Biochemical and genetic analysis of two protein kinases JNKK2 and MEKK3

Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryotic signaling modules consisting of a MAPK, a MAPKK and a MAP3K. MAPK cascades are involved in many cellular responses including proliferation, differentiation, apoptosis, stress and immune responses. ^ The first part of this thesis describes the cloning and biochemical analysis of JNKK2, a member of MAPKK gene family. Our results demonstrate that JNKK2 is a specific JNK activator and activates the JNK-dependent signal transduction pathway in vivo by inducing c-Jun and ATF2-mediated gene expression. We also found that JNKK2 is specifically activated by a MAP3K MEKK2 through formation of MEKK2-JNKK2-JNK1 triple complex module. JNKK2 is likely to mediate specific upstream signals to activate JNK cascade. ^ The second part of this thesis describes biochemical and gene disruption analysis of MEKK3, a member of MAP3K gene family. We showed that overexpression of MEKK3 strongly activates both JNK and p38 MAPKs but only weakly activates ERK. MEKK−/− embryos die at about embryonic day (E) 11. MEKK3−/− embryos displayed defects in blood vessel development in the yolk sacs, and in the myocardium and endocardium development at E9.5. The angiogenesis in the head, intersomitic region and placenta was also abnormal. These results demonstrate that MEKK3, a member of MAP3K MEKK/STE11 subgene family, is essential for early embryonic cardiovascular development. Furthermore, it was found that disruption of MEKK3 did not alter the expression of vascular endothelial growth factor-1 (VEGF-1), angiopoietin-1, -2 and their respective receptors Flt-1, Flk-1, Tie-1, Tie-2. Finally, MEKK3 was shown to activate myocyte-specific enhancer factor 2C (MEF2C), a crucial transcription factor for early embryonic cardiovascular development through the p38 MAPK cascade, suggesting that MEF2C is one of the key targets of the MEEKK3 signaling pathway during early embryonic cardiovascular development. ^

Family stress, coping, communication, and adaptation in unintended adolescent pregnancy

Objective. This study examines post-crisis family stress, coping, communication, and adaptation using the Double ABC-X Model of Family Adaptation in families with a pregnant or postpartum adolescent living at home. ^ Methods. Ninety-eight pregnant and parenting adolescents between ages 14 and 18 years (Group 1 at 20 or more weeks gestation; Group 2 at delivery and 8 weeks postpartum) and their parent(s) completed instruments congruent with the model to measure family stress, coping, communication, and adaptation. Descriptive family data was obtained. Mother-daughter data was analyzed for differences between subjects and within subjects using paired t-tests. Correlational analysis was used to examine relationships among variables. ^ Results. More than 90% of families were Hispanic. There were no significant differences between mother and daughter mean scores for family stress or communication. Adolescent coping was not significantly correlated to family coping at any interval. Adolescent family adaptation scores were significantly lower than mothers' scores at delivery and 8 weeks postpartum. Mean individual ratings of family variables did not differ significantly between delivery and 8 weeks postpartum. Simultaneous multiple regression analysis showed that stress, coping, and communication significantly influenced adaptation for mothers and daughters at all three intervals. The relative contributions of the three independent variables exhibited different patterns for mothers and daughters. Parent-adolescent communication accounted for most of the variability in adaptation for daughters at all three intervals. Daughters' family stress ratings were significant for adaptability (p = .01) during the pregnancy and for cohesion (p = .03) at delivery. Adolescent coping (p = .03) was significant for cohesion at 8 weeks postpartum. Family stress was a significant influence at all three intervals for mothers' ratings of family adaptation. Parent-adolescent communication was significant for mother's perception of both family cohesion (p < .001) and adaptability (p < .001) at delivery and 8 weeks, but not during pregnancy. ^ Conclusions. Mothers' and daughters' ratings of family processes were similar regarding family stress and communication, but were significantly different for family adaptation. Adolescent coping may not reflect family coping. Family communication is a powerful component in family functioning and may be an important focus for interventions with adolescents and parents. ^

Improving the Quality of Life of spousal caregivers of stroke survivors: A cost effectiveness analysis

Back ground and Purpose. There is a growing consensus among health care researchers that Quality of Life (QoL) is an important outcome and, within the field of family caregiving, cost effectiveness research is needed to determine which programs have the greatest benefit for family members. This study uses a multidimensional approach to measure the cost effectiveness of a multicomponent intervention designed to improve the quality of life of spousal caregivers of stroke survivors. Methods. The CAReS study (Committed to Assisting with Recovery after Stroke) was a 5-year prospective, longitudinal intervention study for 159 stroke survivors and their spousal caregivers upon discharge of the stroke survivor from inpatient rehabilitation to their home. CAReS cost data were analyzed to determine the incremental cost of the intervention per caregiver. The mean values of the quality-of-life predictor variables of the intervention group of caregivers were compared to the mean values of usual care groups found in the literature. Significant differences were then divided into the cost of the intervention per caregiver to calculate the incremental cost effectiveness ratio for each predictor variable. Results. The cost of the intervention per caregiver was approximately $2,500. Statistically significant differences were found between the mean scores for the Perceived Stress and Satisfaction with Life scales. Statistically significant differences were not found between the mean scores for the Self Reported Health Status, Mutuality, and Preparedness scales. Conclusions. This study provides a prototype cost effectiveness analysis on which researchers can build. Using a multidimensional approach to measure QoL, as used in this analysis, incorporates both the subjective and objective components of QoL. Some of the QoL predictor variable scores were significantly different between the intervention and comparison groups, indicating a significant impact of the intervention. The estimated cost of the impact was also examined. In future studies, a scale that takes into account both the dimensions and the weighting each person places on the dimensions of QoL should be used to provide a single QoL score per participant. With participant level cost and outcome data, uncertainty around each cost-effectiveness ratio can be calculated using the bias-corrected percentile bootstrapping method and plotted to calculate the cost-effectiveness acceptability curves.^

Longitudinal dyadic data analysis of stroke survivors and their spousal caregivers

Approximately 795,000 new and recurrent strokes occur each year. Because of the resulting functional impairment, stroke survivors are often discharged into the care of a family caregiver, most often their spouse. This dissertation explored the effect that mutuality, a measure of the perceived positive aspects of the caregiving relationship, had on the stress and depression of 159 stroke survivors and their spousal caregivers over the first 12 months post discharge from inpatient rehabilitation. Specifically, cross-lagged regression was utilized to investigate the dyadic, longitudinal relationship between caregiver and stroke survivor mutuality and caregiver and stroke survivor stress over time. Longitudinal meditational analysis was employed to examine the mediating effect of mutuality on the dyads’ perception of family function and caregiver and stroke survivor depression over time.^ Caregivers’ mutuality was found to be associated with their own stress over time but not the stress of the stroke survivor. Caregivers who had higher mutuality scores over the 12 months of the study had lower perceived stress. Additionally, a partner effect of stress for the stroke survivor but not the caregiver was found, indicating that stroke survivors’ stress over time was associated with caregivers’ stress but caregivers’ stress over time was not significantly associated with the stress of the stroke survivor.^ This dissertation did not find mutuality to mediate the relationship between caregivers’ and stroke survivors’ perception of family function at baseline and their own or their partners’ depression at 12 months as hypothesized. However, caregivers who perceived healthier family functioning at baseline and stroke survivors who had higher perceived mutuality at 12 months had lower depression at one year post discharge from inpatient rehabilitation. Additionally, caregiver mutuality at 6 months, but not at baseline or 12 months, was found to be inversely related to caregiver depression at 12 months.^ These findings highlight the interpersonal nature of stress in the context of caregiving, especially among spousal relationships. Thus, health professionals should encourage caregivers and stroke survivors to focus on the positive aspects of the caregiving relationship in order to mitigate stress and depression. ^

Chronic stress promotes tumor growth through increased BDNF production and neo-innervation

Background: Activation of the sympathetic nervous system (SNS) in response to chronic biobehavioral stress results in high levels of catecholamines and persistent activation of adrenergic signaling, which promotes tumor growth and progression. However it is unknown how catecholamine levels within the tumor exceed systemic levels in circulation. I hypothesized that neo-innervation of tumors is required for stress-mediated effects on tumor growth. Results: First, I examined whether sympathetic nerves are present in human ovarian cancer samples as well as orthotopic ovarian cancer models. Immunohistochemical (IHC) staining for neurofilament revealed that catecholaminergic neurons are present within tumor tissue. In order to determine whether chronic stress affects the density of nerves in the tumor, I utilized an orthotopic mouse model of ovarian cancer that was exposed to daily restraint stress. IHC analysis revealed that nerve density in tumors increased by more than three-fold in stressed animals versus non-stressed controls. IHC analysis suggested that this results from both recruitment of existing neurons (axonogenesis) as well as new neuron formation (neurogenesis) within the tumor. To determine how tumors are recruiting nerve growth, I utilized a PCR array analysis of 84 nerve growth related genes and their receptors, which showed that stimulation of the SKOV3 ovarian cancer cell line with norepinephrine (NE) leads to increased expression of several neurotrophins, including brain-derived neurotrophic factor (BDNF). Neurite extension assays showed that media conditioned by ovarian cancer cell lines is capable of inducing neurite outgrowth in differentiated neuron-like PC12 cells, and NE treatment of cancer cells potentiates this effect. Norepinephrine-induced neurite extension was abolished after BDNF silencing by siRNA, suggesting that BDNF is critical to tumor cell-induced nerve growth. in vivo BDNF inhibition resulted in complete abrogation of stress-induced increases in tumor weight and nerve density, as well as downstream markers of stress. Discussion: These studies indicate that adrenergic signalling induced by chronic stress promotes neo-innervation in the tumor microenvironment. This results in a mutually beneficial relationship between the tumor cells and neurons. This work is crucial for providing a link between chronic stress and its effects on the tumor and its microenvironment. The data shown here aims to open new venues that can be used in development of therapies designed to block the stress effects on tumor growth.

Pathway analysis of genome-wide association study data in primary biliary cirrhosisa

Genome-wide association studies (GWAS) have successfully identified several genetic loci associated with inherited predisposition to primary biliary cirrhosis (PBC), the most common autoimmune disease of the liver. Pathway-based tests constitute a novel paradigm for GWAS analysis. By evaluating genetic variation across a biological pathway (gene set), these tests have the potential to determine the collective impact of variants with subtle effects that are individually too weak to be detected in traditional single variant GWAS analysis. To identify biological pathways associated with the risk of development of PBC, GWAS of PBC from Italy (449 cases and 940 controls) and Canada (530 cases and 398 controls) were independently analyzed. The linear combination test (LCT), a recently developed pathway-level statistical method was used for this analysis. For additional validation, pathways that were replicated at the P <0.05 level of significance in both GWAS on LCT analysis were also tested for association with PBC in each dataset using two complementary GWAS pathway approaches. The complementary approaches included a modification of the gene set enrichment analysis algorithm (i-GSEA4GWAS) and Fisher's exact test for pathway enrichment ratios. Twenty-five pathways were associated with PBC risk on LCT analysis in the Italian dataset at P<0.05, of which eight had an FDR<0.25. The top pathway in the Italian dataset was the TNF/stress related signaling pathway (p=7.38×10 -4, FDR=0.18). Twenty-six pathways were associated with PBC at the P<0.05 level using the LCT in the Canadian dataset with the regulation and function of ChREBP in liver pathway (p=5.68×10-4, FDR=0.285) emerging as the most significant pathway. Two pathways, phosphatidylinositol signaling system (Italian: p=0.016, FDR=0.436; Canadian: p=0.034, FDR=0.693) and hedgehog signaling (Italian: p=0.044, FDR=0.636; Canadian: p=0.041, FDR=0.693), were replicated at LCT P<0.05 in both datasets. Statistically significant association of both pathways with PBC genetic susceptibility was confirmed in the Italian dataset on i-GSEA4GWAS. Results for the phosphatidylinositol signaling system were also significant in both datasets on applying Fisher's exact test for pathway enrichment ratios. This study identified a combination of known and novel pathway-level associations with PBC risk. If functionally validated, the findings may yield fresh insights into the etiology of this complex autoimmune disease with possible preventive and therapeutic application.^

Planning for Emotional Labor and Secondary Traumatic Stress in Child Welfare Organizations

This analysis provides an emergent framework that emphasizes a neglected component of both direct practice with families and organizational development. Human emotions, both beneficial (positive emotional labor) and harmful (negative emotional labor), have received short shrift in leadership development, supervision, direct practice preparation and supports, and workforce stabilization, and professionalization. Significantly, a key indicator of negative emotional labor—secondary traumatic stress (STS)—often has been ignored and neglected, despite the fact that it may be endemic in the workforce. STS typically results from traumatic events in practice, but it also stems from workplace violence. Often undetected and untreated, STS is at least a hidden correlate and perhaps a probable cause of myriad problems such as questionable practice with families, life-work conflicts, undesirable workforce turnover, and a sub-optimal organizational climate. Special interventions are needed. At the same time, new organizational designs are needed to promote and reinforce positive emotional labor. Arguably, positive emotional labor and the positive organizational climates it facilitates are requisites for harmonious relations between jobs and personal lives, desirable workforce retention, and better outcomes for children and families. What’s more, specialized interventions for positive emotional labor constitute a key component in the prevention system for STS. A dual design for positive emotional labor and STS (and other negative emotional labor) prevention/intervention is provided herewith. Early detection and rapid response systems for STS, with social work leadership, receive special attention. Guidelines for new organizational designs for emotional labor in child welfare are offered in conclusion.

Analysis of p53 function and regulation

p53 is a tumor suppressor gene that is the most frequent target inactivated in cancers. Overexpression of wild-type p53 in rat embryo fibroblasts suppresses foci formation by other cooperating oncogenes. Introduction of wild-type p53 into cells that lack p53 arrests them at the G1/S boundary and reverses the transformed phenotype of some cells. The function of p53 in normal cells is illustrated by the ability of p53 to arrest cells at G1 phase of the cell cycle upon exposure to DNA-damaging agents including UV-irradiation and biosynthesis inhibitors.^ Since the amino acid sequence of p53 suggested that it may function as a transcription factor, we used GAL4 fusion assays to test that possibility. We found that wild-type p53 could specifically activate transcription when anchored by the GAL4 DNA binding domain. Mutant p53s, which have lost the ability to suppress foci formation by other oncogenes, were not able to activate transcription in this assay. Thus, we established a direct correlation between the tumor suppression and transactivation functions of p53.^ Having learned that p53 was a transcriptional activator, we next sought targets of p53 activation. Because many transcription factors regulate their own expression, we tested whether p53 had this autoregulatory property. Transient expression of wild-type p53 in cells increased the levels of endogenous p53 mRNA. Cotransfection of p53 together with a reporter bearing the p53 promoter confirmed that wild-type p53 specifically activates its own promoter. Deletion analysis from both the 5$\sp\prime$ and 3$\sp\prime$ ends of the promoter minimized the region responsible for p53 autoregulation to 45 bp. Methylation interference identified nucleotides involved in protein-DNA interaction. Mutations within this protected site specifically eliminated the response of the promoter to p53. In addition, multiple copies of this element confer responsiveness to wild-type p53 expression. Thus, we identified a F53 responsive element within the p53 promoter.^ The presence of a consensus NF-$\kappa$B site in the p53 promoter suggested that NF-KB may regulate p53 expression. Gel-shift experiments showed that both the p50 homodimer and the p50/p65 heterodimer bind to the p53 promoter. In addition, the p65 subunit of NF-$\kappa$B activates the p53 promoter in transient transfection experiments. TNF $\alpha$, a natural NF-$\kappa$B inducer, also activates the p53 promoter. Both p65 activation and TNF $\alpha$ induction require an intact NF-$\kappa$B site in the p53 promoter. Since NF-$\kappa$B activation occurs as a response to stress and p53 arrests cells in G1/S, where DNA repair occurs, activation of p53 by NF-$\kappa$B could be a mechanism by which cells recover from stress.^ In conclusion, we provided the first data that wild-type p53 functions as a transcriptional activator, whereas mutant p53 cannot. The correlation between growth suppression and transcriptional activation by p53 implies a pathway of tumor suppression. We have analyzed upstream components of the pathway by the identification of both p53 and NF-$\kappa$B as regulators of the p53 promoter. ^

Functional analysis of GNA -1, a Galphai superfamily member found in the filamentous fungus Neurospora crassa

Heterotrimeric GTP-binding proteins, G proteins, are integral components of eukaryotic signaling systems linking extracellular signals to intracellular responses. Through coupling to seven-transmembrane helix receptors, G proteins convey primary signaling events into multi-leveled cascades of intracellular activity by regulating downstream enzymes, collectively called effectors. The effector enzymes regulated by G proteins include adenylyl cyclase, cAMP phosphodiesterase, phospolipase C-β, mitogen-activated protein kinases, and ion channels. ^ Neurospora crassa is a multicellular, filamentous fungus that is capable of both asexual and sexual reproduction by elaboration of specialized, developmentally controlled structures that give rise to either asexual or sexual spores, respectively. N. crassa possesses at least three heterotrimeric Gα proteins (GNA-1–3) and one Gβ subunit (GNB-1). GNA-1 was the first microbial protein that could be classified in the Gαi superfamily based on its amino acid identity and demonstration that it is a substrate for ADP-ribosylation by pertussis toxin. ^ Experiments were designed to identify the signal transduction pathways and the effector enzymes regulated by GNA-1. Targeted gene-replacement of gna-1 revealed that GNA-1 controls multiple developmental pathways including both asexual and sexual reproduction, maintenance of growth, and resistance to osmotic stress. The Gαi and Gαz members of the Gαi superfamily negatively regulate adenylyl cyclase activity in mammalian cells; therefore, adenylyl cyclase and cAMP levels were measured in Δgna-1 strains and also in strains that were deleted for both gna-1 and gna-2, a second Gα in N. crassa shown to have overlapping functions with GNA-1. Direct measurements of adenylyl cyclase activity revealed that GNA-1, but not GNA-2, was responsible for GTP-stimulated adenylyl cyclase activity in N. crassa. Furthermore, anti-GNA-1 IgG could specifically inhibit GTP-stimulated adenylyl cyclase activity in wild-type strain extracts. These studies also provided evidence that N. crassa possesses feedback mechanisms that control steady-state cAMP levels through indirect regulation of cAMP-phosphodiesterase activity; mutations in gna-1 and gna-2 were additive in their effect on lowering cAMP-phosphodiesterase activity under growth conditions where steady-state cAMP levels were normal but GTP-stimulated adenylyl cyclase activity was reduced 90% in comparison to control strains. ^ Genetic and biochemical epistasis experiments utilizing a Δ gna-1 cr-1 mutant suggest that GNA-1 is essential for female fertility in a cAMP-independent pathway. Furthermore, deletion of gna-1 in a cr-1 background exacerbated many of the defects already observed in the cr-1 strain including more severe growth restriction and developmental defects. However, deletion of gna-1 had no effect on the increased thermotolerance of cr-1, which has been attributed to loss of cAMP. cr-1 possesses GNA-1 protein, and crude membrane fractions from this strain reconstituted GTP-stimulated adenylyl cyclase activity in Δgna-1 membrane fractions. These studies provide direct evidence for the involvement of Gα proteins in the regulation of adenylyl cyclase activity in eukaryotic microbes. ^

Bulk geochemical analysis and planktonic foraminifera of sediment core GeoB7112-5

A multivariable approach utilising bulk sediment, planktonic Foraminifera and siliceous phytoplankton has been used to reconstruct rapid variations in palaeoproductivity in the Peru-Chile Current System off northern Chile for the past 19000 cal. yr. During the early deglaciation (19000-16000 cal. yr BP), our data point to strongest upwelling intensity and highest productivity of the past 19 000 cal. yr. The late deglaciation (16000-13000 cal. yr BP) is characterised by a major change in the oceanographic setting, warmer water masses and weaker upwelling at the study site. Lowest productivity and weakest upwelling intensity are observed from the early to the middle Holocene (13000-4000 cal. yr BP), and the beginning of the late Holocene (<4000 cal. yr BP) is marked by increasing productivity, mainly driven by silicate-producing organisms. Changes in the productivity and upwelling intensity in our record may have resulted from a large-scale compression and/or displacement of the South Pacific subtropical gyre during more productive periods, in line with a northward extension of the Antarctic Circumpolar Current and increased advection of Antarctic water masses with the Peru-Chile Current. The corresponding increase in hemispheric thermal gradient and wind stress induced stronger upwelling. During the periods of lower productivity, this scenario probably reversed.

Analysis of organic matter from DSDP Leg 81 Holes

Organic geochemical and visual kerogen analyses were carried out on approximately 50 samples from Leg 81 (Rockall Plateau, North Atlantic). The sediments are from four sites (Sites 552-555), Pleistocene to Paleocene in age, and represent significantly different depositional environments and sources of organic matter. The Pleistocene glacial-interglacial cycles show differences in sedimentary organic matter based on Rock-Eval pyrolysis, organic phosphorus, and pyrolysis/mass-spectrometry analyses. Glacial samples contain more organic carbon, with a larger proportion of reworked organic matter. This probably reflects increased erosion of continental and shelf areas as a result of low sea level stands. Inter glacial samples contain a larger proportion of marine organic matter as determined by organic phosphorus and pyrolysis analyses. This immature, highly oxidized marine organic matter may be associated with the skeletal organic matrix of calcareous organisms. In addition, Rock-Eval data indicate no significant inorganic-carbonate contribution to the S3 pyrolysis peak. The Pliocene-Miocene sediments consist of pelagic, biogenic carbonates. The organic matter is similar to that of the Pleistocene interglacial periods; a mixture of oxidized marine organic matter and reworked, terrestrial detritus. The Paleocene-Oligocene organic matter reflects variations in source and depositional factors associated with the isolation of Rockall from Greenland. Paleocene sediments contain primarily terrestrial organic matter with evidence of in situ thermal stress resulting from interbedded lava flows. Late Paleocene and early Eocene organic matter suggests a highly oxidized marine environment, with major periods of deposition of terrestrially derived organic matter. These fluctuations in organic-matter type are probably the result of episodic shallowing and deepening of Rockall Basins. The final stage of Eocene/Oligocene sedimentation records the accelerating subsidence of Rockall and its isolation from terrestrial sources (Rockall and Greenland). This is shown by the increasingly marine character of the organic matter. The petroleum potential of sediments containing more than 0.5% organic carbon is poor because of their thermal immaturity and their highly oxidized and terrestrial organic-matter composition.

Stress field of basaltic rocks of ODP Hole 127-794C

We carried out an experiment to estimate in-situ stresses at ODP Hole 794C (water depth: 2809 m) from the basaltic core samples by deformation rate analysis (DRA). Site 794 is located at the northern end of the Yamato Basin and 70 km west of the eastern Japan Sea intraplate or interplate convergent zone. Stress previously applied to a rock specimen is identified in the inelastic strain behavior of the specimen under uniaxial compression by the method used. Natural remanent magnetization of the sample was also measured to get a reference for the orientation of the horizontal stresses. The vertical, maximum, and minimum horizontal in-situ stresses estimated at a depth of 582 mbsf are 36.4, 43.1, and 31.2 MPa, respectively. The average of the largest and the least horizontal stresses is nearly equal in value to the vertical stress. This suggests that the site is in the stress field of the strike slip regime at the depth, while the stress field of the reverse fault regime has been estimated from the focal mechanism solutions of the earthquakes whose hypocenters are located near or on the convergent boundary. The directions of the largest and the least horizontal stress are estimated to be northeast-southwest and in northwest-southeast, respectively, in taking account of rotation tectonics of the Japan Sea since its formation. The directions of the largest and the least horizontal stresses are opposite to those determined from the earthquakes. These discrepancies of our results with those from earthquakes may be due mainly to the fact that the site is not in the convergent zone.

Mesopelagic fish abundance and particle size spectrum analysis from Maria S. Merian cruise MSM26, spring 2013

One of the goals of EU BASIN is to understand variability in production across the Atlantic and the impact of this variability on higher trophic levels. One aspect of these investigations is to examine the biomes defined by Longhurst (2007). These biomes are largely based on productivity measured with remote sensing. During MSM 26, mesopelagic fish and size-spectrum data were collected to test the biome classifications of the north Atlantic. In most marine systems, the size-spectrum is a decay function with more, smaller organisms and fewer larger organisms. The intercept of the size-spectrum has been linked to overall productivity while the slope represents the "rate of decay" of this productivity (Zhou 2006, doi:10.1093/plankt/fbi119). A Laser In-Situ Scattering Transmissometer was used to collect size-spectrum data and net collections were made to capture mesopelagic fish. The relationship among the mesopelagic fish size and abundance distributions will be compared to the estimates of production from the size-spectrum data to evaluate the biomes of the stations occupied during MSM 26.

Flow conditions, bed form dimensions and shear stress experiments

Large asymmetric bed forms commonly develop in rivers. The turbulence associated with flow separation that develops over their steep lee side is responsible for the form shear stress which can represent a substantial part of total shear stress in rivers. This paper uses the Delft3D modeling system to investigate the effects of bed form geometry and forcing conditions on flow separation length and associated turbulence, and bed form shear stress over angle-of-repose (30 lee side angle) bed forms. The model was validated with lab measurements that showed sufficient agreement to be used for a systematic analysis. The influence of flow velocity, bed roughness, relative height (bed form height/water depth), and aspect ratio (bed form height/length) on the variations of the normalized length of the flow separation zone, the extent of the wake region (where the turbulent kinetic energy (TKE) was more than 70% of the maximum TKE), the average TKE within the wake region and the form shear stress were investigated. Form shear stress was found not to scale with the size of the flow separation zone but to be related to the product of the normalized extent of the wake region (extent of the wake region/extent of water body above the bed form) and the average TKE within the wake region. The results add to understanding of the hydrodynamics of bed forms and may be used for the development of better parameterizations of smallscale processes for application in large-scale studies.