Small RNA silencing pathways regulate development, viral defense, and genomic integrity in all kingdoms of life. An Argonaute (Ago) protein, guided by a tightly bound, small RNA or DNA, lies at the core of these pathways. Argonaute uses its small RNA or DNA to find its target sequences, which it either cleaves or stably binds, acting as a binding scaffold for other proteins. We used Co-localization Single-Molecule Spectroscopy (CoSMoS) to analyze target binding and cleavage by Ago and its guide. We find that both eukaryotic and prokaryotic Argonaute proteins re-shape the fundamental properties of RNA:RNA, RNA:DNA, and DNA:DNA hybridization: a small RNA or DNA bound to Argonaute as a guide no longer follows the well-established rules by which oligonucleotides find, bind, and dissociate from complementary nucleic acid sequences. Counter to the rules of nucleic acid hybridization alone, we find that mouse AGO2 and its guide bind to microRNA targets 17,000 times tighter than the guide without Argonaute. Moreover, AGO2 can distinguish between microRNA-like targets that make seven base pairs with the guide and the products of cleavage, which bind via nine base pairs: AGO2 leaves the cleavage products faster, even though they pair more extensively. This thesis presents a detailed kinetic interrogation of microRNA and RNA interference pathways. We discovered sub-domains within the previously defined functional domains created by Argonaute and its bound DNA or RNA guide. These sub-domains have features that no longer conform to the well-established properties of unbound oligonucleotides. It is by re-writing the rules for nucleic acid hybridization that Argonautes allow oligonucleotides to serve as specificity determinants with thermodynamic and kinetic properties more typical of RNA-binding proteins than that of RNA or DNA. Taken altogether, these studies further our understanding about the biology of small RNA silencing pathways and may serve to guide future work related to all RNA-guided endonucleases.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes lethal invasive pulmonary disease in immunocompromised hosts and allergic asthma in sensitized individuals. This dissertation explores how eosinophils may protect hosts from acute infection while driving asthma pathogenesis by co-producing IL-23 and IL-17 in both contexts. In an acute model of pulmonary aspergillosis, eosinophils were observed to associate with and kill A. fumigatus spores in vivo. In addition, eosinopenia was correlated with higher mortality rates, decreased recruitment of inflammatory monocytes to the lungs, and decreased expansion of lung macrophages. As IL-17 signaling must occur on a local level to elicit its stereotypical response, such as the up-regulation of antimicrobial peptides and specific chemokines from stromal cells, eosinophils were discovered to be a significant source of pulmonary IL-17 as well as one of its upstream inducers, IL-23. In the context of asthma, this discovery opens a new paradigm whereby eosinophils might be driving asthma pathogenesis.

Development and aging, two fundamental aspects of life, remain key biological processes that researchers try to understand. Drosophila melanogaster, thanks to its various merits, serves as an excellent model to study both of these processes. This thesis includes two parts. In the first part, I discuss our finding that the presynaptic neuron controls a retrograde signaling pathway by releasing essential components via exosomes. During synaptic development, postsynaptic cells send retrograde signals to adjust the activity and growth of presynaptic cells. It remains unclear what the mechanism is which triggers the release of retrograde signals; and how presynaptic cells are involved in this signaling event. The first part of this thesis demonstrates that a retrograde signal mediated by Synaptotagmin4 (Syt4) depends on the anterograde delivery of Syt4 protein from the presynaptic neuron to the muscle compartment likely through exosomes. This trans-synaptic transfer of Syt4 is required for the retrograde control of activity-dependent synaptic growth at the Drosophila larval neuromuscular junction. In the second part of this thesis, I talk about our discovery that the disruption of nuclear envelope (NE) budding, a novel RNA export pathway, is linked to the loss of mitochondrial integrity and premature aging in Drosophila. We demonstrate that several transcripts, which are essential for mitochondrial integrity and function, use NE-budding for nuclear export. Transgenic Drosophila expressing a LamC mutation modeling progeroid syndrome (PS), a premature aging disorder in humans, displays accelerated aging-related phenotypes including progressive mitochondrial degeneration as well as decreased levels of a specific mitochondrial transcript which is normally enriched at NE-budding site. The PS-modeled LamC mutants exhibit abnormal lamina organization that likely disrupts the egress of these RNAs via NE-budding. These results connect defective RNA export through NE-budding to progressive loss of mitochondrial integrity and premature aging in Drosophila.

Background: Traumatic injury remains a major cause of mortality in the US. Older Americans experience lower rates of injury and higher rates of death at lower injury severity than their younger counterparts. The objectives of this study were to explore pre-injury factors and injury patterns that are associated with post-discharge mortality among injured elderly surviving index hospitalization. Methods: We queried a 5% random sample of Medicare beneficiaries (n=2,002,420) for any hospitalization with a primary ICD-9 diagnosis code for injury. Patients admitted without urgent/emergent admission were excluded, as well as patients presenting from inpatient hospitalization or rehabilitation. The primary endpoint was all-cause mortality. Patients were categorized into three mortality groups: death within 0-30 days, 31-90 days, or 91- 365 days post-discharge from the index hospitalization. These groups were compared with those who survived greater than one year post-discharge. Univariate tests of association and multivariable logistic regression models were utilized to identify factors associated with mortality during the 3 examined periods. Results: 83,439 elderly patients (4.2%) were admitted with new injuries. 63,628 met inclusion criteria. 1,936 patients (3.0%) died during their index hospitalization, 2,410 (3.8%) died within 0-30 days, 3,084 (4.8%) died within 31-90 days, and 5,718 (9.0%) died within 91- 365 days after discharge. In multivariable adjusted models, advanced age, male sex, and higher Elixhauser score were associated with post-discharge mortality. The presence of critical injury had the greatest effect on mortality early after injury (0-30 days, OR 1.81, CI 1.64-2.00). Discharge to anywhere other than home without services was associated with an increased odds of dying. Conclusions: Socio-demographic characteristics, disposition, and co-morbid factors were the strongest predictors of post-discharge mortality. Efforts to reduce injury-related mortality should focus on injury prevention and modification of co-morbidities.

Nicotine dependence is hypothesized to be due to neuroadaptations that ultimately drive compulsive nicotine use. The studies in this thesis aim to understand how the “upregulation” of nicotinic acetylcholine receptors (nAChRs) caused by chronic exposure to nicotine contributes to nicotine reward and nicotine withdrawal. Previous studies have shown that chronic nicotine induces upregulation of nAChRs containing the α4 subunit (α4* nAChR) within the Ventral Tegmental Area (VTA), a brain region critical for the rewarding properties of all illicit drugs. Curiously, α4* nAChR upregulation occurs specifically in the inhibitory GABAergic neuronal subpopulation of the VTA. To determine if increased expression and activation of α4* nAChRs in VTA GABAergic neurons contributes to nicotine dependence behaviors, I devised a viral-mediated, Creregulated gene expression system that selectively expressed α4 nAChR subunits containing a “gain-of-function” point mutation (a leucine mutated to a serine residue at the TM2 9´ position: Leu9´Ser) in VTA GABAergic neurons of adult mice. Sub-reward doses of nicotine were sufficient to activate VTA GABAergic neurons in mice expressing Leu9´Ser α4 nAChR subunits in VTA GABAergic neurons (Gad2VTA: Leu9´Ser mice) and exhibited acute hypolocomotion upon initial injection of low doses of nicotine that developed tolerance with subsequent nicotine exposures compared to control animals. In the conditioned place preference procedure, nicotine was sufficient to condition a significant place preference in Gad2VTA: Leu9´Ser mice at low nicotine doses that failed to condition control animals. I conclude from these data that upregulating α4* nAChRs on VTA GABAergic neurons increases sensitivity to nicotine reward. In a separate study testing the hypothesis that overexpression of Leu9´Ser α4* nAChRs in VTA GABAergic neurons disrupts baseline behavior and promotes anxiety-like behaviors, I found that overexpressing Leu9´Ser α4* nAChRs in VTA GABAergic neurons had a minimal effect on unconditioned anxiety-like behaviors. Drug naïve Gad2VTA: Leu9´Ser and control mice failed to exhibit any behavioral differences in the open-field, marble burying test and elevated plus maze compared to control. Together, these data indicate that overexpression of the “gain-of-function” α4* nAChRs in VTA GABAergic neurons contributes to reward sensitivity without increasing susceptibility to nicotine withdrawal symptoms. My data indicates that nAChRs expressed in VTA GABAergic neurons may be a suitable target for the development of better smoking cessation aids.

Enterohemorrhagic Escherichia coli 0157:H7 (EHEC) and Enteropathogenic E. coli (EPEC) are intestinal pathogens that induce characteristic lesions on mammalian cells called actin pedestals. Attachment to host cells by both EPEC and EHEC is an essential step towards colonization and is associated with the formation of highly organized actin cytoskeletal elements termed as attaching and effacing (AE) lesions beneath bound bacteria. The outer membrane protein intimin is required for the formation of these structures and binds its own translocated mammalian cell receptor called Translocated intimin receptor (Tir). These interactions induce a cascade of events that result in actin pedestal formation. In this thesis, we characterized pedestal formation and the requirements of pedestal formation by host adapted and in vitro cultivated EHEC. Our data indicate that growing EHEC in the mammalian host enhances bacterial cell attachment, expression and translocation of virulence effectors and actin signaling, and this enhancement is likely to entail more than one bacterial activity involved in host cell interactions. We also focused on the interaction between the two key bacterial players involved in pedestal formation, intimin and Tir. We randomly mutagenized the Tir-binding domain of intimin and isolated point mutants that disrupted Tir recognition. The ability of intimin mutants to bind to recombinant Tir correlated with their ability to trigger AE lesions on pre-infected mammalian cells. Half of the mutations fell within the previously identified 50 amino acid C-terminal region of intimin, and alanine scanning mutagenesis of this region identified four residues of EHEC intimin that are critical for Tir recognition. In a model of the EHEC intimin-Tir complex that is based on EPEC intimin and Tir, these four amino acids are predicted to be located at the intimin-Tir interface, indicating that these residues play a functional role in intimin recognition by Tir. To identify critical residues involved in intimin recognition and intimin mediated actin signaling, we generated point mutations in the extracellular domain of EHEC Tir. Based on our data, we conclude that Tir-intimin interaction is essential for triggering actin pedestals, and intimin function in the context of Tir signaling can be replaced by proteins that are entirely unrelated to intimin but that bind to Tir. These data are concordant with the model that intimin functions to cluster Tir in the membrane to induce actin assembly. Finally, as a step to study downstream actin signaling processes after Tir translocation, we mapped the domain of Tir involved in host cell signaling. We found that the clustering of a 12 amino acid stretch of C-terminus encompassing the Nck binding sequence of Tir generated actin nucleation indistinguishable from that mediated by the entire C-terminus, and abrogation of Nck binding by mutation of Y474 to Phenylalanine abolished actin assembly. Although these results do not rule out a role for other domains of Tir involved in actin pedestal formation, this suggests that the essential element of Tir consists of the Nck binding domain.

The development of cancer is a multi-step process which involves a series of events, including activation of oncogenes and loss of tumor suppressor function, leading to cell immortalization and misregulated proliferation. In the last few years, the importance of epigenetic defects in cancer development has become increasingly recognized. While most epigenetic studies focus on silencing of tumor suppressors, this thesis addresses defects in the maintenance of silenced heterochromatin in cancer, particularly breast cancer. Breast cancer is a leading cause of cancer in women and many familial cases have been linked to mutations in the breast cancer susceptibility genes, BRCA1 and BRCA2. BRCA1 has been linked to DNA repair as well as multiple other cellular processes, including cell cycle checkpoints, ubiquitination, centrosome function, and meiotic silencing of the XY body. This work began with a particular interest in the report that BRCA1 was linked to the failed maintenance of random X-inactivation in female somatic cells, via a role in supporting XIST RNA localization to the inactive X chromosome (Xi). XIST RNA is a non-coding RNA that fully coats or “paints” the Xi and induces its silencing. Work presented in Chapter II substantially clarifies the relationship of BRCA1 to XIST RNA, based on several lines of experimentation. Loss of BRCA1 does not lead to loss of XIST RNA in these studies, nor did reconstitution of HCC1937 BRCA1-/- tumor cells with BRCA1 lead to XIST RNA localization on Xi, although an effect on XIST RNA transcription is possible. Studies of BRCA1 localization with Xi showed that BRCA1 has a limited association with the Xi in ~3-10% of cells, it rarely colocalizes with XIST RNA to a significant extent, but rather is in close apposition to a small part of the XIST RNA/Xi territory. Additionally, analysis of several breast cancer cell lines revealed mislocalization of XIST RNA in some breast cancer cell lines. Many studies have examined BRCA1 foci that form following DNA damage and demonstrated that these are sites of repair. However, whether the numerous large foci consistently present in normal S-phase nuclei were storage sites or had any function was unknown. In Chapter III, I demonstrate that the BRCA1 foci in normal S-phase nuclei associate overwhelmingly with specific heterochromatic regions of the genome. More specifically, BRCA1 foci often associate with centromeric or pericentromeric regions in both human and mouse cells. In human cells BRCA1 foci often appear juxtaposed to centromeric signal, whereas in mouse, BRCA1 often rings or paints the large chromocenters, clusters of DAPI-dense pericentric and centric heterochromatin. Using PCNA and BrdU as markers of replication, I demonstrate that BRCA1 preferentially associates with the chromocenters during their replication, although high-resolution analysis indicates that BRCA1 and PCNA foci rarely directly overlap. Interestingly, cells with defects in BRCA1 were found to have lagging chromosomes and DNA bridges which nearly always contained satellite DNA, which is consistent with the possibility that BRCA1 deficit contributes to failed separation of sister chromatids at the centromere. This is consistent with other recent reports that BRCA1 is necessary for DNA decatenation by topoisomerase II during routine replication and with my demonstration that topoisomerase II also accumulates on pericentric heterochromatin (PCH) during replication. Chapter IV presents recent work which reveals that RNA is commonly expressed from the centric/pericentric heterochromatin and appears to be linked to its replication. In mouse cells RNA from heterochromatic sequences is readily detected using a broad molecular cytological assay for repeat transcription (the COT-1 RNA assay). In addition to a more dispersed nucleoplasmic signal from euchromatic nuclear regions, distinct localized foci of repeat RNA are detected with COT1 probe or pancentromeric probe. Further analysis with the minor satellite (centromere proper) and the major satellite (comprising the larger pericentric heterochromatin) reveals that the large RNA foci often contain these satellite sequences, long thought to be essentially silent. These foci generally associate with the PCH of chromocenters, and produce various patterns similar to BRCA1- including a larger signal partially painting or ringing the chromocenter in a fraction of cells. In conjunction again with PCNA staining, it was possible to determine that the major satellite RNAs associate with the chromocenters during replication. While the satellite RNA co-localizes precisely with PCNA, neither of these co-localizes at high resolution with BRCA1, although they all are present on replicating chromocenters contemporaneously. These findings show that satellite RNAs are more widely expressed in normal cells than previously thought and link their expression to replication of centromere-linked heterochromatin. Finally, Chapter V presents three lines of recent results to support a major concept forwarded in this manuscript: that loss of Xi heterochromatin may reflect defects in the broader heterochromatic compartment, which may be manifest at multiple levels. I provide evidence using two new assays that both the peripheral heterochromatic compartment and the expression and silencing of satellite repeats is commonly compromised in cancer, although this appears to vary among cancer lines or types. The final results connect back to the question with which I began: what maintains XIST RNA localization to the chromosome in normal cells. These results demonstrate for the first time that Aurora B Kinase activity, mediated by Protein Phosphatase 1 (PP1) during interphase, controls the interphase retention and mitotic release of XIST RNA from the chromosome, likely linked to chromatin modifications such as H3Ser10 phosphorylation. As Aurora B Kinase is commonly over-expressed in cancer and is linked to chromatin changes, this exemplifies one type of mechanism whereby broad epigenetic changes in cancer may impact XIST RNA localization and the maintenance of heterochromatin more generally. This thesis represents a melding of cancer biology with the study of X inactivation and heterochromatin, with findings of fundamental interest to both of these fields.

Despite the current understanding that sensorimotor circuits function through the action of transmitters and modulators, we have a limited understanding of how the nervous system directs the flow of information necessary to orchestrate complex behaviors. In this dissertation, I aimed to uncover how the nervous system coordinates these behaviors using the escape response of the soil nematode, Caenorhabditis elegans, as a paradigm. C. elegans exhibits a robust escape behavior in response to touch. The worm typically moves forward in a sinusoidal pattern, which is accompanied by exploratory head movements. During escape, the worm quickly retreats by moving backward from the point of stimulus while suppressing its head movements. It was previously shown that the biogenic amine tyramine played an important role in modulating the suppression of these head movmemetns in response to touch. We identified a novel tyramine-gated chloride channel, LGC-55, whose activation by tyramine coordinates motor programs essential for escape. Furthermore, we found that changing the electrical nature of a synapse within the neural circuit for escape behavior can reverse its behavioral output, indicating that the C. elegans connectome is established independent of the nature of synaptic activity or behavioral output. Finally, we characterized a unique mutant, zf35 , which is hyperactive in reversal behavior. This mutant was identified as a gain of function allele of the C. elegans P/Q/N-type voltage-gated calcium channel, UNC-2. Taken together, this work defines tyramine as a genuine neurotransmitter and completes the neural circuit that controls the initial phases of the C. elegans escape response. Additionally, this research further advances the understanding of how the interactions between transmitters and ion channels can precisely regulate neural circuit activity in the execution of a complex behavior.

As sequencing technology continues to produce better quality genomes at decreasing costs, there has been a recent surge in the variety of data that we are now able to analyze. This is particularly true with regards to our understanding of the human genome—where the last decade has seen data advances in primate epigenomics, ancient hominid genomics, and a proliferation of human polymorphism data from multiple populations. In order to utilize such data however, it has become critical to develop increasingly sophisticated tools spanning both bioinformatics and statistical inference. In population genetics particularly, new statistical approaches for analyzing population data are constantly being developed—unfortunately, often without proper model testing and evaluation of type-I and type-II error. Because the common Wright-Fisher assumptions underlying such models are generally violated in natural populations, this statistical testing is critical. Thus, my dissertation has two distinct but related themes: 1) evaluating methods of statistical inference in population genetics, and 2) utilizing these methods to analyze the evolutionary history of humans and our closest relatives. The resulting collection of work has not only provided important biological insights (including some of the first strong evidence of selection on human-specific epigenetic modifications (Shulha, Crisci, Reshetov, Tushir et al. 2012, PLoS Bio), and a characterization of human-specific genetic changes distinguishing modern humans from Neanderthals (Crisci et al. 2011, GBE)), but also important insights in to the performance of population genetic methodologies which will motivate the future development of improved approaches for statistical inference (Crisci et al, in review).

Antigen presented to CD4+ T cells by major histocompatibility complex class II molecules (MHCII) plays a key role in adaptive immunity. Antigen presentation is initiated by the proteolytic cleavage of pathogenic or self proteins and loading of resultant peptides to MHCII. The loading and exchange of peptides to MHCII is catalyzed by a nonclassical MHCII molecule, HLA-DM (DM). It is well established that DM promotes peptide exchange in vitro and in vivo. However, the mechanism of DM-catalyzed peptide association and dissociation, and how this would affect epitope selection in human responses to infectious disease remain unclear. The work presented in this thesis was directed towards the understanding of mechanism of DM-mediated peptide exchange and its role in epitope selection. In Chapter II, I measured the binding affinity, intrinsic dissociation half-life and DM-mediated dissociation half-life for a large set of peptides derived from vaccinia virus and compared these properties to the peptide-specific CD4+ T cell responses. These data indicated that DM shapes the peptide repertoire during epitope selection by favoring the presentation of peptides with greater DM-mediated kinetic stability, and DM-susceptibility is a strong and independent factor governing peptide immunogenicity. In Chapter III, I computationally simulated peptide binding competition reactions and found that DM influences the IC50 (50% inhibition concentration) of peptides based on their susceptibility to DM, which was confirmed by experimental data. Therefore, I developed a novel fluorescence polarization-based method to measure DM-susceptibility, reported as a IC50 (change in IC50 in the absence and presence of DM). Traditional assays to measure DM-susceptibility based on differential peptide dissociation rates are cumbersome because each test peptide has to be individually labeled and multiple time point samples have to be collected. However, in this method developed here only single probe peptide has to be labeled and only single reading have to be done, which allows for fast and high throughput measure of DM-susceptibility for a large set of peptides. In Chapter IV, we generated a series of peptide and MHCII mutants, and investigated their interactions with DM. We found that peptides with non-optimal P1 pocket residues exhibit low MHCII affinity, low kinetic stability and high DM-susceptibility. These changes were accompanied with conformational alterations detected by surface plasmon resonance, gel filtration, dynamic light scattering, small-angle X-ray light scattering, antibody-binding, and nuclear magnetic resonance assays. Surprisingly, all these kinetic and conformational changes could be reversed by reconstitution with a more optimal P9 pocket residue. Taken together, our data demonstrated that conformation of MHCII-peptide complex constrained by interactions throughout the peptide binding groove is a key determinant of DM-susceptibility. B cells recognizing cognate antigen on the virion can internalize and process the whole virion for antigen presentation to CD4+ T cells specific for an epitope from any of the virion proteins. In turn, the epitope-specific CD4+ T cells provide intermolecular (also known as noncognate or heterotypic) help to B cells to generate antibody responses against any protein from the whole virion. This viral intermolecular help model in which CD4+ T cells provide help to B cells with different protein specificities was established in small size influenza virus, hepatitis B virus and viral particle systems. For large and complex pathogens such as vaccinia virus and bacteria, the CD4+ T cell-B cell interaction model may be complicated because B cells might not be able to internalize the large whole pathogen. Recently, a study in mice observed that CD4+ T cell help is preferentially provided to B cells with the same protein specificity to generate antibody responses against vaccinia virus. However, for larger pathogens such as vaccinia virus and bacteria the CD4+ T cell-B cell interaction model has yet to be tested in humans. In Chapter V, I measured in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors the CD4+ T cell responses and antibody responses against four vaccinia viral proteins (A27L, A33R, B5R and L1R) known to be strongly targeted by cellular and humoral responses. We found that there is no direct linkage between antibody and CD4+ T cell responses against each protein. However, the presence of immune responses against these four proteins is linked together within donors. Taken together, our data indicated that individual viral proteins are not the primary recognition unit and CD4+ T cells provide intermolecular help to B cells to generate robust antibody responses against large and complicated vaccinia virus in humans.