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While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain-containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.
SUMO-Targeted Ubiquitin Ligase (STUbL) Slx5 regulates proteolysis of centromeric histone H3 variant Cse4 and prevents its mislocalization to euchromatin
Centromeric histone H3, CENP-ACse4, is essential for faithful chromosome segregation. Stringent regulation of cellular levels of CENP-ACse4 restricts its localization to centromeres. Mislocalization of CENP-ACse4 is associated with aneuploidy in yeast, flies and tumorigenesis in human cells; thus, defining pathways that regulate CENP-A levels is critical for understanding how mislocalization of CENP-A contributes to aneuploidy in human cancers. Previous work in budding yeast has shown that ubiquitination of overexpressed Cse4 by Psh1, an E3 ligase, partially contributes to proteolysis of Cse4. Here, we provide the first evidence that Cse4 is sumoylated by E3 ligases Siz1 and Siz2 in vivo and in vitro. Ubiquitination of Cse4 by Small Ubiquitin-related Modifier (SUMO)-Targeted Ubiquitin Ligase (STUbL) Slx5 plays a critical role in proteolysis of Cse4 and prevents mislocalization of Cse4 to euchromatin under normal physiological conditions. Accumulation of sumoylated Cse4 species and increased stability of Cse4 in slx5 strains suggest that sumoylation precedes ubiquitin-mediated proteolysis of Cse4. Slx5-mediated Cse4 proteolysis is independent of Psh1 since slx5 psh1 strains exhibit higher levels of Cse4 stability and mislocalization compared to either slx5 or psh1 strains. Our results demonstrate a role for Slx5 in ubiquitin-mediated proteolysis of Cse4 to prevent its mislocalization and maintain genome stability.
Implementation of a Computerized Screening Inventory: Improved Usability Through Iterative Testing and Modification
BACKGROUND: The administration of health screeners in a hospital setting has traditionally required (1) clinicians to ask questions and log answers, which can be time consuming and susceptible to error, or (2) patients to complete paper-and-pencil surveys, which require third-party entry of information into the electronic health record and can be vulnerable to error and misinterpretation. A highly promising method that avoids these limitations and bypasses third-party interpretation is direct entry via a computerized inventory.
OBJECTIVE: To (1) computerize medical and behavioral health screening for use in general medical settings, (2) optimize patient acceptability and feasibility through iterative usability testing and modification cycles, and (3) examine how age relates to usability.
METHODS: A computerized version of 15 screeners, including behavioral health screeners recommended by a National Institutes of Health Office of Behavioral and Social Sciences Research collaborative workgroup, was subjected to systematic usability testing and iterative modification. Consecutive adult, English-speaking patients seeking treatment in an urban emergency department were enrolled. Acceptability was defined as (1) the percentage of eligible patients who agreed to take the assessment (initiation rate) and (2) average satisfaction with the assessment (satisfaction rate). Feasibility was defined as the percentage of the screening items completed by those who initiated the assessment (completion rate). Chi-square tests, analyses of variance, and Pearson correlations were used to detect whether improvements in initiation, satisfaction, and completion rates were seen over time and to examine the relation between age and outcomes.
RESULTS: Of 2157 eligible patients approached, 1280 agreed to complete the screening (initiation rate=59.34%). Statistically significant increases were observed over time in satisfaction (F3,1061=3.35, P=.019) and completion rates (F3,1276=25.44, P < .001). Younger age was associated with greater initiation (initiated, mean [SD], 46.6 [18.7] years; declined: 53.0 [19.5] years, t2,155=-7.6, P < .001), higher completion (r=-.20, P < .001), and stronger satisfaction (r=-.23, P < .001).
CONCLUSIONS: In a rapid-paced emergency department with a heterogeneous patient population, 59.34% (1280/2157) of all eligible patients initiated the computerized screener with a completion rate reaching over 90%. Usability testing revealed several critical principles for maximizing usability of the computerized medical and behavioral health screeners used in this study. Further work is needed to identify usability issues pertaining to other screeners, racially and ethnically diverse patient groups, and different health care settings.
The unconventional myosin CRINKLED and its mammalian orthologue MYO7A regulate caspases in their signalling roles
Caspases provide vital links in non-apoptotic regulatory networks controlling inflammation, compensatory proliferation, morphology and cell migration. How caspases are activated under non-apoptotic conditions and process a selective set of substrates without killing the cell remain enigmatic. Here we find that the Drosophila unconventional myosin CRINKLED (CK) selectively interacts with the initiator caspase DRONC and regulates some of its non-apoptotic functions. Loss of CK in the arista, border cells or proneural clusters of the wing imaginal discs affects DRONC-dependent patterning. Our data indicate that CK acts as substrate adaptor, recruiting SHAGGY46/GSK3-beta to DRONC, thereby facilitating caspase-mediated cleavage and localized modulation of kinase activity. Similarly, the mammalian CK counterpart, MYO7A, binds to and impinges on CASPASE-8, revealing a new regulatory axis affecting receptor interacting protein kinase-1 (RIPK1) > CASPASE-8 signalling. Together, our results expose a conserved role for unconventional myosins in transducing caspase-dependent regulation of kinases, allowing them to take part in specific signalling events.
Spontaneous coronary artery dissection in a patient with autosomal dominant polycystic kidney disease: a case report
BACKGROUND: Spontaneous coronary artery dissection is an uncommon syndrome. Its prevalence among patients with polycystic kidney disease is very rare, with no previously reported involvement of the right posterior descending coronary artery.
CASE PRESENTATION: We describe the case of a middle-aged Caucasian woman with polycystic kidney disease who presented with a non-ST elevation myocardial infarction. Cardiac catheterization revealed a dissection of her right posterior descending coronary artery. She was treated with dual antiplatelet therapy and had a favorable outcome.
CONCLUSION: We report a rare and interesting case of spontaneous coronary artery dissection of the right posterior descending coronary artery in a patient with polycystic kidney disease. It is important to consider spontaneous coronary artery dissection in the differential diagnosis of patients with polycystic kidney disease who present with an acute coronary syndrome.
Integrative analyses reveal a long noncoding RNA-mediated sponge regulatory network in prostate cancer
Mounting evidence suggests that long noncoding RNAs (lncRNAs) can function as microRNA sponges and compete for microRNA binding to protein-coding transcripts. However, the prevalence, functional significance and targets of lncRNA-mediated sponge regulation of cancer are mostly unknown. Here we identify a lncRNA-mediated sponge regulatory network that affects the expression of many protein-coding prostate cancer driver genes, by integrating analysis of sequence features and gene expression profiles of both lncRNAs and protein-coding genes in tumours. We confirm the tumour-suppressive function of two lncRNAs (TUG1 and CTB-89H12.4) and their regulation of PTEN expression in prostate cancer. Surprisingly, one of the two lncRNAs, TUG1, was previously known for its function in polycomb repressive complex 2 (PRC2)-mediated transcriptional regulation, suggesting its sub-cellular localization-dependent function. Our findings not only suggest an important role of lncRNA-mediated sponge regulation in cancer, but also underscore the critical influence of cytoplasmic localization on the efficacy of a sponge lncRNA.
BACKGROUND: Persistent thromboxane (TX) generation while receiving aspirin therapy is associated with an increased risk of cardiovascular events. The Reduction in Graft Occlusion Rates (RIGOR) study found that aspirin-insensitive TXA2 generation, indicated by elevated urine 11-dehydro-TXB2 (UTXB2) 6 months after coronary artery bypass graft surgery, was a potent risk factor for vein graft thrombosis and originated predominantly from nonplatelet sources. Our goal was to identify risks factors for nonplatelet TXA2 generation.
METHODS AND RESULTS: Multivariable modeling was performed by using clinical and laboratory variables obtained from 260 RIGOR subjects with verified aspirin-mediated inhibition of platelet TXA2 generation. The strongest variable associated with UTXB2 6 months after surgery, accounting for 47.2% of the modeled effect, was urine 8-iso-prostaglandin (PG)F2alpha, an arachidonic acid metabolite generated nonenzymatically by oxidative stress (standardized coefficient 0.442, P < 0.001). Age, sex, race, lipid therapy, creatinine, left ventricular ejection fraction, and aspirin dose were also significantly associated with UTXB2 (P < 0.03), although they accounted for only 4.8% to 10.2% of the modeled effect. Urine 8-iso-PGF2alpha correlated with risk of vein graft occlusion (odds ratio 1.67, P=0.001) but was not independent of UTXB2. In vitro studies revealed that endothelial cells generate TXA2 in response to oxidative stress and direct exposure to 8-iso-PGF2alpha.
CONCLUSIONS: Oxidative stress-induced formation of 8-iso-PGF2alpha is strongly associated with nonplatelet thromboxane formation and early vein graft thrombosis after coronary artery bypass graft surgery. The endothelium is potentially an important source of oxidative stress-induced thromboxane generation. These findings suggest therapies that reduce oxidative stress could be useful in reducing cardiovascular risks associated with aspirin-insensitive thromboxane generation.
NEMO Prevents RIP Kinase 1-Mediated Epithelial Cell Death and Chronic Intestinal Inflammation by NF-kappaB-Dependent and -Independent Functions
Intestinal epithelial cells (IECs) regulate gut immune homeostasis, and impaired epithelial responses are implicated in the pathogenesis of inflammatory bowel diseases (IBD). IEC-specific ablation of nuclear factor kappaB (NF-kappaB) essential modulator (NEMO) caused Paneth cell apoptosis and impaired antimicrobial factor expression in the ileum, as well as colonocyte apoptosis and microbiota-driven chronic inflammation in the colon. Combined RelA, c-Rel, and RelB deficiency in IECs caused Paneth cell apoptosis but not colitis, suggesting that NEMO prevents colon inflammation by NF-kappaB-independent functions. Inhibition of receptor-interacting protein kinase 1 (RIPK1) kinase activity or combined deficiency of Fas-associated via death domain protein (FADD) and RIPK3 prevented epithelial cell death, Paneth cell loss, and colitis development in mice with epithelial NEMO deficiency. Therefore, NEMO prevents intestinal inflammation by inhibiting RIPK1 kinase activity-mediated IEC death, suggesting that RIPK1 inhibitors could be effective in the treatment of colitis in patients with NEMO mutations and possibly in IBD.
A Delphi process to address medication appropriateness for older persons with multiple chronic conditions
BACKGROUND: Frameworks exist to evaluate the appropriateness of medication regimens for older patients with multiple medical conditions (MCCs). Less is known about how to translate the concepts of the frameworks into specific strategies to identify and remediate inappropriate regimens.
METHODS: Modified Delphi method involving iterative rounds of input from panel members. Panelists (n = 9) represented the disciplines of nursing, medicine and pharmacy. Included among the physicians were two geriatricians, one general internist, one family practitioner, one cardiologist and two nephrologists. They participated in 3 rounds of web-based anonymous surveys.
RESULTS: The panel reached consensus on a set of markers to identify problems with medication regimens, including patient/caregiver report of non-adherence, medication complexity, cognitive impairment, medications identified by expert opinion as inappropriate for older persons, excessively tight blood sugar and blood pressure control among persons with diabetes mellitus, patient/caregiver report of adverse medication effects or medications not achieving desired outcomes, and total number of medications. The panel also reached consensus on approaches to address these problems, including endorsement of strategies to discontinue medications with known benefit if necessary because of problems with feasibility or lack of alignment with patient goals.
CONCLUSIONS: The results of the Delphi process provide the basis for an algorithm to improve medication regimens among older persons with MCCs. The algorithm will require assessment not only of medications and diagnoses but also cognition and social support, and it will support discontinuation of medications both when risks outweigh benefits and when regimens are not feasible or do not align with goals.
BACKGROUND: Microenvironments called niches maintain resident stem cell populations by balancing self-renewal with differentiation, but the genetic regulation of this process is unclear. The niche of the Drosophila testis is well-characterized and genetically tractable, making it ideal for investigating the molecular regulation of stem cell biology. The JAK/STAT pathway, activated by signals from a niche component called the hub, maintains both germline and somatic stem cells.
RESULTS: This study investigated the molecular regulation of the JAK/STAT pathway in the stem cells of the Drosophila testis. We determined that the transcriptional regulator Apontic (Apt) acts in the somatic (cyst) stem cells (CySCs) to balance differentiation and maintenance. We found Apt functions as a negative feedback inhibitor of STAT activity, which enables cyst cell maturation. Simultaneous loss of the STAT regulators apt and Socs36E, or the Stat92E-targeting microRNA miR-279, expanded the somatic stem cell-like population.
CONCLUSIONS: Genetic analysis revealed that a conserved genetic regulatory network limits JAK/STAT activity in the somatic stem cells of Drosophila testis. In these cells, we determined JAK/STAT signaling promotes apt expression. Then, Apt functions through Socs36E and miR-279 to attenuate pathway activation, which is required for timely CySC differentiation. We propose that Apt acts as a core component of a STAT-regulatory circuit to prevent stem cell overpopulation and allow stem cell maturation.
Does the Mutant CAG Expansion in Huntingtin mRNA Interfere with Exonucleolytic Cleavage of its First Exon
BACKGROUND: Silencing mutant huntingtin mRNA by RNA interference (RNAi) is a therapeutic strategy for Huntington's disease. RNAi induces specific endonucleolytic cleavage of the target HTT mRNA, followed by exonucleolytic processing of the cleaved mRNA fragments.
OBJECTIVES: We investigated the clearance of huntingtin mRNA cleavage products following RNAi, to find if particular huntingtin mRNA sequences persist. We especially wanted to find out if the expanded CAG increased production of a toxic mRNA species by impeding degradation of human mutant huntingtin exon 1 mRNA.
METHODS: Mice expressing the human mutant HTT transgene with 128 CAG repeats (YAC128 mice) were injected in the striatum with self-complementary AAV9 vectors carrying a miRNA targeting exon 48 of huntingtin mRNA (scAAV-U6-miRNA-HTT-GFP). Transgenic huntingtin mRNA levels were measured in striatal lysates after two weeks. For qPCR, we used species specific primer-probe combinations that together spanned 6 positions along the open reading frame and untranslated regions of the human huntingtin mRNA. Knockdown was also measured in the liver following tail vein injection.
RESULTS: Two weeks after intrastriatal administration of scAAV9-U6-miRNA-HTT-GFP, we measured transgenic mutant huntingtin in striatum using probes targeting six different sites along the huntingtin mRNA. Real time PCR showed a reduction of 29% to 36% in human HTT. There was no significant difference in knockdown measured at any of the six sites, including exon 1. In liver, we observed a more pronounced HTT mRNA knockdown of 70% to 76% relative to the untreated mice, and there were also no significant differences among sites.
CONCLUSIONS: Our results demonstrate that degradation is equally distributed across the human mutant huntingtin mRNA following RNAi-induced cleavage.
Total Elbow Arthroplasty in the United States: Evaluation of Cost, Patient Demographics, and Complication Rates
Total elbow arthroplasty (TEA) is utilized in the treatment of rheumatoid and post-traumatic elbow arthritis. TEA is a relatively low volume surgery in comparison to other types of arthroplasty and therefore little is known about current surgical utilization, patient demographics and complication rates in the United States. The purpose of our study is to evaluate the current practice trends and associated in-patient complications of TEA at academic centers in the United States. We queried the University Health Systems Consortium administrative database from 2007 to 2011 for patients who underwent an elective TEA. A descriptive analysis of demographics was performed which included patient age, sex, race, and insurance status. We also evaluated the following patient clinical benchmarks: hospital length of stay (LOS), hospital direct cost, in-hospital mortality, complications, and 30-day readmission rates. Our cohort consisted of 3146 adult patients (36.5% male and 63.5% female) with an average age of 58 years who underwent a total elbow arthroplasty (159 academic medical centers) in the United States. The racial demographics included 2334 (74%) Caucasian, 285 (9%) black, 236 (7.5%) Hispanic, 16 (0.5%) Asian, and 283 (9%) other patients. The mean LOS was 4.2+/-5 days and the mean total direct cost for the hospital was 16,300+/-4000 US Dollars per case. The overall inpatient complication rate was 3.1% and included mortality < 1%, DVT (0.8%), re-operation (0.5%), and infection (0.4%). The 30-day readmission rate was 4.4%. TEA is a relatively uncommon surgery in comparison to other forms of arthroplasty but is associated with low in-patient and 30-day perioperative complication rate. Additionally, the 30-day readmission rate and overall hospital costs are comparable to the traditional total hip and knee arthroplasty surgeries.
Structural Variation Discovery and Genotyping from Whole Genome Sequencing: Methodology and Applications: A Dissertation
A comprehensive understanding about how genetic variants and mutations contribute to phenotypic variations and alterations entails experimental technologies and analytical methodologies that are able to detect genetic variants/mutations from various biological samples in a timely and accurate manner. High-throughput sequencing technology represents the latest achievement in a series of efforts to facilitate genetic variants discovery and genotyping and promises to transform the way we tackle healthcare and biomedical problems. The tremendous amount of data generated by this new technology, however, needs to be processed and analyzed in an accurate and efficient way in order to fully harness its potential. Structural variation (SV) encompasses a wide range of genetic variations with different sizes and generated by diverse mechanisms. Due to the technical difficulties of reliably detecting SVs, their characterization lags behind that of SNPs and indels. In this dissertation I presented two novel computational methods: one for detecting transposable element (TE) transpositions and the other for detecting SVs in general using a local assembly approach. Both methods are able to pinpoint breakpoint junctions at single-nucleotide resolution and estimate variant allele frequencies in the sample. I also applied those methods to study the impact of TE transpositions on the genomic stability, the inheritance patterns of TE insertions in the population and the molecular mechanisms and potential functional consequences of somatic SVs in cancer genomes.
Functions of Argonaute Proteins in Self Versus Non-Self Recognition in the C. elegans Germline: A Dissertation
Organisms employ sophisticated mechanisms to silence foreign nucleic acid, such as viruses and transposons. Evidence exists for pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), but the mechanisms that distinguish “self” from “non-self” are not well understood. Our studies on transgene silencing in C. elegans have uncovered an RNA surveillance system in which the PIWI protein, PRG-1, uses a vast repertoire of piRNAs to recognize foreign transcripts and to initiate epigenetic silencing. Partial base pairing by piRNAs is sufficient to guide PRG-1 targeting. PRG-1 in turn recruits RdRP to synthesize perfectly matching antisense siRNAs (22G-RNAs) that are loaded onto worm-specific Argonaute (WAGO) proteins. WAGOs collaborate with chromatin factors to maintain epigenetic silencing (RNAe). Since mismatches are allowed during piRNA targeting, piRNAs could—in theory— target any transcript expressed in the germline, but germline genes are not subject to silencing by RNAe. Moreover, some foreign sequences are expressed and appear to be adopted as “self.” How are “self” transcripts distinguished from foreign transcripts? We have found that another Argonaute, CSR-1, and its siRNAs—also synthesized by RdRP—protect endogenous genes from silencing by RNAe. We refer to this pathway as RNA-mediated gene activation (RNAa). Reducing CSR-1 or PRG-1 or increasing piRNA targeting can shift the balance towards expression or silencing, indicating that PRG-1 and CSR-1 compete for control over their targets. Thus worms have evolved a remarkable nucleic acids immunity mechanism in which opposing Argonaute pathways generate and maintain epigenetic memories of self and non-self nucleotide sequences.
Autophagy-Independent Role for Beclin 1 in the Regulation of Growth Factor Receptor Signaling: A Dissertation
Beclin 1 is a haplo-insufficient tumor suppressor that is decreased in many human tumors. The function of Beclin 1 in cancer has been attributed primarily to its role in the degradative process of autophagy. However, the role of autophagy itself in tumorigenesis is context-dependent and can be both preventive and promoting. Due to its dual function in cancer a better understanding of this process is necessary to develop potential novel cancer therapies. To gain insight into the role of autophagy in breast carcinoma, I analyzed the autophagydependency of different subtypes of breast cancer. My results implicate that triple-negative breast carcinoma cells are more dependent on autophagy than luminal breast carcinoma cells. Chemical inhibition of autophagy decreased the tumorigenicity of triple-negative breast carcinoma cells with regard to proliferation and anchorage-independent growth. However, RNAi-mediated suppression of two autophagy genes, ATG5 and Beclin 1, revealed different outcomes. While suppression of ATG5 decreased glycolysis, Beclin 1 depletion did not affect the glycolytic rates. These results suggest autophagy-independent pro-tumorigenic effects of loss of Beclin 1 in cancer.
Beclin 1 is a core component of the Vps34/Class III PI3K (PI3KC3) and Vps15/p150 complex that regulates multiple membrane trafficking events. I describe a novel mechanism of action for Beclin 1 in breast cancer involving its control of growth factor receptor signaling. I identify a specific stage of early endosome maturation that is regulated by Beclin 1, the transition of APPL1- containing phosphatidyIinositol 3-phosphate-negative (PI3P-) endosomes to PI3P+ endosomes. Beclin 1 regulates PI3P production in response to growth factor stimulation to control the residency time of growth factor receptors in the PI3P-/APPL+ signaling competent compartment. As a result, suppression of BECN1 sustains growth factor stimulated AKT and ERK activation resulting in increased breast carcinoma cell invasion. In human breast tumors, Beclin 1 expression is inversely correlated with AKT and ERK phosphorylation. Taken together my data identify a novel role for Beclin 1 in regulating growth factor signaling and reveal a mechanism by which loss of Beclin 1 expression would enhance breast cancer progression independent of its impact on autophagy.
The Influence of the Insulin-Like Gene Family and Diet-Drug Interactions on Caenorhabditis elegans Physiology: A Dissertation
Aging can be defined as the accumulation of changes affecting the maintenance of homeostatic processes over time, leading to functional decline and increased risk for disease and death. In its simplicity, aging is the systemwide deterioration of an organism. Genetic studies have identified many potential molecular mechanisms of aging including DNA damage, telomere shortening, mitochondrial dysfunction, increased oxidative stress, uncontrolled inflammation, and hormone dysregulation (reviewed in ). However, in reality, aging is likely to be a combination of some (or potentially all) of these mechanisms.
Interestingly, aging and metabolism are tightly coordinated. Aging is a major contributor to metabolic decline and related diseases, including type 2 diabetes, metabolic syndrome, and cancer. One of the best characterized metabolic pathways implicated in aging is the insulin/IGF-1 signaling (IIS) pathway. Downstream signaling components of the IIS pathway receptor have been well studied and include an interconnected network of signaling events that regulate many physiological outputs. However, less is known about the role of upstream signaling components and how intracellular pathways and physiology are regulated accordingly. In Part I, I present my work towards understanding upstream IIS pathway components using a systems biology approach. The goal of this study is to gain insight into the redundancy and specificity of the insulin gene family responsible for initiating IIS pathway activity in Caenorhabditis elegans. The information gained will serve as a foundation for future studies dissecting the molecular mechanisms of this pathway in efforts to uncouple the downstream signaling and physiological outputs.
The clear impact of metabolism on aging and disease stimulated questions regarding the potential of promoting health and longevity through diet and dietary mimetics. Recent findings indicate reduced food intake, meal timing and nutritional modulation of the gut microbiome can ameliorate signs of aging and age-associated diseases. Aging, therefore, is also the result of dynamic and complex interplay between genes of an organism and its environment. In Part II, I will discuss my efforts to gain insight into how diet influences aging. This preliminary study has demonstrated that diet can affect lifespan in the model organism, C. elegans. Additionally, we observe diet-specific effects on drug efficacy that, in turn, modulates C. elegans lifespan and reproduction. The implications of these experiments, while limited, illustrate a potentially greater role in diet- and drug-mediated effects on lifespan.
Regulation of CDK1 Activity during the G1/S Transition in S. cerevisiae through Specific Cyclin-Substrate Docking: A Dissertation
Several cell cycle events require specific forms of the cyclin-CDK complexes. It has been known for some time that cyclins not only contribute by activating the CDK but also by choosing substrates and/or specifying the location of the CDK holoenzyme. There are several examples of B-type cyclins identifying certain peptide motifs in their specific substrates through a conserved region in their structure. Such interactions were not known for the G1 class of cyclins, which are instrumental in helping the cell decide whether or not to commit to a new cell cycle, a function that is non-redundant with B-type cylins in budding yeast. In this dissertation, I have presented evidence that some G1 cyclins in budding yeast, Cln1/2, specifically identify substrates by interacting with a leucine-proline rich sequence different from the ones used by B-type cyclins. These “LP” type docking motifs determine cyclin specificity, promote phosphorylation of suboptimal CDK sites and multi-site phosphorylation of substrates both in vivo and in vitro. Subsequently, we have discovered the substrate-binding region in Cln2 and further showed that this region is highly conserved amongst a variety of fungal G1 cyclins from budding yeasts to molds and mushrooms, thus suggesting a conserved function across fungal evolution. Interestingly, this region is close to but not same as the one implicated in B-type cyclins to binding substrates. We discovered that the main effect of obliterating this interaction is to delay cell cycle entry in budding yeast, such that cells begin DNA replication and budding only at a larger than normal cell size, possibly resulting from incomplete multi-site phosphorylation of several key substrates. The docking-deficient Cln2 was also defective in promoting polarized bud morphogenesis. Quite interestingly, we found that a CDK inhibitor, Far1, could regulate the Cln2-CDK1 activity partly by inhibiting the Cln2-substrate interaction, thus demonstrating that docking interactions can be targets of regulation. Finally, by studying many fungal cyclins exogenously expressed in budding yeast, we discovered that some have the ability to make the CDK hyper-potent, which suggests that these cyclins confer special properties to the CDK. My work provides mechanistic clues for cyclinspecific events during the cell cycle, demonstrates the usefulness of synthetic strategies in problem solving and also possibly resolves long-standing uncertainties regarding functions of some cell cycle proteins.
Inhibiting BCR-ABL kinase activity with tyrosine kinase inhibitors (TKIs) has been the frontline therapy for CML. Resistance to TKIs frequently occurs, but the mechanisms remain elusive.
First, to uncover survival pathways involved in TKI resistance in CML, I conducted a genome-wide RNAi screen in human CML cells to identify genes governing cellular sensitivity to the first generation TKI called IM (Gleevec). I identified genes converging on and activating the MEK/ERK pathway through transcriptional up-regulation of PRKCH. Combining IM with a MEK inhibitor synergistically kills TKI-resistant CML cells and CML stem cells.
Next, I performed single cell RNA-seq to compare expression profiles of CML stem cells and hematopoietic stem cells isolated from the same patient. Among the genes that are preferentially expressed in CML stem cells is PIM2, which encodes a pro-survival serine-threonine kinase that phosphorylates and inhibits the pro-apoptotic protein BAD. Inhibiting PIM2 function sensitizes CML stem cells to IM-induced apoptosis and prevents disease relapse in a CML mouse model.
Last, I devised a CRISPR-Cas9 based strategy to perform insertional mutagenesis at a defined genomic location in murine hematopoietic Ba/F3 cells. As proof of principle, we showed its capability to perform unbiased, saturated point mutagenesis in a 9 amino acid region of BCR-ABL encompassing the socalled “gatekeeper” residue, an important determinant of TKI binding. We found that the ranking order of mutations from the screen correlated well with their prevalence in IM-resistant CML patients.
Overall, my findings reveal novel resistance mechanisms in CML and provide alternative therapeutic strategies.
Systematic Analysis of Duplications and Deletions in the Malaria Parasite P. falciparum: A Dissertation
Duplications and deletions are a major source of genomic variation. Duplications, specifically, have a significant impact on gene genesis and dosage, and the malaria parasite P. falciparum has developed resistance to a growing number of anti-malarial drugs via gene duplication. It also contains highly duplicated families of antigenically variable allelic genes. While specific genes and families have been studied, a comprehensive analysis of duplications and deletions within the reference genome and population has not been performed. We analyzed the extent of segmental duplications (SD) in the reference genome for P. falciparum, primarily by a whole genome self alignment. We discovered that while 5% of the genome identified as SD, the distribution within the genome was partition clustered, with the vast majority localized to the subtelomeres. Within the SDs, we found an overrepresentation of genes encoding antigenically diverse proteins exposed to the extracellular membrane, specifically the var, rifin, and stevor gene families. To examine variation of duplications and deletions within the parasite populations, we designed a novel computational methodology to identify copy number variants (CNVs) from high throughput sequencing, using a read depth based approach refined with discordant read pairs. After validating the program against in vitro lab cultures, we analyzed isolates from Senegal for initial tests into clinical isolates. We then expanded our search to a global sample of 610 strains from Africa and South East Asia, identifying 68 CNV regions. Geographically, genic CNV were found on average in less than 10% of the population, indicating that CNV are rare. However, CNVs at high frequency were almost exclusively duplications associated with known drug resistant CNVs. We also identified the novel biallelic duplication of the crt gene – containing both the chloroquine resistant and sensitive allele. The synthesis of our SD and CNV analysis indicates a CNV conservative P. falciparum genome except where drug and human immune pressure select for gene duplication.