Establishment of a PCR and restriction enzyme assay for the detection of the hemochromatosis gene in normal subjects and diabetic patients

The purpose of this study was to establish a polymerase chain reaction (PCR)-restriction enzyme assay for detecting the hereditary hemochromatosis (HHC) mutation, C282Y, in gestational and gestational diabetic subjects in South Florida. DNA samples from 43 gestational subjects were amplified by PCR, digested with RsaI, and analyzed by electrophoresis. An allelic frequency of 2.33%, or 4.65% heterozygosity, was observed. The assay is successful and applicable to future studies on HHC and gestational diabetes. ^

Prenatal environmental exposure and neurodevelopmentally important gene expression in malformed brain tissue from pediatric intractable epilepsy patients

The primary objective of this proposal was to determine whether mitochondrial oxidative stress and variation in a particular mtDNA lineage contribute to the risk of developing cortical dysplasia and are potential contributing factors in epileptogenesis in children. The occurrence of epilepsy in children is highly associated with malformations of cortical development (MCD). It appears that MCD might arise from developmental errors due to environmental exposures in combination with inherited variation in response to environmental exposures and mitochondrial function. Therefore, it is postulated that variation in a particular mtDNA lineage of children contributes to the effects of mitochondrial DNA damage on MCD phenotype. Quantitative PCR and dot blot were used to examine mitochondrial oxidative damage and single nucleotide polymorphism (SNP) in the mitochondrial genome in brain tissue from 48 pediatric intractable epilepsy patients from Miami Children’s Hospital and 11 control samples from NICHD Brain and Tissue Bank for Developmental Disorders. Epilepsy patients showed higher mtDNA copy number compared to normal health subjects (controls). Oxidative mtDNA damage was lower in non-neoplastic but higher in neoplastic epilepsy patients compared to controls. There was a trend of lower mtDNA oxidative damage in the non-neoplastic (MCD) patients compared to controls, yet, the reverse was observed in neoplastic (MCD and Non-MCD) epilepsy patients. The presence of mtDNA SNP and haplogroups did not show any statistically significant relationships with epilepsy phenotypes. However, SNPs G9804A and G9952A were found in higher frequencies in epilepsy samples. Logistic regression analysis showed no relationship between mtDNA oxidative stress, mtDNA copy number, mitochondrial haplogroups and SNP variations with epilepsy in pediatric patients. The levels of mtDNA copy number and oxidative mtDNA damage and the SNPs G9952A and T10010C predicted neoplastic epilepsy, however, this was not significant due to a small sample size of pediatric subjects. Findings of this study indicate that an increase in mtDNA content may be compensatory mechanisms for defective mitochondria in intractable epilepsy and brain tumor. Further validation of these findings related to mitochondrial genotypes and mitochondrial dysfunction in pediatric epilepsy and MCD may lay the ground for the development of new therapies and prevention strategies during embryogenesis.

Forging professional public health nursing in a southern state: Florida's public health nurses, 1889-1934

From 1889 to 1934, Florida's nurses belonging to a new group of professional women ushered in a pioneering phase of public health nursing in Florida. During this era, the nurses' ability to confront health and professional issues varied a great deal but in quiet and forceful ways they tackled cultural and environmental problems to assist people who were ill or help prevent people from becoming ill. This dissertation places the development of professional public health nursing in its social context by uncovering the relationships public health nurses formed with clubwomen, the medical profession, city leaders, midwives, and others. In 1888, there were few graduate nurses in the state, no state board of health and no organized nursing service to respond to Jacksonville's great yellow fever epidemic. By 1934, national and state leaders of public health nursing had built up the profession to become an essential part of the State Board of Health's service to the community. Between these milestones, in the era of white supremacy and Jim Crow, public health nurses combined their professional training with a pioneer spirit of innovation and risk-taking. In the predominately rural state, the public health nurses' resolve to overcome environmental hazards and cultural obstacles stands out as they attempted to reach those who were unserved or underserved by modern medicine.

Preimplantation genetic diagnosis: Analysis of gene expression, nuclear DNA and cytoplasmic DNA

Preimplantation genetic diagnosis (PGD) following in vitro fertilization (IVF) offers couples at risk for transmitting genetic disorders the opportunity to identify affected embryos prior to replacement. In particular, embryo gender determination permits screening for X-linked diseases of unknown etiology. Analysis of embryos can be performed by polymerase chain reaction (PCR) amplification of material obtained by micromanipulation. This approach provides an alternative to the termination of an established pregnancy following chorionic villi sampling or amniocentesis. ^ Lately, the focus of preimplantation diagnosis and intervention has been shifting toward an attempt to correct cytoplasmic deficiencies. Accordingly, it is the aim of this investigation to develop methods to permit the examination of single cells or components thereof for clinical evaluation. In an attempt to lay the groundwork for precise therapeutic intervention for age related aneuploidy, transcripts encoding proteins believed to be involved in the proper segregation of chromosomes during human oocyte maturation were examined and quantified. Following fluorescent rapid cycle RT-PCR analysis it was determined that the concentration of cell cycle checkpoint gene transcripts decreases significantly as maternal age increases. Given the well established link between increasing maternal age and the incidence of aneuploidy, these results suggest that the degradation of these messages in aging oocytes may be involved with inappropriate chromosome separation during meiosis. ^ In order to investigate the cause of embryonic rescue observed following clinical cytoplasmic transfer procedures and with the objective of developing a diagnostic tool, mtDNA concentrations in polar bodies and subcellular components were evaluated. First, the typical concentration of mtDNA in human and mouse oocytes was determined by fluorescent rapid cycle PCR. Some disparity was noted between the copy numbers of individual cytoplasmic samples which may limit the use of the current methodology for the clinical assessment of the corresponding oocyte. ^

Health care utilization behavior of elders in a multicultural urban environment

The objective of this research is to determine the influences of social, environmental, behavioral, and economic forces on the health care service utilization of four racial/ethnic groups of non-institutionalized elders in a multicultural urban environment. To address these issues this dissertation examines three intertwined themes of culture, aging, and health, using a sample of elders residing in Miami-Dade County, FL in four racial/ethnic groups: white non-Hispanic; black non-Hispanic English speakers; Cuban; and non-Cuban Hispanic. ^ The research questions were analyzed using both quantitative and qualitative data. Data for the quantitative component uses telephone survey data from the Dade County Needs Assessment. The purpose of this component is to develop a more comprehensive model of elder health care utilization behavior. The qualitative component uses data from focus groups from Dade County Needs Assessment, archival data and a literature review of previous ethnographic research. The purpose of this component is to gain a better understanding of the social construction of the terms “age”' and “aging,” as well as to place issues of health and health care in the lives of elders. ^ The findings raised several important issues. First, just because people share a common chronological age does not mean that they are the same in every other respect. Examining elders as a homogeneous group of users of formal health care services in a community is simplistic. Placing “aging” and “health” in a cultural context is important. My findings confirm that the meaning of “aging” and “old” are socially constructed. Further, the term “aging” is NOT synonymous with ill health or frailty. This was a consistent finding in both the quantitative and qualitative results. ^ While all aging individuals share a mutual orientation toward aging (i.e., biological process), they do not age the same way (i.e., social construction of “aging”). Thus, policymakers and others serving the elder population must be aware of the particular cultural context, as well as the previous life experiences of the individuals that they serve. This analysis documents the importance of culture and geographic community in understanding health care service utilization of elders. ^

Horizontal genetic transfer in asexual fungi

Four aspects of horizontal genetic transfer during heterokaryon formation were examined in the asexual pathogen Fusarium oxysporum f.sp. cubense (Foc): (1) variability based on method of heterokaryon formation; (2) differences in nuclear and mitochondrial inheritance; (3) the occurrence of recombination without nuclear fusion; (4) the occurrence of horizontal genetic transfer between distantly related isolates. The use of non-pathogenic strains of Fusarium oxysporum as biocontrol agents warrants a closer examination at the reproductive life cycle of this fungus, particularly if drug resistance or pathogenicity genes can be transmitted horizontally. Experiments were divided into three phases. Phase I looked at heterokaryon formation by hyphal anastomosis and protoplast fusion. Phase II was a time course of heterokaryon formation to look at patterns of nuclear and mitochondrial inheritance. Phase III examined the genetic relatedness of the different vegetative compatibility groups using a multilocus analysis approach. Heterokaryon formation was evident within and between vegetative compatibility groups. Observation of non-parental genotypes after heterokaryon formation confirmed that, although a rare event, horizontal genetic transfer occurred during heterokaryon formation. Uniparental mitochondria inheritance was observed in heterokaryons formed either by hyphal anastomosis or protoplast fusion. Drug resistance was expressed during heterokaryon formation, even across greater genetic distances than those distances imposed by vegetative compatibility. Phylogenies inferred from different molecular markers were incongruent at a significant level, challenging the clonal origins of Foc. Mating type genes were identified in this asexual pathogen Polymorphisms were detected within a Vegetative Compatibility Group (VCG) suggesting non-clonal inheritance and/or sexual recombination in Foc. This research was funded in part by a NIH-NIGMS (National Institutes of Health-National Institute of General Medical Sciences) Grant through the MBRS (Minority Biomedical Research Support), the Department of Biological Sciences and the Tropical Biology Program at FIU. ^

Health beliefs and health care options of disparate black communities in the United States

According to the 1999 U.S. Census, there were approximately thirty-three million African Americans and approximately 1.8 million non-Hispanic black immigrants in the United States. The 1997 U.S. Census estimated that there were as many as 554,000 foreign-born Haitians and 505,000 foreign-born Jamaicans living in the United States, mainly residing in Florida and New York. The U.S. Census claims that blacks are one of the largest emerging ethnic groups in America constituting at least twelve percent of this country's population. Because of this increase, their specific health beliefs and health care options are increasingly nationally significant. ^ In the present intra-black and inter-ethnic study, two hundred seventy African Americans, Haitian immigrants, and Jamaican immigrants residing in South Florida were quantitatively and qualitatively surveyed in order to investigate their health beliefs and health care options. According to the reviewed literature, the three black ethnic groups researched in this study have not been compared or contrasted before in relation to these beliefs and health care choices. ^ As was discovered by the present research, differing health beliefs and health care options were the cultural products of the ethnic differences of the researched communities. It was expected that differing health beliefs among the researched black groups might indicate disparate patterns of health care utilization of either western or non-western models. Additionally, it was projected that by investigating the health beliefs and the health care options of these emerging black ethnic groups, western health care providers in the United States could become better versed in medically servicing growing ethnically-disparate black populations. The study yielded results about the researched groups that supported major findings in the reviewed literature. The data were reported and examined, and theoretical generalizations from the data were discussed. The most important of these findings was that, within a race, health beliefs and health care options were determined by specific ethno-cultural variables dependent on national origins. ^

The health and nutritional status of multiethnic perimenopausal women

An integrated, dual-phase study design assessed the health and nutritional status and practices of African-American (A-A), Caribbean (A-C), and white non-Hispanic (W-A) women during perimenopause (40–55 years). During Phase I, four focus groups (n = 37) of male and female participants discussed the health and social implications of perimenopause. A conceptual framework for the main study (Phase II) was developed from the focus groups' findings, in concert with the main study's specific aims and objectives. ^ The main study, a cross-sectional survey, quantitatively assessed the health and nutritional status of a convenience sample of 109 women (25 A-A, 31 A-C and 53 W-A), who met specific eligibility criteria. Using seven instruments, sociodemographic, dietary, medical, reproductive health, health practice and anthropometric data were collected. ^ The groups were of comparable age, education, and socioeconomic status (SES). Despite these similarities, statistically significant interethnic nutritional status differences were found. Significantly more total energy and energy from fat were consumed by A-A than W-A and A-C women. Also, significantly more A-A and A-C than W-A women were overweight or obese with android-type weight patterning. ^ Overall, iron and calcium Recommended Dietary Allowances (RDA's) were not met by 35% and 68% of participants, respectively. Iron deficiency anemia was reported by 29% of participants while 33% reported heavier menstrual bleeding. Coupled with suboptimal iron intakes, this is likely to present a serious public health problem. Similarly, increased bone demineralization characteristic of perimenopause, coupled with suboptimal calcium intakes could precipitate another public health problem, osteoporosis. ^ Participants had different expectations about the role of medical care during perimenopause. Significantly more white (57%) than black (38% [A-A and AC]) women sought medical attention for symptoms. Whereas Hormone Replacement Therapy (HRT) was prescribed for 25% of them, only 13% were compliant at enrollment. ^ The trends and statistically significant findings of this study have huge public health policy implications. It is imperative that appropriate policies are formulated to ensure that America's ethnically diverse perimenopausal women have ready access to culturally appropriate care. This would optimize their health outcomes, and enhance their quality of life and productive capacities at this critical juncture of their lives. ^

Coronary heart disease risk factors among tri-ethnic students at Florida International University

The present study identified and compared Coronary Heart Disease (CHD) risk factors quantified as “CHD risk point standards” (CHDRPS) among tri-ethnic (White non-Hispanic [WNH], Hispanic [H], and Black non-Hispanic [BNH]) college students. All 300 tri-ethnic subjects completed the Cardiovascular Risk Assessment Instruments and had blood pressure readings recorded on three occasions. The Bioelectrical Impedance Analysis (BIA) was used to measure body composition. Students' knowledge of CHD risk factors was also measured. In addition, a 15 ml fasting blood sample was collected from 180 subjects and blood lipids and Homocysteine (tHcy) levels were measured. Data were analyzed by gender and ethnicity using one-way Analysis of Variance (ANOVA) with Bonferroni's pairwise mean comparison procedure, Pearson correlation, and Chi-square test with follow-up Bonferroni's Chi-square tests. ^ The mean score of CHDRPS for all subjects was 19.15 ± 6.79. Assigned to the CHD risk category, college students were below-average risk of developing CHD. Males scored significantly (p < 0.013) higher for CHD risk than females, and BNHs scored significantly (p < 0.033) higher than WNHs. High consumption of dietary fat saturated fat and cholesterol resulted in a high CHDRPS among H males and females and WNH females. High alcohol consumption resulted in a high CHDRPS among all subjects. Mean tHcy ± SD of all subjects was 6.33 ± 3. 15 μmol/L. Males had significantly (p < 0.001) higher tHcy than females. Black non-Hispanic females and H females had significantly (p < 0.003) lower tHcy than WNH females. Positive associations were found between tHcy levels and CHDRPS among females (p < 0.001), Hs (p < 0.001), H males (p < 0.049), H females (p < 0.009), and BNH females (p < 0.005). Significant positive correlations were found between BMI levels and CHDRPS in males (p < 0.001), females (p < 0.001), WNHs (p < 0.008), Hs (p < 0.001), WNH males (p < 0.024), H males (p < 0.004) and H females (p < 0.001). The mean knowledge of CHD questions of all subjects was 71.70 ± 7.92 out of 100. The mean knowledge of CHD was significantly higher for WNH males (p < 0.039) than BNH males. A significant inverse correlation (r = 0.392, p < 0.032) was found between the CHD knowledge and CHDRPS in WNH females. The researcher's findings indicate strong gender and ethnic differences in CHD risk factors among the college-age population. ^

Mitochondrial DNA, segments I and II, its applications in population genetics and forensic sciences

The investigations of human mitochondrial DNA (mtDNA) have considerably contributed to human evolution and migration. The Middle East is considered to be an essential geographic area for human migrations out of Africa since it is located at the crossroads of Africa, and the rest of the world. United Arab Emirates (UAE) population inhabits the eastern part of Arabian Peninsula and was investigated in this study. Published data of 18 populations were included in the statistical analysis. The diversity indices showed (1) high genetic distance among African populations and (2) high genetic distance between African populations and non-African populations. Asian populations clustered together in the NJ tree between the African and European populations. MtDNA haplotypes database of the UAE population was generated. By incorporating UAE mtDNA dataset into the existing worldwide mtDNA database, UAE Forensic Laboratories will be able to analyze future mtDNA evidence in a more significant and consistent manner. ^

Medical advice, diabetes self-management, and health outcomes of a multi-ethnic population from the National Health and Nutrition Examination Survey 2007--2008

Diabetes self-management, an essential component of diabetes care, includes weight control practices and requires guidance from providers. Minorities are likely to have less access to quality health care than White non-Hispanics (WNH) (American College of Physicians-American Society of Internal Medicine, 2000). Medical advice received and understood may differ by race/ethnicity as a consequence of the patient-provider communication process; and, may affect diabetes self-management. ^ This study examined the relationships among participants’ report of: (1) medical advice given; (2) diabetes self-management, and; (3) health outcomes for Mexican-Americans (MA) and Black non-Hispanics (BNH) as compared to WNH (reference group) using data available through the National Health and Nutrition Examination Survey (NHANES) for the years 2007–2008. This study was a secondary, single point analysis. Approximately 30 datasets were merged; and, the quality and integrity was assured by analysis of frequency, range and quartiles. The subjects were extracted based on the following inclusion criteria: belonging to either the MA, BNH or WNH categories; 21 years or older; responded yes to being diagnosed with diabetes. A final sample size of 654 adults [MA (131); BNH (223); WNH (300)] was used for the analyses. The findings revealed significant statistical differences in medical advice reported given. BNH [OR = 1.83 (1.16, 2.88), p = 0.013] were more likely than WNH to report being told to reduce fat or calories. Similarly, BNH [OR = 2.84 (1.45, 5.59), p = 0.005] were more likely than WNH to report that they were told to increase their physical activity. Mexican-Americans were less likely to self-monitor their blood glucose than WNH [OR = 2.70 (1.66, 4.38), p<0.001]. There were differences among ethnicities for reporting receiving recent diabetes education. Black, non-Hispanics were twice as likely to report receiving diabetes education than WNH [OR = 2.29 (1.36, 3.85), p = 0.004]. Medical advice reported given and ethnicity/race, together, predicted several health outcomes. Having recent diabetes education increased the likelihood of performing several diabetes self-management behaviors, independent of race. ^ These findings indicate a need for patient-provider communication and care to be assessed for effectiveness and, the importance of ongoing diabetes education for persons with diabetes.^

Knowledge, attitudes, and practices affecting health behaviors in the U.S. Army Special Operations Command population of Rangers

Malaria is a threat to United States military personnel operating in endemic areas, from which there have been hundreds of cases reported over the past decade. Each of these cases might have been avoided with proper adherence to malaria chemoprophylaxis medications. Military operations may detract from the strict 100% adherence required of these preventive medications. However, the reasons for non-adherence in military populations are not well understood. This behavior was investigated using a cross sectional study design on a convenience sample of U.S. Army Ranger volunteers (n=150) located at three military instillations. Theoretical support was based on components of the Health Belief Model, the Theory of Reasoned Action/Theory of Planned Behavior, and the Social Cognitive Theory. ^ Data on knowledge, attitudes, and practices, as well as multiple environmental domains was collected using an original yet unvalidated questionnaire. The data was analyzed using bivariate Pearson correlations, binary logistic regression, and moderated logistic regressions employing a 0.05 criterion of statistical significance. Power analyses predicted 96-98% power for this analysis. ^ Multiple significant medium strength Pearson correlation coefficients were identified relative to the two dependent variables Take medications as directed and Intend to take the medications as directed the next time. Binary logistic regression analyses identified multiple variables that may predict behavioral intentions to adhere to these preventive medications, as a proxy for behavioral change. Moderated logistic regression analyses identified Command Support for adherence to these medications as a potential significant moderator that interacts with independent variables within three domains of the survey questionnaire. ^ The findings indicate that there may be potential significant beneficial effects, which may improve this behavior in this population of Rangers through 1) promoting affirmative interpersonal communications that emphasize adherence to these medications, 2) including malaria chemoprophylaxis medications in the mission planning process, and 3) military command support, in the form of including the importance of proper adherence to these medications in the unit safety briefings.^

Gender differences in the influence of protective factors, risk factors, and health risk behaviors on HIV risk behaviors among youth in South Florida

Adolescents engage in a range of risk behaviors during their transition from childhood to adulthood. Identifying and understanding interpersonal and socio-environmental factors that may influence risk-taking is imperative in order to meet the Healthy People 2020 goals of reducing the incidence of unintended pregnancies, HIV, and other sexually transmitted infections among youth. The purpose of this study was to investigate gender differences in the predictors of HIV risk behaviors among South Florida youth. More specifically, this study examined how protective factors, risk factors, and health risk behaviors, derived from a guiding framework using the Theory of Problem Behavior and Theory of Gender and Power, were associated with HIV risk behavior. A secondary analysis of 2009 Youth Risk Behavior Survey data sets from Miami-Dade, Broward, and Palm Beach school districts tested hypotheses for factors associated with HIV risk behaviors. The sample consisted of 5,869 high school students (mean age 16.1 years), with 69% identifying as Black or Hispanic. Logistic regression analyses revealed gender differences in the predictors of HIV risk behavior. An increase in the health risk behaviors was related to an increase in the odds that a student would engage in HIV risk behavior. An increase in risk factors was also found to significantly predict an increase in the odds of HIV risk behavior, but only in females. Also, the probability of participation in HIV risk behavior increased with grade level. Post-hoc analyses identified recent sexual activity (past 3 months) as the strongest predictor of condom nonuse and having four or more sexual partners for both genders. The strongest predictors of having sex under the influence of drugs/alcohol were alcohol use in both genders, marijuana use in females, and physical fighting in males. Gender differences in the predictors of unprotected sex, multiple sexual partners, and having sex under the influence were also found. Additional studies are warranted to understand the gender differences in predictors of HIV risk behavior among youth in order to better inform prevention programming and policy, as well as meet the national Healthy People 2020 goals.

Norm-based comparison of SF -12 physical and mental health scale scores for a sample of employees of Broward County

In the new health paradigm, the connotation of health has extended beyond the measures of morbidity and mortality to include wellness and quality of life. Comprehensive assessments of health go beyond traditional biological indicators to include measures of physical and mental health status, social role-functioning, and general health perceptions. To meet these challenges, tools for assessment and outcome evaluation are being designed to collect information about functioning and well-being from the individual's point of view.^ The purpose of this study was to profile the physical and mental health status of a sample of county government employees against U.S. population norms. A second purpose of the study was to determine if significant relationships existed between respondent characteristics and personal health practices, lifestyle and other health how the tools and methods used in this investigation can be used to guide program development and facilitate monitoring of health promotion initiatives.^ The SF-12 Health Survey (Ware, Kosinski, & Keller, 1995), a validated measure of health status, was administered to a convenience sample of 450 employees attending one of nine health fairs at an urban worksite. The instrument has been utilized nationally which enabled a comparative analysis of findings of this study with national results.^ Results from this study demonstrated that several respondent characteristics and personal health practices were associated with a greater percentage of physical and/or mental scale scores that were significantly "worse" or significantly "better" than the general population. Respondent characteristics that were significantly related to the SF-12 physical and/or mental health scale scores were gender, age, education, ethnicity, and income status. Personal health practices that were significantly related to SF-12 physical and/or mental scale scores were frequency of vigorous exercise, presence of chronic illness, being at one's prescribed height and weight, eating breakfast, smoking and drinking status. This study provides an illustration of the methods used to analyze and interpret SF-12 Health Survey data, using norm-based interpretation guidelines which are useful for purposes of program development and collecting information on health at the community level. ^

Health changes in Hispanic older adults in a Spanish arthritis self -management education program

Arthritis is the most common chronic condition affecting older people and is a major cause of limited activity. Arthritis education programs in English have demonstrated a positive impact on health but these programs have not reached the Hispanic communities where arthritis is the leading cause of disability. Minorities, such as Hispanics, have traditionally been reluctant to pursue self-help programs, and have been identified as an under-served population in terms of medical care. This study examined the effectiveness of one community health adult education program targeting Hispanic older adults with arthritis, the Spanish Arthritis Self Management Education Program (SASMEP), by evaluating changes in the participants' general health, pain, disability, self-efficacy, health perceptions, frequency of physician visits, and exercise. A pre and post control group experimental design and analyses of covariance were used to determine the pre and post differences in health status and health behaviors for a group participating in the SASMEP and a group who did not using gender and age as covariates. A repeated measures design was also used, and repeated measures analyses of variance and post hoc tests were done on health status and health behavior data collected pre, post and one-year post education to determine long-term differences. ^ Results indicated the participants' health status significantly improved in general health, significantly decreased in pain, and significantly decreased in arthritic disability immediately following the education. Self-efficacy and health perceptions increased for both groups but not significantly. The participants' health behaviors showed significantly fewer physician visits and significantly increased time spent performing stretching and strengthening exercise and time spent performing aerobic exercise. No group differences were found in the frequency of arthritis physician visits. ^ The improvements seen immediately after the SASMEP participation were not reflected in the post one-year scores. No significant differences were found for the participants' health status or health behaviors one year following the education. Health status and health behaviors did not return below baseline scores after one year suggesting the participants' health, although not improved, did not deteriorate. Therefore, the SASMEP education provided short-term health benefits for older Hispanic adults with arthritis, but not long-term health benefits. ^