DNA mismatch repair is one process in the preservation of genomic integrity. It has been found in Archeae, bacteria, plants, yeast and mammals. The mismatch repair system is highly conserved among species and allows the strand-specific elimination of base-base mispairs, chemical base modifications, as well as short insertion/deletion loops following DNA replication. The repair system also has important effects on homeologous recombination, contributing to the frequency of reciprocal exchanges. In humans, defects in the repair system have been found to be associated with tumorigenesis. In Escherichia coli, this pathway was originally called long patch repair before being renamed the methyl-directed mismatch repair system. It is unique in that it utilizes a DNA methylation pattern to discriminate between the parental DNA strand and the newly synthesized daughter DNA strand. The current model for the initiation of methyl-directed mismatch repair is that the mispaired bases are recognized and bound by the MutS protein with MutL as a helper protein for binding. MutL also assists the MutH protein to bind, thereby forming the completed initiation complex of MutS, MutL and MutH. In the presence of ATP, there is evidence for translocation ofthe complex along the DNA forming alpha loops. At a d(GATC) site the MutH protein binds and nicks the unmethylated daughter DNA strand 5' to the d(G) (by recognizing the N6-d(A) methylation of the parental DNA strand which it is unable to cut). This completes the initiation of the repair system and allows the hydrolysis and resynthesis of the daughter DNA strand. MutH is a monomer of 25.5 kD in solution and contains a latent Mg2+-dependent endonuclease activity. Unmethylated DNA is nicked without any discrimination on one of the two strands and fully methylated DNA is resistant to cleavage by MutH even though the protein is able to bind the d(GATC) site. The structure of MutH was recently solved and compared to a group of restriction endonucleases that share a structural common core domain with similarly placed catalytic residues. The MutH protein is comprised of two major domains that are able to pivot and rotate with respect to one another. The cleft between the two domains is large enough for double-strand DNA to bind. This research started with the determination of the MutH structure before it was known. After crystallizing the protein and collecting several heavy atom data sets, it was found that the electron density maps were too discontinuous to trace the structure of the protein. Following that work, site-directed mutagenesis was performed on several areas of MutH based on the similarity of MutH and PvuII structural models. The aims were to identify DNA binding residues (in two flexible loop regions), to determine if MutH has the same mechanism for DNA binding and catalysis as PvuII (MutH histidines 112 and 115), and to localize the residues responsible for MutH stimulation by MutL (MutH C-terminal tail region). An in-vivoscreen based on the mutator phenotype was used to select for functionally defective MutH mutants. These bacteria accumulate mutations at a greater frequency than wild-type and this was monitored by selection on plates with rifampicin. Three MutH mutants were identified from this screen (K48A, G49A, and Δ214). They were purified and assayed for total activity and binding ability. Four other mutants with wild-type phenotypic screen results were also chosen to confirm they were not involved in any MutH function (D47A, H112A, H115A, and Δ224). No DNA binding residues (such as D47A) were identified in the two flexible loop regions of MutH, although similar loops in PvuII are involved in DNA binding. The purified D47A MutH protein showed wild-type biochemical activity. Instead, the lysine residue (K48) in the first flexible loop was found to function in catalysis together with the three presumed catalytic amino acids (Asp70, Glu77, and Lys79). This purified MutH protein (K48A) had wild-type binding ability but no endonuclease activity without MutL. In the presence of MutL, the K48A protein had only a three-fold reduction in endonuclease activity. This research has shown that MutL stimulates the wild-type MutH activity by 1000-fold. The wild-type MutH stimulation by MutL for binding was only shown to be 16-fold. The G49A MutH mutant interferes with the proper functioning of the protein but is not informative about the mechanism of action. The binding ability of this mutant was the same as wild-type and the endonuclease activity was down 30-fold with a 10-fold stimulation by MutL. The extra methyl group of the alanine may cause slight structural changes in the lysine 48 side chain that slows catalysis. The two histidines (H112 and H115) in MutH that are in a similar position as the two histidines (H84 and H85) in PvuII (that signal for DNA binding and catalysis) were changed to alanines, but had wild-type activity both in-vivo and in-vitro. These results indicate that the MutH signal for DNA binding and catalysis remains unknown. The two deletion mutations (MutHΔ224 and MutHΔ214) in the C-terminal end of the protein, localized the MutL stimulation region to five amino acids (Ala220, Leu221, Leu222, Ala223, and Arg224). Mutant MutHΔ224 had wild-type MutL stimulation activity, while MutHΔ214 showed no MutL stimulation. Another deletion mutant, MutHΔ119, from another laboratory was shown to have wild-type MutL stimulation also. This leaves one (or more) of the remaining five residues as important for MutL stimulation.

In previous work I described the purification and properties of the microtubule-based mechanochemical ATPase cytoplasmic dynein. Cytoplasmic dynein was found to produce force along microtubules in the direction corresponding to retrograde axonal transport. Cytoplasmic dynein has been identified in a variety of eukaryotes including yeast and human, and there is a growing body of evidence suggesting that this "molecular motor" is responsible for the transport of membranous organelles and mitotic chromosomes. The first part of this thesis investigates the molecular basis of microtubule-activation of the cytoplasmic dynein ATPase. By analogy with other mechanoenzymes, this appears to accelerate the rate-limiting step of the cross-bridge cycle, ADP release. Using limited proteolysis, site-directed antibodies, and N-terminal microsequencing, I identified the acidic C-termini of α and β-tubulin as the domains responsible for activation of the dynein ATPase. The second part of this thesis investigates the structure of the 74 kDa subunit of cytoplasmic dynein. The amino acid sequence deduced from cDNA clones predicts a 72,753 dalton polypeptide which includes the amino acid sequences of nine peptides determined by microsequencing. Northern analysis of rat brain poly(A) revealed an abundant 2.9 kb mRNA. However, PCR performed on first strand cDNA, together with the sequence of a partially matching tryptic peptide, indicate the existence of three isoforms. The C-terminal half is 26.4% identical and 47.7% similar to the product of the Chlamydomonas ODA6 gene, a 70 kDa subunit of flagellar outer arm dynein. Based on what is known about the Chlamydomonas70 kDa subunit, I suggest that the 74 kDa subunit is responsible for targeting cytoplasmic dynein to membranous organelles and kinetochores of mitotic chromosomes. The third part of this thesis investigates a 50 kDa polypeptide which co-purifies with cytoplasmic dynein on sucrose density gradients. Monoclonal antibodies were produced against the 50 kDa subunit and used to show that it is a component of a distinct 20S complex which contains additional subunits of 45 and 150 kDa. Moreover, like cytoplasmic dynein, the 50 kDa polypeptide localizes to kinetochores of metaphase chromosomes by light and electron microscopy. The 50 kDa-associated complex is reported to stimulate cytoplasmic dynein-mediated organelle motility in vitro. The complex is, therefore, a candidate for modulating cytoplasmic dynein activity during mitosis.

DNA is packaged within the cells' nucleus as a highly compact chromatin structure ranging between 100-400 nm fibers. The organization and alteration of this structure is mandatory in order to arbitrate DNA-mediated processes of the cell, including transcription, DNA replication, recombination and repair. Many different kinds of enzymes modify chromatin components and, in turn, regulate the accessibility of DNA. These multi-subunited enzymes have emerged as key regulators for several processes of the cell. Central to understanding how DNA-mediated processes are regulated is to comprehend the consequences of these modifications of chromatin, which lead to altered states of either activation or inactivation. One class of factors known to modify chromatin structure is the ATP-dependent chromatin remodeling enzymes. This class of enzymes encompasses evolutionarily conserved multi-subunited enzymes, which appear to function by using the energy of ATP hydrolysis to disrupt histone-DNA interactions. The prototype of ATP-dependent chromatin remodelers is the Saccharomyces cerevisiae SWI/SNF complex. The yeast SWI/SNF complex is required for the full functioning of several transcriptional activators and for the expression of a subset of yeast genes, a notable number being inducible and mitotic genes. The purified complex is comprised of the following eleven different polypeptides: Swi2p/Snf2p, Swi1p, Swi3p, Snf5p, Snf6p, Swp73p, Arp7p, Arp9p, Swp82p, Swp29p and Snf11 p. It has been established that a core of homologous subunits (Swi2p, Swi3p, Swp73p, Snf5p and the Arp proteins) is conserved among the SWI/SNF-related complexes from several organisms (yRSC, hSWI/SNF, hRSC, DrosophilaBrahma). However, the functional contribution of these polypeptides in the complexes for altering chromatin structure is largely unknown. In this study, biochemistry is used to examine the structure of the complex and function of individual subunits of the yeast SWI/SNF complex to understand better how these proteins are acting in concert to remodel chromatin. In addition, we examine a role for SWI/SNF complex in the process of DNA replication. The relative stoichiometry of the SWI/SNF complex subunits was determined by in vitrobiochemical studies. Co-immunoprecipitation has demonstrated that there is only one copy of Swi2p/Snf2p per complex. Subsequent radioactive labeling of the purified complex revealed that the complex contains one copy of each subunit per complex with the exception of Swi3p and Snf5p, which are present in two copies per complex. The subunit organization of SWI/SNF complex has been more clearly defined by determining direct subunit-subunit interactions in the complex. The Swi3p component has previously been shown to be critical for complex function in vivo and essential for the integrity of the complex in vitro, and this study demonstrates that Swi3p serves as a scaffolding protein that nucleates SWI/SNF complex assembly. In vitrobinding studies with Swi3p have revealed that Swi3p displays self-association, as well as direct interactions with the Swi2p, Snf5p, Swp73p, Swi1p and Snf6p members of the complex. The direct interactions of the yeast SWI/SNF subunits with transcriptional activators, thought to be important for yeast SWI/SNF targeting, were examined. In vitrobinding assays demonstrate that individual SWI/SNF subunits, Snf5p, Snf6p and Swi1p, and sub-complexes Swi2p/Swi3p and Swp73p/Swi3p can directly interact with specific domains of transcriptional activators of either the Swi5p zinc-finger DBD or VP16 acidic activation domain. This work begins to characterize the functional contribution of individual subunits, and cooperative sub-complexes that are critical for the SWI/SNF complex functional activities. The yeast SWI/SNF complex was investigated for the ability to playa role in DNA replication. Interestingly, plasmid stability assays reveal that minichromosomes that contain DNA replication origin ARS121 is weakened when the SWIISNF complex is non-functional. ARS121's SWI/SNF dependency is overcome by the over-expression of DNA replication regulatory protein, Cdc6p. Thus, this suggests SWI/SNF may either indirectly effect DNA replication by effecting the expression of Cdc6p, or has a redundant function with Cdc6p. In addition, several crippled derivatives of ARS1 acquire SWI/SNF dependence, and it is found that the SWI/SNF complex requires a transcriptional activation domain to enhance ARS1 function. These results reinforce the view that SWI/SNF play a role in two chromatin-mediated processes', transcription and DNA replication.

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)