Animal Experimentation and Research Commons^™

Magnesium-Based Metal Matrix Nanocomposite Implants And Nanobubble-Based Delivery Systems: Integrated Strategies For Bone Loss Treatment With Cytocompatibility Evaluation, Lucas José Martinho Bizuti

Honors Undergraduate Theses

This thesis intends to investigate the development of different multimodal therapeutic strategies for combating bone loss. Most specifically, this paper will emphasize the utilization of Magnesium-Based Metal Matrix Nanocomposite Implants (MMNC) and Nanobubble-Based Delivery Systems as options for future treatment focus. The effects of biodegradable magnesium implants were evaluated for cytotoxicity and cytocompatibility via Hematoxylin and Eosin, and Masson’s Trichrome staining, as well as radiological images. The Nanobubble-Based Delivery System is being developed and optimized as a delivery system to target osteoclast activity. This paper focused on optimizing experimental parameters, which include Rotational speeds for nanobubble stimulation under microgravity conditions …

A Cross-Species Meta-Analysis Of Environmental Enrichment Effects On Cognition, Greg S. Fitzgerald

Electronic Theses & Dissertations (2024 - present)

Environmental enrichment --providing animals or people with stimulating, complex surroundings --consistently improves cognitive performance in laboratory rodents. But when similar interventions are tested in humans, the benefits are much smaller. Understanding why matters: if researchers overestimate how well rodent findings will translate, it can mislead expectations for human cognitive interventions.

This dissertation presents the first cross-species meta-analysis comparing environmental enrichment effects on cognition between rodent and human studies. Drawing on 121 studies (71 rodent, 50 human) contributing 478 effect sizes, the analysis used multilevel models with variance correction to account for the statistical dependence created when studies report multiple outcomes. …

Hepatocyte-Specific Lrp1 Silencing Exacerbates Brain Amyloidosis Without Compensatory Ldl Receptor Family Involvement, Brian Carson, Josephine Chu, Devaraj V. Chandrashekar, Jerome Garcia, Rachita K. Sumbria, Derick Han

Pharmacy Faculty Articles and Research

Background

LDL receptor related protein 1 (LRP1) is a major hepatic receptor involved in lipoprotein metabolism, protease degradation, transmembrane receptor modulation, and clearance of excess protein, such as Aβ. Decreased expression of LRP1 due to liver injury can impair receptor-mediated clearance, increasing Aβ availability in the periphery. We investigated the effects of hepato-specific silencing of LRP1 on Aβ deposition in the brain. Additionally, other LDL family members (LRP5, LRP6, and LDLR) that may participate in Aβ clearance were monitored in the liver to ensure non-compensatory effects of LRP1 silencing.

Method

4-month-old male double transgenic (APP/PS1) AD mice were injected with …

The Mitochondria Is The Source Of Hepatic Amyloid Precursor Protein And Peripheral Amyloid Beta: Implications Of Alcohol-Induced Liver Steatosis In Alzheimer's Disease, Josephine Chu, Ross A. Steinberg, Brian Carson, Devaraj Venkatapura Chandrashekar, Rachita K. Sumbria, Derick Han

Pharmacy Faculty Articles and Research

Background

Alcohol-induced liver injury occurs in the pericentral region of the liver and can induce mitochondrial remodeling and exacerbate Alzheimer's Disease (AD) progression. Subpopulations of mitochondria: general mitochondria (GM), peridroplet mitochondria (PDM) and endoplasmic reticulum(ER)-bound mitochondria (ERM) maintain cellular homeostasis via energy synthesis, lipid homeostasis, and regulation of intracellular calcium. Hepatic amyloid precursor protein (APP) is a source of peripheral amyloid beta (aB) and affects AD pathology in the brain. Understanding the localization and expression of hepatic APP in mitochondrial subpopulations are critical to understanding aB metabolism and may play a role in identifying a potential mechanism for metabolic dysfunction. …

Just "Beet" It? A Comparison Of Road Salt Effects On The Life History Of Daphnia Magna, Koby Sellner

The Journal of Purdue Undergraduate Research

In areas that experience snow and ice, deicing reagents such as road salts are often used on roads to promote safer driving conditions. However, road salts run off into local vegetation, soil systems, and (my focus) freshwater systems. The impacts of excess salt and chloride in freshwater have been studied extensively on a broad diversity of organisms under different conditions, but previous studies have rarely used different types of road salts. Zooplankton are primary consumers in many freshwater systems, feeding on phytoplankton and serving as prey for predator species higher in the trophic chain. These animals generally are impacted negatively …

Two Major Ontario Hospital Labs Bought Research Dogs From U.S. Breeder With Troubled Pastdogs Bred For Scientific And Medical Testing Suffer Abuse As The Scientific Industry Increasingly Turns To Alternatives For Research., Allison Hunter, Mary Beech, Sami Marshak, Kate Morano

Capstones

The purpose-bred dog breeding industry for research is little known. Each year, thousands of dogs are bred for scientific and medical testing, supplied to research facilities by a handful of major commercial breeders. But gaps in enforcement and regulation have allowed abuses to persist, even as the industry increasingly turns to alternatives to animal testing. Animal rights advocates have been decrying the treatment of these dogs. A federal Inspector General report found lax oversight of the industry, raising further concerns about how dogs are treated. This story was reported in collaboration with the Investigative Journalism Bureau, based in Toronto, and …

The Timing Of Pubertal Onset Affects Adults Anxiety Behavior In A Sex-Specific Manner, Evan Raubenolt

Honors Projects

This study aims to look at the relationship between the timing of pubertal onset and its long-term effects on adult anxiety behavior in rats. Though the topic of pubertal onset has been frequently discussed in developmental research, its exact effects have been inconsistent. Human models have shown that early and late maturing males are associated with a notable increase in adult anxiety behavior, while in rat models, similar sex-specific effects regarding anxiety have been demonstrated, though those studies focus on a causal relationship between exposure to stress and age of pubertal onset. This study first aims to examine that relationship …

Longitudinal Pharmacokinetic And Safety Studies Of An Antibody-Erythropoietin Fusion Protein For Alzheimer's Disease, Rudy Chang, Devaraj Venkatapura Chandrashekar, G. Chuli Roules, Nataraj Jagadeesan, Emi Iwasaki, Adenike Oyegbesan, Hayk Davtyan, Rachita K. Sumbria

Pharmacy Faculty Articles and Research

Erythropoietin (EPO) shows promise for Alzheimer's disease (AD) but has poor brain penetration, necessitating high doses that cause hematopoietic side effects. To improve brain delivery, EPO was fused to a transferrin receptor monoclonal antibody (TfRMAb), and this study evaluated the pharmacokinetics (PK), safety, and efficacy of repeated TfRMAb-EPO dosing in mice to further its preclinical development. C57BL/6J male mice (10 weeks old, n = 4–5/dose) received subcutaneous (SQ) low (1 mg/kg), mid (3 and 6 mg/kg), or high (20 mg/kg) TfRMAb-EPO doses for 4 weeks. The 1 mg/kg dose showed no adverse effects and resulted in sustained brain and plasma …

Impacts Of Nutrient Addition And Disturbance On Coastal Dune Arthropod Communities And Trophic Interactions, Kayla Beitzel

Graduate Theses and Dissertations (2019 - present)

Coastal dune ecosystems face ongoing threats from human-mediated disturbance and altered resource availability. Because these dunes provide critical habitat for many organisms, including shorebirds, beach mice, and arthropods, changes in disturbance regimes or resource availability can impact plant communities and higher trophic levels by altering plant richness and productivity, which in turn influence arthropod abundance and diversity. To examine how disturbance and nutrient addition affect arthropod communities, we established twenty 5×5 m plots on Dauphin Island, crossing mechanical disturbance with a 10 g m⁻² addition of nitrogen, phosphorus, and potassium, replicated across five experimental blocks. Arthropods were sampled using three …

Prediction Equations For In Vitro Nutrient Utilization Of Bakery Byproducts For Pigs, Junghyun Oh, Hansol Kim, Yoon Soo Song, Noa Park, Youl-Chang Baek, Chung Hyun Kim, Beob Gyun Kim

The Philippine Agricultural Scientist

This study aimed to develop equations for predicting the in vitro ileal disappearance (IVID) of dry matter (DM) and crude protein (CP) and in vitro total tract disappearance (IVTTD) of DM and organic matter (OM) in bakery byproducts based on their chemical composition, including neutral detergent fiber (NDF). Ten sources of bakery byproducts were tested for IVID and IVTTD of nutrients using a 2-step procedure simulating the digestion and absorption in the stomach and small intestine and a 3-step procedure simulating the digestion and absorption in the stomach, small intestine, and large intestine of pigs. The prediction models for IVID …

Factors Affecting Beef Average Daily Gain And Carcass Traits In Smallholder Farms In Thailand, China Supakorn, Sumalee Supant, Warinthorn Maneerat, Ya-Shiou Chang, Monticha Putsakhum

The Philippine Agricultural Scientist

This study examined the effects of farm management, body weight, fattening duration, breed, and estimated age on average daily gain (ADG) and meat quality traits, such as hot carcass weight (HCW) and dressing percentage (DP). The dataset comprised 3,286 steers from the Livestock Cooperative in Sakon Nakhon province, Thailand, spanning 10 yr (from 2013 to 2023). The mean initial and slaughter weights were 392.92 ± 29.18 kg and 600.75 ± 60.73 kg, respectively. The ADG during the fattening period was 0.95 ± 0.17 kg/d, while HCW and DP averaged 328.86 ± 40.79 kg and 54.75 ± 1.41%, respectively. The beef …

Host-Intrinsic Factors Determine Anti-Tumor Efficacy Of Exercise In Pancreatic Ductal Adenocarcinoma, Sumedha Pareek

Dissertations and Theses (Open Access)

Pancreatic Ductal Adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths with a low 5-year relative survival rate of approximately 13.3% for all stages combined. We have previously shown that exercise can improve quality of life and enhance functional capacity among patients with PDAC. Exercise induces a variety of changes in the tumor microenvironment with beneficial effects in several tumor types. However, our understanding of the clinical effects of exercise and the mechanisms that mediate anti-tumor effects are limited in pancreatic cancer.

In this project, using C57BL/6 mice from Taconic Biosciences (Tac) and Jackson Laboratories (Jax), we established a …

Bricks, Bushes, And Bones: Urbanization Influence On Western Fence Lizard (Sceloporus Occidentalis) Morphology And Performance, Daisy P. Xiong

Electronic Theses, Projects, and Dissertations

With increasing urbanization due to rising human populations, organisms may be forced to adapt rapidly to changing environments. Urbanization is replacing natural surfaces with more impervious substrates which can result in morphological changes affecting organismal locomotion, then also impact fitness. This study compares urban and natural habitat impact on Western Fence Lizards (Sceloporus occidentalis) and their adaptations in such settings. We captured lizards from three locations in inland Southern California with both urbanized and natural habitats in order to compare the morphological differences between lizards, and then measured their performance to determine the possible impacts from their morphology. …

Improved Model For Neurodegeneration In C. Elegans, Jean-Pierre Arditi

Graduate Theses and Dissertations (2019 - present)

During Alzheimer's disease (AD), insoluble amyloid beta (AP) peptides accumulate to form extracellular aggregates (plaques). The direct cause of neuronal dysfunction observed in AD has been broadly investigated. The amyloid hypothesis states that AP plaques are neurotoxic, but recent studies support the AP oligomer hypothesis, which states that intracellular AP (iAP) is neurotoxic. To test this hypothesis, I used CRISPR-Cas9 gene editing to generate two transgenic Caenorhabditis elegans (C. elegans) strains. I generated a strain (RSLl 11) using the rab-3 promoter to co-express GFP in neurons, which showed no gross behavioral changes but had a 50% reduced egglaying rate. RSLl …

Citronellol Reduces Sepsis-Induced Renal Inflammation Via Ap-1/Nf-Κb/Tnf-Α Pathway, Huda Rashid Atiyah, Sarmed H. Kathem, Surya M. Nauli

Pharmacy Faculty Articles and Research

Sepsis is characterized by the over-production of pro-inflammatory cytokines. Cecal ligation and puncture (CLP) is a well-accepted model for recreating sepsis-induced renal injury in mice. The current study investigates how citronellol, a naturally occurring substance with a variety of biological characteristics, can prevent acute kidney inflammation brought on by CLP. In the CLP mouse model, citronellol was administered orally at doses of 50 and 100 mg/kg. Serum levels of creatinine and urea were used as markers of renal function, and the Murine Sepsis Score (MSS) was used to assess the severity of sepsis. According to our findings, CLP caused a …

Animal Welfare Body Terms Of Reference, Munster Technological University, Ireland

Policies & Procedures Documents

The University recognises the necessity to uphold animal health and welfare in its education and research activities. As part of this obligation, mindful of the legal regulatory requirements , the University has set up the Animal Welfare Body (AWB) to oversee and enforce the protection of animals and promote a culture of animal care among researchers and educators.

The AWB is given the powers by the Vice President Research & Innovation to fully uphold the principles of Scientific Animal Protection within the University, ensure compliance with the Health Products Regulatory Authority (HPRA), and provide records of animal engagement for research …

Understanding Animal Introductions And Welfare In Zoos: A Scoping Review, Claudia C. Martinez, Valerie James-Aldridge, Todd J. Mcwhorter, Eduardo J. Fernandez

Psychological Science Faculty Publications

Managing captive zoo species often requires modifying their social groups, but these changes can increase stress and aggression. Specifically, introductions of new animals to existing exhibits with established animals is commonplace, yet little has been published to systematically evaluate this process. This review used the PRISMA framework to evaluate published research on introductions of unfamiliar individuals in zoos, aquariums, and sanctuaries. Peer-reviewed studies from PubMed, Web of Science, and Scopus were screened and extracted using Covidence software. Studies were included if they provided quantitative data and before-and-after comparisons. Of 25 studies meeting criteria, most focused on primates, especially western lowland …

Evaluation Of Semen Characteristics And The Impact Of Storage Time In Dong Tao And Ho Indigenous Chickens, Phan Nhan, Nguyen Thi My Phuong

The Philippine Agricultural Scientist

This study aimed to evaluate the semen quality of 2 indigenous Vietnamese chicken breeds, Dong Tao and Ho, and to assess their reproductive potential for breeding and conservation programs. Semen samples were collected from 10 roosters of each breed and analyzed for macroscopic and microscopic traits. The results indicated that both breeds exhibited semen quality within acceptable physiological ranges. Mean semen volume was 0.47 mL in Dong Tao and 0.48 mL in Ho roosters; pH values were 7.47 and 7.42, respectively. Semen color scores (2.39 vs. 2.35), sperm concentrations (2.11 vs. 1.92 × 109/mL), and mass activity scores (3.79 vs. …

Polyphenols For The Mitigation Of Vascular Calcification; In Vitro And In Vivo Studies, Abdullah Altuhami

All Theses

Globally, cardiovascular diseases, in most cases associated with calcification, are the main cause of death. However, currently, the only effective cure is to undergo surgery to augment or receive a healthy donor heart, which could be very difficult due to the long waiting list and the associated risks. The possibility of body rejection of the foreign organs is attributed to immunoreaction or the risk of infection, since the donor must be on immunosuppressant drugs that lower the body's immunity and facilitate the body's acceptance of the new organ as one of its own. Taking that into consideration, using tissue-engineered vascular …

Impact Of S-Phase Kinase Protien 2 Blockades On Hematopoetic Stem Cell Metabolism, Nicole Elmaraghy

Electronic Theses, Projects, and Dissertations

Hematopoietic stem and progenitor cells (HSPCs) quiescence is vital for the success of bone marrow transplantation, as it preserves long term self- renewal and prevents premature exhaustion (Takubo et al., 2013; Wilson et al., 2008). However, bone marrow transplant (BMT) failure remains a clinical challenge, often due to lack of long-term engraftment and insufficient stress reliance. Both of these characteristics are tightly linked to disrupted stem cell quiescence and metabolic imbalance (Anso et al., 2017; Vannini et al., 2016). One key player is S-phase kinase protein (SKP2), an E3 ubiquitin ligase that targets cell cycle inhibitors, like p27, for proteosome …

Microbial Metabolite Regulation Of Microglial Ahr Signaling In Alzheimer’S Disease, Ghalya E. Alrousan

Dissertations and Theses (Open Access)

Introduction: Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by amyloid-β plaque accumulation, neuroinflammation, and cognitive decline. Recent evidence suggests that systemic factors, particularly the gut microbiota and its metabolites, play a significant role in shaping brain immune responses, including microglial activation. However, the mechanisms linking gut dysbiosis to microglial dysfunction in AD are poorly understood. The aryl hydrocarbon receptor (AHR), a ligand-activated transcription factor responsive to tryptophan metabolites, has recently gained attention as a key regulator of neuroinflammation and microglial function. I hypothesize that the loss of beneficial microbial AHR ligands, specifically indole derivatives, during aging and AD …

Role And Electrophysiological Properties Of Sexually Dimorphic Oxytocin Receptor Expressing Neurons In The Anteroventral Periventricular Nucleus Of The Female Mice, Armita Abdollahi Govar

LSU Doctoral Dissertations

Oxytocin is a neurohypophyseal hormone that plays a crucial role in regulating mother-infant bonding and maternal behavior. Oxytocin modulates sex-specific parental behaviors by binding to the oxytocin receptor (OXTR)-expressing neurons in the brain. This study investigates the sexually dimorphic OXTR neurons in the anteroventral periventricular nucleus (AVPV) to understand how oxytocin modulates behavior in females. Specifically, we explored the behavioral roles, neural projections, and intrinsic electrophysiological characteristics of these neurons in the female mice. Using immunohistochemistry revealed that the number of OXTR-expressing neurons was enhanced in postpartum females compared with virgin females, suggesting that these neurons are involved in female-specific …

Cilia In The Brain Display Region-Dependent Oscillations Of Length And Orientation, Roudabeh Vakil Monfared, Sherif Abdelkarim, Pieter Derdeyn, Kiki Chen, Hanting Wu, Kenneth Leong, Tiffany Chang, Justine Lee, Sara Versales, Surya M. Nauli, Kevin Beier, Pierre Baldi, Amal Alachkar

Pharmacy Faculty Articles and Research

In this study, we conducted high-throughput spatiotemporal analysis of primary cilia length and orientation across 22 mouse brain regions. We developed automated image analysis algorithms, which enabled us to examine over 10 million individual cilia, generating the largest spatiotemporal atlas of cilia. We found that cilia length and orientation display substantial variations across different brain regions and exhibit fluctuations over a 24-h period, with region-specific peaks during light-dark phases. Our analysis revealed unique orientation patterns of cilia, suggesting that cilia orientation within the brain is not random but follows specific patterns. Using BioCycle, we identified rhythmic fluctuations in cilia length …

Murine Tbk1 Regulates Mpp3-Type Hspcs And Circulating Leukocytes In Normal Hematopoiesis And Flt3+ Lscs In Mll-Af9-Driven Leukemia, Austin P. Runde, Joseph Cannova, Ryan Mack, Kanak Joshi, Mark Sellin, Rohit Thalla, Allan Youmaran, Mattias Lenz, Peter Breslin, Wei Wei, Jiwang Zhang

School of Medicine

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic cancer with a notoriously bleak prognosis; for non-M3 AML, the overall 5-year survival rate is ∼30%. While 60-70% of newly diagnosed AML patients will achieve complete remission (CR), half of these patients will experience relapse (secondary resistance) by three years from their diagnosis. Moreover, 30-40% of AML patients present with refractory disease (primary resistance) and cannot respond to frontline treatments. Leukemia stem cells (LSCs) are implicated in both primary and secondary resistance, and their eradication is necessary to maintain CR. LSCs have unique transcriptomes and immunophenotypes, thus can be identified relatively …

The Role Of G6pd Variants And 3d Genomic Structure In The Development Of Pulmonary Hypertension, Christina M. Signoretti Ph.D.

NYMC Student Theses and Dissertations

Pulmonary hypertension is an under-recognized global health epidemic which is estimated to affect 1% of the population. It is associated with sustained mean pulmonary artery pressure greater than 20 mmHg, pulmonary artery remodeling, smooth muscle and endothelial cell proliferation and subsequent increased right ventricle hypertrophy due to increased afterload. Glucose-6-phosphate dehydrogenase (G6PD) is one of the key enzymes in the pentose phosphate pathway and has been previously linked to the development of pulmonary hypertension. G6PD deficiency is the most common human enzymopathy which affects approximately 400 million people worldwide and is a result of agricultural and epidemiological evolution in different …

Mechanisms Of Placental Impairment And The Upregulation Of Fetuin-B During Maternal Undernourishment, Mia Camilliere

NYMC Student Theses and Dissertations

Proper nutrition is crucial during pregnancy to support fetal and placental development. Failure to meet these nutritional needs can result in adverse health outcomes for both mother and baby. Despite its high prevalence in underdeveloped countries, maternal undernourishment is a public health issue worldwide. There are several possible causes of maternal undernourishment, and these can occur alone or in combination, such as poor access to nutrition, hyperemesis gravidarum, and drug use. This condition can adversely affect placental development and function to the extent of placental insufficiency, perturbing the entire pregnancy. The placenta is responsible not only for hormonally and physically …

Ammonia Regulation In Aedes Taeniorhynchus, A Salt-Water Mosquito, Joseph Edmonds

Undergraduate Honors Theses

The salt tolerant mosquito species Aedes taeniorhynchus has been observed in high salinity and ammonia conditions. Previous studies indicate Na+/K+ ATPase (NKA), the proton pump vacuolar-type H+ ATPase (VHA) and sodium-dependent cation-chloride cotransporters (NKCC’s) in sodium secretion in larval Aedes taeniorhynchus. However ammonia transporter such as Rh-protein, Amt1, and Amt2 have only been characterized in Aedes taeniorhynchus’s fresh water relative Aedes aegypti. Our research exhibits the expression of these transporters in A.taeniorhynchus using immunohistochemistry techniques. Evidence of VHA and NKA function in ammonia transport was characterized in vivo using drug antagonists in varying saline and ammonia concentrations. Ammonia flux rates …

Sickle Cell Mice Exhibit Elevated Plasma Bilirubin And Altered Intracranial Cerebral Blood Velocities That Are Exacerbated By Hypoxia-Reoxygenation, Francisco J. Nunez, Ashraf M. Mohieldin, Amy Y. Pan, Sean P. Palecek, Rahima Zennadi, Ramani Ramchandran, Kevin R. Rarick, Surya M. Nauli

Pharmacy Faculty Articles and Research

Sickle cell disease (SCD) is a genetic disorder characterized by sickle red blood cells (RBCs). Sickle RBCs cause cerebral vasculopathies including vaso-occlusive events, leading to ischemia-reperfusion injury and hypoxic tissue environment. To date, the physiological blood flow velocities in cerebral vessels of preclinical SCD models has not been evaluated under hypoxic-reoxygenation. In our study, we used transcranial ultrasound techniques to measure abnormal blood flow velocities in the internal carotid (ICA) and middle cerebral arteries (MCA) of transgenic sickle cell mice (SS) challenged with hypoxia-reoxygenation. Our study showed that SS mice that underwent hypoxic stress exhibited lower relative mean velocities in …

Peripheral Neuroimmune Mechanisms Of Chronic Back Pain, Aleyah Esther Goins

Biomedical Sciences ETDs

Chronic back pain (CBP) affects 1 in 10 people worldwide, reducing mobility, productivity, and quality of life. Despite its prevalence, non-addictive treatments remain limited due to an incomplete understanding of chronic pain mechanisms. This dissertation examines peripheral neuroimmune interactions in CBP using a urokinase-type plasminogen activator (uPA)-induced mouse model (uPA-CBP). uPA, a pro-inflammatory serine protease, was injected into the L2/L3 lumbar spinous ligament, inducing prolonged mechanical and cold hypersensitivity. Flow cytometry of dorsal root ganglia (DRG) showed increased CD45+CD11b+ myeloid cells in males and CD45+CD3+CD4+ lymphocytes in females. DRG neurons from male uPA-CBP mice exhibited heightened action potential firing. Pharmacological …

Ghosts In Glass: Ghost Crabs (Ocypode Quadrata) As Judges Of Glass Sand For Coastal Restoration, Emily Parrish

Poster Presentations

Coastal erosion is heavily impacting coastal ecosystems across the Gulf. For instance, the state of Louisiana loses a football field worth of land every 100 minutes (Couvillion, 2017). The drastic loss of coastline highlights the dire need for restoration in the Northern Gulf Coast. An unexpected solution to this sediment loss may solve two major environmental issues at once by diverting millions of tons of glass waste from entering America’s landfills each year. By crushing glass to create sand and gravel for coastal restoration, we may generate a consistent and sustainable source of substrate. Our goal is to determine the …