From Caregiver to Computer Geek: The Design of a Web Resource for Novice Clinical Informaticians

Introduction: Throughout the United States, there are massive initiatives in place to reform healthcare through the implementation of electronic health records. The goals are to improve patient care through improved access to records, the improvement of business and reimbursement processes, streamlining of clinician workflows for increased efficiency, and reducing the variability in the delivery of patient care. [See PDF for complete abstract]

Characterization and optimization of antigen-specific T cell responses during ex vivo expansion of melanoma tumor-infiltrating lymphocytes

Treatment of metastatic melanoma with tumor reactive T cells (adoptive T cell therapy, ACT) is a promising approach associated with a high clinical response rate. However, further optimization of this treatment modality is required to increase the clinical response after this therapy. ACT in melanoma involves an initial phase (pre-REP) of tumor-infiltrating lymphocyte (TIL) expansion ex vivo from tumor isolates followed by a second phase, “rapid expansion protocol” (REP) generating the billions of cells used as the TIL infusion product. The main question addressed in this thesis was how the currently used REP affected the responsiveness of the CD8+ T cells to defined melanoma antigens. We hypothesized that the REP drives the TIL to further differentiate and become hyporesponsive to antigen restimulation, therefore, proper cytokine treatment or other ways to expand TIL is required to improve upon this outcome. We evaluated the response of CD8+ TIL to melanoma antigen restimulation using MART-1 peptide-pulsed mature DC in vitro. Post-REP TILs were mostly hypo-responsive with poor proliferation and higher apoptosis. Phenotypic analysis revealed that the expression of CD28 was significantly reduced in post-REP TILs. By sorting experiment and microarray analysis, we confirmed that the few CD28+ post-REP TILs had superior survival capacity and proliferated after restimulation. We then went on to investigate methods to maintain CD28 expression during the REP and improve TIL responsiveness. Firstly, IL-15 and IL-21 were found to synergize in maintaining TIL CD28 expression and antigenic responsiveness during REP. Secondly, we found IL-15 was superior as compared to IL-2 in supporting the long-term expansion of antigen-specific CD8+ TIL after restimulation. These results suggest that current expansion protocols used for adoptive T-cell therapy in melanoma yield largely hyporesponsive products containing CD8+ T cells unable to respond in vivo to re-stimulation with antigen. A modification of our current approaches by using IL-15+IL-21 as supporting cytokines in the REP, or/and administration of IL-15 instead of IL-2 after TIL infusion, may enhance the anti-tumor efficacy and long-term persistence of infused T cells in vivo.

A Web Services architecture for UMLS Knowledge Sources.

A web service is a collection of industry standards to enable reusability of services and interoperability of heterogeneous applications. The UMLS Knowledge Source (UMLSKS) Server provides remote access to the UMLSKS and related resources. We propose a Web Services Architecture that encapsulates UMLSKS-API and makes it available in distributed and heterogeneous environments. This is the first step towards intelligent and automatic UMLS services discovery and invocation by computer systems in distributed environments such as web.

Comparing communication technology on Chinese, English, and Spanish diabetes web sites.

Technological and cultural factors influence access to health information on the web in multifarious ways. We evaluated structural differences and availability of communication services on the web in three diverse language and cultural groups: Chinese, English, and Spanish. A total of 382 web sites were analyzed: 144 were English language sites (38%), 129 were Chinese language sites (34%), and 108 were Spanish language sites (28%). We did not find technical differences in the number of outgoing links per domain or the total availability of communication services between the three groups. There were differences in the distribution of available services between Chinese and English sites. In the Chinese sites, there were more communication services between consumers and health experts. Our results suggest that the health-related web presence of these three cultural groups is technologically comparable, but reflects differences that may be attributable to cultural factors.

CHARACTERIZATION OF ALPHA-GALACTOSYLCERAMIDE AS A MUCOSAL ADJUVANT

Adjuvants are essential components of vaccine formulations that enhance adaptive immune responses to antigens, particularly for immunizations targeting the tolerogenic mucosal tissues, which are more biologically relevant for protective immunity against pathogens transmitted by the mucosal routes. Adjuvants possess the inherent capacity to bridge innate and adaptive immune responses through activating innate immune mediators. Here evidence is presented in support of the effectiveness of a synthetic glycolipid, alpha-Galactosylceramide (-GalCer), as an adjuvant for mucosal immunization with peptide and protein antigens, by oral and intranasal routes, to prime antigen-specific immune responses in multiple systemic and mucosal compartments. The adjuvant activity of -GalCer delivered by the intranasal route was manifested in terms of potent activation of NKT cells, an important innate immunity mediator, along with the activation of dendritic cells (DC) which serve as the professional antigen-presenting cells. Data from this investigation provide the first evidence for mucosal delivery as an effective means to harness the adjuvant potential of α-GalCer for priming as well as boosting cellular immune responses to co-administered immunogens. Unlike systemic administration where a single dose of α-GalCer leads to anergy of responding NKT cells and thus hinders delivery of booster immunizations, we demonstrated that administration of multiple doses of α-GalCer by the intranasal route affords repeated activation of NKT cells and the induction of broad systemic and mucosal immunity. This is specifically advantageous, and may be even essential, for vaccination regimens against mucosal pathogens such as the human immunodeficiency virus (HIV) and the human papillomavirus (HPV), where priming of durable protective immunity at the mucosal portals of pathogen entry would be highly desirable.

Characterization of Optically Stimulated Luminescent Detectors in Photon & Proton Beams for Use in Anthropomorphic Phantoms

This study investigated characteristics of optically stimulated luminescent detectors (OSLDs) in protons, allowing comparison to thermoluminescent detectors, and to be implemented into the Radiological Physics Center’s (RPC) remote audit quality assurance program for protons, and for remote anthropomorphic phantom irradiations. The OSLDs used were aluminum oxide (Al2O3:C) nanoDots from Landauer, Inc. (Glenwood, Ill.) measuring 10x10x2 mm3. A square, 20(L)x20(W)x0.5(H) cm3 piece of solid water was fabricated with pockets to allow OSLDs and TLDs to be irradiated simultaneously and perpendicular to the beam. Irradiations were performed at 5cm depth in photons, and in the center of a 10 cm SOBP in a 200MeV proton beam. Additionally, the Radiological Physics Center’s anthropomorphic pelvic phantom was used to test the angular dependence of OSLDs in photons and protons. A cylindrical insert in the phantom allows the dosimeters to be rotated to any angle with a fixed gantry angle. OSLDs were irradiated at 12 angles between 0 and 360 degrees. The OSLDs were read out with a MicroStar reader from Landauer, Inc. Dose response indicates that at angles where the dosimeter is near parallel with the radiation beam response is reduced slightly. Measurements in proton beams do not show significant angular dependence. Post-irradiation fading of OSLDs was studied in proton beams to determine if the fading was different than that of photons. The fading results showed no significant difference from results in photon beams. OSLDs and TLDs are comparable within 3% in photon beams and a correction factor can be posited for proton beams. With angular dependence characteristics defined, OSLDs can be implemented into multiple-field treatment plans in photons and protons and used in the RPC’s quality assurance program.

Characterization of the Agrobacterium tumefaciens VirB2 pilin of the VirB/D4 Type IV Secretion System

The Agrobacterium tumefaciens VirB/D4 type IV secretion system (T4SS) delivers oncogenic T-DNA and effector proteins to susceptible plant cells. This leads to the formation of tumors termed Crown Galls. The VirB/D4 T4SS is comprised of 12 subunits (VirB1 to VirB11 and VirD4), which assemble to form two structures, a secretion channel spanning the cell envelope and a T-pilus extending from the cell surface. In A. tumefaciens, the VirB2 pilin subunit is required for assembly of the secretion channel and is the main subunit of the T-pilus. The focus of this thesis is to define key reactions associated with the T4SS biogenesis pathway involving the VirB2 pilin. Topology studies demonstrated that VirB2 integrates into the inner membrane with two transmembrane regions, a small cytoplasmic loop, and a long periplasmic loop comprised of covalently linked N and C termini. VirB2 was shown by the substituted cysteine accessibility method (SCAM) to adopt distinct structural states when integrated into the inner membrane and when assembled as a component of the secretion channel and the T-pilus. The VirB4 and VirB11 ATPases were shown by SCAM to modulate the structural state of membrane-integrated VirB2 pilin, and evidence was also obtained that VirB4 mediates extraction of pilin from the membrane. A model that VirB4 functions as a pilin dislocase by an energy-dependent mechanism was further supported by coimmunoprecipitation and osmotic shock studies. Mutational studies identified two regions of VirB10, an N-terminal transmembrane domain and an outer membrane-associated domain termed the antennae projection, that contribute selectively to T-pilus biogenesis. Lastly, characterization of a VirB10 mutant that confers a ‘leaky’ channel phenotype further highlighted the role of VirB10 in gating substrate translocation across the outer membrane as well as T-pilus biogenesis. Results of my studies support a working model in which the VirB4 ATPase catalyzes dislocation of membrane-integrated pilin, and distinct domains of VirB10 coordinate pilin incorporation into the secretion channel and the extracellular T-pilus.

EXPRESSION AND IMMUNOLOGICAL CHARACTERIZATION OF HERV-K TRANSMEMBRANE ENVELOPE PROTEIN IN HUMAN BREAST CANCER

The human endogenous retrovirus K (HERV-K) env gene encodes envelope protein comprising surface (SU) and transmembrane (TM) domains. Having shown the exclusive expression of SU in human breast cancer and the stimulation of SU-specific immune responses in patients with breast cancer, our research here confirmed and extended the data by investigating the expression of HERV-K TM envelope domain and the induction of specific immune responses against TM in breast cancer patients. We found HERV-K TM mRNA and protein expression only in human breast cancer cells but not in normal controls. The specific immune responses against TM domain were induced in mice determined by enzyme-linked immunosorbent assay (ELISA) and IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay. Furthermore, ELISA detected higher titers of anti-HERV-K TM Env IgG antibodies in sera of breast cancer patients. In addition, the magnitude of the anti-HERV TM B cell response was correlated with the disease stage. Peripheral blood mononuclear cells (PBMCs) before and after in vitro stimulation (IVS) with HERV-K TM from patients with breast cancer as well as healthy controls were tested for T cell responses against HERV-K TM domain by ELISPOT assay. Breast cancer patients (n=21) had stronger HERV-K TM-specific cellular responses than healthy controls (n=12) (P < 0.05). These findings suggest, for the first time, that HERV-K TM expression was enhanced in human breast cancer cells and was able to induce specific B cell and T cell immune responses in breast cancer patients. This study provides support for HERV-K TM as a promising source of antigen for anti-tumor immunotherapy, prevention, diagnosis, and prognosis.

IDENTIFICATION AND CHARACTERIZATION OF DISTINCT POPULATIONS OF CLONOGENIC BONE MARROW STROMAL CELLS CAPABLE OF TRANSFERRING THE HEMATOPOIETIC MICROENVIRONMENT IN VIVO AND SUPPORTING LT-HSCs IN VITRO

Bone marrow (BM) stromal cells are ascribed two key functions, 1) stem cells for non-hematopoietic tissues (MSC) and 2) as components of the hematopoietic stem cell niche. Current approaches studying the stromal cell system in the mouse are complicated by the low yield of clonogenic progenitors (CFU-F). Given the perivascular location of MSC in BM, we developed an alternative methodology to isolate MSC from mBM. An intact ‘plug’ of bone marrow is expelled from bones and enzymatically disaggregated to yield a single cell suspension. The recovery of CFU-F (1917.95+199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32+1.9) by at least 2 orders of magnitude (P<0.001; N = 8) with an accompanying 196-fold enrichment of CFU-F frequency. Purified BM stromal and vascular endothelial cell populations are readily obtained by FACS. A detailed immunophenotypic analysis of lineage depleted BM identified PDGFRαβPOS stromal cell subpopulations distinguished by their expression of CD105. Both subpopulations retained their original phenotype of CD105 expression in culture and demonstrate MSC properties of multi-lineage differentiation and the ability to transfer the hematopoietic microenvironment in vivo. To determine the capacity of either subpopulation to support long-term multi-lineage reconstituting HSCs, we fractionated BM stromal cells into either the LinNEGPDGFRαβPOSCD105POS and LINNEGPDGFRαβPOSCD105LOW/- populations and tested their capacity to support LT-HSC by co-culturing each population with either 1 or 10 HSCs for 10 days. Following the 10 day co-culture period, both populations supported transplantable HSCs from 10 cells/well co-cultures demonstrating high levels of donor repopulation with an average of 65+23.6% chimerism from CD105POS co-cultures and 49.3+19.5% chimerism from the CD105NEG co-cultures. However, we observed a significant difference when mice were transplanted with the progeny of a single co-cultured HSC. In these experiments, CD105POS co-cultures (100%) demonstrated long-term multi- lineage reconstitution, while only 4 of 8 mice (50%) from CD105NEG -single HSC co-cultures demonstrated long-term reconstitution, suggesting a more limited expansion of functional stem cells. Taken together, these results demonstrate that the PDGFRαβCD105POS stromal cell subpopulation is distinguished by a unique capacity to support the expansion of long-term reconstituting HSCs in vitro.

Functional Characterization of AtxA, the Bacillus anthracis Virulence Regulator

Coordinated expression of virulence genes in Bacillus anthracis occurs via a multi-faceted signal transduction pathway that is dependent upon the AtxA protein. Intricate control of atxA gene transcription and AtxA protein function have become apparent from studies of AtxA-induced synthesis of the anthrax toxin proteins and the poly-D-glutamic acid capsule, two factors with important roles in B. anthracis pathogenesis. The amino-terminal region of the AtxA protein contains winged-helix (WH) and helix-turn-helix (HTH) motifs, structural features associated with DNA-binding. Using filter binding assays, I determined that AtxA interacted non-specifically at a low nanomolar affinity with a target promoter (Plef) and AtxA-independent promoters. AtxA also contains motifs associated with phosphoenolpyruvate: sugar phosphotransferase system (PTS) regulation. These PTS-regulated domains, PRD1 and PRD2, are within the central amino acid sequence. Specific histidines in the PRDs serve as sites of phosphorylation (H199 and H379). Phosphorylation of H199 increases AtxA activity; whereas, H379 phosphorylation decreases AtxA function. For my dissertation, I hypothesized that AtxA binds target promoters to activate transcription and that DNA-binding activity is regulated via structural changes within the PRDs and a carboxy-terminal EIIB-like motif that are induced by phosphorylation and ligand binding. I determined that AtxA has one large protease-inaccessible domain containing the PRDs and the carboxy-terminal end of the protein. These results suggest that AtxA has a domain that is distinct from the putative DNA-binding region of the protein. My data indicate that AtxA activity is associated with AtxA multimerization. Oligomeric AtxA was detected when co-affinity purification, non-denaturing gel electrophoresis, and bis(maleimido)hexane (BMH) cross-linking techniques were employed. I exploited the specificity of BMH for cysteine residues to show that AtxA was cross-linked at C402, implicating the carboxy-terminal EIIB-like region in protein-protein interactions. In addition, higher amounts of the cross-linked dimeric form of AtxA were observed when cells were cultured in conditions that promote toxin gene expression. Based on the results, I propose that AtxA multimerization requires the EIIB-like motif and multimerization of AtxA positively impacts function. I investigated the role of the PTS in the function of AtxA and the impact of phosphomimetic residues on AtxA multimerization. B. anthracis Enzyme I (EI) and HPr did not facilitate phosphorylation of AtxA in vitro. Moreover, markerless deletion of ptsHI in B. anthracis did not perturb AtxA function. Taken together, these results suggest that proteins other than the PTS phosphorylate AtxA. Point mutations mimicking phosphohistidine (H to D) and non-phosphorylated histidine (H to A) were tested for an impact on AtxA activity and multimerization. AtxA H199D, AtxA H199A, and AtxA H379A displayed multimerization phenotypes similar to that of the native protein, whereas AtxA H379D was not susceptible to BMH cross-linking or co-affinity purification with AtxA-His. These data suggest that phosphorylation of H379 may decrease AtxA activity by preventing AtxA multimerization. Overall, my data support the following model of AtxA function. AtxA binds to target gene promoters in an oligomeric state. AtxA activity is increased in response to the host-related signal bicarbonate/CO2 because this signal enhances AtxA multimerization. In contrast, AtxA activity is decreased by phosphorylation at H379 because multimerization is inhibited. Future studies will address the interplay between bicarbonate/CO2 signaling and phosphorylation on AtxA function.

CHARACTERIZATION AND TREATMENT OF A NOVEL MOUSE MODEL OF TSC-ASSOCIATED AUTISM

Tuberous sclerosis complex (TSC) is a dominant tumor suppressor disorder caused by mutations in either TSC1 or TSC2. The proteins of these genes form a complex to inhibit the mammalian target of rapamycin complex 1 (mTORC1), which controls protein translation and cell growth. TSC causes substantial neuropathology, often leading to autism spectrum disorders (ASDs) in up to 60% of patients. The anatomic and neurophysiologic links between these two disorders are not well understood. However, both disorders share cerebellar abnormalities. Therefore, we have characterized a novel mouse model in which the Tsc2 gene was selectively deleted from cerebellar Purkinje cells (Tsc2f/-;Cre). These mice exhibit progressive Purkinje cell degeneration. Since loss of Purkinje cells is a well-reported postmortem finding in patients with ASD, we conducted a series of behavior tests to assess if Tsc2f/-;Cre mice displayed autistic-like deficits. Using the three chambered social choice assay, we found that Tsc2f/-;Cre mice showed behavioral deficits, exhibiting no preference between a stranger mouse and an inanimate object, or between a novel and a familiar mouse. Tsc2f/-;Cre mice also demonstrated increased repetitive behavior as assessed with marble burying activity. Altogether, these results demonstrate that loss of Tsc2 in Purkinje cells in a haploinsufficient background lead to behavioral deficits that are characteristic of human autism. Therefore, Purkinje cells loss and/or dysfunction may be an important link between TSC and ASD. Additionally, we have examined some of the cellular mechanisms resulting from mutations in Tsc2 leading to Purkinje cell death. Loss of Tsc2 led to upregulation of mTORC1 and increased cell size. As a consequence of increased protein synthesis, several cellular stress pathways were upregulated. Principally, these included altered calcium signaling, oxidative stress, and ER stress. Likely as a consequence of ER stress, there was also upregulation of ubiquitin and autophagy. Excitingly, treatment with an mTORC1 inhibitor, rapamycin attenuated mTORC1 activity and prevented Purkinje cell death by reducing of calcium signaling, the ER stress response, and ubiquitin. Remarkably, rapamycin treatment also reversed the social behavior deficits, thus providing a promising potential therapy for TSC-associated ASD.

BIOCHEMICAL CHARACTERIZATION OF BINDING PARTNERS OF TWO HSP70 CO-CHAPERONES IN SACCHAROMYCES CEREVISIAE

Cells are exposed to a variety of environmental and physiological changes including temperature, pH and nutrient availability. These changes cause stress to cells, which results in protein misfolding and altered cellular protein homeostasis. How proteins fold into their three-dimensional functional structure is a fundamental biological process with important relevance to human health. Misfolded and aggregated proteins are linked to multiple neurodegenerative diseases, cardiovascular disease and cystic fibrosis. To combat proteotoxic stress, cells deploy an array of molecular chaperones that assist in the repair or removal of misfolded proteins. Hsp70, an evolutionarily conserved molecular chaperone, promotes protein folding and helps maintain them in a functional state. Requisite co-chaperones, including nucleotide exchange factors (NEFs) strictly regulate and serve to recruit Hsp70 to distinct cellular processes or locations. In yeast and human cells, three structurally non-related cytosolic NEFs are present: Sse1 (Hsp110), Fes1 (HspBP1) and Snl1 (Bag-1). Snl1 is unique among the cytosolic NEFs as it is localized at the ER membrane with its Hsp70 binding (BAG) domain exposed to the cytosol. I discovered that Snl1 distinctly interacts with assembled ribosomes and several lines of evidence indicate that this interaction is both independent of and concurrent with binding to Hsp70 and is not dependent on membrane localization. The ribosome-binding site is identified as a short lysine-rich motif within the amino terminus of the Snl1 BAG domain distinct from the Hsp70 interaction region. In addition, I demonstrate ribosome association with the Snl1 homolog in the pathogenic fungus, Candida albicans and localize this putative NEF to a perinuclear/ER membrane, suggesting functional conservation in fungal BAG domain-containing proteins. As a first step in determining specific domain architecture in fungal BAG proteins, I present the preliminary steps of protein purification and analysis of the minimal Hsp70 binding region in in both S.cerevisiae and C. albicans Snl1. Contrary to previous in vitro evidence which showed the Fes1 NEF to interact with both cytosolic Hsp70s, Ssa and Ssb, Fes1 is shown to interact specifically with Ssa when expressed under normal cellular conditions in S. cerevisiae. This is the first reported evidence of Hsp70 binding selectivity for a cytosolic NEF, and suggests a possible mechanism to achieve specificity in Hsp70-dependent functions. Taken together, the work presented in this dissertation highlights the striking divergence among Hsp70 co-chaperones in selecting binding partners, which may correlate with their specific roles in the cell.

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS

CHARACTERIZATION OF THE COUNT RATE PERFORMANCE AND EVALUATION OF THE EFFECTS OF HIGH COUNT RATES ON MODERN GAMMA CAMERAS Michael Stephen Silosky, B.S. Supervisory Professor: S. Cheenu Kappadath, Ph.D. Evaluation of count rate performance (CRP) is an integral component of gamma camera quality assurance and measurement of system dead time (τ) is important for quantitative SPECT. The CRP of three modern gamma cameras was characterized using established methods (Decay and Dual Source) under a variety of experimental conditions. For the Decay method, input count rate was plotted against observed count rate and fit to the paralyzable detector model (PDM) to estimate τ (Rates method). A novel expression for observed counts as a function of measurement time interval was derived and the observed counts were fit to this expression to estimate τ (Counts method). Correlation and Bland-Altman analysis were performed to assess agreement in estimates of τ between methods. The dependencies of τ on energy window definition and incident energy spectrum were characterized. The Dual Source method was also used to estimate τ and its agreement with the Decay method under identical conditions and the effects of total activity and the ratio of source activities were investigated. Additionally, the effects of count rate on several performance metrics were evaluated. The CRP curves for each system agreed with the PDM at low count rates but deviated substantially at high count rates. Estimates of τ for the paralyzable portion of the CRP curves using the Rates and Counts methods were highly correlated (r=0.999) but with a small (~6%) difference. No significant difference was observed between the highly correlated estimates of τ using the Decay or Dual Source methods under identical experimental conditions (r=0.996). Estimates of τ increased as a power-law function with decreasing ratio of counts in the photopeak to the total counts and linearly with decreasing spectral effective energy. Dual Source method estimates of τ varied as a quadratic with the ratio of the single source to combined source activities and linearly with total activity used across a large range. Image uniformity, spatial resolution, and energy resolution degraded linearly with count rate and image distorting effects were observed. Guidelines for CRP testing and a possible method for the correction of count rate losses for clinical images have been proposed.

Development and Characterization of Novel Ras Inhibitors

Ras genes are mutated in 15% of human cancers. Ras GTPases operate as molecular switches regulating cellular processes including proliferation, differentiation, and apoptosis. The three main isoforms of Ras – H-Ras, K-Ras, and N-Ras – inhabit distinct nanodomains of the plasma membrane and intracellular compartments including the Golgi. However, the role of single endogenous Ras isoforms on these compartments remains unclear as most studies have utilized ectopically expressed and mutant forms of Ras proteins. In an effort to develop novel tools that will allow us to abrogate individual endogenous Ras isoforms, we targeted the catalytic domain of p120RasGAP to the plasma membrane with the hypervariable region (HVR) of H-Ras (GAP-CTH) or K-Ras (GAP-CTK) and to the Golgi using the HVR of H-Ras with insertion of a point mutation (GAP-CTH181S). We performed GST-RBD pull-downs on cells expressing each GAP construct and stimulated with epidermal growth factor (EGF). We found that GAP-CTH and GAP-CTK specifically inhibited H-Ras or K-Ras, respectively. However, we did not detect any effect of GAP-CTH181S on Ras activation. Additionally, we used confocal microscopy to verify the ability of GAP constructs to abrogate Ras activation in distinct sub-cellular compartments. We found that GAP-CTH inhibits H-Ras activation on the plasma membrane, while GAP-CTK inhibits K-Ras activation on the plasma membrane. On the contrary, GAP-CTH181S inhibited H-Ras activation on the Golgi. We also analyzed the effects of these GAP constructs on the activation of ERK and Akt in response to EGF stimulation. We found that EGF stimulation of the MAPK pathway was inhibited by GAP-CTK but none of the other GAP constructs, while Akt activation was not inhibited by any GAP construct. Finally, we assayed cellular proliferation and differentiation. We found that GAP-CTK and GAP-CTH were equipotent inhibitors of cellular growth, whereas GAP-CTH181S was less potent. We also found that GAP-CTK and GAP-CTH inhibited differentiation with similar potency, while GAP-CTH181S was more potent. This approach may be adapted to investigate any Ras-dependent signaling pathway. Therefore, it has the potential to become a powerful tool for studying Ras isoform-specific signaling outputs.

Characterization of a Novel Role for the Tousled- like Kinase in Kinetochore Assembly and Function in Caenorhabditis elegans

Chromosome segregation is a critical step during cell division to avoid aneuploidy and promote proper organismal development. Correct sister chromatid positioning and separation during mitosis helps to achieve faithful transmission of genetic material to daughter cells. This prevents improper chromosome partitioning that can potentially result in extrachromosomal fragments, increasing the tumorigenic potential of the cells. The kinetochore is a protenaicious structure responsible for the initiation and orchestration of chromosome movement during mitosis. This highly conserved structure among eukaryotes is required for chromosome attachment to the mitotic spindle and failure to assemble the kinetochore results in aberrant chromosome segregation. Thus elucidating the mechanism of kinetochore assembly is important to have a better understanding of the regulation that controls chromosome segregation. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as a mitotic kinase and depletion of TLK-1 results in embryonic lethality, characterized by nuclei displaying poor mitotic chromosome alignment, lagging chromosome, and chromosome bridges during anaphase. Additionally, previous studies from our group revealed that TLK-1 is phosphorylated independently by Aurora B at serine 634, and by CHK-1 at threonine T610. The research presented herein reveals that both phosphorylated forms of TLK-1 associate with the kinetochore during mitosis. Moreover, by systematic depletion of kinetochore proteins, I uncovered that pTLK-1 is bona fide kinetochore component that is located at the outer kinetochore layer, influencing the microtubule-binding interface. I also demonstrated that TLK-1 is necessary for the kinetochore localization of the microtubule interacting proteins CLS-2 and LIS-1 and I show that embryos depleted of TLK-1 presented an aberrant twisted kinetochore pattern. Furthermore, I established that the inner kinetochore protein KNL-2 is an in vitro substrate of TLK-1 indicating a possible role of TLK-1 in regulating centromeric assembly. Collectively, these results suggest a novel role for the Tousled-like kinase in regulation of kinetochore assembly and microtubule dynamics and demonstrate the necessity of TLK-1 for proper chromosome segregation in C. elegans.