The outer dynein arm-docking complex (ODA-DC) specifies the outer dynein arm-binding site on the flagellar axoneme. The ODA-DC of Chlamydomonas contains equimolar amounts of three proteins termed DC1, DC2, and DC3 (Takada et al., 2002). DC1 and DC2 are predicted to be coiled-coil proteins, and are encoded by ODA3 and ODA1, respectively (Koutoulis et al., 1997; Takada et al., 2002). Prior to this work, nothing was known about DC3. To fully understand the function(s) of the ODA-DC, a detailed analysis of each of its component parts is necessary. To that end, this dissertation describes the characterization of the smallest subunit, DC3. In Chapter II, I report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 D protein with four EF hands that is a member of the CTER (Calmodulin, Troponin C, Essential and Regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming due to loss of some but not all, outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly. As mentioned above, DC3 has four EF-hands: two fit the consensus pattern for calcium binding and one contains two cysteine residues within its binding loop. To determine if the consensus EF-hands are functional, I purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of DTT and Mg2+. As reported in Chapter III, the protein bound one calcium ion with an affinity (Kd) of ~1 x 10-5 M. Calcium binding was observed only in the presence of DTT and thus is redox sensitive. DC3 also bound Mg2+ at physiological concentrations, but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium-binding activity of the bacterially expressed protein. To investigate the role of the EF hands in vivo, I transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the E to Q mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains obscure. In Chapter IV, I report the initial characterization of a DC3 insertional mutant having a phenotype intermediate between that of the DC3-deletion strain and wild type. Furthermore, I suggest future experiments that may help elucidate the specific role of DC3 in outer arm assembly and ODA-DC function. Lastly, I speculate that the ODA-DC may play a role in flagellar regeneration.

Many polypeptide growth factors act by binding to cell surface receptors that have intrinsic tyrosine kinase activity. Binding of these growth factors to their cognate receptors results in the initiation of mitogenic signals which then get transduced to the interior of the cell. A critical target for extracellular signals is the nucleus. A plethora of recent evidence indicates that extracellular signals can affect nuclear gene expression by modulating transcription factor activity. In this study, I have determined that the transactivation domain of c-Myc (protein product of the c-myc proto-oncogene) is a direct target of mitogen-activated signaling pathways involving protein kinases. Further, my study demonstrates that transactivation of gene expression by c-Myc is regulated as a function of the cell cycle. c-Myc is a sequence-specific DNA binding protein that forms leucine zipper complexes and can act as a transcription factor. Although, significant progress has been made in understanding the cellular properties of c-Myc, the precise molecular mechanism of c-Myc function in oncogenesis and in normal cell growth is not known. I have focused my attention on the property of c-Myc to function as a sequence-specific transcription factor. In my studies, I have employed a fusion protein strategy, where the transactivation domain of the transcription factor c-Myc is fused to the DNA binding domain and nuclear localization signal of the yeast transcription factor GAL4. This fusion protein was expressed together with a plasmid consisting of specific GAL4 binding sites cloned upstream of a minimal E1b promoter and a reporter gene. The activity of the c-Myc transactivation domain was measured as reporter gene activity in cell extracts. This experimental approach enabled me to directly monitor the activity of the c-Myc transactivation domain. Results listed in Chapter II demonstrate that the transactivation domain of c-Myc at Ser-62 is a target of regulation by mitogen-stimulated signaling pathways. Furthermore, I have determined that a mitogen activated protein kinase, p41mapk, can phosphorylate the c-Myc transactivation domain at Ser-62. Phosphorylation at this site results in a marked increase in transactivation of gene expression. A point mutation at the MAP kinase phosphorylation site (Ser-62) causes a decrease in transactivation. c-Myc expression is altered in many types of cancer cells, strongly implicating c-myc as a critical gene in cell growth control. The molecular mechanisms by which c-Myc regulates cellular proliferation are not understood. For instance, it is not clear where in the cell cycle c-Myc functions and what regulates its activity. In exponentially growing cells, the expression levels of c-Myc remain unchanged as the cells progress through the cell cycle. The function of c-Myc may therefore be regulated by a mechanism involving a post-translational modification, such as phosphorylation. Results described in chapter IV demonstrate that the level of c-Myc mediated transactivation oscillates as cells progress through the cell cycle and was greatly increased during the S to G2/M transition. Furthermore, mutation of the phosphorylation site Ser-62 in the c-Myc transactivation domain diminishes this effect, suggesting a functional role for this phosphorylation site in the cell cycle-specific regulation of c-Myc activity. Taken together, my dissertation study reveals a molecular mechanism for the regulation of nuclear gene expression in response to mitogenic stimuli.

Depression is a common comorbidity in rheumatoid arthritis (RA), yet it may not be adequately recognized during routine clinical care. RA symptoms may confer a risk for depression, and vice versa; depression may affect RA disease activity and response to treatment. The study aims were to compare patient- and physician-reported depression measures, evaluate the temporal bi-directional association between RA disease activity and depressive symptomology, and assess depression as a moderator of RA treatment. Patients were identified using a national RA registry sample (Consortium of Rheumatology Researchers of North America; CORRONA). Depression prevalence and incidence rates were estimated, and concordance and disagreement using measures reported separately by patients and physicians, as well as baseline cross-sectional associations between RA disease and a history of depression. A survival analysis was conducted to temporally predict the incident onset of self-reported depressive symptoms using the different metrics of RA disease activity. Also, mixed effects models were used to assess prospective changes in RA disease activity by prevalent and incident depressive symptom status. Lastly, logistic regression models compared the likelihood of clinical response to RA treatment during follow-up in those with and without depression when starting biologic disease modifying anti-rheumatic drug (DMARD) therapy. Patient-reported depression rates were much higher and significantly different from physician based comorbidity estimates. Patient and physician RA disease activity measures were associated with an increased risk for depression onset, but not laboratory-reported serum biomarkers. Similarly, depression was temporally associated with significantly slower rates of decline regarding every patient-reported disease activity measure, some physician-reported metrics, but not acute phase reactants. Moreover, there was a significantly lower probability of achieving clinical remission among those with depression on a biologic DMARD after 6 months and an analogous effect at 12-months that was slightly lower in magnitude, which did not reach statistical significance. Rheumatologists under-reported the occurrence of prevalent and incident depressive symptoms, and thus are likely unaware of its presence in their RA patients. Further, the results suggest the bi-directional effects between these conditions are related to the cognitive and behavioral aspects of depression and their interactions with disease activity, rather than shared immunological mechanisms in the context of cell-mediated immunity. When also considering the impact on clinical response to biologic DMARDS, the findings collectively imply that rheumatologists must address any challenges due to depression to provide the best care to their patients.

Attention Deficit/Hyperactivity Disorder (ADHD) is a common psychiatric disorder of childhood that is characterized by symptoms of inattention, impulsivity/hyperactivity, or a combination of both. Intrinsic brain dysfunction in ADHD can be examined through various methods including resting state functional Magnetic Resonance Imaging (rs-fMRI), which investigates patients’ functional brain connections in the absence of an explicit task. To date, studies of group differences in resting brain connectivity between patients with ADHD and typically developing controls (TDCs) have revealed reduced connectivity within the Default Mode Network (DMN), a resting state network implicated in introspection, mind-wandering, and day-dreaming. However, few studies have addressed the use of resting state connectivity measures as a diagnostic aide for ADHD on the individual patient level. In the current work, we attempted first to characterize the differences in resting state networks, including the DMN and three attention networks (the salience network, the left executive network, and the right executive network), between a group of youth with ADHD and a group of TDCs matched for age, IQ, gender, and handedness. Significant over- and under-connections were found in the ADHD group in all of these networks compared with TDCs. We then attempted to use a support vector machine (SVM) based on the information extracted from resting state network connectivity to classify participants as “ADHD” or “TDC.” The IFGmiddle temporal network (66.8% accuracy), the parietal association network (86.6% specificity and 48.5% PPV), and a physiological noise component (sensitivity 39.7% and NPV 69.6%) performed the best classifications. Finally, we attempted to combine and utilize information from all the resting state networks that we identified to improve classification accuracy. Contrary to our hypothesis, classification accuracy decreased to 54-55% when this information was combined. Overall, the work presented here supports the theory that the ADHD brain is differently connected at rest than that of TDCs, and that this information may be useful for developing a diagnostic aid. However, because ADHD is such a heterogeneous disorder, each ADHD patient’s underlying brain deficits may be unique making it difficult to determine what connectivity information is diagnostically useful.

The CBFbeta-SMMHC fusion protein is expressed in acute myeloid leukemia (AML) samples with the chromosome inversion inv(16)(p13;q22). This fusion protein binds the transcription factor RUNX with higher affinity than its physiological partner CBFbeta and disrupts the core binding factor (CBF) activity in hematopoietic stem and progenitor cells. Studies in the Castilla laboratory have shown that CBFbeta-SMMHC expression blocks differentiation of hematopoietic progenitors, creating a pre-leukemic progenitor that progresses to AML in cooperation with other mutations. However, the combined function of cumulative cooperating mutations in the pre-leukemic progenitor cells that enhance their expansion to induce leukemia is not known. The standard treatment for inv(16) AML is based on the use of non-selective cytotoxic chemotherapy, resulting in a good initial response, but with limited long-term survival. Therefore, there is a need for developing targeted therapies with improved efficacy in leukemic cells and minimal toxicity for normal cells. Here, we used conditional Nras+/LSL-G12D; Cbfb+/56M; Mx1Cre knock-in mice to show that allelic expression of oncogenic N-RasG12D expanded the multi-potential progenitor (MPP) compartment by 8 fold. Allelic expression of Cbfbeta-SMMHC increased the MPPs and short-term hematopoietic stem cells (ST-HSCs) by 2 to 4 fold both alone and in combination with N-RasG12D expression. In addition, allelic expression of oncogenic N-RasG12D and Cbfbeta-SMMHC increases survival of pre-leukemic stem and progenitor cells. Differential analysis of bone marrow cells determined that Cbfb+/MYH11 and Nras+/G12D; vii Cbfb+/MYH11 cells included increased number of blasts, myeloblasts and promyelocytes and a reduction in immature granulocytes, suggesting that expression of N-RasG12D cannot bypass Cbfbeta-SMMHC driven differentiation block. N-RasG12D and Cbfbeta-SMMHC synergized in leukemia, in which Nras+/G12D; Cbfb+/MYH11 mice have a shorter median latency than Cbfb+/MYH11 mice. In addition, the synergy in leukemogenesis was cell autonomous. Notably, leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC showed higher (over 100 fold) leukemia-initiating cell activity in vivo than leukemic cells expressing Cbfbeta-SMMHC (L-IC activity of 1/4,000 and 1/528,334, respectively). Short term culture and biochemical assays revealed that pre-leukemic and leukemic cells expressing N-RasG12D and Cbfbeta-SMMHC have reduced levels of pro-apoptotic protein Bim compared to control. The Nras+/G12D; CbfbMYH11 pre-leukemic and leukemic cells were sensitive to pharmacologic inhibition of MEK/ERK signaling pathway with increasing apoptosis and Bim protein levels but not sensitive to PI3K inhibitors. In addition, knock-down of Bcl2l11 (Bim) expression in Cbfbeta-SMMHC pre-leukemic progenitors decreased their apoptosis levels. In collaboration with Dr. John Bushweller’s and other research laboratories, we recently developed a CBFbeta-SMMHC inhibitor named AI-10-49, which specifically binds to CBFbeta-SMMHC, prevents its binding to RUNX proteins and restores CBF function. Biochemical analysis in human leukemic cells showed that AI-10-49 has significant specificity in reducing the viability of leukemic cells expressing CBFbeta-SMMHC (IC50= 0.83μM), and negligible toxicity in normal cells. Likewise, mouse Nras+/G12D; viii Cbfb+/MYH11 leukemic cells were sensitive to AI-10-49 (IC50= 0.93μM). By using the NrasLSL-G12D; Cbfb56M mouse model, we also show that AI-10-49 significantly prolongs the survival of mice bearing the leukemic cells. Preliminary mechanistic analysis of AI-10-49 activity has shown that AI-10-49 increased BCL2L11 transcript levels in a dose and time dependent manner in murine and human leukemic cells, suggesting that the viability through BIM-mediated apoptosis may be targeted by both oncogenic signals. My thesis study demonstrates that Cbfbeta-SMMHC and N-RasG12D promote the survival of pre-leukemic myeloid progenitors primed for leukemia by activation of the MEK/ERK/Bim axis, and define NrasLSL-G12D; Cbfb56M mice as a valuable genetic model for the study of inv(16) AML targeted therapies. For instance, the novel CBFbeta-SMMHC inhibitor AI-10-49 shows a significant efficacy in this mouse model. This small molecule will serve as a promising first generation drug for targeted therapy of inv(16) leukemia and also a very useful tool to understand mechanisms of leukemogenesis driving by CBFbeta-SMMHC.

Depression is the leading cause of disability among women of reproductive age worldwide. Upwards of 1 in 5 women suffer from perinatal depression. This condition has deleterious effects on several birth outcomes, infant attachment, and children’s behavior/development. Maternal suicide causes 20% of postpartum deaths in depressed women. Although the vast majority of perinatal women are amenable to being screened for depression, screening alone does not improve treatment rates or patient outcomes. Obstetrics/Gynecology (Ob/Gyn) clinics need supports in place to adequately address depression in their patient populations. The primary goal of this thesis is to develop, refine, and pilot test a new low-cost and sustainable stepped care program for Ob/Gyn clinics that will improve perinatal women’s depression treatment rates and outcomes. We developed and beta tested the Rapid Access to Perinatal Psychiatric Care in Depression (RAPPID) Program, to create a comprehensive intervention that is proactive, multifaceted, and practical. RAPPID aims to improve perinatal depression treatment and treatment response rates through: (1) access to immediate resource provision/referrals and psychiatric telephone consultation for Ob/Gyn providers; (2) clinic-specific implementation of depression care, including training support and toolkits; and (3) proactive depression screening, assessment, and treatment in OB/Gyn clinics. RAPPID builds on a low-cost and widely disseminated population-based model for delivering psychiatric care in primary care settings. Formative data and feedback from key stakeholders also informed the development of RAPPID. Our formative and pilot work in real-world settings suggests RAPPID is feasible and has the potential to improve depression detection and treatment in Ob/Gyn settings. The next step will be to compare two active interventions, RAPPID vs. enhanced usual care (access to resource provision/referrals and psychiatric telephone consultation) in a cluster-randomized trial in which we will randomize 12 Ob/Gyn clinics to either RAPPID or enhanced usual care.

BACKGROUND: Latinos report higher food insecurity than the national average, and food insecurity has been associated with adverse health outcomes wherein Latinos experience disparities. This study quantified the independent effects of language-speaking proficiency and citizenship on increased food insecurity among a predominantly immigrant Caribbean Latino sample in Lawrence, Massachusetts. METHODS: The analytic sample comprised 574 participants aged 21-83 who visited a community health center in 2011-2013. Food insecurity was assessed via the 6-item US Household Food Security Survey. Multivariable logistic modeling (adjusted for self-reported age group, gender, education, and marital status) examined the independent associations between language proficiency and citizenship on increased food insecurity. RESULTS: One-third of participants were classified as food insecure. Most respondents were citizens (59.5%), foreign-born (92.4%; 70.3% from the Dominican Republic), and spoke monolingual Spanish (72.8%). Monolingual Spanish-speakers had marginally increased odds of food insecurity (odds ratio (OR) = 1.50, 95% confidence interval (CI): 1.00 to 2.26), compared to bilingual participants; however after adjustment this relationship was attenuated (OR = 1.25, 95% CI: 0.79 to 2.00). Non-citizenship was not associated with increased odds of food insecurity (OR=1.18, 95% CI: 0.82 to 1.68). CONCLUSION: Food insecurity in this predominantly immigrant Caribbean Latino sample was higher than the national average for Latinos. Future research on food insecurity among different Latino ethnicities is needed in order to inform targeted interventions that promote food security.

A hierarchy of different chromosome conformations plays a role in many biological systems. These conformations contribute to the regulation of gene expression, cellular development, chromosome transmission, and defects can lead to human disease. The highest functional level of this hierarchy is the partitioning of the genome into compartments of active and inactive chromatin domains (1’s -10’s Mb). These compartments are further partitioned into Topologically Associating Domains (TADs) that spatially cluster co-regulated genes (100’s kb – 1’s Mb). The final level that has been observed is long range loops formed between regulatory elements and promoters (10’s kb – 100’s Mb). At all of these levels, mechanisms that establish these conformations remain poorly understood. To gain new insights into processes that determine chromosome folding I used the mating type switching system in budding yeast to study the chromosome conformation at length scales analogous to looping interaction. I specifically examined the role in chromosome conformation in the mating type switching system. Budding yeast cells can have two sexes: MATa and MATα. The mating types are determined by allele-specific expression of the MAT locus on chromosome III. The MATa allele encodes for transcription factors responsible for the MATa mating type and the MATα allele encodes transcription factors responsible for the MATα mating type. Yeast cells can switch their mating type by a process that repairs a break at MAT using one of two silent loci, HML or HMR, as a donor to convert the allele at the MAT locus. When MATa cells switch they prefer to use HML, which contains the MATα allele, located at the end of the left arm. MATα cells prefer to use HMR, which contains the MATa allele, located on the end of the right arm of chromosome III. The sequences of the HM loci are not important for donor preference. Instead the cell chooses the donor on the left arm in MATa cells and chooses the donor on the right arm in MATα cells. This lack of sequence specificity has led to the hypothesis that the conformation of the chromosome may play a role in donor preference. I found that the conformation of chromosome III is, indeed, different between the two mating types. In MATa cells the chromosomes displays a more crumpled conformation in which the left arm of the chromosome interacts with a large region of the right arm which includes the centromere and the MAT locus. In MATα cells, on the other hand, the left arm of the chromosomes displays a more extend conformation. I found that the Recombination Enhancer (RE), which enhances recombination along the left arm of the chromosome in MATa cells, is responsible for these mating type-specific conformations. Deleting the RE affects the conformation of the chromosomes in both MATa and MATα cells. The left portion of the RE, which is essential for donor preference during the switching reaction in MATa cells, does not contribute to the conformation in MATa. This region does have a minor effect on the conformation in MATα cells. However, I found that the right portion of the RE is responsible for the conformation of chromosome III in both mating types prior to initiation of switching. This work demonstrates that chromosome conformation is determined by specific cis regulatory elements that drive cell-type specific chromosome conformation.

Neurons comprise the main information processing cells of the nervous system. To integrate and transmit information, neurons elaborate dendritic structures to receive input and axons to relay that information to other cells. Due to their intricate structures, dendrites and axons are susceptible to damage whether by physical means or via disease mechanisms. Studying responses to axon injury, called Wallerian degeneration, in the neuronal processes of Drosophila melanogaster has allowed the identification of genes that are required for injury responses. Screens in Drosophila have identified dsarm and highwire as two genes required for axon degeneration; when these genes are mutated axons fail to degenerate after injury, even when completely cut off from the neuronal cell body. We found that these genes are also required for dendrite degeneration after injury in vivo. Further, we reveal differences between axon and dendrite injury responses using in vivo timelapse recordings and GCaMP indicators of intracellular and mitochondrial calcium transients. These data provide insights into the neuronal responses to injury, and better define novel targets for the treatment of neurodegenerative diseases.

Retinitis Pigmentosa (RP) is an inherited photoreceptor degenerative disease that leads to blindness and affects about 1 in 4000 people worldwide. The disease is predominantly caused by mutations in genes expressed exclusively in the night active rod photoreceptors; however, blindness results from the secondary loss of the day active cone photoreceptors, the mechanism of which remains elusive. Here, we show that the mammalian target of rapamycin complex 1 (mTORC1) is required to delay the progression of cone death during disease and that constitutive activation of mTORC1 is sufficient to maintain cone function and promote cone survival in RP. Activation of mTORC1 increased expression of genes that promote glucose uptake, retention and utilization, leading to increased NADPH levels; a key metabolite for cones. This protective effect was conserved in two mouse models of RP, indicating that the secondary loss of cones can be delayed by an approach that is independent of the primary mutation in rods. However, since mTORC1 is a negative regulator of autophagy, its constitutive activation led to an unwarranted secondary effect of shortage of amino acids due to incomplete digestion of autophagic cargo, which reduces the efficiency of cone survival over time. Moderate activation of mTORC1, which promotes expression of glycolytic genes, as well as maintains autophagy, provided more sustained cone survival. Together, our work addresses a long-standing question of non-autonomous cone death in RP and presents a novel, mutation-independent approach to extend vision in a disease that remains incurable.