Biology Commons^™

New Frontiers For The Nfil3 Bzip Transcription Factor In Cancer, Metabolism And Beyond, Megan Keniry, Robert K. Dearth, Michael W. Persans, Ramon Parsons

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The bZIP transcription factor NFIL3 (Nuclear factor Interleukin 3 regulated, also known as E4 binding protein 4, E4BP4) regulates diverse biological processes from circadian rhythm to cellular viability. Recently, a host of novel roles have been identified for NFIL3 in immunological signal transduction, cancer, aging and metabolism. Elucidating the signaling pathways that are impacted by NFIL3 and the regulatory mechanisms that it targets, inhibits or activates will be critical for developing a clearer picture of its physiological roles in disease and normal processes. This review will discuss the recent advances and emerging issues regarding NFIL3-mediated transcriptional regulation of CEBPb and …

Projected Future Distributions Of Vectors Of Trypanosoma Cruzi In North America Under Climate Change Scenarios, Miroslava Garza, Teresa Patricia Feria-Arroyo, Edgar A. Casillas, Victor Sanchez-Cordero, Chissa-Louise Rivaldi, Sahotra Sarkar

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Background

Chagas disease kills approximately 45 thousand people annually and affects 10 million people in Latin America and the southern United States. The parasite that causes the disease, Trypanosoma cruzi, can be transmitted by insects of the family Reduviidae, subfamily Triatominae. Any study that attempts to evaluate risk for Chagas disease must focus on the ecology and biogeography of these vectors. Expected distributional shifts of vector species due to climate change are likely to alter spatial patterns of risk of Chagas disease, presumably through northward expansion of high risk areas in North America.

Methodology/Principal Findings

We forecast the future …

Rates Of Evolution And Point Mutations Of Bacterial Plant Pathogens Compared To Bacterial Vertebrate Pathogens, Alejandro Cantu

Theses and Dissertations - UTB/UTPA

The relationship between a pathogen and its host is a constant interaction; as the host evolves defense mechanisms to protect itself from the pathogen, the pathogen will evolve in order to evade them. During this evolutionary process, the genomes of bacterial pathogens can change due to different selection pressures exerted by the immune system of their respective hosts. This study aimed to determine how differences in selection pressures affect the evolution of plant and vertebrate pathogens. The focus of this study was to analyze population level changes in conserved genes, as well as species level changes in the Type III …

Implications Of Climate Change On The Distribution Of The Tick Vector Ixodes Scapularis And Risk For Lyme Disease In The Texas-Mexico Transboundary Region, Teresa Patricia Feria-Arroyo, Ivan Castro-Arellano, Guadalupe Gordillo Pérez, Ana L. Cavazos, Margarita Vargas-Sandoval, Abha Grover, Javier Torres, Raul F. Medina, Adalberto A. Pérez De León, Maria Dolores Esteve-Gassent

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Background: Disease risk maps are important tools that help ascertain the likelihood of exposure to specific infectious agents. Understanding how climate change may affect the suitability of habitats for ticks will improve the accuracy of risk maps of tick-borne pathogen transmission in humans and domestic animal populations. Lyme disease (LD) is the most prevalent arthropod borne disease in the US and Europe. The bacterium Borrelia burgdorferi causes LD and it is transmitted to humans and other mammalian hosts through the bite of infected Ixodes ticks. LD risk maps in the transboundary region between the U.S. and Mexico are lacking. Moreover, …

Visual Responses Of Adult Asian Citrus Psyllid (Hemiptera: Liviidae) To Colored Sticky Traps On Citrus Trees, Mamoudou Setamou, A. Sanchez, R. R. Saldaña, J. M. Patt, Kenneth R. Summy

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The effects of five differently-colored sticky traps in capturing adult Diaphorina citri were evaluated in citrus orchards. Trap catches of D. citri were monitored fortnightly on blue, green, red, white and yellow sticky cards placed on three citrus varieties during D. citri active flight period from April to July in south Texas. Evaluation of mean trap catches of each color by repeated measures analysis of variance produced three separate groups: yellow traps caught significantly more D. citri adults than the other four traps; red and green traps caught significantly more D. citri than blue and white traps, which were not …

Prepubertal Exposure To Arsenic(Iii) Suppresses Circulating Insulin-Like Growth Factor-1 (Igf-1) Delaying Sexual Maturation In Female Rats, Michael P. Reilly, James C. Saca, Alina Hamilton, Rene F. Solano, Jesse R. Rivera, Wendy Innis-Whitehouse, Jason Parsons, Robert K. Dearth

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Arsenic (As) is a prevalent environmental toxin readily accessible for human consumption and has been identified as an endocrine disruptor. However, it is not known what impact As has on female sexual maturation. Therefore, in the present study, we investigated the effects of prepubertal exposure on mammary gland development and pubertal onset in female rats. Results showed that prepubertal exposure to 10 mg/kg of arsenite (As(III)) delayed vaginal opening (VO) and prepubertal mammary gland maturation. We determined that As accumulates in the liver, disrupts hepatocyte function and suppresses serum levels of the puberty related hormone insulin-like growth factor 1 (IGF-1) …

The Vibrio Cholerae Type Vi Secretion System Employs Diverse Effector Modules For Intraspecific Competition, Daniel Unterweger, Sarah T. Miyata, Verena Bachmann, Teresa M. Brooks, Travis Mullins, Benjamin Kostiuk, Daniele Provenzano, Stefan U. Pukatzki

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Vibrio cholerae is a Gram-negative bacterial pathogen that consists of over 200 serogroups with differing pathogenic potential. Only strains that express the virulence factors cholera toxin (CT) and toxin-coregulated pilus (TCP) are capable of pandemic spread of cholera diarrhoea. Regardless, all V. cholerae strains sequenced to date harbour genes for the type VI secretion system (T6SS) that translocates effectors into neighbouring eukaryotic and prokaryotic cells. Here we report that the effectors encoded within these conserved gene clusters differ widely among V. cholerae strains, and that immunity proteins encoded immediately downstream from the effector genes protect their host from neighbouring bacteria …

Environmental Correlates Of Assemblages Of Fishes In The Tilostoc River Of Central Mexico, Juan Manuel Rivas-González, Felipe De Jesús Rodríguez-Romero, María De Lourdes Ruiz-Gómez, Diego Alfonso Viveros-Guardado, Christopher M. Taylor

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Ordination, correlation, and regression analyses were used to identify trophic and taxonomic distributional patterns of fishes in the Tilostoc River (located west of Valle de Bravo Lake in the Middle Balsas Basin, central Mexico) and associations of these patterns with environmental variables. Nonmetric multidimensional scaling analysis indicated that trophic and taxonomic assemblages corresponded similarly to measured environmental gradients, especially elevation and velocity of currents. We also compared the trophic structure of assemblages of fishes of the Tilostoc River to that of Terreros Creek, a temperate system of similar size and species richness but with a taxonomically different group of species. …

Conservation Genetics Of Cyprinid Fishes In The Upper Nueces River Basin In Central Texas, Evan W. Carson, Ashley H. Hanna, Gary P. Garrett, Robert J. Edwards, John R. Gold

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Sequences of the mitochondrial (mt) NADH dehydrogenase subunit 5 gene (ND5) were acquired to assess genetic diversity and female effective population size (Nef) of two forms of Cyprinella (C. lepida and C. sp. cf lepida) and two species of Dionda (D. serena and D. texensis) in headwaters of three rivers in the upper Nueces River basin in central Texas. As documented in prior studies, two divergent clades of haplotypes of mtDNA were found in both genera: one in the Frio and Sabinal rivers, representing C. lepida and D. serena; one in the …

The Genetic Drift Inventory: A Tool For Measuring What Advanced Undergraduates Have Mastered About Genetic Drift, Rebecca M. Price, Tessa C. Andrews, Teresa L. Mcelhinny, Louise S. Mead, Joel K. Abraham, Anna Thanukos, Kathryn E. Perez

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Understanding genetic drift is crucial for a comprehensive understanding of biology, yet it is difficult to learn because it combines the conceptual challenges of both evolution and randomness. To help assess strategies for teaching genetic drift, we have developed and evaluated the Genetic Drift Inventory (GeDI), a concept inventory that measures upper-division students’ understanding of this concept. We used an iterative approach that included extensive interviews and field tests involving 1723 students across five different undergraduate campuses. The GeDI consists of 22 agree–disagree statements that assess four key concepts and six misconceptions. Student scores ranged from 4/22 to 22/22. Statements …

Environmental Correlates Of Assemblages Of Fishes In The Tilostoc River Of Central Mexico, Juan Manuel Rivas-González, Felipe De Jesús Rodríguez-Romero, María De Lourdes Ruiz-Gómez, Diego Alfonso Viveros-Guardado, Christopher M. Taylor

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Ordination, correlation, and regression analyses were used to identify trophic and taxonomic distributional patterns of fishes in the Tilostoc River (located west of Valle de Bravo Lake in the Middle Balsas Basin, central Mexico) and associations of these patterns with environmental variables. Nonmetric multidimensional scaling analysis indicated that trophic and taxonomic assemblages corresponded similarly to measured environmental gradients, especially elevation and velocity of currents. We also compared the trophic structure of assemblages of fishes of the Tilostoc River to that of Terreros Creek, a temperate system of similar size and species richness but with a taxonomically different group of species. …

Predominant And Substoichiometric Isomers Of The Plastid Genome Coexist Within Juniperus Plants And Have Shifted Multiple Times During Cupressophyte Evolution, Wenhu Guo, Felix Grewe, Amie Cobo-Clark, Weishu Fan, Zelin Duan, Robert P. Adams, Andrea E. Schwarzbach, Jeffrey P. Mower

Health & Biomedical Sciences Faculty Publications

Most land plant plastomes contain two copies of a large inverted repeat (IR) that promote high-frequency homologous recombination to generate isomeric genomic forms. Among conifer plastomes, this canonical IR is highly reduced in Pinaceae and completely lost from cupressophytes. However, both lineages have acquired short, novel IRs, some of which also exhibit recombinational activity to generate genomic structural diversity. This diversity has been shown to exist between, and occasionally within, cupressophyte species, but it is not known whether multiple genomic forms coexist within individual plants. To examine the recombinational potential of the novel cupressophyte IRs within individuals and between species, …

Painted Redstarts In South Texas, 2013-2014, Timothy Brush

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

No abstract provided.

Changes In Fish Populations In The Lower Canyons Of The Rio Grande, Gary P. Garrett, Robert Edwards

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The Lower Canyons of the Rio Grande suffer from environmental degradation that has negatively impacted native fish populations and their distributions. Macrhybopsis aestivalis (speckled chub), Notropis jemezanus (Rio Grande shiner), Rhinichthys cataractae (longnose dace) and Cycleptus elongatus (blue sucker) populations appear to have suffered recent declines. Although diminished water quantity is likely an important factor in these declines, related changes in channel morphology precipitated by massive stands of Arundo donax (giant reed) and Tamarix sp. (salt cedar) may also be responsible. These invasive exotics have essentially channelized the river, disrupted normal sediment distribution and reduced shallow, low-velocity habitats. Much of …

Vibrio Cholerae As A Predator: Lessons From Evolutionary Principles, Stefan Pukatzki, Daniele Provenzano

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Diarrheal diseases are the second-most common cause of death among children under the age of five worldwide. Cholera alone, caused by the marine bacterium Vibrio cholerae, is responsible for several million cases and over 120,000 deaths annually. When contaminated water is ingested, V. cholerae passes through the gastric acid barrier, penetrates the mucin layer of the small intestine, and adheres to the underlying epithelial lining. V. cholerae multiplies rapidly, secretes cholera toxin, and exits the human host in vast numbers during diarrheal purges. How V. cholerae rapidly reaches such high numbers during each purge is not clearly understood. We propose …

Distribution And Abundance Of Anopheles Spp. In The Lower Rio Grande Valley, South Texas, Norma Hermelinda Martinez

Theses and Dissertations - UTB/UTPA

This study investigated the relationship between Anopheles abundance, collection sites and environmental variables in the Lower Rio Grande Valley, South Texas. In addition, species composition in the Lower Rio Grande Valley was determined in 2011. A total of 6772 female mosquitoes were identified to six genera and 27 species. The most prevalent genera collected were Culex (53.9%), Ochlerotatus (25.6%) and Aedes (13.6%). Anopheles mosquitoes were collected using resting boxes during summer 2012 at multiple locations along the Lower Rio Grande Valley. ArcGIS was used to identify land cover characteristics and nearest water sources at mosquito collection sites. Estero Llano Grande …

A Survey Of Heavy Metals In Water, Soil And The Surrounding Plants In The Lower Rio Grande Valley, Erving W. Morelius

Theses and Dissertations - UTB/UTPA

Heavy metal pollution of the environment in recent years has become a major issue of importance for both human and environmental health. Samples were investigated for the potential pollution of the area as it is a vital produce producing area. During the study plant, soil and water samples were collected from the Donna Reservoir and Canal System to test the concentration of various heavy metals. The heavy metals tested included both potentially toxic and micronutrients in the samples which included arsenic, cadmium, chromium, copper, iron, lead, nickel and sulfur. During the study, a sixty day sampling period was used to …

Abundance And Size Of Sand Crabs, Lepidopa Benedicti (Decapoda: Albuneidae), In Southern Texas, Jessica H. Murph, Zen Faulkes

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Albuneid sand crabs are found in sandy beaches around the world, but little is known about the basic biology of any species in the family. We sampled sand crabs, Lepidopa benedicti, for 2 years at South Padre Island, Texas, at two locations: one developed site near the town, where recreational use is high and one undeveloped site away from the town, where recreational use is lower. We hypothesized that sand crabs would be less abundant and smaller at the developed site than at the undeveloped one. Densities were highest in summer and lowest in winter but did not differ between …

Decomposition Patterns And Nitrogen Dynamics Of Black Mangrove (Avicennia Germinans)Leaf Litter In Disturbed Estuaries Linked To The Lower Laguna Madre, Texas, Mario Alberto Marquez

Theses and Dissertations - UTB/UTPA

Estuaries are among the most productive aquatic systems in the world but are subject to both anthropogenic and natural disturbances. With increasing environmental concerns, efforts have become commonplace in assessing the status and trends of environmental conditions. In this study, an assessment of ecosystem status of various estuaries affected by different disturbances, was attempted through the examination of key functional processes such as leaf litter decomposition and nutrient dynamics during decay. Three estuaries located along the Brownsville Ship Channel near the southern terminus of the Lower Laguna Madre in Texas were studied. The overlying goal of this study was to …

Rapid Protocol For Preparation Of Electrocompetent Escherichia Coli And Vibrio Cholerae, Miguel F. Gonzales, Teresa Brooks, Stefan U. Pukatzki, Daniele Provenzano

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of …

Getting To Evo-Devo: Concepts And Challenges For Students Learning Evolutionary Developmental Biology, Anna Hiatt, Gregory K. Davis, Caleb Trujillo, Mark Terry, Donald P. French, Rebecca M. Price, Kathryn E. Perez, Diane Ebert-May

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

In this study we used surveys of evo-devo experts to identify the core concepts of evo-devo and outline an underlying conceptual framework. We also use interviews and surveys of conceptual difficulties with these concepts.

To examine how well biology majors have achieved the necessary foundation in evolution, numerous studies have examined how students learn natural selection. However, no studies to date have examined how students learn developmental aspects of evolution (evo-devo). Although evo-devo plays an increasing role in undergraduate biology curricula, we find that instruction often addresses development cursorily, with most of the treatment embedded within instruction on evolution. Based …

Thorns Of Native Lower Rio Grande Valley Plants Are Colonized By Potentially Pathogenic Bacteria, Felicia A. Charles

Theses and Dissertations - UTB/UTPA

Epiphytic pathogenic bacteria on the thorns of native LRGV plants may provide a defense mechanism for the plants. To test this, plant thorns were collected from LRGV plant species and aerobic and anaerobic bacteria were isolated in thioglycollate medium. Tests were performed to characterize the bacterial cultures. The 16S rRNA gene was sequenced to allow for species identification. Sixty-nine bacterial cultures were isolated, including gram-positive and gram-negative cell types. All utilized a variety of carbon sources for nutrients and 23% of isolates displayed hemolysis. Bacterial identification included members of the genus Bacillus, Pseudomonas, Serratia, and others. Most were opportunistic pathogens. …

Examination Of Dietary Habits And Fruit And Vegetable Intake In A Chronic Disease Program, Qian Kuang

Theses and Dissertations - UTB/UTPA

The AHB program is a chronic disease prevention program which provides resources for community health centers along the U.S.-Mexico border. Healthy dietary habits and fruit/ vegetable intake play important roles in maintaining human health. Eight factors were used to evaluate the success of the AHB program. Another twenty factors were considered to predict dietary habits and fruit/vegetable intake status. Chi-square and multinomial logistic regression were used to study their correlation. Results from analysis showed that factors like birth country, employment, diabetes, baseline HbA1c, limited by problems, overall program quality, group, and program duration were correlated to the dietary habits, and …

Sfmtp1 And Spmtp1 Cloning And Expression In Saccharomyces Cerevisiae: Potential For The Genetic Engineering Of Arabidopsis Thaliana, Elyssa Rae Garza

Theses and Dissertations - UTB/UTPA

To evaluate metal tolerance conferred by the overexpression of Metal Tolerance Protein 1 (MTP1) in transgenic non-accumulator plants such as Arabidopsis thaliana, a model system was developed in Saccharomyces cerevisiae containing either SfMTP1 cDNA, from the metal tolerant plant Streptanthus farnsworthianus, or SpMTP1 cDNA, from the hyperaccumulator Streptanthus polygaloides. Nonquantitative RT-PCR showed constitutive expression of MTP1 in both plant species. Alignment of SfMTP1 and SpMTP1 protein sequences shows a gap in SpMTP1 in a variable histidine region known to affect metal transport. Disk assays revealed SfMTP1 cDNA imparted greater Ni tolerance to yeast than SpMTP1 cDNA; however, SpMTP1 showed higher …

Biochemical, Nutrient And Inhibitory Characteristics Of Streptomyces Cultured From A Hypersaline Estuary, The Laguna Madre (Texas), Luis E. Espinoza, Anita L. Davelos Baines, Kristine L. Lowe

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Streptomyces are common soil bacteria that produce secondary metabolites, including several antibiotics; however, the characteristics of marine Streptomyces are largely unknown. Sediment samples were taken from 3 sites in the Laguna Madre to isolate marine Streptomyces. Sediment was diluted, spread onto synthetic seawater media to estimate the total bacterial density of the samples and spread onto starch casein agar to isolate Streptomyces. Isolated Streptomyces were tested for salinity tolerance and optimal growth pH. Isolates were assayed using API 20E® test strips and BIOLOG™ plates to construct biochemical profiles and assess nutrient utilization abilities of the bacteria, respectively. Individual Streptomyces were …

Predicted Effects Of Climate Change On The Distribution Of The Invasive Grass Dichanthium Annulatum, Cynthia Isabel Garcia

Theses and Dissertations - UTB/UTPA

Kleberg bluestem (Dichanthium annulatum) is an invasive grass species native to Africa but now found in southern United States, Mexico, and other tropical and subtropical countries throughout the world. Using the modeling software MaxEnt, climatic variables from WorldClim, the Intergovernmental Panel on Climate Change (IPCC) scenario A1B, and two General Circulatory Models: the Canadian model (Canadian Centre for Climate Modelling and Analysis or CCCMA), and the Australian model (Commonwealth Scientific and Industrial Research Organisation or CSIRO), several models were developed to determine the possible implications of climatic change on the suitable habitat for D. annulatum, in the year 2050. Models …

The Extracellular Polysaccharide Pel Makes The Attachment Of P. Aeruginosa To Surfaces Symmetric And Short-Ranged, Benjamin J. Cooley, Travis W. Thatcher, Sara M. Hashmi, Guillaume L'Her, Daniel A. Hurwitz, Daniele Provenzano, Ahmed Touhami, Vernita D. Gordon

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Biofilms are surface-mounted, multicellular communities of microbes. Biofilms are often associated with chronic infections that resist treatment, evade the immune system, and damage host tissue. An essential characteristic of the biofilm state is that constituent organisms are bound in a polymeric matrix. This matrix gives the system spatial structure and clusters bacteria near each other, facilitating intercellular interactions. The Pseudomonas aeruginosa strain PAO1 is widely studied as a model biofilm-forming organism. The polymeric matrix of PAO1 biofilms is dominated by two bacteria-produced extracellular polymers, Pel and Psl. We use a combination of optical and atomic force microscopy to examine the …

The Evodevoci: A Concept Inventory For Gauging Students’ Understanding Of Evolutionary Developmental Biology, Kathryn E. Perez, Anna Hiatt, Gregory K. Davis, Caleb Trujillo, Donald P. French, Mark Terry, Rebecca M. Price

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

The American Association for the Advancement of Science 2011 report Vision and Change in Undergraduate Biology Education encourages the teaching of developmental biology as an important part of teaching evolution. Recently, however, we found that biology majors often lack the developmental knowledge needed to understand evolutionary developmental biology, or “evo-devo.” To assist in efforts to improve evo-devo instruction among undergraduate biology majors, we designed a concept inventory (CI) for evolutionary developmental biology, the EvoDevoCI. The CI measures student understanding of six core evo-devo concepts using four scenarios and 11 multiple-choice items, all inspired by authentic scientific examples. Distracters were designed …

Avifauna Of The Mache Chindul Ecological Reserve, Northwest Ecuador, Luis Carrasco, Karl S. Berg, Jennifer Litz, Andrew Cook, Jordan Karubian

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

We report on the avifauna of the 120,000 ha Mache Chindul Ecological Reserve (REMACH), northwest Ecuador. The study area is located in a poorly studied transition zone between three major Neotropical biogeographic regions-the Chocó, Tumbesian, and Tropical Andes-each of which contains exceptional diversity and endemism in birds and other organisms. We collected data from 1998–99 and 2004–11 from the Bilsa Biological Station (a 3500 ha private reserve) and several farms, forest fragments, and communities distributed across the central portion of REMACH using observations (aural and visual), audio recordings, mist netting, point counts and photographs. We recorded 360 species of bird …

Factors Shaping The Ontogeny Of Vocal Signals In A Wild Parrot, Karl S. Berg, Steven R. Beissinger, Jack W. Bradbury

School of Integrative Biological & Chemical Sciences (Formerly Dept. of Biology)

Parrots rely heavily on vocal signals to maintain their social and mobile lifestyles. We studied vocal ontogeny in nests of wild green-rumped parrotlets (Forpus passerinus) in Venezuela. We identified three successive phases of vocal signaling that corresponded closely to three independently derived phases of physiological development. For each ontogenetic phase, we characterized the relative importance of anatomical constraints, motor skills necessary for responding to specific contexts of the immediate environment, and the learning of signals that are necessary for adult forms of communication. We observed shifts in the relative importance of these three factors as individuals progressed from …