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NCI - National Cancer Institute

Breast tissue is dynamic and undergoes significant structural changes throughout a woman's life. The breast tissue architecture is maintained by a population of stem cells with self-renewal capacity which are essential for tissue repair and remodeling. Recently, the role of stem cells in breast carcinogenesis has been recognized as a high priority for translational breast cancer (BCa) research. The stem cell hypothesis of breast carcinogenesis suggests that breast cancer development might be directly related to the size of the stem cell pool and its mitotic activity. Further, in the mammary gland, stem cells are the only cell subpopulation that has capacity to accumulate all the oncogenic alterations. This project will fill gaps in our understanding of the role of stem cells in the early stages of breast carcinogenesis and their interplay with BCa risk factors by focusing on co-stained stem cell marker combinations that are reflective of high-risk stem cell lineages. Specifically, we will explore: 1) the associations of high-risk stem cell lineages (CD44+/CD24-/low, EpCAM-/low/CD49fhigh and ALDH1A1high/CD44+/CD133+) in histologically normal breast tissue with mammographic percent density and AI- based mammographic features (n=1,290); 2) the associations of high-risk stem cell lineages with subsequent BCa risk in women with a previous benign biopsy (444 cases/1,003 controls); and 3) the associations of hormone-related factors (oral contraceptives, menopausal hormone therapy, alcohol use, body mass index, weight gain since age 18, and reproductive factors) with high-risk stem cell lineages (n=1,834). We will use prospectively collected data/samples/ mammograms from cancer-free women with benign breast disease (BBD) within the Nurses' Health Study (NHS), NHS II, Washington University's Women's Health Repository (WHR), and a unique collection of data/samples/mammograms from healthy women in the Komen Tissue Bank, with the final study population representative of US demographics. Stem cell markers will be stained on study tissue microarrays (specifically constructed from histologically normal breast tissue regions) with commercially available antibodies using multiplex immunofluorescence, and the staining results will be evaluated with automated image analysis. We propose a highly novel investigation that will comprehensively examine the role of stem cell markers in breast carcinogenesis. The study aims to shed light on molecular pathways behind the observed associations of risk factors with BCa risk as well as to identify markers that could advance future risk prediction in a large segment of high-risk women undergoing routine breast biopsies and those with high-risk mammographic features. This proposal could pave the way for novel personalized breast cancer prevention and surveillance strategies. As stem cell activity is potentially modifiable via a variety of targeted therapies, the findings could translate into stem cell -directed pharmaceutical interventions aimed at BCa risk reduction in high-risk women with BBD and/or high mammographic breast density in whom novel prevention strategies are urgently needed.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT The motor symptoms associated with Parkinson’s Disease (PD) are caused by progressive loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Currently, there is no explanation for what causes this selective cell vulnerability. In addition to age, environmental factors (e.g. exposure to neurotoxins) also increase the risk for PD, but the mechanisms contributing to vulnerability are poorly understood. Further, there are no available treatment options that effectively prevent or slow DA neuron loss or the progression of related symptoms. Thus, there is a significant need for molecular dissection of the key pathways involved in regulating susceptibility to degeneration in order to identify novel potential drug targets and develop improved treatment options for PD patients. Myocyte enhancer factor 2A (MEF2A) was identified as a genetic master regulator whose activity is decreased in the DA neurons of MPTP treated mice. Dysregulation of MEF2A activity is thought to underlie DA neuron vulnerability. Inactivation of MEF2A can occur through phosphorylation of serine 408 (S408) in the carboxy terminal of the protein. The kinases involved in this phosphorylation are known to be activated by environmental exposure to neurotoxins which disrupt mitochondrial function and energy production. My preliminary data in stem cell-derived midbrain DA neurons indicates that in the absence of MEF2A, these cells experience significant changes in the expression of genes associated with synaptic signaling, maintenance of membrane potential, regulation of cell cycle processes, and DNA metabolic processes. My central hypothesis is that MEF2A is a critical regulator of vulnerability because it regulates the expression of other genes necessary for proper DA neuronal function, and that dysregulation of MEF2A activity underlies the DA neuron vulnerability associated with PD pathogenesis. To test this hypothesis, I will carry out two Aims. In Aim 1, I will determine how MEF2A affects intrinsic DA neuron vulnerability. Using in vitro human embryonic stem cell (hESC)-derived midbrain DA neurons, I have generated an inducible MEF2A knockout cell line. I will use this line, along with wild type (WT) DA neurons, to define the role of MEF2A in cell vulnerability using cell death assays, immunohistochemistry, senescence assays, patch clamp electrophysiology, live Ca2+ imaging, and fluorescent mitochondrial ROS assays. In Aim 2, I will look at how environmental factors contribute to MEF2A activity dysregulation and SNpc DA neuron vulnerability. For this, I will use in vitro neurotoxin and kinase inhibitor treatments along with cell death assays and quantitative western blotting to look at changes in survival as well as levels of phosphorylated MEF2A (p-MEF2A) in both WT and MEF2A-KO DA neurons. This proposed research will provide crucial insights into the molecular and cellular mechanism underlying SNpc DA neuron vulnerability and inform future avenues of investigation for the development of preventative treatments for PD patients.

NCI - National Cancer Institute

Project summary/abstract Richter Transformation (RT) is an aggressive, therapy-refractory large cell lymphoma that arises in up to 10% of patients with chronic lymphocytic leukemia (CLL). RT represents a major unmet need in CLL management, as patients are largely refractory to existing therapies and have a median overall survival of less than 12 months. Immunotherapies, including immune checkpoint blockade (ICB), have shown promise in RT, but responses often display limited duration. The objective of this study is to elucidate the molecular function of the MAX dimerization protein MGA, a gene that is disrupted by inactivating mutations leading to loss-of-function (LOF) in ~29% of RT cases. Our long-term goal is to improve the efficacy of immunotherapy-based combination treatments for this currently incurable malignancy, tailored to the molecular characteristics of patients. We hypothesize that MGALOF regulates tumor cell fitness by controlling growth programs and inflammatory signaling, thereby promoting B-cell clonal expansion and immune evasion. This hypothesis is supported by preliminary data showing that MGA-LOF leads to de-repression of transcription factors involved in proliferation and inflammatory cytokine production in both human tumors and murine models. In murine models faithful to the CLL-to-RT progression, Mga-LOF drives clonal selection of malignant B cells and promotes the formation of a PD-1+ T-cell rich tumor microenvironment (TME). In this study, we will pursue two specific aims: 1) define the mechanisms through which Mga-LOF favors B-cell fitness during the CLL transformation into RT; 2) determine how Mga-LOF driven inflammatory signaling rewires the RT-TME, predisposing Mga-LOF RT to anti-PD-1 ICB-based combination therapies. These questions will be addressed through integrated studies of faithful murine models of the CLL-to- RT evolution, isogenic human cell lines, and genetically annotated patient samples collected longitudinally during CLL progression or at time of RT. As primary RT samples are generally scarce and mostly available as fixed, non-viable, tissue, they preclude ex vivo manipulation and longitudinal functional studies. Our novel immunocompetent murine models provide a necessary and physiologically relevant platform to investigate the molecular mechanisms underlying the stepwise CLL-to-RT evolution, enabling the preclinical evaluation of novel therapeutic strategies. We have assembled a multidisciplinary team to support this work, including experts in computational biology (Landau), stem cell biology and epigenetics (Apostolou), and tumor immunology (Zappasodi). Collaborations with clinicians (Furman, Thompson, Inghirami, Parry) will provide access to genetically annotated patient specimens and spatial transcriptomics datasets from ongoing immunotherapy clinical trials. This research is significant because it will reveal critical biological mechanisms underlying CLL transformation and provide a preclinical foundation for personalized therapies for RT patients.

NIA - National Institute on Aging

Project Summary: Hutchinson-Gilford progeria syndrome (HGPS) is a rapid, progressive aging disease caused be a de novo point mutation in the LMNA gene. The mutation causes missplicing of LMNA and production of a mutant mRNA and truncated protein called progerin. Progerin accumulation exerts a dominant negative effect on wild type LMNA, leading to DNA damage, proinflammatory gene activation, premature senescence and ultimately cell death. Children with HGPS die of accelerated atherosclerotic vascular disease. A notable pathologic feature of HGPS vascular disease is progressive loss of vascular smooth muscle cells with a simultaneous increase in inflammatory cells in the tunica adventitia and extensive periadventitial fibrosis. Some of these features are shared with other aging-associated cardiovascular diseases including hypertension and arteriosclerosis. Thus, a better understanding of how adventitial inflammation amplified vascular disease in HGPS may have implications for more common forms of aging in the cardiovascular system. In preliminary data we have found that HGPG aortas from a mouse model of disease harbor accumulation of Mac2 positive macrophage cells in the adventitia. On single-cell RNA sequencing we observe a significant increase in proinflammatory macrophage gene expression with enrichment for pathways involving interferon and tumor necrosis alpha pathways. Our central hypothesis is that progerin expression in bone-marrow derived myeloid cells promote vascular inflammation in HGPS. To test this hypothesis we will pursue the following two Specific Aims: 1) determine the contribution of myeloid-expressed mutant Lamin A (i.e. progerin) in adventitial fibrosis and vascular disease; 2) develop in vitro human stem cell models of HGPS to dissect the impact of Lamin dysfunction in myeloid cell differentiation and function. This project will explore a novel mechanism in HGPS- associate vascular disease, which has broader implications for how adventitial inflammation derived from bone- marrow cells may promote more common forms of atherosclerosis in humans.

NHLBI - National Heart Lung and Blood Institute

Abstract Hematopoietic stem cell (HSC) transplantation is a potent therapeutic strategy for the treatment of many blood disorders. Therefore, the development of methods for deriving large quantities of HSCs is a major focus in regenerative medicine. Unlike HSCs, human induced pluripotent stem cells (hiPSCs) can be grown indefinitely. Despite hiPSCs being an extremely scalable source, HSCs derived from hiPSCs using current protocols are largely devoid of long-term blood reconstitution potential. These shortcomings are suggestive of knowledge gaps surrounding HSC biology. In the Camargo lab we leveraged our in-vivo barcoding mouse model to show definitively that HSCs arise from both intraembryonic (aorta) and extraembryonic (umbilical and vitelline arteries) sites during native hematopoiesis, and that these sites play differential roles in blood production. Our findings suggest for the first time that umbilical and vitelline (UV) artery HSCs are more short-lived than aortic HSCs, and that the UV artery engages potently in embryonic lymphopoiesis. By performing single cell RNA-seq on murine blood producing endothelial cells, I observed differential NOTCH signaling strength and the presence of NOTCH inhibitors GPR183 and DLK1 in the aorta and UV arteries respectively. While it is vastly appreciated that a transient reduction in NOTCH signaling strength is required for hematopoiesis to occur, no study has detailed differential mechanisms of NOTCH inhibition at spatially distinct HSC-producing sites. Cross-referencing this site-specific data with scRNA-seq on a commonly used hiPSC hematopoietic differentiation protocol, I identified for the first time the exclusive prevalence of the UV-like hemogenic endothelium in vitro. To develop methods of producing long-lived HSCs with adult-like lymphoid potential, we plan to study the Notch pathway as a regulator of site-specific hematopoiesis and modulate NOTCH signaling strength to produce more aortic-like hemogenic endothelium from hiPSCs. From this preliminary data, we hypothesize that differential NOTCH signaling strength is crucial for producing distinct hematopoietic programs in the UV arteries and the aorta. To test this central hypothesis, we plan to pursue the following specific aims: (1) characterize the role of GPR183 in aortic hematopoiesis through murine loss of function studies, (2) describe the role of DLK1 in UV hematopoiesis by murine loss of function, and (3) determine the function of DLK1 in controlling the hiPSC spatial hematopoietic program. From these experiments we expect to elucidate the role of these NOTCH inhibitors in spatially defined hematopoiesis. By leveraging our site-specific scRNA-seq dataset, we are uniquely positioned to produce methods of deriving aortic-like blood cells. These novel blood populations have the potential to revolutionize the therapeutics landscape.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY The intestinal microbiota is a complex community that modulates immune responses and host metabolism. However, colonization of this niche by pathobionts, particularly early in life, can perturb this homeostasis and cause intestinal damage and inflammation. Further, because the neonatal intestinal microbiota, immunity, and barriers are not fully developed, infants are particularly susceptible to pathogen barrier breach and systemic bloodstream infection. Group B Streptococcus (GBS) is one such pathobiont that colonizes the neonatal gastrointestinal tract following aspiration of infected amniotic fluid and/or breast milk. This intestinal colonization serves as a reservoir for “late-onset” GBS systemic disease (LOD), which presents at least one week following birth (typically as bacteremia and/or meningitis) and causes ~15% mortality regardless of antibiotic treatment. Despite this, the mechanisms underlying GBS infant intestinal colonization and resulting systemic neonatal disease remain largely unknown. Type VII secretion systems (T7SS) export effector proteins with functions in virulence, toxicity, or interbacterial killing, and we recently characterized this system in GBS. Our previous work showed that GBS T7SS and its effectors promote virulence in murine models of meningitis by pore-forming effectors as well as female genital tract colonization by toxin-mediated interbacterial competition and immune evasion. We recently found that GBS T7SS is also important for neonatal systemic infection stemming from intestinal colonization and that GBS T7SS effectors are toxic to intestinal epithelium. Based on these data, this proposal will investigate the role of the GBS T7SS in neonatal intestinal dysbiosis, including disruption of the microbiota, impairment of immune responses, barrier breakdown, and ultimately systemic bacterial spread/bacteremia. These questions will be addressed with human cohort data as well as in vitro and in vivo models of GBS intestinal colonization and systemic dissemination in the following aims: AIM 1: Investigate GBS perturbation of the intestinal microbiota during infant intestinal colonization. AIM 2: Evaluate anti-GBS mucosal immune responses during infant intestinal colonization. AIM 3: Determine mechanisms of GBS-mediated inflammation and barrier damage in intestinal epithelium in vitro and during infant intestinal colonization in vivo. This proposal will investigate the role of GBS T7SS effectors on newborn intestinal colonization and barrier loss, which may afford novel targets and alternative therapeutic strategies to treat and prevent neonatal infections.

NCI - National Cancer Institute

PROJECT SUMMARY/ABSTRACT Squamous cell carcinoma (SCC) is one of the most common cancers worldwide, yet the early events that drive mutated epithelial stem cells toward malignancy remain poorly understood. While cellular plasticity has emerged as a key process enabling clinically normal mutation-harboring skin to acquire oncogenic potential and undergo malignant transformation, the non-genetic variables that regulate this process and ultimately drive tumorigenesis remain largely unknown. These gaps in knowledge can largely be attributed to a dearth of tools and models that capture pre-oncogenic stem cell identity landscape in intact tissues. In this project, I will investigate how the lymphatic vascular niche, increasingly recognized as a key regulator of epithelial stem cell, controls cancer stem cell fate transitions during early tumor initiation and malignant progression in skin SCC. Using deep imaging and sequencing approaches, we discovered that lymphatic vascular insufficiency predisposes stem cells to malignant transformation, while oncogenic plasticity propelling the transition from benign to metastatic carcinoma is preceded by dynamic lymphatic remodeling. The central hypothesis is that lymphatic niches evolve during tumor progression, initially supporting tolerance to oncogenic stress and later driving epigenetic rewiring that enables malignant transformation. Aim 1 will determine how lymphatic regression influences tolerance to oncogenic stress and promotes malignant transformation using in vivo lymphatic ablation models, chromatin accessibility profiling and organotypic culture systems. Aim 2 will define the molecular and spatial interactions between tumor-initiating cells and lymphatic niches during malignant progression. Leveraging enhancer-based proximity sensors, I will identify lymphatic-derived signals that promote stem cell plasticity potential. This work is expected to reveal context-dependent lymphatic cues that regulate early tumor initiation and malignant transition, while generating new tools to study vascular–stem cell interactions in vivo. These studies aim to uncover actionable pathways that can be targeted to intercept skin cancer before it becomes invasive, offering new opportunities for early intervention in a disease that affects millions. My training will take place in the Gur-Cohen Lab at UC San Diego and the Sanford Stem Cell Institute, a highly interdisciplinary environment with access to state-of-the-art tools in imaging, genomics, and computational biology. In this fellowship, I will gain advanced expertise in 3-dimensional tumor imaging, epigenomic analysis, and the development of experimental tools to interrogate the cancer stem cell interactome in real time. Under the guidance of a dedicated mentoring team, I will also strengthen my skills in scientific communication, grant writing, and mentorship. This integrated research and training plan will prepare me for an independent academic career focused on understanding how systemic signals influence stem cell fate and oncogenic potential.

NHLBI - National Heart Lung and Blood Institute

Project Summary Clonal hematopoiesis (CH) a premalignant condition affects 20% of people above age 60, increases risk for developing blood cancers like Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), and cardiovascular disease. CH-mutant clones originate in somatically-mutated hematopoietic stem cells (HSCs), with 80% of mutations occurring in epigenetic regulators, predominantly, DNMT3A. However, the molecular mechanisms that support clonal fitness remain incompletely understood. Since CH emerges with age, we reasoned that age-related changes in HSC regulation might create selective vulnerabilities that CH clones exploit for competitive advantage. Employing multiome single-cell sequencing, we recently identified zinc finger and BTB domain containing 1 (Zbtb1) as an essential transcription factor for HSC function during hematopoietic regeneration. In our preliminary analysis, while aged HSCs exhibit decreased Zbtb1 expression, DNMT3A- mutant HSCs exhibit increased Zbtb1 with hypomethylation at the Zbtb1 locus, suggesting epigenetic dysregulation drives aberrant expression. We further observed that Zbtb1 loss eliminates clonal advantage of DNMT3A-mutant HSCs. Finally, we identified direct ZBTB1 binding to the promoters of metabolic regulators, Got2 and Impdh2, as potential downstream mediators of ZBTB1 function in HSCs, and as targets in DNMT3A- mutant CH. Therefore, we hypothesize that epigenetic derepression of ZBTB1 drives clonal advantage in DNMT3A-mutant HSCs through transcriptional reprogramming of downstream targets regulating metabolic and other cellular processes essential for clonal fitness. This career development program will address two specific aims: (1) Determine the role of ZBTB1 in normal hematopoiesis and DNMT3A-mutated CH, (2) Define the epigenomic and metabolic functions of ZBTB1 in normal and DNMT3A-mutant HSCs. During the award period, the candidate, Dr. Harold Elias, MD, will conduct research at Memorial Sloan Kettering Cancer Center under the mentorship of Dr. Michael Kharas, an expert in stem cell RNA regulation, and co-mentor Dr. Marcel van den Brink, a leader in translational hematology. Both mentors have outstanding records guiding trainees to independence, complemented by advisors with expertise in epigenetics, metabolism, computational biology, and CH. He will build on his HSC biology foundation to develop critical skills for independence as an NIH-funded laboratory-based physician-scientist. His training will focus on four key areas: advanced epigenetic and metabolomics analysis, in vivo genetic screening techniques, and prime-editing optimization for in vivo CH modeling in primary human HSPCs. This comprehensive training will enable him to develop clinically relevant disease models and enhance his computational expertise in sequencing-based approaches, to generate preclinical validation data. Completion of this project will provide the candidate with the training and mentorship required to establish his academic career as an independent laboratory-based physician-scientist whose mechanistic discoveries will provide the foundation for therapeutic strategies targeting CH and its complications.

NINDS - National Institute of Neurological Disorders and Stroke

Abstract: Neurodevelopmental disorders associated with autism, intellectual disability, and cortical malformations are frequently caused by rare genetic abnormalities. Recently rare de-novo mutations in MAST1 have been identified in multiple patients with severe intellectual disability and cortical malformations. MAST1 is a microtubule- associated serine/threonine protein kinase-1 that is specifically expressed in neurons during brain development. However, little is known about the roles of MAST1 in regulating neuronal properties, and how MAST1 mutations affect the cellular and molecular mechanisms to cause neurodevelopmental deficits. The goal of this project is to elucidate the role of MAST1 in regulating neurodevelopment under normal conditions and disease states. In our preliminary experiments, we generated telencephalic brain organoids and neurons from control as well as patient and engineered stem cells with MAST1 mutations. We found microcephaly-associated size deficits in MAST1-mutated organoids due to reduced number of neurons and smaller neuropil size. We also observed elevated apoptosis, impaired neurites, and mitochondrial abnormalities in MAST1 mutated cortical neurons as compared to control neurons. We hypothesize that MAST1 regulates mitochondria trafficking in neurites and that mutated MAST1 causes neurite outgrowth deficits by disrupting mitochondria transport, ATP synthesis, and interactions with its binding partners. Collectively, these deficits lead to hyperactive mitochondria, overproduction of reactive oxygen species, neuronal apoptosis, and microcephaly-associated deficits in MAST1-mutated organoids. We will test this hypothesis in the following Specific Aims: (1) We will perform live-cell imaging on control and MAST1-mutated neurons to characterize mitochondria trafficking and ATP synthesis in axons and dendrites. (2) We will conduct structural-functional studies to determine how MAST1 interacts with mitochondria in neurons. Finally, (3) we will investigate how patient MAST1 mutations affect MAST1 interaction with its binding partners. The proposed research is significant because it is expected to substantially advance understanding of the cellular and molecular mechanisms disrupted in patient with neurodevelopmental disorders caused by MAST1 mutations and other brain disorders associated with mitochondrial abnormalities.

NCI - National Cancer Institute

PROJECT SUMMARY The main goal of the RUNX1 Research Program 10th Annual Scientific Conference and Patient Meeting is to create a dynamic convening to foster collaboration and provide opportunities for knowledge and data sharing among a diverse set of researchers, clinicians, and patients and their families for a rare disease called RUNX1 familial platelet disorder with predisposition to hematologic malignancies (RUNX1-FPD or FPDMM). Individuals with the disorder have a 35-50% lifetime risk of developing a hematologic malignancy. Of those who develop a malignancy, over 50% are diagnosed with AML. It is the only convening of its kind and continues to be an important part of deepening the communities’ understanding of the disease, of driving collaborations, and facilitating the discovery of future therapies for patients. Equally important is the opportunity for early career investigators, posts, and graduate students to present their research and receive feedback from a diverse, multidisciplinary community, a critical aspect of their training. RRP is committed to cultivating the next generation of RUNX1-FPD basic, translational, and clinical researchers. Conference participation has grown since our first conference in 2017, which had only 18 attendees. By 2021 we had over 181 attendees for the virtual conference. In 2022, we had 130 individuals - 40 patients and 90 scientists, clinicians, and genetic counselors - who attended in person in Princeton, NJ. Thanks to NCATS/NIH funding, we were able to award five (5) deserving young investigators with travel scholarships to attend the conference in 2022 and present at the poster sessions. In 2023, we returned to a virtual format with 397 registrants and a total of 219 participants. Once again, we held a poster session with four (4) young investigators.At our 2024 in-person scientific conference in Princeton, NJ we had 84 scientists, clinicians, and genetic counselors. As a result of NCI funding for the conference, we awarded travel scholarships to six (6) trainees.In 2024, we did not host a formal patient meeting alongside the scientific conference because we decided to pilot a smaller regional patient meeting in Houston to better reach patients outside of the northeast. This decision was influenced by a patient community-wide survey we conducted. Regardless, there were still seven patients and patient family members who attended the scientific conference. In 2025, our scientific conference and patient meeting will be held in September. The agenda includes research-specific sessions open to all stakeholders as well as a track dedicated to RUNX1-FPD patients and family members. The objective is to promote and drive patient-engaged research forward, with direct patient input in the development and planning of the sessions via our 23-member Research Guided by Patients Committee (RGPC). The research-focused portion of the meeting will bring together experts from the fields of hematology, immunology, oncology, cell therapy, and basic science who are committed to uncovering the mechanisms underlying the pathogenesis of RUNX1-FPD with the goal of developing therapeutic interventions that impact the bleeding and immune dysfunction issues and most importantly the predisposition to malignancy. The audience will include investigators devoted to studying RUNX1, leukemia progression, clonal hematopoiesis, gene editing, and hematopoietic stem cell transplants. It has become increasingly clear through the NHGRI-sponsored natural history study of RUNX1-FPD that immune dysfunction and inflammatory disorders are common. RUNX1-FPD includes a unique patient population where there may be a nexus between immunology and oncology based entirely on the reduced and/or altered activity of a single transcription factor, RUNX1.

NCI - National Cancer Institute

Abstract Multiple myeloma (MM) is an incurable plasma cell malignancy. Despite therapeutic advances, nearly all MM patients are expected to relapse and develop refractory disease, underscoring the need to improve the efficacy of first line therapy to prevent disease relapse. The current standard of care for newly diagnosed MM includes a myeloablative dose of the cytotoxic agent melphalan prior to autologous stem cell transplant (ASCT). Cytotoxic drugs are known to trigger therapy induced senescence (TIS) in normal cells. Several solid tumors have been shown to exhibit features of TIS following chemotherapy, and these markers in tumor cells are associated with superior progression free survival (PFS). One potential mechanism for this benefit is the activation of anti-tumor immune responses through the senescence associated secretory phenotype (SASP), which recruits and stimulates immune cells. Consistent with the findings in solid tumors, our analysis of longitudinal MM patient bone biopsies revealed a significant increase in senescence in MM cells in patients with PFS ≥2.5 years following ASCT. In contrast, patients that relapsed in <2.5 years did not exhibit increased tumor cell TIS. Additionally, patients that relapsed showed a positive correlation between MM burden post-ASCT and PFS, such that patients with the lowest plasma cell burden post-ASCT relapsed earliest. These data support that the ability of MM cells to evade apoptosis and enter a senescence-like, stable growth arrest in response to cytotoxic stress may protect against early relapse. To investigate the mechanisms underlying this phenomenon, we developed in vitro and in vivo models in which high-dose melphalan (HDM) consistently induces stable growth arrest and TIS features in human and mouse MM cell lines. Interestingly, HDM-MM cells also develop a myeloid gene signature. This myeloid gene expression was linked to functional plasticity, as HDM-MM cells cultured with osteoclast differentiation factors developed tartrate-resistant acid phosphatase (TRAcP5b) activity and multinucleation, two defining hallmarks of osteoclasts. Osteoclast-MM hybrids have previously been identified in MM patient biopsies, and our post-ASCT biopsy analysis revealed that nearly all patients had multinucleated CD138+ cells, including cells with osteoclast-like morphology on bone surfaces. Therefore, we hypothesize that TIS in MM cells activates anti-tumor immunity and drives MM-myeloid plasticity, enabling novel interactions with the bone niche that contribute to persistence and relapse. This hypothesis will be tested through the following specific aims: 1) Investigate the senescence-associated and immunological properties of MM cells that persist following cytotoxic therapy; 2) Define how TIS-myeloid plasticity supports MM cell survival and bone niche engagement. These studies will integrate our novel HDM in vitro and in vivo models with longitudinal patient biopsies. Because TIS and MM-myeloid plasticity are shaped by spatial and cellular interactions within the bone marrow microenvironment, in vivo models are essential to define how growth-arrested MM cells persist, engage the bone niche, and contribute to relapse. We expect these studies to reveal novel therapeutic strategies to target persistent, growth-arrested MM cells, ultimately preventing MM disease relapse.

NIGMS - National Institute of General Medical Sciences

We seek funding to acquire a Thermogravimetric Analyzer (TGA) with Evolved Gas Analysis (EGA) capabilities via selective sampling for GC-MS, to advance materials research and STEM education at the University of North Carolina at Pembroke (UNCP). Currently, UNCP lacks a TGA system, limiting capacity for advanced thermal analysis, reducing research productivity, and requiring reliance on external collaborations that introduce cost and delays. UNCP is a primarily undergraduate institution serving a large student population in a resource-limited region. The proposed instrument will directly support three faculty-led research initiatives—including projects led by early-career investigators—focused on biomass pyrolysis, plastics recycling, and the development of polymeric adsorbents for environmental remediation. The TGA-EGA system will enable precise measurements of decomposition temperatures, small molecule desorption profiles, and trace impurities, which are essential for characterizing complex materials. The instrument will also be integrated into upper-level chemistry courses, including organic, analytical, instrumental, and physical chemistry, providing hands-on training in thermal analysis and macromolecular characterization. These experiences will enhance preparation for careers in STEM and graduate study, strengthening the scientific workforce. The TGA-EGA will be housed in a shared research facility with broad access across departments, supporting interdisciplinary collaboration and student-faculty research. Its acquisition aligns with institutional goals to expand research infrastructure and increase access to advanced instrumentation.

Toyota USA Foundation

Supports innovative STEM education programs that develop students' skills for future careers, with emphasis on project-based learning.

NIGMS - National Institute of General Medical Sciences

Project Summary Signals that cells receive over time from a small set of pathways (e.g., BMP, Wnt, and TGFβ) shape their fate and phenotype during development, regeneration, and disease. Despite their central importance, signaling histories of individual cells are often inaccessible to direct observation, hindering quantitative analysis and obscuring their connection to eventual cell fate. This challenge is particularly pronounced in mammalian systems, where limited optical access and the constraints of size and timescale often render live imaging impractical. To address this issue, we have developed an approach to reconstruct the history of signaling activity in single cells based on endpoint fluorescence images. This is achieved by regulating CRISPR base editors to generate mutations in engineered target sites at rates proportional to the signal of interest. These mutations create a heritable record of signaling activity in the genome, which can be read out at a later time, together with the gene expression profile of the cells. Using this approach, we demonstrated that cells retain a memory of their past response level to BMP signaling for up to 18 days, providing a mechanism for long-term interactions between signals that can facilitate coordination of developmental processes over time. In this proposal, we will expand the scope and utility of our signal recording approach by extending its dynamic range to capture the broad spectrum of in vivo signal intensities and enabling simultaneous recording of the sequence and timing of two signaling pathways. We will also engineer mouse embryonic stem cells to record three key developmental pathways: BMP, Wnt, and Nodal. This will allow us to generate stem cell-derived embryo models and chimeric embryos to link cell fate and spatial organization at the onset of organogenesis with signaling activity at different time windows earlier in development. Additionally, we will investigate mechanisms that enable long-term changes in BMP responsiveness following an initial stimulation, without requiring differentiation. We will then test whether similar mechanisms exist in Wnt and Nodal pathways and assess their role in mediating long-term crosstalk between pathways. To achieve these goals, we will take an interdisciplinary approach combining gene editing, quantitative imaging, epigenomic assays, computational analysis, and generation of developmental models. The proposed goals build on my prior publications, recent preliminary data from our lab, and collaborations I have established since launching my lab. This research program will substantially advance the state of the art in molecular recording, transforming it into a technology that can be used in vivo, in mammalian systems to drive biological discovery. Our long term vision is to identify how signaling history controls cellular decision making during development, and how instructions that cells receive are coordinated over time to produce tissues with the correct number, types, and spatial arrangement of cells. Ultimately, this knowledge will inform strategies for tissue engineering, and open new avenues for understanding and treating diseases driven by dysregulated signaling.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Abstract In sexually reproducing organisms, the fusion of an oocyte and a sperm forms a totipotent zygote capable of generating every cell type in the body. In several model organisms such as Drosophila melanogaster, early asymmetries in the oocyte established before fertilization determine future developmental trajectories. However, the mechanisms that break symmetry and guide early cell fate biases in mammals remain poorly understood. Classical views held that mammalian embryos remain developmentally equivalent until the 16-cell stage, when inside and outside cells begin to emerge. Yet, recent studies suggest that asymmetries can arise as early as the 2-cell stage. At this early stage, both blastomeres are capable of generating all embryonic and extraembryonic lineages, but they often display reproducible biases in lineage contributions. One blastomere tends to favor epiblast fate, which gives rise to the embryo proper, while the other more often contributes to extraembryonic lineages like the trophectoderm. This phenomenon has also been observed in human embryos, implying conserved mechanisms underlying early cell fate biases. When these cells are physically separated at the 2-cell stage and cultured independently, only one consistently forms a robust inner cell mass, suggesting inherent differences in developmental potential. We have observed that in 2-cell stage mouse embryos, the cell with higher epiblast contribution correlates with the cell that inherits the sperm entry point. This research aims to identify the molecular and structural cues introduced at fertilization—such as sperm-derived components and the second polar body—that influence early fate biases in the mammalian embryo. Understanding these early symmetry-breaking events has broad implications for reproductive biology, stem cell research, and our fundamental understanding of developmental potential.

NIAID - National Institute of Allergy and Infectious Diseases

PROJECT SUMMARY Aging leads to widespread changes in the immune system, including increased inflammation, diminished vaccine responsiveness, and greater susceptibility to infection and chronic disease. However, older adults vary significantly in their immune trajectories—some maintain robust function, while others experience immune decline. The biological mechanisms driving this heterogeneity remain poorly understood, particularly the role of upstream hematopoietic stem and progenitor cells (HSPCs), which continually regenerate the immune system throughout life. This project tests the central hypothesis that epigenetic remodeling of HSPCs contributes to immune-related hallmarks of aging and variability in these phenotypes, shaping both inflammatory set points and immune responsiveness in older adults. To address this, we leverage a well-characterized, longitudinal cohort of 62 older adults, each of whom received a different influenza vaccine over three consecutive seasons. Using our novel PBMC-PIE platform—developed to enrich and profile circulating HSPCs from cryopreserved PBMCs at single-cell resolution—we can interrogate progenitor cell states and their progeny without requiring bone marrow biopsies. This enables unprecedented, longitudinal insight into how human HSPCs are reprogrammed with age and vaccination. In Aim 1, we will identify molecular and epigenomic features in HSPCs and innate immune cells that distinguish strong from poor vaccine responders. In Aim 2, we will determine how different influenza vaccine platforms (high-dose, adjuvanted, recombinant) shape the chromatin landscape and lineage bias of HSPCs and their progeny over time. In Aim 3, we will test whether maladaptive “aged” HSPC states can be rejuvenated in vitro using candidate immunomodulatory factors identified in Aims 1–2. By linking blood-based epigenetic signatures to real-world immune outcomes, this work will reveal fundamental mechanisms of immune aging, identify biomarkers of immune resilience, and inform the development of targeted strategies to restore immune function in older adults.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY Advances in regenerative medicine continue to transform the way we treat human diseases. To maintain our prominence in this important field, this application seeks to establish a new Training Program in Developmental, Stem Cell and Regenerative Biology (DSCRB). Despite tremendous interdisciplinary strength in this area at UW-Madison, there is no programmatic training in this field. Our Training Program, which is built around a recently inauguration curriculum that provides students a PhD Minor in DSCRB, is designed to fill this gap. The Minor requires 9 class credits, which are drawn from required and elective courses offered by the 8 PhD programs from which we draw trainees; therefore, students can complete the Minor without taking additional coursework beyond the requirements of their PhD programs. While the program’s administrative home is the Department of Cell and Regenerative Biology, our 55 trainers are from 17 departments and 6 schools/colleges at UW-Madison. Trainers are selected on the relevance of their research, their training and mentorship record, and the robustness of their research program, and more junior associate trainers are mentored by a more senior trainer. We seek to attract and train a cohort of 14 predoctoral trainees, matriculating 7 trainees per year for a two-year training program, with the goal of providing rigorous intellectual, technical, and interdisciplinary training. We will engage outstanding predoctoral trainees in the second semester of their first year, immediately after research rotations are complete. At this point, each student will join a trainer’s laboratory while they continue didactic training. To build cohesion, monthly Trainee Meetings, a student-led Journal Club, and a student mentorship program will bring together participants to share their research and interact. Career development activities and ethics training will be an important focus of these meetings, emphasizing reproducible and responsible conduct of research; ethical use of animal models, stem cells, fetal tissue, and human subjects in regenerative medicine research; and how to incorporate experimental rigor. The Training Program also leverages Stem Cell and Regenerative Medicine Center (SCRMC) activities, including the SCRMC Fall Conference, and the Wisconsin Stem Cell Symposium (international symposium sponsored jointly with Promega). These symposia offer our trainees opportunities to participate in meeting organizations, host external speakers, engage in career development activities, and present their research to a broad community. The PIs of this proposal have considerable combined experience in T32 training program administration and graduate education, and there is strong institutional support. The DSCRB Training Program will provide rigorous, cutting-edge training to produce the next generation of leaders in the field, enabling breakthroughs and empowering research that will lead to transformative regenerative therapies.

NIGMS - National Institute of General Medical Sciences

Project Summary The University of Missouri-Columbia (MU) proposes to direct a Trans-disciplinary Basic Biomedical Science (TDS) T32 training program for predoctoral students in PhD programs. The mission of our training program is to facilitate the transformation of our trainees into independent scientists who are active producers of new biomedical knowledge and are adept at solving the complex problems of disease and illness that adversely affect human health. Our training program accomplishes this mission by bringing together four PhD programs including Biochemistry, Biological Sciences, Biological Engineering, and Translational Biosciences. The latter program includes parallel emphasis areas (tracks) covering specialties including microbiology & virology, gene & stem cell therapy, immunology, physiology, nutrition & exercise science, cancer biology, and epidemiology & precision health. We propose to use science identity as our organizing principle, with its composition of community belonging, self-efficacy, and achieved deliverables. Objective #1: To strengthen the safe, supportive, and nurturing nature of our community by providing training to improve the quality of the peer- and mentor-mentee relationships that are so critical to career success and satisfaction. Objective #2: To expedite fluency in the scientific method, we will institute a Critical Thinking Workshop to be followed by a new course, Biomedical Literature Colloquium, for primary literature critique, presentation, and writing. Objective #3: To promote professional communication skills and increase achieved deliverables in the form of external fellowship wins and peer-reviewed publications. Objective #4: To improve the PhD completion rate of trainees in the participating biomedical doctoral programs from the current level of 74-81% to 85%. Our active program evaluation structure will allow independent, professional assessment of trainees’ sense of science self- efficacy, science identity, perceived support, and satisfaction with mentoring relationships, all of which are predictors of academic satisfaction and persistence outcomes among predoctoral students. The TDS T32 program will implement a wide-ranging set of programmatic activities to develop the technical, operational, and professional skills that will enable our trainees to flourish in their independent scientific careers.

NIGMS - National Institute of General Medical Sciences

Project Summary Double-strand breaks (DSBs) in DNA represent one of the most severe threats to genomic integrity, with improper repair leading to mutations, chromosomal translocations, and oncogenic transformation. Cells have evolved robust pathways, including non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), to repair DSBs. While much is known about protein-mediated DSB repair mechanisms, the role of transcript RNA in modulating repair remains poorly understood. Yet, the potential contribution of RNA molecules, particularly transcript RNA with sequence complementarity to DNA ends, remains largely unexplored. Recent discoveries from the PI’s laboratory demonstrate that RNA transcripts can bridge broken DNA ends and facilitate repair via NHEJ and MMEJ in both human and yeast systems, suggesting a conserved and fundamental role for RNA in genome maintenance. This project seeks to systematically uncover the mechanisms, regulatory proteins, and biological consequences of transcript RNA involvement in DSB repair across multiple contexts in human cells. Aim 1 establishes isogenic human embryonic kidney (HEK293T) and diploid retinal pigment epithelial (RPE-1) cell lines expressing spliced or non-spliced RNAs from the same adeno-associated virus integration site 1 (AAVS1) safe harbor locus and investigates how these transcripts modulate end-joining repair (NHEJ and MMEJ) of chromosomal DSBs induced by CRISPR/Cas9, followed by deep sequencing and bioinformatics analysis. Aim 2 identifies the key proteins and molecular processes involved in RNA-mediated end-joining repair in human cells. This goal will be achieved by inhibiting core NHEJ and MMEJ factors using chemical inhibitors and siRNAs, and by selectively reducing nascent spliced and non-spliced RNA transcripts using CRISPRi. These perturbations will define how individual proteins and transcript RNAs contribute to the efficiency, fidelity, and pathway choice of RNA-mediated end joining. Aim 3 examines whether transcript RNA differentially modulates DSB repair in exonic and intronic sequences of endogenous human genes in both HEK293T and RPE-1 cells, including breaks induced by CRISPR/Cas9 and mutagens, to understand RNA’s role in maintaining genome integrity. The significance of this work lies in establishing transcript RNA as a previously unrecognized regulator of DSB repair pathways, revealing new dimensions of genome stability control and potential therapeutic targets. The results could provide insights to improve genome editing technologies, inform strategies to reduce mutagenesis and carcinogenesis, and contribute fundamentally to understanding how RNA functions extend beyond traditional regulatory roles to directly safeguarding the genome. The outcomes will also lay groundwork for exploring RNA-mediated DNA repair in stem cells, non-dividing cells, aging models, and disease states, potentially transforming approaches to cancer prevention and therapy.

NCI - National Cancer Institute

Abstract PAX3 is an indispensable upstream regulator of normal melanocyte survival, population expansion, and migration, and these functions are subverted in melanoma proliferation, resistance to apoptosis, and metastasis. Despite significant advances in treatment, melanoma is still a deadly cancer with considerable mortality rates, and PAX3 contributes to this by supporting cancer aggressiveness, resistance to therapies, and relapse. While PAX3 is expressed in developing melanoblasts and melanocyte stem cells, downregulation of PAX3 during terminal differentiation is required. In melanoma, PAX3 expression is overexpressed, maintained, and essential for cancer progression. Melanoma cells rely on PAX3 function; indeed, a loss of PAX3 expression leads to catastrophic loss of melanoma cell growth, migration, and viability. Due to the significance of PAX3 in melanoma, it is surprising how much is unknown about PAX3 function. Classically defined as a transcription factor, only a handful of PAX3 downstream target genes in melanoma are known. Our lab and others have uncovered select downstream effector genes that can explain some but not all of the wide roles that PAX3 has on melanoma survival and progression. To uncover clues on other functions of PAX3, our laboratory performed an unbiased immunoprecipitation and mass spectroscopy (IP/MS) screen to identify PAX3 binding partners. Unexpectedly, we discovered that PAX3 interacted with several proteins involved with RNA surveillance, decay, and splicing. Further, our preliminary data support that this role in RNA regulation is independent of the transcriptional role of PAX3. The goal of this proposal is to uncover how PAX3 regulates these cell functions through downstream effector genes, by acting directly on genetic regulation or indirectly through other molecular means. To address the significant gaps in knowledge into PAX3 function, the hypothesis guiding this proposal is that PAX3 can dynamically act on downstream genes through canonical and noncanonical transcription factor functions, with outcomes modified by binding partners, tumor stage, and small molecule inhibitors. The major scientific impact of this proposal is to reveal a PAX3 dependent genomic enhancer map in melanoma connected to regulated genes, discover mechanisms for modulating this signature, and exploit these pathways as targets for therapy. The innovation of this proposal lies in a more detailed focus on PAX3, in terms of what genes are controlled and how. We propose the innovative hypothesis that PAX3 is not purely a canonical transcription factor and can function both transcriptionally and post-transcriptionally. This work is significant since the PAX3 transcriptional signature in melanoma is still undiscovered, and it is not clear if it is static or adaptive at different tumor stages or drug treatments. This work will uncover PAX3-dependent pathways driving melanoma progression and reveal potential molecular weak points that can be targeted by therapeutics. Use of mice in this proposal: The rationale for utilizing mouse models is that normal and pathological cells behave differently in vivo due to a complex microenvironment that cannot be replicated in other model systems.

NIMH - National Institute of Mental Health

Project Summary Mutations that reduce or alter the activity of repressive chromatin modifiers are frequent causes of neurodevelopmental disorders. The inaccessibility of the developing and the dynamic nature of neurogenesis so far have prevented an adequate understanding of how molecular pathologies arise downstream of the loss of specific chromatin regulators. This knowledge gap represents a hurdle for developing therapeutic interventions for neurodevelopmental disease. This proposal aims to combine targeted protein depletion with highly efficient directed differentiation regimens for human pluripotent stem cells to dissect gene regulatory functions of the autism-associated chromatin repressor EHMT1 during human cortical neurogenesis. Based on the extensive characterization of a novel, multipurpose (degradation/immunoprecipitation/visualization) degron allele, we hypothesize that interactions with cell type-specific co-factors allow EHMT1 to control the expression of stage- specific target genes during neurogenesis, resulting in the accumulation of molecular alterations and cortical neuron (CN) dysfunction when EHMT1 is lost from early development onwards. To systematically test this hypothesis, we will first determine whether molecular alterations caused by EHMT1 deficiency from earlier stages of neurogenesis onwards accumulate in CNs and to what degree dysregulated gene loci and CN function remain responsive to restoring physiological EHMT1 levels (Aim 1). We will then combine genomics, proteomics, and genetic approaches to identify how EHMT1, together with candidate recruiters and co-factors, regulates distinct gene loci at specific stages of cortical neurogenesis (Aim 2). Finally, we will expand our degron approach to dissect the functional interplay of different autism-associated H3K9 methylation regulators during human neurogenesis to identify interactions between these proteins that could be clinically exploited (Aim 3). Our experiments will determine currently unknown gene regulatory functions of disease-associated chromatin repressors at critical stages of human cortical neurogenesis. By generating mechanistic insight into how deficiencies of EHMT1 and other H3K9 methylation regulators introduce molecular pathologies in CNs, we anticipate revealing new opportunities for therapeutic interventions with specific neurodevelopmental diseases.

NEI - National Eye Institute

PROJECT SUMMARY/ABSTRACT Vision loss caused by the death of photoreceptors is a leading cause of irreversible blindness worldwide, yet therapeutic options remain limited. For this reason, the NEI’s Retinal Disease Program has identified the development of strategies for the treatment of retinal degenerations as a core program goal. Recently, several laboratories have derived photoreceptors from stem cells, making cell-replacement therapies particularly promising. Additionally, important advances have been made into manipulations that could stimulate retinal regeneration from the retinal Müller glia. The critical barrier for the success of such therapies is to understand the factors required to direct fate decisions in progenitors towards fully differentiated cell subtypes that are also capable of properly rewiring into retinal circuits. Although key transcription factors have been identified as essential for generating retinal cell classes, the target genes required to generate each retinal cell subtype are still undetermined. TBX2 is a central transcriptional regulator of all photoreceptor subtypes and is highly conserved across vertebrates. Our main hypothesis for this proposal is that retinal progenitors express TBX2 to repress the identity of photoreceptor subtypes that are not UV cones through two different mechanisms, and our main goal is to identify these two roles. Three Specific Aims are proposed: Specific Aim 1 will test the hypothesis that tbx2a and tbx2b have subdivided functions and their roles in generating photoreceptor subtypes are different. Specific Aim 2 will identify candidate factors downstream of TBX2 important for the generation of photoreceptor subtypes. Specific aim 3 will test the hypothesis that differences in Tbx2a and Tbx2b function are caused by differences in the repression domain. At the successful completion of this project, I will have identified the subdivided functions of tbx2a and tbx2b and subsequent targets to generate and maintain photoreceptor subtypes. This proposal benefits from the experimental accessibility of the retina and our deep knowledge of retinal cell types and circuits, but our approach has the potential to impact the study of other neuronal degenerative diseases. In addition to my proposed research, I designed a holistic training plan that will help me advance toward my goal of leading an independent research lab. The work planned for the F31 award period will be valuable in terms of both research and training opportunities.

U.S. National Science Foundation

TSL combines interests and resources of separate programs in the Division of Research on Learning in Formal and Informal Settings (DRL) to explore the opportunities and challenges implied by innovative visions of the future for STEM learning.?? The TSL program invites interdisciplinary teams of STEM content specialists, experts in relevant technologies, STEM formal and informal education specialists, researchers with expertise in the learning sciences, and specialists in education research and evaluation methods to submit proposals for research projects that (1) Study efficacy of existing prototypes for innovations like virtual schools, special STEM schools, and educational programs that combine opportunities of formal and informal learning resources in their communities; or (2) Design and conduct exploratory development of new potentially transformative models for STEM learning environments.?? The cross-cutting proposals will draw from work in the four primary DRL programs: Discovery Research K-12 (DR K-12), Informal Science Education (ISE), Research and Evaluation on Education in Science and Engineering (REESE), and Innovative Technology Experiences for Students and Teachers (ITEST).?? However, proposals submitted in response to this solicitation must have a scope that extends well beyond any of those programs individually.

U.S. National Science Foundation

The Transforming Undergraduate Education in Science, Technology, Engineering, and Mathematics (TUES) program seeks to improve the quality of science, technology, engineering, and mathematics (STEM) education for all undergraduate students. This solicitation especially encourages projects that have the potential to transform undergraduate STEM education, for example, by bringing about widespread adoption of classroom practices that embody understanding of how students learn most effectively. Thus transferability and dissemination are critical aspects for projects developing instructional materials and methods and should be considered throughout the project's lifetime.?? More advanced projects should involve efforts to facilitate adaptation at other sites.The program supports efforts to create, adapt, and disseminate new learning materials and teaching strategies to reflect advances both in STEM disciplines and in what is known about teaching and learning.?? It funds projects that develop faculty expertise, implement educational innovations, assess learning and evaluate innovations, prepare K-12 teachers, or conduct research on STEM teaching and learning. It also supports projects that further the work of the program itself, for example, synthesis and dissemination of findings across the program. The program supports projects representing different stages of development, ranging from small, exploratory investigations to large, comprehensive projects.