Preparative mass spectrometry applications in nanomaterials and organic synthesis

Preparative mass spectrometry was traditionally a vacuum based experiment where mass selected ion beams were deposited onto surfaces; however, recently ions have been manipulated in the open air to perform accelerated synthesis. The work of this dissertation focuses on the generation of charged ions from microdroplets and their use in organic synthesis and the preparation of atomically precise metal nanoclusters. First the mechanism for charging in the zero volt paper spray will be presented. This works builds a computational model based on the statistical fluctuation of positive and negative charges based on the theory of Dodd. It is found through experiment and simulation that in zero volt paper spray and presumably other zero volt methods the most surface active species are preferentially ionized. Charged microdroplets are exploited in this dissertation for two very different applications but in seemingly similar ways. First metal salts are electrosprayed, heated, and subjected to harsh in-source conditions to cause the reduction of the metal salts into atomically precise metal clusters. This method is capable of producing atomically precise silver cluster cations and anions as well as bimetallic silver-palladium complexes. Microdroplets are also exploited to screen chemical pathways to common pharmaceutical including atropine and diphenhydramine. The two step synthesis of diphenhydramine from benzhydrol was performed in charged microdroplets and confirmed in microfluidics. While the fundamental mechanism of reaction in microdroplets and microfluidics might differ useful connections were found in the cases of atropine, diphenhydramine, and diazepam. It is envisioned that this methodology could impact drug discovery as a rapid platform for the screening of chemical pathways to both old and new molecules. The droplet reaction was extended to a “super reagent” form where a reagent of choice is sprayed onto a variety of substrate contained in a well plate or on paper. This method allows for rapid screening with hopes of screening 1000’s of reaction conditions per hour

Immunohistochemical panel to characterize canine prostate carcinomas according to aberrant p63 expression

<div><p>An unusual variant of prostate adenocarcinoma (PC) expressing nuclear p63 in secretory cells instead of the typical basal expression has been reported in men. Nevertheless, the biological behavior and clinical significance of this phenomenon is unknown. In dogs, this unusual PC subtype has not been described. In this study, p63 immunoexpression was investigated in 90 canine PCs and 20 normal prostate tissues (NT). The p63 expression pattern in luminal or basal cells was confirmed in a selected group of 26 PCs and 20 NT by immunohistochemistry and/or Western blotting assays. Eleven canine PC samples aberrantly expressing p63 (p63+) in secretory cells were compared with 15 p63 negative (p63-) cases in the context of several molecular markers (high molecular weight cytokeratin-HMWC, CK8/18, CK5, AR, PSA, chromogranin, NKX3.1, PTEN, AKT and C-MYC). P63+ samples were positive for CK5, HMWC and CK8/18 and negative for PSA, NKX3.1, PTEN and chromogranin. Five p63+ PCs were negative for AR, and the remaining six samples had low AR expression. In contrast, p63- PC showed AR and PSA positive expression in all 15 samples. Only five p63- PCs were positive for CK5. Both p63+ and p63- PC samples showed higher cytoplasmic AKT expression and nuclear C-MYC staining in comparison with normal tissues. Metastatic (N = 12) and non-metastatic (N = 14) PCs showed similar immunoexpression for all markers tested. In contrast to human PC, canine PC aberrantly expressing p63 showed higher expression levels of HMWC and CK5 and lower levels of NKX3.1. Canine p63+ PC is a very rare PC group showing a distinct phenotype compared to typical canine PC, including AR and PSA negative expression. Although in a limited number of cases, p63 expression was not associated with metastasis in canine PC, and cytoplasmic p63 expression was observed in animals with shorter survival time, similar to human PC cases.</p></div

Immunohistochemical panel evaluated in normal canine prostate tissues and neoplastic prostate cells according to p63 expression.

<p>Immunohistochemical panel evaluated in normal canine prostate tissues and neoplastic prostate cells according to p63 expression.</p

Characterization of basal cell markers in normal prostate samples, canine p63+ PC and canine p63- PC.

<p>(A) p63 immunohistochemical expression in a normal prostate gland. Positive basal cells were observed in a discontinuous basal cell layer. (B) p63+ tumor showing diffuse nuclear p63 expression by neoplastic cells. (C) p63- tumor presenting no p63 expression (blue staining). Few non-neoplastic basal cells with nuclear p63 expression (internal control–black arrows) were observed. (D) Immunohistochemical staining of high molecular weight cytokeratin (HMWC) in a normal prostate gland. Positive staining was observed in few basal cells (brown staining—white arrows) establishing a discontinuous basal cell layer. (E and F) Diffuse HMWC expression in p63+ and p63- tumors, respectively. (G) CK5 immunohistochemical expression in canine normal samples. There was a discontinuous basal cell layer with few CK5 positive cells (brown staining). (H) p63+ tumor showing diffuse CK5 expression (brown staining) in all neoplastic cells. (I) p63 tumor showing no CK5 expression by neoplastic cells (blue staining). An atrophic prostatic gland (black arrow) was observed presenting diffuse CK5 expression (internal control). (J) CK8/18 expression in normal prostate. Positive cytoplasmic expression was observed in the epithelial luminal cells (brown staining). p63+ (K) and p63 (L) tumors presented diffuse cytoplasmic staining in all neoplastic cells (brown staining). The negative staining can be observed as a blue signal.</p

Immunohistochemical staining for NKX3.1, AR, PTEN, AKT AND C-MYC in canine prostate cancer.

<p>(A) Normal prostatic tissue presented strong expression of NKX3.1. All p63+ and p63- PCs were negative for NKX3.1 staining (B and C). D: Strong expression of AR was observed in normal prostatic tissue. (E and F) Few neoplastic and basal cells were positive for AR in p63+ PC and p63- PC. (G) Normal prostatic tissue presented strong expression of PTEN. p63+ PC (H) and p63- PC (L) showed low PTEN expression. (J) Normal prostatic tissue showed AKT negative PTEN expression (blue staining). (K and L) Strong expression in p63+ and P63- PC was noted. (M) Normal prostatic tissue showed negative C-MYC expression, and higher expression was noted in both p63+ (N) and p63- (O) tumors.</p

Photomicrograph showing the canine prostate cancer.

<p><b>Double immunohistochemical staining for p63 and CK8/18.</b> (A) PC with p63-/CK8/18+ phenotype. It is possible to note cytoplasmic CK8/18 expression (red staining—arrow) in neoplastic cells and nuclear negative p63 staining in neoplastic cells. There is a p63+/CK8/18- basal cell (arrowhead) (internal control). Negative signal can be observed as the blue color. (B) PC with p63+/CK8/18+ phenotype. Note the neoplastic doubled stained cells (arrows). It is also observed p63-/CK8/18+ cells (arrowhead) and p63+/CK8/18- basal cells (doted circle).</p

Western blotting analysis.

<p>There was no difference in p63 expression in tumor samples compared to normal samples (A and C) and there was higher expression of HMWC in tumor samples compared with normal samples (B and D).</p