Specific Aims

1. Specific Aims • The Specific Aims are the first part of the proposal that the reviewer (committee member) reads. • This section should clearly state the hypotheses being tested. • The aims should clearly and succinctly outline the proposed research to test the hypotheses.

2. Specific Aims (from NIH) • State concisely the goals of the proposed research and summarize the expected outcome, including the impact that the results of the proposed research will exert on the research field involved. • List succinctly the specific objectives of the research proposed, e.g., to test a stated hypothesis, create a novel design, solve a specific problem, challenge an existing paradigm or address a critical barrier to progress in the field. • Specific Aims are limited to one page

3. Useful Tips on Specific Aims • Do include a brief description of the problem being investigated and a short background summary of what is known to educate the reviewer (One paragraph). • Do state the long term goal of the research. • Do state the hypothesis being tested. • Do state the aims as a bulleted list with a brief description of the approaches to be used after each aim.

4. Brief summary of background Long term goal Hypothesis Specific Aims Bulleted list Impact

5. 2. Specific Aims The epilepsies are a large group of disorders characterized by abnormal electrical activity in the CNS, affecting up to 3% of the population (Hauser et al., 1993). Approximately 40% of this group are considered idiopathic, in which the underlying cause is most likely a genetic abnormality (Steinlein, 2002). The specific abnormality has been identified in only a small minority of cases, most of which are channelopathies. Even in those cases, the mechanisms by which the defects result in epilepsy are not understood. One class of channelopathies that cause epilepsy are mutations in voltage-gated sodium channels, which cause a number of different syndromes including Generalized Epilepsy with Febrile Seizures Plus (GEFS+) and Severe Myoclonic Epilepsy of Infancy (SMEI). GEFS+ is caused by missense mutations in the SCN1A gene encoding the Nav1.1 sodium channel. SMEI, which has a more severe phenotype, is most commonly caused by nonsense and frameshift mutations in the same gene. We and others have shown that GEFS+ mutations have many different effects in heterologous expression systems. However, we have also shown that those effects do not represent the phenotype in native neurons, and that at least one GEFS+ mutation decreases channel activity in inhibitory neurons in mice (Progress Report). This result is consistent with the observation that loss of one Scn1a allele in knockout mouse models of SMEI decreases sodium channel activity in inhibitory neurons (Yu et al., 2006;Ogiwara et al., 2007). Based on these results, we have developed the following hypothesis: Sodium channel mutations that cause GEFS+ decrease Nav1.1 activity in inhibitory neurons, particularly parvalbumin-positive basket cells, leading to decreased inhibition on excitatory neurons. The goal of the proposed research is to test that hypothesis and determine the mechanism by which two sodium channel mutations cause GEFS+. Aim 1. To determine which neuronal populations express the R1648H GEFS+ mutant sodium channels and to characterize the mutant channels in those cells. Our analysis of transgenic and knock-in mice expressing the R1648H GEFS+ mutation demonstrated decreased sodium channel activity in bipolar inhibitory neurons, similar to the observations of Yu et al. (2006) in Scn1a knockout mice. Because those studies were carried out using dissociated cells, it was not possible to distinguish among the many different populations of interneurons. Ogiwara et al. (2007) showed that Nav1.1 was highly expressed in parvalbumin-positive interneurons, but they did not test other populations of interneurons. Our first aim is to determine the specific populations of inhibitory neurons affected by the GEFS+ mutations. We will identify the specific interneuron cell types expressing the R1648H mutant sodium channels and characterize the properties of those channels by recording from dissociated neurons from mice in which specific populations are tagged with fluorescent proteins. Aim 2. To determine the mechanism by which altered sodium channel activity resulting from the R1648H mutation leads to greater seizure susceptibility in GEFS +. The second prediction of our working hypothesis is that the R1648H mutation decreases somatic inhibition on excitatory pyramidal neurons. We will test that prediction using three approaches. First, we will determine how the mutation affects the firing properties of specific classes of interneurons by current clamp recordings. Second, we will test for epileptiform discharges in field recordings using techniques to increase excitability. We will also test if manipulations that decrease inhibition mimic the effects of the mutation in wild-type mice, and if manipulations that increase inhibition restore normal network stability in R1648H mice. Finally, we will determine the effects on synaptic connectivity by whole cell patch-clamp recordings combined with laser scanning photostimulation, in collaboration with Dr. Xiangmin Xu (Xu and Callaway, 2009). Aim 3. To determine if a second GEFS+ mutation (D1866Y) also causes decreased firing of inhibitory neurons in the CNS. The D1866Y mutation also increased sodium channel activity in oocytes, but did so by weakening the interaction between the C-terminus of the  subunit and the 1 subunit (Spampanato et al., 2004b). Therefore, this mutation represents a second case in which increased sodium channel activity was observed in oocytes, but due to a different mechanism. We will determine if the D1866Y mutation also decreases sodium channel activity in inhibitory neurons leading to decreased firing, and examine the role of the 1 subunit interaction in that effect.

6. What is the long term goal of the proposed project? • Describe the problem you are investigating • What is known • What isn’t known • Why is the problem significant • What do you hope to accomplish in your studies

7. Badly written long term goal Recent evidence has shown that substitution mutations in the nuclear export signal of HTLV-1 rex protein results in a delay in the export kinetics of rex. This delay in export activity will be further analyzed and is the overall goal of this project.

8. Badly written goals • Latent infection of Epstein Barr Virus (EBV) has been linked to human malignancies of lymphoid and epithelial cell lineage. The latent virus expresses a limited set of viral genes that contribute to host-cell growth transformation and viral genome survival. EBNA1 is a latency-associated gene product that is consistently expressed in all EBV-associated tumor cells and is essential for latent genome maintenance. • The goals of this application are to further our understanding of EBNA1 in viral latent cycle, DNA replication and episome maintenance. We will also investigate possible strategies for EBNA1 and OriPfunctions essential for maintaining the episomal state during latency.

9. Well written long term goals • Influenza vaccines are safe but have varying effectiveness due to antigenic variation of circulating influenza viruses in the human population. Influenza vaccine is updated every year with new viruses but due to the time required to determine that a new strain is spreading and to adapt it for vaccine use, the vaccine is usually a year behind the virus. • The long term goal of our work is to develop a vaccine strategy for influenza that is more effective against antigenic drift variants than the current updating of vaccine viruses, which is typically a year behind the circulating viruses. The overall goal of the work proposed is to characterize the antigenic sites recognized by antibodies in human sera that are induced by vaccination or infection and to determine how antigenic variants are selected by polyclonal antibodies in the human population.

10. What is the hypothesis being tested? • After you state the long term goal of the project, clearly define the hypothesis that you are proposing and that you will be testing in your studies.

11. What is the hypothesis?

12. What is the hypothesis? Previously, we have investigated replication of measles virus in human lymphoid cells, i.e., T cells as well as B cells. We propose to extend these studies and determine the effects of infection on the ability of B cells to present specific antigens to T cell lines. The importance of these studies will determine how infection of lymphocytes by measles virus can alter functional responses to antigen.

13. What is the hypothesis? • The proposed studies will combine molecular cell biology, biochemistry, and virology methods to provide a greater understanding of the molecular basis of host evasion strategies used by two medically important Paramyxoviruses, measles virus and mumps virus. It is anticipated that investigation of these host evasion strategies will also reveal fundamental principles of cellular antiviral responses, and should also lead to new therapies for the treatment and prevention of these medically important virus infections.

14. Well written hypothesis • The Alphavirus genus in the Togaviridae family contains a number of significant human and animal pathogens that are widely distributed on all continents, excluding the Antarctic regions. In the present proposal, we will focus on further understanding the mechanism of alphavirus RNA replication, with particular emphasis on cellular protein functioning in this process. The proposed research plan is aimed at testing the hypothesis that alphavirus replicative complexes (RCs) are directly associated with cellular cytoskeletal fibrils, and that cellular endosomes are involved in transporting the RCs inside eukaryotic cells to points of RNA synthesis and nucleocapsid assembly.

15. Clear Hypothesis • The retinoblastoma susceptibility protein (pRb) plays a critical role in control of cellular proliferation, differentiation, and apoptosis, in part by regulating E2F transcription factors. It is a key target of oncoproteins expressed by DNA tumor viruses. Despite little prior evidence that RNA viruses regulate pRb function, we have discovered that pRb abundance is negatively regulated post-translationally in cells infected with hepatitis C virus (HCV), a human virus that is strongly associated with hepatocellular carcinoma. • Our central hypothesis is that the HCV NS protein-dependent degradation of pRb benefits the viral life cycle by promoting cellular proliferation or otherwise favorably altering the intracellular environment for viral RNA replication, and that changes in cellular homeostasis associated with pRb loss contribute to the carcinogenic potential of HCV infection.

16. Well written hypothesis • The Specific Aims for this proposal are based on the hypothesis that epitopes are not equally immunogenic and the bulk of the antibody response may be to a small subset of antigenic sites. Accumulation of mutations during antigenic drift may cause particular antigenic sites to become less or more immuno-dominant.

17. Specific Aims • What do you intend to do?

18. Specific Aims • The Specific Aims should clearly and succinctly outline the proposed research. • The Specific Aims should be a bulleted list.

19. Poorly written specific aims What are the Specific Aims?

20. The Specific Aims should clearly and succinctly outline the proposed research. The specific aims should be a bulleted list not long narratives.

21. Badly written specific aims Specific Aims • Aim 1: Viruses expressing substitution mutations in the nuclear export sequence will be used in time course experiments. At 4, 8, and 12-hours after infection, co-localization studies will be performed between rex and CRM1. If necessary, the time course will be prolonged to include later time points. This study will be furthered by observing the trafficking of rex using live cell imaging. • Aim 2: The results will be further corroborated with co-immunoprecipitation studies.

22. Clearly written specific aims • The hypothesis that underlies this proposal is that an improved understanding of herpesvirus DNA packaging and the biochemical activities of the terminase will facilitate development of novel antivirals that selectively inhibit those activities. • Aim 1. Define the cis-acting cleavage and packaging sequences of HCMV • Aim 2. Identify proteins that interact with cis-acting DNA sequences and define the DNA sequence elements important for these interactions • Aim 3. Determine the in vitro biochemical activities of purified HCMV terminase, including the identification of cellular and viral proteins that interact with one or more subunits • Aim 4. Evaluate the impact of terminase mutations on viral replication

23. The specific aims of this proposal are designed to test the hypothesis that treatment of selected host immune response molecules can be useful for resolving persistent infections and to develop a mechanism to enhance a more successful vaccination strategy. The specific aims also seek to define the mechanism and host cell(s) responsible for the IL-10 effect. • Specific Aim #1. Determine the optimal therapeutic dose and kinetics of antibody against IL-10R alone or combined with antibody to PD-1. • 1i. Assay the effectiveness of treatment with antibody to IL-10R during chronic phases of infection when virus-specific T cells are non-responsive. • 1ii. Assay results of combined therapy with antibody to IL-10R and antibody to PD-1. • Specific Aim #2. Detail the mechanism of action of IL-10 during persistent viral infection. • 2i. Use of IL-10 knock out mice in which IL-10 GFP has been knocked-in and use of antibody to IL-10 to determine the cell source of IL-10. • 2ii. Determine if increased IL-10 expression results from direct viral infection of host cells and define the biologic target of IL-10. • Specific Aim #3. Explore a role for IL-10 blockade in generation of a more effective vaccine. • 3i. Prophylactic vaccination. • 3ii. Therapeutic vaccination.

24. The goals of this proposal are to identify cellular targets of HPV E6 and E7 that contribute to their oncogenic function. We will also investigate the role of the Notch signaling pathway in cervical carcinogenesis, as this pathway has been shown to play a role in tumorigenesis in epithelial tissues and is dysregulated in cervical cancers. We hypothesize that Notch signaling contributes to HPV carcinogenesis. We shall use HPV16 transgenic mice expressing E6 and E7 in stratified epithelia, together with knock-in and knock-out mice altered in their expression of Notch pathway genes, to evaluate the role of viral and cellular genes in cervical carcinogenesis. The specific aims of the proposal are: • Aim 1. Mechanisms of Action of HPV16 E7 in Cervical Cancer • 1A. Determine whether inactivation of more than one pocket protein is sufficient to account for E7's role in cervical carcinogenesis • 1B. Define importance of cellular targets other than the pocket proteins in mediating E7's role in the cervical cancer Aim 2. Mechanisms of Action of HPV16 E6 in Cervical Carcinogenesis • 2A. Identify PDZ partners of E6 important in cervical carcinogenesis • 2B. Define the role of p53 as an essential target in E6-mediated cervical carcinogenesis • Aim 3: Role of Notch Signaling in Cervical Carcinogenesis • 3A. Test the requirements for Notch1 in Cervical Carcinogenesis • 3B. Determine the mechanisms by which E6 and E7 activate Notch1

25. Important Tip • The specific aims should be interrelated but should not totally depend on the success of one aim to perform the others. • Example: • Aim 1 proposes to identify and clone the cellular receptor that restricts HXV infection to humans. • Aim 2 plans to construct transgenic mice expressing the receptor to develop an animal model for HXV to study pathogenesis. • What if there is more than one receptor? Or what if you are not successful in identifying putative receptors? Or what if infection is also blocked at a stage past entry?