A biochemical and structural characterization of Drosophila neuroglian

A number of cell-cell interactions in the nervous system are mediated by immunoglobulin gene superfamily members. For example, neuroglian, a homophilic neural cell adhesion molecule in Drosophila, has an extracellular portion comprising six C- 2 type immunoglobulin-like domains followed by five fibronectin type III (FnIII) repeats. Neuroglian shares this domain organization and significant sequence identity with Ll, a murine neural adhesion molecule that could be a functional homologue. Here I report the crystal structure of a proteolytic fragment containing the first two FnIII repeats of neuroglian (NgFn 1,2) at 2.0Å. The interpretation of photomicrographs of rotary shadowed Ng, the entire extracellular portion of neuroglian, and NgFnl-5, the five neuroglian Fn III domains, is also discussed.

The structure of NgFn 1,2 consists of two roughly cylindrical β-barrel structural motifs arranged in a head-to-tail fashion with the domains meeting at an angle of ~120, as defined by the cylinder axes. The folding topology of each domain is identical to that previously observed for single FnIII domains from tenascin and fibronectin. The domains of NgFn1,2 are related by an approximate two fold screw axis that is nearly parallel to the longest dimension of the fragment. Assuming this relative orientation is a general property of tandem FnIII repeats, the multiple tandem FnIII domains in neuroglian and other proteins are modeled as thin straight rods with two domain zig-zag repeats. When combined with the dimensions of pairs of tandem immunoglobulin-like domains from CD4 and CD2, this model suggests that neuroglian is a long narrow molecule (20 - 30 Å in diameter) that extends up to 370Å from the cell surface.

In photomicrographs, rotary shadowed Ng and NgFn1-5 appear to be highly flexible rod-like molecules. NgFn 1-5 is observed to bend in at least two positions and has a mean total length consistent with models generated from the NgFn1,2 structure. Ng molecules have up to four bends and a mean total length of 392 Å, consistent with a head-to-tail packing of neuroglian's C2-type domains.

Statistical and econometric analysis of the treatment of HIV/AIDS

The epidemic of HIV/AIDS in the United States is constantly changing and evolving, starting from patient zero to now an estimated 650,000 to 900,000 Americans infected. The nature and course of HIV changed dramatically with the introduction of antiretrovirals. This discourse examines many different facets of HIV from the beginning where there wasn't any treatment for HIV until the present era of highly active antiretroviral therapy (HAART). By utilizing statistical analysis of clinical data, this paper examines where we were, where we are and projections as to where treatment of HIV/AIDS is headed.

Chapter Two describes the datasets that were used for the analyses. The primary database utilized was collected by myself from an outpatient HIV clinic. The data included dates from 1984 until the present. The second database was from the Multicenter AIDS Cohort Study (MACS) public dataset. The data from the MACS cover the time between 1984 and October 1992. Comparisons are made between both datasets.

Chapter Three discusses where we were. Before the first anti-HIV drugs (called antiretrovirals) were approved, there was no treatment to slow the progression of HIV. The first generation of antiretrovirals, reverse transcriptase inhibitors such as AZT (zidovudine), DDI (didanosine), DDC (zalcitabine), and D4T (stavudine) provided the first treatment for HIV. The first clinical trials showed that these antiretrovirals had a significant impact on increasing patient survival. The trials also showed that patients on these drugs had increased CD4+ T cell counts. Chapter Three examines the distributions of CD4 T cell counts. The results show that the estimated distributions of CD4 T cell counts are distinctly non-Gaussian. Thus distributional assumptions regarding CD4 T cell counts must be taken, into account when performing analyses with this marker. The results also show the estimated CD4 T cell distributions for each disease stage: asymptomatic, symptomatic and AIDS are non-Gaussian. Interestingly, the distribution of CD4 T cell counts for the asymptomatic period is significantly below that of the CD4 T cell distribution for the uninfected population suggesting that even in patients with no outward symptoms of HIV infection, there exists high levels of immunosuppression.

Chapter Four discusses where we are at present. HIV quickly grew resistant to reverse transcriptase inhibitors which were given sequentially as mono or dual therapy. As resistance grew, the positive effects of the reverse transcriptase inhibitors on CD4 T cell counts and survival dissipated. As the old era faded a new era characterized by a new class of drugs and new technology changed the way that we treat HIV-infected patients. Viral load assays were able to quantify the levels of HIV RNA in the blood. By quantifying the viral load, one now had a faster, more direct way to test antiretroviral regimen efficacy. Protease inhibitors, which attacked a different region of HIV than reverse transcriptase inhibitors, when used in combination with other antiretroviral agents were found to dramatically and significantly reduce the HIV RNA levels in the blood. Patients also experienced significant increases in CD4 T cell counts. For the first time in the epidemic, there was hope. It was hypothesized that with HAART, viral levels could be kept so low that the immune system as measured by CD4 T cell counts would be able to recover. If these viral levels could be kept low enough, it would be possible for the immune system to eradicate the virus. The hypothesis of immune reconstitution, that is bringing CD4 T cell counts up to levels seen in uninfected patients, is tested in Chapter Four. It was found that for these patients, there was not enough of a CD4 T cell increase to be consistent with the hypothesis of immune reconstitution.

In Chapter Five, the effectiveness of long-term HAART is analyzed. Survival analysis was conducted on 213 patients on long-term HAART. The primary endpoint was presence of an AIDS defining illness. A high level of clinical failure, or progression to an endpoint, was found.

Chapter Six yields insights into where we are going. New technology such as viral genotypic testing, that looks at the genetic structure of HIV and determines where mutations have occurred, has shown that HIV is capable of producing resistance mutations that confer multiple drug resistance. This section looks at resistance issues and speculates, ceterus parabis, where the state of HIV is going. This section first addresses viral genotype and the correlates of viral load and disease progression. A second analysis looks at patients who have failed their primary attempts at HAART and subsequent salvage therapy. It was found that salvage regimens, efforts to control viral replication through the administration of different combinations of antiretrovirals, were not effective in 90 percent of the population in controlling viral replication. Thus, primary attempts at therapy offer the best change of viral suppression and delay of disease progression. Documentation of transmission of drug-resistant virus suggests that the public health crisis of HIV is far from over. Drug resistant HIV can sustain the epidemic and hamper our efforts to treat HIV infection. The data presented suggest that the decrease in the morbidity and mortality due to HIV/AIDS is transient. Deaths due to HIV will increase and public health officials must prepare for this eventuality unless new treatments become available. These results also underscore the importance of the vaccine effort.

The final chapter looks at the economic issues related to HIV. The direct and indirect costs of treating HIV/AIDS are very high. For the first time in the epidemic, there exists treatment that can actually slow disease progression. The direct costs for HAART are estimated. It is estimated that the direct lifetime costs for treating each HIV infected patient with HAART is between $353,000 to $598,000 depending on how long HAART prolongs life. If one looks at the incremental cost per year of life saved it is only $101,000. This is comparable with the incremental costs per year of life saved from coronary artery bypass surgery.

Policy makers need to be aware that although HAART can delay disease progression, it is not a cure and HIV is not over. The results presented here suggest that the decreases in the morbidity and mortality due to HIV are transient. Policymakers need to be prepared for the eventual increase in AIDS incidence and mortality. Costs associated with HIV/AIDS are also projected to increase. The cost savings seen recently have been from the dramatic decreases in the incidence of AIDS defining opportunistic infections. As patients who have been on HAART the longest start to progress to AIDS, policymakers and insurance companies will find that the cost of treating HIV/AIDS will increase.

Hyperfine interactions and nuclear structure

An automatic experimental apparatus for perturbed angular correlation measurements, capable of incorporating Ge(Li) detectors as well as scintillation counters, has been constructed.

The gamma-gamma perturbed angular correlation technique has been used to measure magnetic dipole moments of several nuclear excited states in the osmium transition region. In addition, the hyperfine magnetic fields, experienced by nuclei of 'impurity' atoms embedded in ferromagnetic host lattices, have been determined for several '4d' and '5d' impurity atoms.

The following magnetic dipole moments were obtained in the osmium transition region μ_2⁺(¹⁹⁰Os) = 0.54 ± 0.06 nm μ_4⁺(¹⁹⁰Os) = 0.88 ± 0.48 nm μ_2⁺(¹⁹²Os) = 0.56 ± 0.08 nm μ_2⁺(¹⁹²Pt) = 0.56 ± 0.06 nm μ_2^+’(¹⁹²Pt) = 0.62 ± 0.14 nm.

These results are discussed in terms of three collective nuclear models; the cranking model, the rotation-vibration model and the pairing-plus-quadrupole model. The measurements are found to be in satisfactory agreement with collective descriptions of low lying nuclear states in this region.

The following hyperfine magnetic fields of 'impurities' in ferromagnetic hosts were determined; H_int(Cd Ni) = - (64.0 ± 0.8)kG H_int(Hg Fe) = - (440 ± 105)kG H_int(Hg Co) = - (370 ± 78)kG H_int(Hg Ni) = - (86 ± 22)kG H_int(Tl Fe) = - (185 ± 70)kG H_int(Tl Co) = - (90 ± 35)kG H_int(Ra Fe) = - (105 ± 20)kG H_int(Ra Co) = - (80 ± 16)kG H_int(Ra Ni) = - (30 ± 10)kG, where in H_int(AB); A is the impurity atom embedded in the host lattice B. No quantitative theory is available for comparison. However, these results are found to obey the general systematics displayed by these fields. Several mechanisms which may be responsible for the appearance of these fields are mentioned.

Finally, a theoretical expression for time-differential perturbed angular correlation measurement, which duplicates experimental conditions is developed and its importance in data analysis is discussed.

The geochronology of the plutonic and metamorphic rocks of New Zealand

Extensive Rubidium-Strontium age determinations on both mineral and total rock samples of the crystalline rocks of New Zealand, which almost solely crop out in the South Island, indicate widespread plutonic and metamorphic activity occurred during two periods, one about 100-118 million years ago and the other about 340-370 million years ago. The former results date the Rangitata Orogeny as Cretaceous. They associate extensive plutonic activity with this orogeny which uplifted and metamorphosed the rocks of the New Zealand Geosyncline, although no field association between the metamorphosed geosynclinal rocks and plutonic rocks has been found. The Cretaceous plutonic rocks occur to the west in the Foreland Province in Fiordland, Nelson, and Westland, geographically separated from the Geosynclinal Province. Because of this synchronous timing of plutonic and high pressure metamorphic activity in spatially separated belts, the Rangitata Orogeny in New Zealand is very similar to late Mesozoic orogenic activity in many other areas of the circum-Pacific margin (Miyashiro, 1961).

The 340-370 million year rocks, both plutonic and metamorphic, have been found only in that part of the Foreland Province north of the Alpine Fault. There, they are concentrated along the west coast over a distance of 500 km, and appear scattered inland from the coast. Probably this activity marks the outstanding Phanerozoic stratigraphic gap in New Zealand which occurred after the Lower Devonian.

A few crystalline rocks in the Foreland Province north of the Alpine Fault with measured ages intermediate between 340 and 120 million years have been found. Of these, those with more than one mineral examined give discordant results. All of these rocks are tentatively regarded as 340-370 million year old rocks that have been variously disturbed during the Rangitata Orogeny, 100-120 million years ago.

In addition to these two periods, plutonic activity, dominantly basic and ultrabasic, but including the development of some rocks of intermediate and acidic composition, occurred along the margin of the Geosynclinal Province at its border with the Foreland Province during Permian times about 245 million years ago, and this activity possibly extended into the Mesozoic.

Evidence from rubidium-strontium analyses of minerals and a total rock, and from uranium, thorium, and lead analyses of uniform euhedral zircons from a meta-igneous portion of the Charleston Gneiss, previously mapped as Precambrian, indicate that this rock is a 350-370 million year old plutonic rock metamorphosed 100 million yea rs ago during the Rangitata Orogeny. No crystalline rocks with primary Precambrian ages have been found in New Zealand. However, Pb²⁰⁷/Pb₂₀₆ ages of 1360 million years and 1370 million years have been determined for rounded detrital zircons separated from each of two hornfels samples of one of New Zealand's olde st sedimentary units, the Greenland Series. These two samples were metamorphosed 345- 370 million years ago. They occur along the west coast, north of the Alpine Fault, at Waitaha River and Moeraki River, separated by 135 km. The Precambrian measured ages are most likely minimum ages for the oldest source area which provided the detrital zircons because the uranium, thorium and lead data are highly discordant. These results are of fundamental importance for the tectonic picture of the Southwest Pacific margin and demonstrate the existence of relatively old continental crust of some lateral extent in the neighborhood of New Zealand.