Exon involvement in the regulation of MuSVts110 RNA splicing

MuSVts110 is a conditionally defective mutant of Moloney murine sarcoma virus which undergoes a novel tmperature-dependent splice event at growth temperatures of 33$\sp\circ$C or lower. Relative to wild-type MuSV-124, MuSVts110 contains a 1487 base deletion spanning from the 3$\sp\prime$ end of the p30 gag coding region to just downstream of the first v-mos initiation codon. As a result, the gag and mos genes are fused out of frame and no v-mos protein is expressed. However, upon a shift to 33$\sp\circ$C or lower, a splice event occurs which removes 431 bases, realigns the gag and mos genes, and allows read-through translation of a P85gag-mos transforming protein. Interestingly, while the cryptic splice sites utilized in MuSVts110 are present and unaltered in MuSV-124, they are never used. Due to the 1487 base deletion, the MuSV-124 intron was reduced from 1919 to 431 bases suggesting that intron size might be involved in the activation of these cryptic splice sites in MuSVts110. Since the splicing phenotype of the MuSVts110 equivalent (TS32 DNA) which contains the identical 1487 base deletion introduced into otherwise wild-type MuSV-124 DNA, was indistinguishable from authentic MuSVts110, it was concluded that this deletion alone is responsible for activation of the cryptic splice sites used in MuSVts110. These results also confirmed that thermodependent splicing is an intrinsic property of the viral RNA and not due to some cellular defect. Furthermore, analysis of gag gene deletion and frameshift MuSVts110 mutants demonstrated that viral gag gene proteins do not play a role in regulation of MuSVts110 splicing. Instead, cis-acting viral sequences appear to mediate regulation of the splice event.^ Our initial observation that truncation of the MuSVts110 transcript, leaving only residual amounts of the flanking exon sequences, completely abolished splicing activity argued that exon sequences might participate in the regulation of the splice event.^ Analysis of exon sequence involvement has also identified cis-acting sequences important in the thermodependence of the splice event. Data suggest that regulation of the MuSVts110 splice event involves multiple interactions between specific intron and exon sequences and spliceosome components which together limit splicing activity to temperatures of 33$\sp\circ$C or lower while simultaneously restricting splicing to a maximum of 50% efficiency. (Abstract shortened with permission of author.) ^

Intron involvement in the regulation of MuSVts110 RNA splicing

The murine sarcoma virus MuSVts110 exhibits an alternative RNA splicing pattern. Like other simple retroviruses, MuSVts110 pre-mRNA splicing is balanced to allow the production of both spliced and unspliced RNA during the replicative cycle. In addition to balance, MuSVts110 RNA splicing exhibits a unique growth-temperature restriction to splicing; temperatures below 33$\sp\circ$C are permissive for splicing while temperatures of 37$\sp\circ$C or above are non-permissive. Previous work has established that this thermosensitive splicing phenotype is mediated in cis by viral transcript features. Here we show that at least three sequence elements regulate the MuSVts110 splicing phenotype. First, the MuSVts110 branchpoint (BP) and poly-pyrimidine tract (PPT) were found to be determinants of overall splicing efficiency. Wild-type MuSVts110 possesses a weak BP and PPT adjacent to the 3$\sp\prime$ splice site. Introduction of a strong BP caused MuSVts110 splicing to proceed to virtual completion in vivo, thus losing any vestige of balance or thermosensitivity. In in vitro splicing extracts, the strong BP overcame a blockade to wt MuSVts110 splicing at both the first and second catalytic steps. Weakening the consensus nature of the strong BP allowed the recovery of thermosensitive splicing in vivo, and reinstated the blockades to splicing in vitro, arguing that a suboptimal BP is an unusual manifestation of the proportional splicing pattern of retroviruses. The PPT is essential for accurate recognition of the BP sequence by the splicing machinery. Lengthening the PPT of MuSVts110 from 9 to 19 consecutive pyrimidines increased the overall efficiency of splicing in vivo dramatically, but was less effective than the strong BP in overriding the restriction on splicing imposed by high growth temperatures. Finally, decreasing gradually the overall size of the intron unexpectedly reduced splicing efficiency at growth temperatures permissive for splicing, suggesting that non-conserved sequences within the intron of MuSVts110 participate in splicing regulation as well. Taken together, these results suggest a mechanism of control in which MuSVts110 splicing is modulated by the entire intron, but principally by suboptimal signals at the splice acceptor site. Furthermore, this retroviral system provides a powerful genetic method for selection and analysis of mutations that affect splicing. ^

Cerebellar cortex involvement in classical eyelid conditioning

A model for cerebellar involvement in motor learning was tested using classical eyelid conditioning in the rabbit. Briefly, we assume that modifications of the strength of granule cell synapses at Purkinje cells in the cerebellar cortex and mossy fiber (MF) synapses at cerebellar interpositus nuclei are responsible for the acquisition, adaptively-timed expression, and extinction of conditioned eyelid responses (CRs). A corollary of these assumptions is that the cerebellar cortex is necessary for acquisition and extinction. This model also suggests a mechanism whereby the cerebellar cortex can discriminate different times during a conditioned stimulus (CS) and thus mediate the learned timing of CRs. Therefore, experiments were done to determine the role of the cerebellar cortex in the timing, extinction, and acquisition of CRs. Lesions of the cerebellar cortex that included the anterior lobe disrupted the learned timing of CRs such that they occurred at extremely short latencies. Stimulation of MFs in the middle cerebellar peduncle as the CS could support differently timed CRs in the same animal. These data indicate that synaptic plasticity in the cerebellar cortex mediates the learned timing of CRs. These short-latency CRs which resulted from anterior lobe damage did not extinguish, while CRs in animals receiving lesions which did not include the anterior lobe extinguished normally. Preliminary data suggests that lesions of the anterior lobe which produce short-latency responses prevent the acquisition of CRs to a novel CS. These findings indicate that the anterior lobe of cerebellar cortex is necessary for eyelid conditioning. The involvement of the anterior lobe in eyelid conditioning has not been previously reported, however, the anterior lobe has generally been spared in lesion studies examining cerebellar cortex involvement in eyelid conditioning due to its relatively inaccessible location. The observation that the anterior lobe of the cerebellar cortex is not always required for the basic expression of CRs, but is necessary for response timing, extinction, and acquisition, is consistent with the hypothesis that eyelid conditioning can involve plasticity in both the cerebellar cortex and interpositus nucleus and that plasticity in the nucleus is controlled by Purkinje cell activity. ^

EVIDENCE OF HUMAN ENDOGENOUS RETROVIRUS K INVOLVEMENT IN HUMAN CANCER

Many lines of clinical and experimental evidence indicate a viral role in carcinogenesis (1-6). Our access to patient plasma, serum, and tissue samples from invasive breast cancer (N=19), ductal carcinoma in situ (N=13), malignant ovarian cancer (N=12), and benign ovarian tumors (N=9), via IRB-approved and informed consent protocols through M.D. Anderson Cancer Center, as well as normal donor plasmas purchased from Gulf Coast Regional Blood Center (N=6), has allowed us to survey primary patient blood and tissue samples, healthy donor blood from the general population, as well as commercially available human cell lines for the presence of human endogenous retrovirus K (HERV-K) Env viral RNA (vRNA), protein, and viral particles. We hypothesize that HERV-K proteins are tumor-associated antigens and as such can be profiled and targeted in patients for diagnostic and therapeutic purposes. To test this hypothesis, we employed isopycnic ultracentrifugation, a microplate-based reverse transcriptase enzyme activity assay, reverse transcription – polymerase chain reaction (RT-PCR), cDNA sequencing, SDS-PAGE and western blotting, immunofluorescent staining, confocal microscopy, and transmission electron microscopy to evaluate v HERV-K activation in cancer. Data from large numbers of patients tested by reverse transcriptase activity assay were analyzed statistically by t-test to determine the potential use of this assay as a diagnostic tool for cancer. Significant reverse transcriptase enzyme activity was detected in 75% of ovarian cancer patients, 53.8% of ductal carcinoma in situ patient, and 42.1% of invasive breast cancer patient samples. Only 11.1% of benign ovarian patient and 16.7% of normal donor samples tested positive. HERV-K Env vRNA, or Env SU were detected in the majority of cancer types screened, as demonstrated by the results shown herein, and were largely absent in normal controls. These findings support our hypothesis that the presence of HERV-K in patient blood circulation is an indicator of cancer or pre-malignancy in vivo, that the presence of HERV-K Env on tumor cell surfaces is indicative of malignant phenotype, and that HERV-K Env is a tumor-associated antigen useful not only as a diagnostic screening tool to predict patient disease status, but also as an exploitable therapeutic target for various novel antibody-based immunotherapies.

Parents Too Can Evaluate Family Preservation Services - or –Involving Parents In Evaluation of Family Preservation Services

In line with the long-standing emphasis in the human services on involving clients in program evaluation, in this essay I focus on principles and strategies for involving parents in evaluation of family preservation services. In particular, I delineate the crucial roles that parents can play as partners in the helping process within a family-centered context.

An evaluation of the impact of Inner City Development's Cooperative Home School: An educational alternative program to increase the quality and level of education in San Antonio's West Side community

The research study was intended to evaluate the effectiveness of Inner City Development's (I.C.D.) Cooperative Home School, an educational alternative program to the Title I public schools of San Antonio's West Side community. The study investigated students', parents' and tutors' perception of parental involvement and educational resources. The study also investigated each student's academic achievement. ^ The study found that students progressed toward expected math proficiency at a faster rate than they did in reading proficiency. However, because the target population size was small and a comparison group was not used, the results of this study are only suggestive. This research also indicated that study subjects believed students' quality and level of education increased substantially since program exposure. Study subjects mainly attributed the students' strides in academic performance to the increased amount of individualized attention students received in the small twelve-student class size. Study subjects were more satisfied with the home school's educational resources than those of the Title I public schools. Study subjects also perceived that parental involvement both at home and at school increased since enrollment in the home school program because: (1) there were more opportunities for involvement in the home school; and (2) parents felt closer to the tutors than the teachers in public school. ^ This evaluation also suggested improvements to program operations. With the help of additional volunteers, I.C.D. program operators could improve collection and organization of academic records. Furthermore, as suggested by program participants, science could be added to the curriculum. Lastly, a formal tutor orientation could be implemented to familiarize and train tutors on classroom management procedures. ^

AN EVALUATION OF THE CONSISTENCY OF IMRT PATIENT-SPECIFIC QA TECHNIQUES

To ensure the integrity of an intensity modulated radiation therapy (IMRT) treatment, each plan must be validated through a measurement-based quality assurance (QA) procedure, known as patient specific IMRT QA. Many methods of measurement and analysis have evolved for this QA. There is not a standard among clinical institutions, and many devices and action levels are used. Since the acceptance criteria determines if the dosimetric tools’ output passes the patient plan, it is important to see how these parameters influence the performance of the QA device. While analyzing the results of IMRT QA, it is important to understand the variability in the measurements. Due to the different form factors of the many QA methods, this reproducibility can be device dependent. These questions of patient-specific IMRT QA reproducibility and performance were investigated across five dosimeter systems: a helical diode array, radiographic film, ion chamber, diode array (AP field-by-field, AP composite, and rotational composite), and an in-house designed multiple ion chamber phantom. The reproducibility was gauged for each device by comparing the coefficients of variation (CV) across six patient plans. The performance of each device was determined by comparing each one’s ability to accurately label a plan as acceptable or unacceptable compared to a gold standard. All methods demonstrated a CV of less than 4%. Film proved to have the highest variability in QA measurement, likely due to the high level of user involvement in the readout and analysis. This is further shown by how the setup contributed more variation than the readout and analysis for all of the methods, except film. When evaluated for ability to correctly label acceptable and unacceptable plans, two distinct performance groups emerged with the helical diode array, AP composite diode array, film, and ion chamber in the better group; and the rotational composite and AP field-by-field diode array in the poorer group. Additionally, optimal threshold cutoffs were determined for each of the dosimetry systems. These findings, combined with practical considerations for factors such as labor and cost, can aid a clinic in its choice of an effective and safe patient-specific IMRT QA implementation.

CHARACTERIZING AND TREATING THE NEUROPATHOLOGY OF TUBEROUS SCLEROSIS COMPLEX IN THE MOUSE

Tuberous sclerosis complex (TSC) is a multisystem, autosomal dominant disorder affecting approximately 1 in 6000 births. Developmental brain abnormalities cause substantial morbidity and mortality and often lead to neurological disease including epilepsy, cognitive disabilities, and autism. TSC is caused by inactivating mutations in either TSC1 or TSC2, whose protein products are known inhibitors of mTORC1, an important kinase regulating translation and cell growth. Nonetheless, neither the pathophysiology of the neurological manifestations of TSC nor the extent of mTORC1 involvement in the development of these lesions is known. Murine models would greatly advance the study of this debilitating disorder. This thesis will describe the generation and characterization of a novel brain-specific mouse model of TSC, Tsc2flox/ko;hGFAP-Cre. In this model, the Tsc2 gene has been removed from most neurons and glia of the cortex and hippocampus by targeted Cre-mediated deletion in radial glial neuroprogenitor cells. The Tsc2flox/ko;hGFAP-Cre mice fail to thrive beginning postnatal day 8 and die from seizures around 23 days. Further characterization of these mice demonstrated megalencephaly, enlarged neurons, abnormal neuronal migration, altered progenitor pools, hypomyelination, and an astrogliosis. The similarity of these defects to those of TSC patients establishes this mouse as an excellent model for the study of the neuropathology of TSC and testing novel therapies. We further describe the use of this mouse model to assess the therapeutic potential of the macrolide rapamycin, an inhibitor of mTORC1. We demonstrate that rapamycin administered from postnatal day 10 can extend the life of the mutant animals 5 fold. Since TSC is a neurodevelopmental disorder, we also assessed in utero and/or immediate postnatal treatment of the animals with rapamycin. Amazingly, combined in utero and postnatal rapamycin effected a histologic rescue that was almost indistinguishable from control animals, indicating that dysregulation of mTORC1 plays a large role in TSC neuropathology. In spite of the almost complete histologic rescue, behavioral studies demonstrated that combined treatment resulted in poorer learning and memory than postnatal treatment alone. Postnatally-treated animals behaved similarly to treated controls, suggesting that immediate human treatment in the newborn period might provide the most opportune developmental timepoint for rapamycin administration.

Chipping Away at the Monolith: Dispelling the Myth of Father Noninvolvement in Children's Early Literacy Development

Current research stresses the importance of parent involvement in their children 's academic development. Parents reading and writing with their young children is shown to prepare them for the benefits of for ma I education. Studies completed on parent participation in early literacy activities have tended to look at mothers ' role. Few researchers have investigated the contributions fathers have made. The results of a study completed on father-child early literacy practices are presented. Fathers reported engaging in reading and writing activities with their children for three reasons: To prepare their children for school, to bond with their children, and to assist their children in language skill development. Recommendations are provided on how to encourage fathers to participate in early literacy practices

Integrin function in T cell activation recapitulated in tumor growth

An in vitro model using highly purified freshly isolated T cells demonstrated that immobilized ligands for the integrin $\alpha4\beta1$ could cooperate to enhance mitogen signals delivered by coimmobilized anti-CD3 specfic monoclonal antibody OKT3. Costimulation through $\alpha4\beta1$ integrin lead to enhanced proliferation which depended on expression of both IL-2 as well as IL-2 receptor. The transcription factors NF-AT, AP-1, and NF-$\kappa$B, which are involved in the regulation of IL-2 as well as other cytokine genes, were weakly induced by anti-CD3 stimulation alone in electromobility shift assays, but were augmented significantly with $\alpha4\beta1$ costimulation. These results suggested that $\alpha4\beta1$ ligands delivered a growth promoting signal which could synergize with signals induced by engagement of the TCR/CD3 complex, and also suggested a dual function for integrins in both localization and subsequent delivery of a growth promoting signal for T lymphocytes. Integrin involvement in lymphocyte trafficking has been employed as a model for understanding tumor cell metastasis. Therefore we have extended the duality of integrin function in both homing and subsequent delivery of a growth promoting signal to include a role for integrins in providing growth stimulation for tumor cells. Using a gastric derived tumor line, inhibition of adhesion to substrate leads to G0/G1 cell cycle arrest, reduced cyclin A expression, and reduced phospholipid synthesis. This effect could be reversed upon $\alpha2\beta1$ integrin mediated reattachment to collagen. These observations demonstrated a role for an integrin in the growth regulation of a tumor line. The small GTP-binding protein Rho, implicated in phospholipid synthesis, can be inactivated by the ADP-ribosylation exoenzyme C3 from C. botulinum. Addition of C3 to cell cultures inhibited the growth promoting effect due to integrin mediated adhesion. Taken together, these results are consistent with a model for cooperative interaction between integrins and Rho leading to enhanced phospholipid synthesis and mitogen signaling. This model may provide a basis for understanding the phenomena of integrin costimulation in T cell activation. ^

The role of specific regions of ribosomal RNAs in translation termination

The ribosome is a molecular machine that produces proteins in a cell. It consists of RNAs (rRNAs) and proteins. The rRNAs have been implicated in various aspects of protein biosynthesis supporting the idea that they function directly in translation. In this study the direct involvement of rRNA in translation termination was hypothesized and both genetic and biochemical strategies were designed to test this hypothesis. As a result, several regions of rRNAs from both ribosomal subunits were implicated in termination. More specifically, the mutation G1093A in an RNA of the large subunit (23S rRNA) and the mutation C1054A in the small subunit RNA (16S rRNA) of the Escherichia coli ribosome, were shown to affect the binding of the proteins that drive termination, RF1 and RF2. These mutations also caused defects in catalysis of peptidyl-tRNA hydrolysis, the last step of termination. Furthermore, the mutations affected the function of RF2 to a greater extent than that of RF1, a striking result considering the similarity of the RFs. The major defect in RF2 function was consistent with in vivo characteristics of the mutants and can be explained by the inability of the mutant rRNA sites to activate the hydrolytic center, that is the catalytic site for peptidyl-tRNA hydrolysis. Consistent with this explanation is the possibility of a direct interaction between the G1093-region (domain II of 23S rRNA) and the hydrolytic center (most likely domains IV–VI of 23S rRNA). To test that interaction hypothesis selections were performed for mutations in domains IV–VI that compensated for the growth defects caused by G1093A. Several compensatory mutations were isolated which not only restored growth in the presence of G1093A but also appeared to compensate for the termination defects caused by the G1093A. Therefore these results provided genetic evidence for an intramolecular interaction that might lead to peptidyl-tRNA hydrolysis. Finally, a new approach to the study of rRNA involvement in termination was designed. By screening a library of rRNA fragments, a fragment of the 23S rRNA (nt 74-136) was identified that caused readthrough of UGA. The antisense RNA fragment produced a similar effect. The data implicated the corresponding segment of intact 23S rRNA in termination. ^

Cytochrome P450 2D18 in the mammalian brain: Recombinant expression, purification, and characterization of xenobiotic and endobiotic metabolism

The cytochromes P450 (P450) comprise a superfamily of hemoproteins that function in concert with NADPH-cytochrome P450 reductase (P450-reductase) to metabolize both endogenous and exogenous compounds. Many pharmacological agents undergo phase I metabolism by this P450 and P450-reductase monooxygenase system. Phase I metabolism ensures that these highly hydrophobic xenobiotics are made more hydrophilic, and hence easier to extrude from the body. While the majority of phase I metabolism occurs in the liver, metabolism in extrahepatic organ-systems like the intestine, kidney, and brain can have important roles in drug metabolism and/or efficacy. ^ While P450-mediated phase I metabolism has been well studied, investigators have only recently begun to elucidate what physiological roles P450 may have. One way to approach this question is to study P450s that are highly or specifically expressed in extrahepatic tissues. In this project I have studied the role of a recently cloned P450 family member, P450 2D18, that was previously shown to be expressed in the rat brain and kidney, but not in the liver. To this end, I have used the baculovirus expression system to over-express recombinant P450 2D18 and purified the functional enzyme using nickel and hydroxylapatite chromatography. SDS-PAGE analysis indicated that the enzyme was purified to electrophoretic homogeneity and Western analysis showed cross-reactivity with rabbit anti-human P450 2D6. Carbon monoxide difference spectra indicated that the purified protein contained no denatured P450 enzyme; this allowed for further characterization of the substrates and metabolites formed by P450 2D18-mediated metabolism. ^ Because P450 2D18 is expressed in brain, we characterized the activity toward several psychoactive drugs including the antidepressants imipramine and desipramine, and the anti-psychotic drugs chlorpromazine and haloperidol. P450 2D18 preferentially catalyzed the N-demethylation of imipramine, desipramine, and chlorpromazine. This is interesting given the fact that other P450 isoforms form multiple metabolites from such compounds. This limited metabolic profile might suggest that P450 2D18 has some unique function, or perhaps a role in endobiotic metabolism. ^ Further analysis of possible endogenous substrates for P450 2D18 led to the identification of dopamine and arachidonic acid as substrates. It was shown that P450 2D18 catalyzes the oxidation of dopamine to aminochrome, and that the enzyme binds dopamine with an apparent KS value of 678 μM, a value well within reported dopamine concentration in brain dopaminergic systems. Further, it was shown that P450 2D18 binds arachidonic acid with an apparent KS value of 148 μM, and catalyzes both the ω-hydroxylation and epoxygenation of arachidonic acid to metabolites that have been shown to have vasoactive properties in brain, kidney, and heart tissues. These data provide clues for endogenous roles of P450 within the brain, and possible involvement in the pathogenesis of Parkinson's disease. ^

14-3-3zeta overexpression in multiple human cancers plays a critical role in cancer progression

14-3-3 is a family of highly conserved and ubiquitously expressed proteins in eukaryotic organisms. 14-3-3 isoforms bind in a phospho-serine/threonine-dependent manner to a host of proteins involved in essential cellular processes including cell cycle, signal transduction and apoptosis. We fortuitously discovered 14-3-3 zeta overexpression in many human primary cancers, such as breast, lung, and sarcoma, and in a majority of cancer cell lines. To determine 14-3-3 zeta involvement in breast cancer progression, we used immunohistochemical analysis to examine 14-3-3 zeta expression in human primary invasive breast carcinomas. High 14-3-3 zeta expression was significantly correlated with poor prognosis of breast cancer patients. Increased expression of 14-3-3 zeta was also significantly correlated with elevated PKB/Akt activation in patient samples. Thus, 14-3-3 zeta is a marker of poor prognosis in breast cancers. Furthermore, up-regulation of 14-3-3 zeta enhanced malignant transformation of cancer cells in vitro. ^ To determine the biological significance of 14-3-3 zeta in human cancers, small interfering RNAs (siRNA) were used to specifically block 14-3-3 zeta expression in cancer cells. 14-3-3 zeta siRNA inhibited cellular proliferation by inducing a G1 arrest associated with up-regulation of p27 KIP1 and p21CIP1 cyclin dependent kinase inhibitors. Reduced 14-3-3 zeta inhibited PKB/Akt activation while stimulating the p38 signaling pathway. Silencing 14-3-3 zeta expression also increased stress-induced apoptosis by caspase activation. Notably, 14-3-3 zeta siRNA inhibited transformation related properties of breast cancer cells in vitro and inhibited tumor progression of breast cancer cells in vivo. 14-3-3 zeta may be a key regulatory factor controlling multiple signaling pathways leading to tumor progression. ^ The data indicate 14-3-3 zeta is a major regulator of cell growth and apoptosis and may play a critical role in the development of multiple cancer types. Hence, blocking 14-3-3 zeta may be a promising therapeutic approach for numerous cancers. ^

Pediatric antiretroviral adherence and child disclosure in Botswana

The scale-up of antiretrovirals (ARVs) to treat HIV/AIDS in Africa has been rapid over the last five years. Botswana was the first African nation to roll out a comprehensive ARV program, where ARVs are available to all citizens who qualify. Excellent adherence to these ARVs is necessary to maintain HIV suppression and on-going health of all individuals taking them. Children rely almost entirely on their caregivers for the administration of these medications, and very little research has been done to examine the factors which affect both adherence and disclosure to the child of their HIV status. ^ Methods. This cross-sectional study used multiple methods to examine adherence, disclosure, and stigma across various dimensions of the child and caregiver's lives, including 30 caregiver questionnaires, interviewer-administered 3-day adherence recalls, pharmacy pill counts, and chart reviews. Fifty in-depth interviews were conducted with caregivers, male caregivers, teenagers, and health care providers. ^ Results. Perceived family stigma was found to be a predictor of excellent adherence. After controlling for age, children who live with their mothers were significantly less likely to know their HIV status than children living with any other relative (OR=0.403, p=0.014). Children who have a grandmother living in the household or taking care of them each day are significantly more likely to have optimal adherence than children who don't have grandmother involvement in their daily lives. ^ Discussion. Visible illness plays an intermediary role between adherence and perceived family stigma: Caregivers know that ARVs suppress physical manifestations of HIV, and in an effort to avoid unnecessary disclosure of the child's status to family members, therefore have children with excellent adherence. Grandmothers play a vital role in supporting the care and treatment of children in Botswana. ^