A challenge faced by investigators who use Adeno Associated Virus (AAV) vectors lies in verifying successful assembly of the genetic construct developed for a given experimental manipulation. Failure at this step can introduce mutations that produce non-functional proteins and failure to obtain the desired expression. For these reasons, quality control measures support generation of valuable data and prevention of resource loss. Using molecular techniques such as PCR and Sanger Sequencing, we developed a quality control pipeline to amplify and identify viral sequences. Viruses were selected from stock for an identity assay based on relevance to planned optogenetics procedures, and PCR amplicons were designed to contain functional regions of each viral construct. Amplification was carried out using universal primers targeting 5'ITR and WPRE regions, which were common to all assembled viruses. Sanger sequencing was used to confirm that the amplicon sequence matched the viral template. Using the outlined procedure, we can now amplify, purify, and sequence the vector genome to gain nucleotide level confirmation of coding gene sequences prior to use in experiments.

Adeno-Associated Virus (AAV) has been used as a vector to deliver DNA to cells for transgene expression. When used to target cells in the brain, these injections are typically made directly into that tissue. Several factors determine whether injection of AAV causes expression of the transgene. A neutralizing antibody response may impede transgene expression through the binding of antibodies to viral vectors, which would prevent gene delivery. Different AAV serotypes have different epitopes, or binding regions for antibodies. However, epitopes may be shared between closely-related AAV serotypes. AAV-PHP.eB is a modification of AAV9 with improved ability to cross the blood brain barrier. This serotype could be injected intravenously to transfect cells in the brain, allowing for less invasive gene delivery as compared to brain injection. It is unclear whether a neutralizing antibody response to AAV9 would also prevent AAV-PHP.eB from infecting neurons. To test this, I will infect human embryonic kidney (HEK293) cells with AAV-PHP.eB in the presence and absence of blood serum taken from macaques before and after injection with AAV9, AAV-PHP.eB and other AAV serotypes. I hope to determine whether blood serum from macaques that have been injected with either AAV9 or AAV-PHP.eB, but not both, contains neutralizing antibodies that reduce the efficacy of the AAV serotype that was not injected. While blood serum from some macaques indicates the presence of antibodies for AAV serotypes other than the ones they were injected with, further analysis must be done before drawing conclusions. This study has implications for researchers conducting gene therapy research both in non-human primates and in human clinical trials, as the choice of serotype may determine whether or not the gene product is effectively delivered.

The Civil Rights Movement of the 1960s in the United States galvanized millions of Americans to fight for a more free and democratic society, banding together to protest racial segregation and other forms of systemic racism such as police violence against minorities. Newspapers covered these actions extensively, spreading the message of civil rights across the US. People eager for change in cities far from the centers of civil rights activism in the South, such as Seattle, responded to this national political fervor by fighting for change locally. In Seattle, activists sought an end to job and housing discrimination, de facto school segregation, and police violence through non-violent direct action. Seattle’s major print newspapers, The Seattle Times and the Seattle Post-Intelligencer covered these issues extensively, spreading news and controversial developments to their readers. In my research, I analyze newspaper coverage on activism and cases of police violence which garnered a strong public demand for justice. With the support of other sources, such as the biographies of Seattle activists, the histories of local civil rights organizations, and studies on media coverage of the police, I construct an analysis of how these newspapers shifted their coverage of civil rights activism and police violence throughout the 1960s as a response to community activism. This critical angle focuses on how the actions of Seattle’s activist community influenced newspaper media, prompting the newspapers to include more activist perspectives in their news coverage. This research, therefore, displays the power that local activists held in influencing print media coverage of their actions, and with that, the influence that activists had to shift the public perspective towards activism in the 1960s in Seattle.