional form. In mouse embryos, the identity of the molecule that breaks the bilateral symmetry and sets in motion gastrulation remains elusive. The Wnt signaling pathway plays a pivotal role during axial specification and gastrulation in metazoans. Loss-of-function experiments have demonstrated a requirement of Wnt3 for gastrulation in mice. But because Wnt3 is expressed sequentially in two tissues, the visceral endoderm and the epiblast, its tissue specific requirements remain uncertain. Here, we report that embryos lacking Wnt3 specifically in the visceral endoderm do not form a primitive streak, mesoderm, endoderm or any derivatives. Visceral endoderm-specific Wnt3 mutants also lack primordial germ cells. Moreover, we provide data demonstrating that Wnt3 carries out its actions in the epiblast via the canonical Wnt pathway. Together, these data suggest that the posterior visceral endoderm via Wnt3, regulates the development of mouse embryos in a similar fashion to the amphibian Nieuwkoop center. Next, we conditionally ablated Wnt3 locus in the epiblast to investigate whether Wnt3 expression is also required in that tissue. Embryos lacking Wnt3 expression in the epiblast, but retaining its expression in the visceral endoderm, show delayed but not absent gastrulation. We conclude that the expression of Wnt3 in the epiblast is required for maintenance but not initiation of gastrulation in mouse embryos. Furthermore, we used in vitro and in vivo approaches to demonstrate that the Wnt3-mediated activation of the canonical Wnt pathway leads to β-catenin occupancy followed by transcription of key loci, including the Wnt3 locus itself, during gastrulation in mice. Our data indicate the presence of an autoregulatory loop in which Wnt3 controls its own expression and orchestrates the process of gastrulation in the mouse embryo.

Dengue virus (DENV) causes over 500,000 infections annually with a spectrum of clinical diseases ranging from subclinical infection to dengue, a mild febrile illness, to life-threatening severe dengue. Vascular leakage without endothelial cell damage is the hallmark symptom of severe dengue illness and is proposed to be directly mediated by soluble inflammatory mediators IL-8 and TNFα. IL-8 production occurs in response to DENV infection, is elevated during severe dengue, is proposed to inhibit interferon, and could potentially recruit target cells to sites of infection. We previously showed that expression of DENV NS5 activates the IL-8 promoter, induces IL-8 transcription, and induces IL-8 protein production in HepG2 and HEK293A cell lines. As multiple DENV proteins are reported to interact with important signaling pathways, we hypothesized that other DENV proteins could contribute to the activation of IL-8. We found that plasmids expressing prM-E together, the GPI-linked variant of NS1 (NS1G), the carboxyl-terminal 112 amino acids of NS4B, as well as NS5 each induced expression from an IL-8 promoter-driven reporter plasmid. Expression of NS5 also induced activation of a RANTES promoter construct and TNFα mRNA expression. Further, we found that the carboxyl-terminal polymerase domain of NS5 was sufficient to induce IL-8 secretion but polymerase function was not required. Like NS5, prM-E and NS1G induced luciferase expression from an AP-1-driven reporter plasmid. We further tested whether activation of the IL-8 promoter depended on any single transcription factor within IL-8 using IL-8 promoter-driven plasmids mutated at the AP-1, C/EBP or NF-κB binding sites. We found that activation of the IL-8 promoter by prM-E, NS1G and NS4B did not depend on activation of any single transcription factor. Our data suggested that AP-1 may be both positively and negatively inducing transcription, fitting with previous theories that DENV regulates IL-8 induction. However, we did not observe any differences in activation of AP-1 subunit c-Jun, or the inhibitory subunits Fra-1 or Fra-2 between DENV and mock-infected cells. These data support a model in which multiple DENV proteins activate the IL-8 promoter, provide a potential basis of IL-8 induction by DENV in multiple cell types, and further supports a mechanism by which DENV contributes to severe dengue illness.

<p>Allosteric modulators are emerging as a new class of therapeutics for the treatment of complex disorders, including psychiatric illnesses such as schizophrenia. The disease is marked by hyperdopaminergic signaling in the striatum, which plays a role in the development of positive symptoms like delusions, hallucinations, and paranoia. Conventional antipsychotic drug therapy typically employs dopamine D2 receptor antagonists that compete with endogenous dopamine at the orthosteric, or dopamine-binding site, in an attempt to normalize these psychotic symptoms. However, they are often associated with adverse motor and metabolic side effects. Furthermore, only some antipsychotic drugs are able to treat the negative symptoms of schizophrenia, which include social withdrawal and anhedonia, and there is currently no treatment for the cognitive impairment associated with the disease.</p> <p>Allosteric modulators are safer alternatives to conventional orthosteric therapeutics as they interact with their receptor at a novel binding site and their mechanism involves modulation of endogenous signaling. Therefore, levels of endogenous ligand limit the activity of an allosteric modulator. Our lab has synthesized and evaluated over 185 compounds for their activity at the dopamine D2 receptor. Of these compounds, PAOPA is the most potent allosteric modulator, and has been shown to be effective in treating the MK-801 induced preclinical animal model of schizophrenia without causing the adverse effects induced by currently prescribed antipsychotic drugs. The objective of this study was to evaluate PAOPA’s ability to treat behavioural abnormalities in an amphetamine-sensitized model of schizophrenia.</p> <p>Four groups (n=10/group) of male Sprague Dawley rats received intraperitoneal injections three days per week on alternate days over three weeks. Group A received saline, group B received D-amphetamine (1mg/kg during week one, 2mg/kg during week two, 3mg/kg during week three), group C received PAOPA (1mg/kg), and group D received the same doses of amphetamine as group B with PAOPA (1mg/kg). Following a three-week withdrawal, each group was tested for prepulse inhibition, social interaction, and locomotor activity. Amphetamine-sensitized rats were subjected to the same tests following PAOPA administration (1mg/kg). To assess whether behavioural changes were associated with changes in brain chemistry, post-mortem dopamine levels were measured in the striatum, nucleus accumbens, and medial prefrontal cortex. Data were analyzed by one-way ANOVA or paired t test where appropriate.</p> <p>Amphetamine sensitization induced schizophrenic-like behavioural abnormalities, including deficits in prepulse inhibition and social interaction, as well as increased locomotor activity and sensitivity to amphetamine challenge. Concurrent amphetamine and PAOPA treatment prevented all amphetamine- induced behavioural abnormalities. Furthermore, amphetamine-induced deficits in prepulse inhibition and social interaction were reversed one hour following PAOPA treatment. PAOPA treatment alone had no effect on behaviour or post-mortem striatal dopamine. Behavioural changes in amphetamine-sensitized rats were accompanied by a reduction in post-mortem striatal dopamine levels. In correlation with behavioural results, PAOPA administration during amphetamine sensitization prevented this biochemical change.</p> <p>These results demonstrate that PAOPA can prevent and reverse behavioural and associated biochemical abnormalities in amphetamine-sensitized rats. PAOPA is a candidate for the development of treatments for schizophrenia.</p> / Master of Science (MSc)

<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.